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Metalloporphyrins for Medical Imaging Applications
CHAPTER FOUR
Metalloporphyrins for Medical Imaging Applications Francesca Bryden1, Ross W. Boyle Department of Chemistry, University of Hull, Hull, United Kingdom 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Fluorescence Imaging 2.1 Phthalocyanine Dyes 2.2 Nanoscale Structures 2.3 FRET Systems 3. Raman Imaging 4. Gamma Imaging 4.1 Indium-111 4.2 Technecium 99m 4.3 Gallium 67 4.4 Other Radionuclides 5. Positron Imaging (PET) 5.1 Copper 64 5.2 Gallium 68 5.3 Other Isotopes 6. Magnetic Resonance Imaging 6.1 Gadolinium 6.2 Manganese 7. Photo-Acoustic Imaging 8. Multimodal Imaging 8.1 SPECT–Fluorescence 8.2 Photo-Acoustic/Fluorescence 8.3 PET–Fluorescence 8.4 MRI/Fluorescence 8.5 PET/MRI 9. Conclusions References
Advances in Inorganic Chemistry, Volume 68 ISSN 0898-8838 http://dx.doi.org/10.1016/bs.adioch.2015.09.003
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2016 Elsevier Inc. All rights reserved.
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Abstract In this account, the synthesis and biological evaluation of metalloporphyrins and related structures for use in biomedical imaging applications are outlined. The structural design of these tetrapyrrolic metal chelators is discussed, with evaluation of strategies designed to facilitate more rapid chelation under milder conditions, improve water solubility, and allow for conjugation to targeting groups and macromolecular structures. Particular emphasis is given to the application of these metalloporphyrin structures to clinically relevant imaging techniques including magnetic resonance imaging, fluorescence imaging, radio-imaging, and Raman and photo-acoustic imaging, with a broad overview of the progress made in these areas. The future of research in this area is also examined, with the emerging research interest in more modern multimodal imaging set to dominate the future of biomedical imaging.
1. INTRODUCTION Porphyrins are a class of macrocycles comprised of four pyrrole units conjugated through methine bridges, with this highly conjugated structure giving intense absorption in both the UV and visible regions of the electromagnetic spectrum. This in turn leads to both the characteristic purple color of these structures and their name; the word porphyrin is derived from the Greek porphyra, meaning purple. Modifications to the porphyrin structure including alterations to the degree of conjugation and number of heteroatoms can be carried out to produce a host of tetrapyrrole structures, including the naturally occurring chlorins and bacteriochlorins (Figure 1).
Porphyrin
Chlorin
N
N
N
HN
N NH
N
N
N
N HN
Bacteriochlorin
N
NH N
NH
HN
N
HN
N
N
NH
N
NH
N
HN
N HN
N
N
N
Phthalocyanine
Porphyrazine
NH HN
Corrole
Figure 1 Skeleton structures of porphyrin and a range of other tetrapyrrolic molecules.
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Porphyrins are most well known for their essential roles within a number of biological processes, most notably as the iron-containing heme which permits hemoglobin oxygen transportation in the blood of all vertebrates. It is therefore unsurprising that the first examples of porphyrins described in the literature were produced from endogenous sources, with Nencki and Zeleski first describing the isolation of hematoporphyrin from hemoglobin in 1900 (1). Subsequently, a range of both endogenous and synthetic exogenous porphyrins have been described, in addition to numerous alternative tetrapyrrolic structures; while these structures are of interest due to their enhanced photophysical characteristics and altered reactivity, their challenging synthesis and functionalization make them less commonly utilized in comparison to porphyrins. Although porphyrins and related structures have been exploited in many applications including light harvesting (2) and the development of supramolecular structures (3), they are best known for their use in medical applications, in particular as photosensitizers in photodynamic therapy. These photosensitizers are essentially nontoxic in the absence of light; however, upon irradiation these structures can undergo a cascade of photochemical processes to produce cytotoxic reactive oxygen species, capable of triggering cell death and leading to tumor eradication. However, there is also a growing interest in porphyrins and other tetrapyrroles for use in diagnostic medical applications, with the inherent fluorescence, facile derivatization, low cytotoxicity in the absence of light, and preferential tumor uptake of these structures, making them highly attractive as diagnostic modalities as well as therapeutics. To date, the vast majority of research into porphyrins for medical applications has exploited the use of free-base porphyrins; these unmetallated structures are generally easier to synthesize, and chelation of paramagnetic metals into the porphyrin core is incompatible with many uses, causing quenching of the porphyrin excited state and associated loss of both fluorescence and therapeutic activity. Despite this, metalloporphyrins also offer significant research interest; porphyrins are highly efficient metal chelators, with the planar porphyrin ring distorting to allow chelation to metals which are either too small or too large to fit well into the porphyrin cavity, and to date complexes hosting a range of at least 50 metals have been described in the literature (4). In particular, the utility of metalloporphyrins as imaging agents is a relatively unappreciated but growing field, with the metallation of porphyrins offering application of the natural tumor affinity of porphyrins to imaging techniques including fluorescence, magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT).
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2. FLUORESCENCE IMAGING Fluorescence imaging was one of the earliest medical imaging techniques to be adopted, exploiting the innate property of some chromophores to absorb light at a specific wavelength and subsequently reemit light at a longer wavelength. Autofluorescence of hematoporphyrin in a rat tumor upon irradiation with UV light was first observed in 1921 (5), while the first deliberate application of fluorescence to tumor imaging was carried out in 1942, with administered hematoporphyrin showing localization in both tumor tissue and lymph nodes (6). Despite this long history, fluorescence imaging is still highly relevant to clinical imaging applications today, allowing both in vivo and in vitro imaging on the micro- and macroscale. In addition, fluorescence imaging offers high sensitivity, rapid imaging, and continuous real-time monitoring without high cost or the need for ionizing radiation, and as a result, it is still one of the most popular imaging techniques. Despite this, the use of light to both activate and detect the imaging agent presents challenges; both resolution (2–3 mm) and imaging depth (<1 cm) are limited, and obtaining quantitative data is difficult (7). Due to the well-understood photophysical properties of porphyrins, much of the early literature focusses on the use of free-base porphyrins as fluorescence imaging agents. However, metalloporphyrins and derivatives, in particular metallophthalocyanines, are also of significant interest for optical imaging. Although metallation can affect the wavelength and intensity of both the UV–vis and fluorescence spectra of tetrapyrrolic structures, it allows greater flexibility in the construction of synthetic reaction schemes as well as optimization of photophysical characteristics of the fluorescence imaging agent.
2.1 Phthalocyanine Dyes Phthalocyanines show particular promise in the area of fluorescence imaging due to their favorable optical properties, with their high molar absorptivity (ε > 200,000 M1 cm1) (8) at wavelengths greater than 680 nm allowing for good quality fluorescent images to be produced using exciting light in the red and near-infrared portion of the electromagnetic spectrum. As a result of this shift in wavelength, both sensitivity of the technique and light penetration in human tissue increase significantly due to a simultaneous reduction in both autofluorescence from endogenous fluorophores, and light scattering in the near-infrared region. Metallophthalocyanines
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incorporating diamagnetic metals such as aluminum, zinc, and gallium are of particular interest in this application due to their increased fluorescence quantum yield and increased stability, while no significant quenching of fluorescence is observed, in contrast to paramagnetic metals such as copper. Chloroaluminum sulfophthalocyanine was the first metallophthalocyanine utilized for optical imaging, with its excellent photostability and long emission lifetimes (ca. 7.5 ns), making it an ideal chromophore for time-delayed fluorescence imaging. Although its use in vivo was not explored, in vitro tests in a model tissue phantom demonstrated excellent potential as a fluorescence imaging agent, operating well over a range of concentrations (9). However, despite these early promising results and the commercial availability of the sulfonated aluminum phthalocyanine derivative Photosens® as a therapeutic agent in Russia, no attempt to utilize this phthalocyanine in fluorescence imaging has been published. In contrast, zinc phthalocyanine derivatives have been widely explored as optical imaging agents, particularly in the imaging of neoplastic conditions. The first example of this was carried out by Mantavera et al., who utilized a range of cationic zinc phthalocyanines to image B16 melanoma; a highly pigmented cancer which cannot be imaged easily with traditional porphyrin imaging agents due to the strong visible absorption of the highly pigmented melanoma. Functionalization of cationic phthalocyanines with varying lengths of alkyl chain produced a range of phthalocyanines with varying amphiphilicity which retained photophysical properties of the parent phthalocyanine, demonstrating excellent light absorption outside the absorption range of melanin. In vitro uptake of all three dyes into melanoma cells over time was monitored; with 2 demonstrating the best uptake profile, attributed to the highly amphiphilic nature of the molecule. In vivo imaging also demonstrated promising results, with a mouse model showing good (2.4–3.8 higher fluorescence) differentiation between normal skin and tumors measuring 3–5 mm, although fluorescence of smaller tumors was minimal (Figure 2) (10). Imparting water solubility on zinc phthalocyanines has also been demonstrated through peripheral functionalization with a range of mono- and oligosaccharides. In addition to improving hydrophilicity, these saccharides may also demonstrate a tumor-targeting effect, with the overexpression of glucose transporting and galactose receptors reported on the cell surface of some neoplastic tissues (11). This strategy was first utilized in the synthesis of fluorescent probes by Liu et al., who utilized a click chemistry to conjugate a zinc phthalocyanine to four azide-functionalized glucose groups (4).
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R N+ 1
N+ R
R=
O
O N 2
R=
N N
4 3
N Zn
N
R=
N N
N 10
O
O
R N+ N+ R
Figure 2 Cationic zinc phthalocyanines 1–3, with differing aliphatic chain lengths allowing modification of amphiphilicity.
Analysis of 4 in DMSO demonstrated retention of clinically relevant photophysical properties: absorbance at 618 and 682 nm, fluorescence at 690 nm with a high fluorescence quantum yield (0.48), and no significant photobleaching on timescales relevant to imaging. Despite the observation of significant aggregation of the photosensitizer and corresponding complete fluorescence quenching during in vitro imaging, imaging in an animal model showed no fluorescence quenching, with the difference between in vivo and in vitro results attributed to disaggregation of 4 following administration. While 4 demonstrated clear liver and kidney uptake and no associated organ toxicity, tumor uptake was found to be negligible, with fluorescence shown to be no higher than in control animals (Figure 3) (12). As a result of the demonstrated limited tumor affinity of glucose moieties, the same group subsequently utilized an alternative saccharide, galactose, for targeting liver cancer. Synthesis of a series of phthalocyanines bearing between 1 and 4 galactose groups was carried out (5–8), with only 7 and 8 exhibiting complete water solubility. As for 4, while aggregation in aqueous media led to weak fluorescence in vitro, all phthalocyanines demonstrated good in vivo fluorescence. Optical imaging in a mouse model demonstrated clear fluorescence of the tumor in comparison to surrounding tissue when utilizing phthalocyanines 7 and 8, with the best images obtained between 12 and 24 h after administration. In contrast, imaging agents 5 and 6 showed no significant increase in tumor fluorescence compared to the
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OH HO
HO O
HO
OH
O
O
N N N
N OH
HO HO
O
N N N
OH
OH
N N
N
O
O
N Zn
N
HO HO
N N N
N
N
OH
HO
O
N N N
4
O
OH OH
Figure 3 Structure of glucose-functionalized zinc phthalocyanine 4.
R1
R4
N
OH
Zn
N N
N R3
O
HO N
N
HO
O
N N
R=
R2
OH
5: R1 = R, R2 = H, R3 = H, R4 = H 6: R1 = R, R2 = H, R3 = R, R4 = H 7: R1 = R, R2 = R, R3 = R, R4 = H 8: R1 = R, R2 = R, R3 = R, R4 = R
Figure 4 Zinc phthalocyanines 5–8, with the differing levels of saccharide functionalization shown to have significant effects on both water solubility and pharmacokinetic character of the imaging agents.
control, with this lack of fluorescence attributed to poor solubility and an associated enhanced potential for aggregation (Figure 4) (13). An alternative to galactose for the targeting of liver cancer is the disaccharide lactose; similar to galactose, it allows active targeting due to the hepatic asialoglycoprotein receptors overexpressed on the surface of many liver tumors, while also offering improved water solubility. A click conjugation strategy was again used by Lv et al. to produce the saccharidesubstituted phthalocyanine 9, with four lactose units attached per phthalocyanine. While photophysical properties were found to be similar to previously synthesized optical imaging agents 4–8, in contrast 9 also showed limited cytotoxicity and improved photostability. In vivo evaluation in a mouse model demonstrated good selectivity, with a clearly visible signal
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R N N N
Francesca Bryden and Ross W. Boyle
O
O N N N
HO OH O
R= HO
OH
HO O
O
OH
N Zn
N
N R N N
OH
N N
N R N N N
O
O
N R N N
9
Figure 5 Saccharide-substituted zinc phthalocyanine 9. The use of the disaccharide lactose was shown to allow improved potential for active targeting of ASGP receptor overexpression on some tumors.
from the tumor and limited fluorescence in nontarget organs, while nontarget tumors, including lung and melanoma models, showed no uptake over a 12-h period (Figure 5) (14). The utility of this lactose-functionalized phthalocyanine has also been demonstrated in sentinel lymph node mapping. In vivo imaging utilizing both lactose-functionalized 9 and glucose-functionalized 4 was carried out in mice, with 9 showing strong sentinel lymph node fluorescence after just 10 min with visible fluorescence persisting for over 30 min, while only background levels of fluorescence were observed in nontarget muscle and fat. In contrast, 4 was found to show no accumulation in the lymph nodes, with fluorescence no higher than the control (15).
2.2 Nanoscale Structures An alternative solution to the problem of imaging agent solubility is the use of nanoscale delivery vehicles, with the literature describing a plethora of recently developed nanoparticles including biological macromolecules, inorganic and organic nanotubes and nanoparticles, and liposome-like structures. All of the described structures allow for incorporation of fluorescent imaging agents via either encapsulation or conjugation, in both cases aiding solubility and allowing delivery of imaging agents directly to tumor tissue. In addition to aiding solubility, these macromolecular structures can also reduce aggregation and allow for greater concentrations of fluorophores through multiple loading onto the delivery platform, allowing for increased fluorescence signal and improved contrast ratios. The use of serum components, such as cholesterol as delivery vehicles, has been demonstrated, exploiting the increased catabolism and
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receptor-mediated uptake of cholesterol in some tumor cells (16). Wu et al. demonstrated the use of LDL cholesterol as a delivery platform for transportation of NIR-absorbing oligoporphyrin chromophores, for the treatment of B16 melanoma. Four examples of fluorescent probes were synthesized, all showing broad-spectrum light absorption and highly lipophilic character, with the aim of preventing leakage from the cholesterol in vivo. However, only 10 and 11 were successfully incorporated into LDL, with an average of 30 chromophores per LDL unit. In vitro studies demonstrated no toxicity even after 24 h, with both 10 and 11 showing rapid internalization into target cells, while the corresponding untargeted chromophores showed no uptake or fluorescence. Fluorescent imaging compared to a glucose-targeted pyropheophorbide control showed excellent fluorescent image quality for both 10 and 11, even at low dye concentrations (ca. 60 nM dye concentration), while the control required around 20 times higher dye concentrations to achieve the same image quality (Figure 6) (17). While highly conjugated oligoporphyrin species represent a novel methodology to allow increased NIR absorbance of optical imaging agents, use of the biomolecule LDL as a delivery vehicle limits structural flexibility as functionalization and derivatization of this natural biomolecule are complex processes. An alternative is the use of polymersomes, synthetic liposome-like structures which utilize amphiphilic block copolymers to form a vesicle membrane, allowing development of delivery vehicles with excellent synthetic flexibility and biocompatibility. Polymersomes allow transportation of the imaging agent via incorporation into the polymer shell, making delivery of large numbers of fluorophores to each cell possible, while fluorophore spacing within the polymersome can be carefully controlled, limiting fluorescence quenching. Four examples of NIR-emitting zinc oligoporphyrin structures were synthesized (14–17), with varying peripheral chain composition and length, and all structures were incorporated into diblock copolymers comprised of polyethylene-oxide and polybutadiene. Self-assembly of the copolymers in aqueous solution results in the formation of the polymersome structures. All synthesized polymersomes showed excellent stability under physiological conditions, with no dye leakage and minimal photobleaching observed in aqueous conditions. In vivo imaging efficacy was examined in a rat tumor model, with ex vivo imaging of excised tumors carried out to confirm localization of the polymersomes. In all cases, the polymersomes demonstrated excellent potential as imaging agents, showing signal:noise ratios of at least 10:1 and retention in tumor tissue following direct injection (18).
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R1
N
R2
N
N
N
N
Zn
R1
N
N
N
R1
N Zn
Zn N
N
R2
N
R1
O 10
R1 =
12
R1 =
O
O
O
COOEt R2 =
3
O
R2 =
O
O 3
11
13 R1 =
R1, R2 = O
O R2 =
O
O NH
O O
Figure 6 Oligoporphyrin optical imaging agents 10–13 showing the diversity of peripheral functionalization produced on these structures.
Subsequent work in this area has demonstrated the synthetic flexibility of the polymersome system, demonstrating modification of both the polymersomes and zinc porphyrin fluorophores to allow optimization of both biological and photophysical properties. Modulating the amphiphilicity, substitution, and size of the oligoporphyrin imaging agents was shown to alter both absorption and fluorescence maxima (19). Changing porphyrin loading ratios was shown to affect significantly polymersome stability (20), and the
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alteration of porphyrin and polymer functionalization allowed alterations to bulk optical properties (21) and excited-state dynamics (22) of the imaging modality (Figure 7). The formation of synthetic porphyrin-based micelles has also been demonstrated without the use of block copolymers. Nakamura et al. exploited the highly hydrophobic nature of zinc protoporphyrin IX to form a conjugate with the hydrophilic HPMA; the resulting amphiphilic structure 18 spontaneously formed micelles of 30–80 nm in aqueous solution. Micelles were synthesized containing ca. 20% zinc porphyrin by weight and, unlike the parent porphyrin, demonstrated excellent water solubility. While the intact micellar form demonstrated minimal fluorescence and singlet oxygen generation, in vitro fluorescence was observed following internalization of the micelles, suggesting that intracellular disintegration of the micelles allowed reduction of the observed fluorescence quenching. The efficacy of these micelles as imaging agents was demonstrated in vivo, with good tumor uptake and minimal fluorescence of surrounding tissues observed. Micelles of 18 demonstrated tumor:normal tissue contrast ratios of 5:1 and greater, while the control zinc protoporphyrin IX showed no tumor uptake and rapid clearance from the body (Figure 8) (23). As well as facilitating transportation of lipophilic porphyrins, porphyrin nanogels also allow containment of porphyrins, reducing damage caused by generation of reactive oxygen species during irradiation. The first example of metalloporphyrin hydrogel nanoparticles was obtained through the reaction of the bis azide PEG chain (20) and the gallium tetraalkyne porphyrin 19 to produce a cross-linked structure, with the size of the nanoparticles formed controlled by the quantity of emulsifier added to produce particles between 30 and 110 nm. UV–vis analysis was conducted on both the gallium porphyrin nanogels and the demetallated free-base porphyrin derivative. In all cases, the gallium porphyrin showed improved photophysical characteristics, with a significant red shift in both the absorption band (682 nm) and the fluorescence emission (725 nm) (24). Synthesis of tumor-targeted nanogels was also demonstrated utilizing a folate-functionalized bis azide PEG (21), producing uniform nanoparticles of 120 nm diameter. While photophysical evaluation showed minimal change in both the fluorescence and absorption spectra in comparison to the hydroxyl-functionalized nanogels, the targeting ability of the folate functionalization was not evaluated in a tumor model (Figure 9) (24). The use of nanoscale structures also allows the incorporation of multiple fluorescent imaging agents into a single structure, allowing exploitation of a
3
3
O
O
N
N
Zn N
O
O
O
N
N
Zn N
N
O
O
O
O
N
N
Zn N
N
O
O
N
N
Zn N
N
O
O
N Zn
N
N
O
O 14
O
O
3
N
O
O
3
O
O
O
N
O
O 15
Figure 7 Oligoporphyrin optical imaging agents 14–17, with structures containing between 3 and 5 zinc porphyrin units to allow for improved NIR imaging efficacy.
O
O
N
O
O
N
N
Zn N
O
N
N
Zn N
N
O
O
O
O
N
N
Zn N
N
O
O
16
Figure 7—Cont'd
O
O
N Zn
N
N
O
O
N
O
N
O
N
N
O
O
O
3
N
N
N
N
N
O
O
O
3
N
N
O
N
Zn N
N
O
O
O
O
Zn
O 17
Figure 7—Cont'd
3
3
N
Zn
3
Zn
O
O
O
O
N Zn
N
N
O
3
N
O
O
3
N
O
O
O
3
O
O
O
3
O
3
3
3
O
O
O
O
N
O
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NH O
N
O
S O
CN HN
O
N
N Zn
N
N
O O
HO
O N
N Zn
N
N O OH
18
Figure 8 Amphiphilic diporphyrin monomer 18, which in aqueous solutions spontaneously forms micelles with improved tumor uptake and optical imaging potential.
OH O
O
N3
O
O OH 20
N N
Ga
N3
45
N
N FA O
O
O
O
N3
N3
45
FA 21
19
Figure 9 The synthesis of both tumor-targeted and untargeted nanogels for optical imaging application was demonstrated utilizing precursors zinc tetraalkyne porphyrin 19 and diazide chains 20 and 21 via a click chemistry methodology.
greater proportion of visible light, as well as development of theranostic agents. Incorporation of both zinc tetrasulfophthalocyanine and an adamantyl-nitroaniline fluorescent imaging agent into an epichlorohydrinβ-cyclodextrin demonstrated both red and green fluorescence, as well as therapeutic singlet oxygen and NO generation. No interaction between
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the two chromophores was observed following encapsulation, with all photophysical properties of the single fluorophores retained. The suitability of the nanoconstructs as biocompatible vehicles with dual-color imaging capacity was demonstrated in melanoma cells, with fluorescence observed at both 457 and 640 nm (25). Imaging in human squamous carcinoma cells and skin tissue ex vivo was also carried out, with the nanostructure demonstrating cellular accumulation and visible fluorescence in human tissue (26).
2.3 FRET Systems The development of dual-chromophore fluorescence resonance energy transfer (FRET) systems as optical imaging probes is an area of growing research interest. FRET systems are comprised of two conjugated chromophores: a donor and an acceptor. When light excites the donor chromophore, energy is then transferred to the acceptor chromophore, which emits light through fluorescence. The transfer of energy in this way has numerous advantages, principally the ability to allow a very large Stokes shift between exciting and emitted light, reducing interference, and improving image quality. Chlorin–bacteriochlorin dyads were synthesized and explored as FRET fluorescence imaging agents, with either a zinc (22) or free-base chlorin (23) as the donor chromophore and the bacteriochlorin as the acceptor. While a large Stokes shift was observed for both structures, chelation of zinc led to a significant enhancement (110 nm) in comparison to 23 (85 nm), with good fluorescence quantum yields (0.19) and fluorescent lifetimes (5.3 ns) observed in both cases. Evaluation of the dyads in a tissue phantom in comparison to commercial cyanine dyes showed brighter images for the commercial dyes and significant quenching of the dyad fluorescence in polar solvents; however, both 22 and 23 demonstrated improved excitation and detection selectivity and longer fluorescence lifetimes, suggesting that they could be developed as promising alternatives to current commercial dyes (Figure 10) (27). Alternatively, conjugation of the chlorin donor to NIR-emitting lanthanide–DOTA acceptors has been demonstrated by Laakso et al., utilizing Nd- and Yb-chelated DOTA and both zinc and free-base chlorins. Photophysical properties of all synthesized dyads were evaluated, with shorter emission lifetimes observed for Nd than for Yb, and comparable results obtained for both zinc and free-base chlorins; however, no in vitro evaluation of these imaging agents was carried out (Figure 11) (28).
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N
N
N
N
NH
HN
M N
22 23
O N
M = Zn M = 2H
Figure 10 FRET imaging agents 22 and 23, with the combination of both chlorin and bacteriochlorin structures into a single agent allowing for significant enhancement of Stokes shift and fluorescence lifetimes in both structures.
Subsequently, the same group carried out a systematic analysis of a range of similar chlorin-functionalized DOTA–lanthanide dyads, evaluating the effect of altering both linker properties and metallation of the chlorin and cyclam units. Examination of photophysical properties of the synthesized metallochlorins demonstrated increasing absorption coefficients in the order 2H Ni < Pd < Zn < Cu, with a hypsochromic shift (30–50 nm) of absorption observed in all cases. Photophysical evaluation of the zinc chlorin dyads was also carried out, with the absorption spectra of the chlorin varying significantly based on the lanthanide attached; however, the very weak lanthanide emission could not be detected. In addition, the solubility of all complexes was found to be an issue, with all poorly soluble in water and insoluble in methanol (Figure 12) (29). Cyanine dyes have also been utilized as acceptor chromophores for metallochlorin donors; Ethirajan et al. demonstrated the synthesis of this FRET pair, conjugating a cyanine dye to nickel purpurinimide. Photophysical evaluation of the dyad suggested that FRET was taking place, with fluorescence emission observed from the cyanine dye following irradiation of the chlorin. Nickel metallation was also found to have a significant impact on the dyad, quenching singlet oxygen production and significantly increasing the photostability of the imaging agent in comparison to the free-base
O NH
N O
N
NH
NH
O
N O
HN N O
24
O
N Ln
N
N
NH
HN N
O
25
N
O
O O
O N
O
O
O
N O
Zn N
Ln N
O
O Ln = Nd, Yb
N
O
N
NH
N N 26
O
O
N Nd
N
O N
O O
O
Figure 11 DOTA–lanthanide/ chlorin FRET imaging agents 24–26, with varying metallation of both macrocycles shown to alter photophysical properties of the structures.
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N
N O
Zn
N N N
N
N
N 27
O
N Yb
O
O
N
N
O O N
N
O
N O
Zn N
O
N 28
N
N N
N O
Ln
O N
N O
Ln = Yb, Gd, Nd
O
O
Figure 12 Structures of DOTA–lanthanide/zinc chlorin NIR FRET imaging agents 27 and 28. −
O3S N+
29 30
M = 2H M = Ni
O S
N
HN
N M
N
N
N+ −
O3S
O MeOOC
N
O
hexyl
Figure 13 Metallochlorin-cyanine FRET structures 29 and 30. Nickel metallation of 30 allowed for improved photostability of the structure and improved pharmacokinetic profile during imaging.
derivative 29. Fluorescence imaging was carried out in a mouse tumor model, with 30 showing good tumor specificity and uptake, allowing clear visualization of the tumor. In comparison, 29 showed a significantly higher uptake in the liver, kidneys, and spleen, and poor tumor fluorescence, confirming the importance of the metalloporphyrin unit (Figure 13) (30).
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3. RAMAN IMAGING The application of Raman spectroscopy in medical imaging is a novel technique of growing interest and is currently showing promise in both cellular and in vivo models. Surface-enhanced Raman scattering (SERS) techniques utilize highly conjugated chromophores as imaging agents, with the numerous highly polarizable double bonds making them Raman active. In addition, conjugation of these chromophores to metallic nanoparticles increases the intensity of the inelastically scattered photons by orders of magnitude, allowing improved limits of detection. Metalloporphyrins in many ways represent ideal optimal imaging agents for SERS, with the highly conjugated porphyrin being strongly Raman active, while the use of an appropriate chelated metal allows for quenching of any interfering fluorescent signals. The first example of the synthesis of a metalloporphyrin system for Raman imaging was developed by Tam et al., utilizing a manganese pyropheophorbide-a–lipid conjugate 31 as a surface coating for gold nanoparticles, allowing stabilization of the nanoparticle while acting as a Raman reporter. Surface coating of the nanoparticles was shown to be facile, producing a coating of 4–7 nm, with the Raman reporting properties of the metalloporphyrin retained without requiring direct contact with the nanoparticle. The nanoparticles were shown to have excellent serum stability, minimal cytotoxicity, and excellent potential as imaging agents, with in vitro studies demonstrating clear visualization of the nanoparticles in cells utilizing Raman microscopy (Figure 14) (31). Subsequently, the same group utilized the same porphyrin–lipid structure, but exchanged the manganese core for palladium (32); this change in the metallation allowed for the same reduction in porphyrin fluorescence, while preserving singlet oxygen generation and potential as a therapeutic agent. In vitro imaging demonstrated retention of the Raman reporter properties of the structure, as well as the potential of the structure to act as a theranostic agent, suggesting a possible use in the monitoring of the effects of photodynamic therapy in vivo (32).
4. GAMMA IMAGING While the use of gamma-emitting isotopes to study biological processes in living systems was first demonstrated in 1923 by de Hevesy, the
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O
O O O
O O
P O O−
N+
O
31 32
M = Mn M = Pd
N
N M
N
N
Figure 14 Metalloporphyrin-lipid structures chelating either manganese (31) or palladium (32). Coating of gold nanoparticles with these structures was shown to produce effective Raman imaging agents, while the palladium metallation of 32 also allows for potential tumor treatment via photodynamic therapy.
major breakthrough in the use of radioisotopes in medical imaging came in 1948 with the discovery of the NaI(Tl) scintillation detector. The enhanced efficiency with which this technology could detect gamma rays allowed practical clinical detection of radiation inside the body for the first time (33). Early planar gamma imaging operated on the principle of the administration of a gamma-emitting radioisotope followed by detection by scintigraphy to produce a 2D image. Subsequent improvements in imaging technology allowed for the development of SPECT, which uses a gamma camera which rotates around the patient, taking images from multiple angles to produce a true 3D image. These 3D images can be utilized to provide functional medical imaging and can be combined with SPECT/CT imaging to provide co-registered images, which allow overlay of the functional information obtained from SPECT over the gross anatomical CT image. Although SPECT shows poorer image quality and more limited resolution in comparison to more modern imaging techniques, it has not been superseded largely due to its use of cheaper, more long-lived isotopes than its counterpart PET, and subsequent lower cost. Modern gamma imaging is dominated by the use of the generatorproduced 99m-technetium, with over 80% of clinical SPECT imaging carried out utilizing this isotope (34). This popularity is due to the ease of its generation from 99Mo/99mTc generators, allowing the isotope to be produced effectively and reliably on-site in hospitals at low cost. While 99mTc is largely utilized in the form of ionic species such as the pertechnetate anion [99mTcO4], porphyrins offer an attractive option as chelators for 99mTc
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and other gamma-emitting metals, reducing undesirable uptake resulting from free ions and offering increased control over tumor localization.
4.1 Indium-111 111
In was the first metal used to label porphyrins for gamma imaging, decaying primarily by electron capture to emit two gamma photons of 173 and 247 keV. The stability of indium porphyrins makes them an attractive synthetic target, with the first examples of 111In porphyrins synthesized in 1972, with radiolabeling carried out to produce radiolabeled indium derivatives of tetraphenylporphyrin (TPP), tetratolylporphyrin (TTP), and tetra(4-methoxyphenyl)porphyrin (35). Subsequent publications also demonstrated the potential of 111In radiolabeling endogenous porphyrins, with indium-labeled structures including mesoprotoporphyrin IX (36) and hematoporphyrin derivative (HPD) synthesized (37). The first synthesis of an 111In porphyrin for biological use was carried out in 1975; Merrick et al. examined the biodistribution of indium TPP in rats, with the porphyrin displaying high liver uptake in comparison to other indium chelators, which was attributed to the high lipophilicity of the structure (38). The lipophilicity of this structure was also found to limit biological applicability in its use in imaging platelets in vivo; while platelet labeling in greater than 30% RCY was achieved and conjugates showed good plasma stability, during human trials after 24 h a 50% redistribution of 111In to erythrocytes was observed (39). Despite limited success of imaging with 111In-TPP, synthesis and biological evaluation of a number of alternative 111In porphyrins with improved biological relevance have been carried out. 111In-labeled HPD has been trialed successfully in animal breast cancer models, demonstrating clear visualization of malignant tumors by nuclear scintigraphy, with limited radiotracer uptake observed in either benign tumors or abdominal cysts. Good selectivity in uptake was also observed, with around 2.5% of the injected dose localizing in malignant tumors, and tumor to blood, heart, and lung ratios averaging 50:1, 10:1, and 3:1, respectively (40). A number of hydrophilic exogenous porphyrins have also been developed for biological use. 111In labeling of tetra(methyl-4-pyridyl)porphyrin was first carried out by Vaum et al. for the development of lymph node imaging agents. Imaging in a rat model demonstrated excellent lymph node selectivity compared to both muscle and bone (ratios of 72:1 and 16:1, respectively), despite the challenging nature of selective lymph node
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localization (41). Following the successful use of 33 in lymph node imaging, its efficacy was compared to two alternative water-soluble 111In porphyrins 34 and 35. Initial biodistribution studies in a rat model demonstrated slowest blood clearance for 34, with superior lymph node-to-muscle uptake ratios observed for 35. In vivo imaging in a rabbit model was carried out utilizing a scintillation camera, with the image quality varying significantly dependent on the radiotracer utilized. While porphyrin 35 demonstrated clear delineation of the popliteal nodes from 4 h after administration, the nodes were only faintly visualized after 24 h utilizing 33, with significant skeletal uptake also observed, and 34 showing no node uptake (Figure 15) (42). In addition to lymph node imaging, 33 has also been demonstrated to show promise as an imaging agent for melanoma. In vivo imaging in a hamster melanoma model demonstrated high uptake into tumor tissue (3.3% of ID); however, interestingly only poor localization in the necrotic core of the tumor was observed. Despite this, clear images of both the primary tumor and kidney metastases were obtained after 6 h, with the viable periphery
N+
−O
N+
SO3−
3S
N
N N
111
In
111
In
N
N
N
N
N
N+
N+
−O
33
3S
34
N+
N+
N N
111In
N
N
N+
Figure 15 Charged hydrophilic
35 111
N+
In porphyrins 33–35.
SO3−
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clearly delineated (43). Subsequently, this porphyrin was also utilized for imaging of human melanoma cells in a mouse model, with uptake and imaging potential compared to both free gallium citrate and a 67Ga-radiolabeled porphyrin. Rapid tumor uptake of 33 was observed, showing higher tumor activity than the 67Ga porphyrin at all times. While absolute tumor uptake of 33 was substantially lower than that of 67Ga citrate, rapid blood clearance of the porphyrin resulted in substantially higher tumor-to-blood ratios, with imaging after 6 h showing good contrast with the tumor clearly visible (44). In contrast, uncharged hydrophilic porphyrins have only recently been evaluated as radiotracers, with their pharmacokinetics being largely dominated by noncovalent interactions with serum proteins. 111In radiolabeling was carried out on three examples of hydroxyl-functionalized porphyrins by heating at 80 °C for 60 min, with the resulting structures demonstrating excellent RCP (>99%) and good serum stability over 2 days. Biodistribution in rats differed significantly in comparison to free indium; all porphyrins accumulated in both the liver and kidneys to some extent, although the rapid renal clearance and reduced liver accumulation observed for 38 made it the most suitable for further biological use. This pattern was confirmed by SPECT imaging in rats, with all three porphyrins showing clear visualization of spleen, liver, and kidneys, although uptake was not evaluated in a tumor model (Figure 16) (45). Alternatively, synthesis of bioconjugatable 111In porphyrins allows for attachment to targeting groups such as tumor-associated antibodies, allowing improved uptake into tumor tissue and reducing uptake into nontarget organs. The first example of this strategy was carried out by Bedel-Cloutour et al.; chelation of 111In to produce porphyrin 39 was carried out in 97% RCY, with subsequent peptide coupling of the unpurified mixture to both IgG and F(ab0 )2 antibody fragments. Conjugates were produced bearing 9–9.5 porphyrins per Mab and 1.5 porphyrins per F(ab0 )2, with 90–95% RCY and full retention of immunoreactivity in all cases (Figure 17) (46). Synthesis and conjugation of a range of water-soluble 111In porphyrins were then demonstrated, with metallation carried out using a mixture of both 111In and 115In to produce carrier-added derivatives in RCYs of between 53% and 90%. Subsequent bioconjugation was carried out to BSA and an anti-CEA MAb, with specific activities varying considerably between 7.4 and 25.9 MBq mg1 of protein. Synthesis of conjugates with improved specific activity was also achieved through removal of the isotopic dilution step, to obtain conjugates with 9.5–10 porphyrins per antibody, and average specific activity of 73 MBq mg1. All porphyrin–antibody
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OH
OH
HO
HO
HO
OH N
N N
111In
N
N
111In
N
N
N
HO HO
OH
OH
36
OH MeO
MeO
37
HO
OMe
MeO N N
111In
N
N
OMe MeO
OMe
OMe
38
Figure 16 Uncharged synthetic hydrophilic
111
In porphyrins 36–38.
R1
R2
N+
N N
111
In
N
Cl N
N
N
O
111In
N
N N+
39
N+
O 40: R1 = NO2
R2 = F
41: R1 = H
R2 = F
42: R1 = H
R2 = OCH2COOH
43: R1 = H
R2 = OCH2COO(C6H4)SCH3
Figure 17 Structures of bioconjugatable In porphyrins 39–43. Conjugation to both antibody fragments and full monoclonal antibodies was demonstrated via peptide coupling to allow for targeted gamma imaging. 111
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conjugates demonstrated good stability in aqueous solution at room temperature over 48 h; however, no biological evaluation of these conjugates was carried out (47). Although promising results have been obtained by 111In-chelating porphyrin radiotracers, synthesis of 111In radiotracers can also be carried out through the conjugation of metalloporphyrins to alternative 111In chelators; this allows exploitation of the tumor localization of porphyrins while allowing RCY and reaction times to be optimized through the use of nonmacrocyclic chelators. Nakajima et al. first demonstrated this methodology utilizing a gallium porphyrin attached to the commercially available chelator DTPA for 111In chelation. Biodistribution was evaluated in pancreatic cancer and lung cancer models, with 44 showing significantly higher tumor: blood ratios than the gallium citrate control (84:1 and 4:1, respectively), with good tumor:organ ratios also observed. Low levels of accumulation of 44 were seen in a control inflammation animal model, while uptake of gallium citrate remained high. Phototoxicity of 44 was also low in contrast to the commercial photosensitizer Photofrin, minimizing the risk of skin sensitivity after administration. Tumor imaging also showed improved results with 44; imaging after 72 h showed visible tumor tissue, while tumor tissue could not be distinguished using the gallium citrate control (48). In subsequent work by the same group an alternative metalloporphyrin conjugated to DTPA was utilized, with the latter structure allowing very rapid chelation of 111In at room temperature. Whole-body autoradiography was carried out in hamsters utilizing gallium citrate as a control. While both 45 and gallium citrate showed recticuloendothelial uptake, bone accumulation was observed only with gallium citrate. Uptake was also more selective with 45, showing equal or better tumor:organ ratios in all cases, and tumor: blood ratios up to 17 times higher than those observed for the control. In addition, while both radiotracers produced clear images with good contrast between tumor and normal tissue, 45 accumulated in viable tumor tissue only, while gallium citrate accumulated in both necrotic and viable tumor tissue, thus demonstrating that 45 shows specific affinity for tumor tissue rather than for all inflammation (49). While 45 demonstrated excellent affinity for tumor tissue, the phototoxic effects associated with the gallium porphyrin are undesirable for an imaging agent. As a result, modification of the porphyrin to produce manganese derivative 46 was carried out, which demonstrated low triplet lifetimes and no phototoxicity. In vivo imaging was carried out in three animal tumor models, with 46 giving significantly clearer images of tumor after
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O O N
HO
O
O N
N
O
111ln
N
O
O
O
O
O
O
O
N Ga
N
HOOC
N
44
COOH
H2O
OOC
O N
N M N
N
O
O
N COO
COO N
111ln
N OOC
45 M = Ga 46 M = Mn HOOC
COOH
Figure 18 Metalloporphyrin structures 44–46, with the use of 111In chelators external to the porphyrin structure allowing for improved radiochemical yields and reduced reaction times in comparison to porphyrin chelators.
72 h than the control gallium citrate. Selective uptake was also improved for 46, with all tumors having tumor:blood ratios of >50:1 and tumor:muscle ratios of >4:1, and two of the three tumor models showing higher tumor uptake and lower organ uptake than gallium citrate (Figure 18) (50).
4.2 Technecium 99m The metastable isotope of technetium, 99mTc, is one of the most widely utilized isotopes in gamma imaging, with its facile production from 99Mo generators, and relatively short half-life allowing for low patient radiation exposure and high clinical applicability. As for 111In, the larger size of 99m Tc means that chelation into porphyrins can be difficult, with longer reaction times and poorer chelate stability, and as a result porphyrin structure must be carefully controlled to optimize radiotracer parameters.
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The first example of the synthesis of a 99mTc porphyrin radiotracer was carried out in 1982, with radiolabeling of HPD carried out at ambient temperature for 30 min with 97.3% RCY. However, stability of the complex was limited, with storage for longer than 3 h at room temperature showing significant loss of 99mTc (51). Despite this, in vivo evaluation of the 99mTcHPD was carried out in an adenocarcinoma animal model, with selective uptake into primary tumors as well as in metastatic lesions present in the lungs and liver observed. In contrast, uptake into a benign tumor model was low and could only be visualized by fluorescence (52). Radiolabeling has also been demonstrated on Photosan, a commercial variant of HPD. While radiolabeling was found to proceed well under mild reaction conditions, stability was again found to be limited, with loss of chelated 99mTc after 2 h in vitro. In contrast, the conjugate displayed good in vivo stability for up to 24 h and scintigraphy imaging demonstrated good tumor uptake, particularly into small, vascularized tumors, and maximum tumor: muscle ratios of 7.9:1 (53). In contrast, labeling of tetra(4-sulfonatophenyl)porphyrin (TPPS) with 99m Tc has been shown to generate stable complexes. While radiolabeling required more forcing conditions, with heating to 100 °C for 30 min producing a 90% RCY, the generated complex was found to be stable in ambient conditions for 14 h, with this improved stability attributed to conformational changes in the porphyrin following chelation. While in vitro evaluation showed rapid uptake into Hep2 tumor cells, SPECT imaging in mice showed no uptake into the tumor and rapid clearance of the porphyrin from the body (54). 99m Tc-TPPS has also been exploited in the imaging of nonneoplastic conditions, including the imaging of inflammatory response. The first example of this was demonstrated by Zanelli et al., with 99mTc-TPPS showing accumulation at the site of injections of histamine, a well-known mediator of the inflammatory response (55). Subsequent work demonstrated the use of this inflammatory uptake for imaging of osteomyelitis, with 99mTc-TPPS compared to the commercially available 111InCl and 111In-WBC in a rabbit model. While all radiotracers demonstrated increased uptake in inflamed tissue, 99mTc-TPPS showed the best clinical relevance, with more selective uptake, improved image quality and reduced time between administration and scanning (56). More recently, the radiolabeling of a pheophorbide-a derivative with 99m Tc was carried out by Ocakoglu et al. with the aim of exploiting the antimicrobial effect of pheophorbide to produce an infection imaging agent.
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Heating to 60–70 °C for 25 min produced the 99mTc derivative 47 in 87% RCY; however, again in vitro serum stability was shown to be limited to 2 h or less. Uptake was evaluated in both a bacterially infected muscle model and a sterile inflamed control; while both showed uptake of 47, maximum target: nontarget muscle uptake varied significantly between the models, with maximum ratios of 1.29:1 and 5.61:1 for the control and infection models, respectively, allowing the potential for distinguishing infection from inflammation (Figure 19) (57). Due to the instability of 99mTc chelates, attempts have also been made to increase stability utilizing an alternative chelation methodology, chelation of the radioisotope by peripheral functionalities of the porphyrin rather than the central core. This was first demonstrated using UV–vis analysis by Shetty et al., with the radiolabeling of novel porphyrins 48 and 49 carried out in 30 min at room temperature. Radiolabeling of porphyrin derivatives containing Mn in the central core confirmed 99mTc binding to the peripheral carboxylic acid functionalities rather than the porphyrin core. In vivo imaging using 48 was carried out in sarcoma-bearing mice, with good visualization of both the primary tumor and brain metastases observed (58). Subsequent work confirmed improved in vitro stability of 48, with the porphyrin showing good stability at room temperature for up to 4 h. Evaluation in an animal model was carried out in comparison to the commercial radiotracers 99mTc(V)-DMSA, 99mTc-citrate, and 201TlCl, with 48 showing the highest tumor:muscle ratios (5.87), but tumor:liver and tumor:kidney ratios of below 1, due to both rapid blood and muscle clearance, and slow renal and hepatobiliary excretion (Figure 20) (59). Evaluation of the chlorin analogue 50 was also carried out by the same group, with this analogue offering identical chelation properties but enhanced potential to be utilized as a theranostic for photodynamic therapy.
N
N
99mTc
N
O
Figure 19 Structure of
99m
O N
O O
47
Tc-labeled pheophorbide-a derivative 47.
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COOH
HOOC O
O
COOH
O O
COOH
N HN
NH N O
HOOC
O
HOOC
O
O 48
COOH
HOOC
HOOC O
O
COOH
N HN
NH N
HOOC
O
O 49
COOH
Figure 20 Chelation of 99mTc utilizing the peripheral carboxylic acid functionalization present on 48 and 49 allows for the development of gamma imaging agents with improved chelate stability in comparison to conventional 99mTc porphyrin structures.
Radiolabeling was carried out in 15 min at room temperature, with the resulting complex showing in vitro stability for up to 4 h at room temperature. Pharmacokinetics in a rat model were similar to 48, showing rapid blood clearance and high liver and kidney uptake, while in vivo imaging demonstrated higher tumor:muscle uptake ratios in comparison to 99mTc(V)-DMSA and 201TlCl in all tumor models, although uptake did not differ significantly between 50 and 99mTc-citrate (Figure 21) (60). The carboxylic acid-functionalized tetra-(4-carbomethoxyphenyl)porphyrin was also used for noncore chelation of 99mTc, with UV analysis confirming chelation by the peripheral functionalities only. Biodistribution
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HOOC O
COOH O
O O
COOH COOH
N NH
HN N
HOOC HOOC
O O
50
O HOOC
O COOH
Figure 21 The structure of chlorin 50 allows for chelation of 99mTc via the peripheral carboxylic acid moieties rather than the core of the tetrapyrrole structure, while also offering enhanced absorption at clinically relevant wavelengths in comparison to porphyrin analogues.
studies demonstrated limited uptake in most organs; however, very high liver accumulation was observed, with associated short-term liver damage also observed (61). Most recently, development of the externally chelating porphyrin 51 was carried out by Santos et al. with the aim of producing a radiotracer with high structural similarities to natural porphyrins, but with a simple structure to allow for rapid clearance. 99mTc incorporation was carried out with 92.5% RCY, with UV–vis analysis confirming chelation of the metal outside of the porphyrin core. 51 demonstrated significantly improved serum stability, with no loss of 99mTc after 6 h at 37 °C. In vivo biodistribution in a mouse model demonstrated rapid clearance from the body, with low activity in all organs after 6 h; however, initial high uptake in the spleen, kidney, and liver was observed, possibly limiting the use for imaging colorectal tumors. Imaging by a gamma camera in tumor-bearing mice showed some selective tumor uptake, with tumor:muscle ratios of 3:1 and higher obtained in two tumor lines (Figure 22) (62).
4.3 Gallium 67 Use of 67Ga in gamma imaging radiotracers is an attractive prospect due to both its two gamma photopeaks (185 and 300 keV) and its long half-life (t1/2 ¼ 78 h), allowing increased manipulation and complex purification
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O NH
HOOC
N
O N
O
HN
O O O Tc 99m O O O
HOOC 51
Figure 22 Structure of 51 showing
99m
Tc chelation via peripheral functionalization.
steps without significant loss of activity. Despite this, its use has been largely superseded by the use of the positron-emitting 68Ga isotope for PET, which allows improved resolution and reduced patient exposure to radiation due to its short half-life. 67 Ga radiolabeling of a tetrapyrrole structure was first demonstrated by Rousseau et al. in 1985, utilizing both 67Ga and 99mTc to label tetrasulfophthalocyanine. Radiolabeling was shown to be significantly more efficient for 67Ga, with a maximum RCY of 63% obtained in comparison to an RCY of 19% utilizing 99mTc. In vivo distribution of both phthalocyanines was evaluated, with the 99mTc phthalocyanine showing high kidney accumulation and no significant tumor accumulation within the 24-h period of study. In contrast, the 67Ga phthalocyanine showed a significantly different distribution, with lower kidney uptake and higher tumor:blood and tumor: muscle ratios, although overall tumor uptake was lower than the gallium citrate control (63). Low overall tumor uptake was also observed with 67Ga-radiolabeled tetra(methyl-4-pyridyl)porphyrin. While radiolabeling was shown to proceed in high RCY (94.7%) after 30 min at 120 °C, absolute tumor uptake of the 67G-labeled porphyrin was substantially lower than that of both gallium citrate and an analogous 111In-labeled porphyrin at all time points. Despite this, rapid blood clearance of the 67Ga-labeled porphyrin over a period of 48 h resulted in tumor:blood ratios which were substantially higher than that of 67Ga citrate (44). Most recently, radiolabeling of a porphyrin with 67Ga was carried out by Paknafas et al., with 52 produced with an excellent radiochemical purity after heating to 100 °C for 60 min. While 52 demonstrated good in vitro stability over 24 h, imaging in a rat model showed skeletal and myocardial uptake after 2 h, attributed to release of gallium in vivo (Figure 23) (64).
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MeO
OMe
OMe OMe N
N
67Ga
N
N MeO MeO
Figure 23 Structure of
67
52
MeO
OMe
Ga-radiolabeled porphyrin 52.
4.4 Other Radionuclides Although dwarfed in popularity by the positron-emitting isotope 64Cu, 67 Cu has found use as both a therapeutic beta emitter and a diagnostic gamma emitter. The long half-life of 67Cu makes it particularly appropriate for imaging in combination with antibodies due to their long biological half-life, a strategy demonstrated by Bhalgat et al. utilizing n-benzyl functionalized porphyrins, which demonstrate improved metal chelate stability and more facile incorporation than porphyrins with unfunctionalized cores. Two examples of hydrophilic porphyrins were synthesized, and attachment to an antirenal cell carcinoma antibody was carried out via peptide coupling in 25–30% yield, with two to three porphyrins attached per antibody. Subsequent radiometallation was carried out in 41% RCY, yielding conjugates with a specific activity of 18.5 MBq mg1 protein, with all products having greater than 70% stability serum over 7 days. In vivo biodistribution in a mouse tumor model showed significant differences between targeted and untargeted porphyrins; free porphyrins showed high liver and kidney uptake, and modest tumor:blood selectivity (1.8–2:1). In contrast, the antibody conjugate demonstrated excellent tumor selectivity, with maximum tumor:blood ratios of 16.4:1 after 45 h (Figure 24) (65). Despite the prevalence of endogenous iron porphyrins, only a single example of the development of iron porphyrins for gamma imaging is known in the literature. Thaller et al. carried out radiolabeling on a number of both endogenous and exogenous porphyrins with 59Fe and 52Fe, evaluating tumor tissue uptake in an ex vivo model. Highest tumor tissue uptake was observed for endogenous deuteroporphyrins, while good uptake was
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HO O
O
O
S
S
O
O
OH
OH
OH
O
N N
67Cu
N N
N
HO O
53
N
N
N
O2N
67Cu
O
S
O OH
HO
O O
54
HO
Figure 24 Cu-labeled metalloporphyrins 53 and 54, with attachment to targeting antibodies carried out via peptide-coupling reactions with the peripheral carboxylic acid functionalities of the porphyrins. 67
also observed for exogenous porphyrins tetra(methyl-4-pyridyl)porphyrin and tetra-(4-carbomethoxyphenyl)porphyrin. Despite this, in vivo tumor imaging in rat models with 52Fe porphyrins demonstrated no tumor uptake at either 3 or 24 h time points (66). Similarly, while paramagnetic manganese has found favor in porphyrin imaging agents for MRI, gamma-emitting 54Mn has only been utilized once for the labeling of HPD, with heating to 150 °C for 60 min producing an RCY of 86.7%. In vivo biodistribution in mice showed no significant difference from free-base porphyrins, with limited selective accumulation in tumor tissue and high liver uptake (67). While 201Tl has largely been superseded by 99mTc due to its ease of generation, recently radiolabeling of tetrakis(pentafluorophenyl)porphyrin with 201 Tl was carried out by Fazaeli et al., producing the complex with greater than 99% purity and good specific activity (13–14 GBq mmol1). Despite this, clinical relevance of the conjugate was limited, with poor in vivo stability, limited tumor uptake, and rapid clearance observed (68). Although a gamma-emitting isotope of cobalt does exist, the extremely long half-life (271 days) of 57Co means that the use in gamma imaging is of little clinical relevance, with the only clinically developed variant being the radiolabeled vitamin B12 mimic 57Co-cyanocobalamin. The only example of the development of 57Co metalloporphyrins for medical imaging was the synthesis of 57Co hematoporphyrin by Angileri; however, in vivo studies showed limited applicability as an imaging agent, with normal and tumor-bearing mice showing no significant differences in uptake (69).
Metalloporphyrins for Medical Imaging Applications
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5. POSITRON IMAGING (PET) While PET also exploits radioisotopes for imaging purposes, it differs from SPECT in that the detected emissions are not produced directly from the radioisotope decay. Isotopes which decay to emit positrons are utilized, with the generated positrons undergoing an annihilation event with an electron to produce two coincident 511 keV gamma rays. These gamma rays are then detected by a ring of scintillation detectors around the patient, with only gamma photons originating from a single coincident event detected, removing the need for the collimators required by SPECT. As for SPECT, almost all clinical PET scanning today is carried out utilizing combined PET/CT scanners, which provide both functional and morphological imaging to aid in image registration. PET offers much higher sensitivity than SPECT, as well as improved spatial resolution, and resulting improved image quality. In addition, the shorter half-life of many clinically relevant positron-emitting isotopes means that radiation doses to patients are reduced; however, this also means that many isotopes require expensive on-site cyclotron generation, while even isotopes with comparatively longer half-lives (i.e., 18F) require rapid automated synthesis equipment in order to produce radiotracers with clinically relevant specific activities following transportation. Radiolabeling of biologically relevant structures with positron-emitting radioisotopes allows for functional imaging of disease states, with most well-known example being the current clinical gold-standard, 18F-FDG (fluorodeoxyglucose). Enhanced glucose uptake of many tumor cells as a result of the Warburg effect leads to increased uptake of this 18F-labeled glucose analogue, with subsequent phosphorylation preventing cellular egress and causing accumulation of 18F-FDG in target tissue. Since clinical approval, 18F-FDG has been utilized for diagnosis and staging of a plethora of malignant diseases, including digestive, lung, and breast cancers (70). Despite this, uptake of 18F-FDG occurs only as a result of enhanced metabolic activity and does not provide information about receptor expression in these cancers. As a result, other radioligands have been developed which allow assessment of tumor hypoxia, apoptosis, and expression of receptors including somatostatin, steroid hormone receptors, and epidermal growth factor receptors (71). Radiolabeling of porphyrins has also been widely explored, and while there is some interest in labeling via covalent attachment of nonmetal isotopes including 18F (72) and 124I (73), the real utility of
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porphyrin radiotracers is in their ability to efficiently chelate metallic radioisotopes to produce stable metal complexes.
5.1 Copper 64 Despite the popularity of 18F in the development of radiotracers, the first examples of PET imaging carried out with metalloporphyrin radiotracers utilized 64Cu and were first used in preclinical in vivo studies in 1951 (74). The popularity of 64Cu in radiotracers can be attributed to both the long half-life and favorable decay characteristics of this isotope (t1/2 ¼ 12.7 h, decay: β+ (61%) β (39%), energy β+max ¼ 656 keV), as well as the ready cyclotron preparation from proton bombardment of the stable 64 Ni target. Copper also offers good compatibility with porphyrins, with several studies showing that the pharmacokinetics of metalloporphyrin radiotracers are dominated by the chelating porphyrin rather than the coordinated metal (75, 76) although the paramagnetic nature of the copper alters the photophysical properties of the porphyrin, quenching both fluorescence and singlet oxygen generation. Early imaging using copper porphyrin radiotracers utilized endogenous porphyrins including hematoporphyrin, uroporphyrin, and coproporphyrin and showed mixed results; while tumor uptake was observed in mice, localization in human tumors was insufficient to allow imaging, with high liver uptake observed in all cases suggesting hepatobiliary metabolism (77). Use of commercially available derivatives of endogenous porphyrins including HPD was found to be more effective, showing a threefold reduction in liver uptake; however, addition of the copper was found to alter the composition of the HPD, and uptake ratios varied considerably between animals (78). As a result, subsequent work has utilized a range of exogenous porphyrins and phthalocyanines, with these synthetic structures allowing facile tuning of the porphyrin properties to improve chelation, improve tumor localization, and limit risk of in vivo degradation. Radiolabeling of tetra(pentafluorophenyl)porphyrin with 64Cu has been carried out by Fazaeli et al., with the aim of producing porphyrin radiotracers with an improved pharmacokinetic profile. Forcing radiolabeling conditions and long reaction times (100 °C for 60 min) were required, with the radiotracer showing good biostability over a 48-h period. In vivo pharmacokinetic studies showing minimal liver uptake, with most excreted via the kidneys, however, no imaging or tumor uptake studies were carried out (79).
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Sulfonated phthalocyanines also offer improved solubility profiles as well as improved biological stability in comparison to endogenous porphyrins. Radiolabeling of a mixture of sulfonated phthalocyanines bearing between one and four sulfonate groups was carried out by Soucy-Faulkner. While comparison of the pharmacokinetics of 55 and free copper acetate suggested no in vivo phthalocyanine demetallation, significant uptake into both liver and kidneys was observed. Additionally, while the tumor could be distinguished during imaging, tumor activity was low, with these poor results attributed to the complexity of the radiolabeled mixture (Figure 25) (80). For this reason, subsequent work by this group utilized purified fractions of this mixture, bearing 2, 3, or 4 sulfonate groups, as well as phthalocyanine 59, designed to show improved amphiphilic character. Radiolabeling of all phthalocyanines was rapid, with microwave irradiation of 150 W for 1 min producing the desired 64Cu metallophthalocyanines. In vivo studies demonstrated marked differences in the pharmacokinetics, with significant kidney and bladder uptake, and no tumor activity observed for the highly hydrophilic phthalocyanines 56 and 57. In contrast, the more lipophilic 58 and 59 showed liver uptake in addition to good tumor localization, with the best PET images, showing clear tumor delineation, obtained for phthalocyanine 59 (Figure 26) (81). The targeting ability of a range of biomolecules has also been exploited in the synthesis of porphyrin radiotracers, with the aim of improving tumor uptake and enhancing PET image quality. 64Cu-radiolabeled porphyrins in particular offer good compatibility with large biomolecules such as proteins and antibodies, with the long half-life of 64Cu corresponding well with the slow tumor accumulation and blood clearance of these biomacromolecules. NaO3S
R N N N
N Cu64
N
R = H or SO3Na
N
N N R
R 55
Figure 25 Representative structure of 55, which comprises a mixture of sulfonated phthalocyanines bearing between one and four of these peripheral water-solubilizing functionalities.
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NaO3S
SO3Na
NaO3S
N N N
N
N Cu64
N
N
N Cu64
N
N
N
N
N N
N
N SO3Na
NaO3S
NaO3S
SO3Na
56
57 NaO3S
C6H11
N N N
N
N Cu64
N
N N 58
N Cu64
N
N
N
N SO3Na
NaO3S
N N
N
NaO3S
SO3Na 59
(mixture of cis and trans isomers)
Figure 26 64Cu-radiolabeled phthalocyanines 56–59, with varying levels of peripheral sulfonation.
Roberts et al. synthesized a range of antibody-targeted radiotracers utilizing N-benzyl porphyrin 60, which allows the incorporation of 64Cu more rapidly and at temperatures more appropriate for antibody radiolabeling. Radiolabeling was shown to proceed well under mild reaction conditions (40 °C for 30–60 min), with the macrocyclic structure of 60 showing significantly enhanced stability in comparison to the linear chelator DTPA. Conjugation of 60 to antibodies was achieved via peptide coupling, with subsequent 64Cu insertion carried out in approximately 60% RCY. An average of two porphyrins was attached per antibody with specific activity of ca. 18 MBq mg1, with all conjugates showing good biostability (70% after 7 days). However, poor regiocontrol of porphyrin conjugation was observed, and antibody immunoreactivity was reduced to around 60% of that of the unmodified antibody (Figure 27) (82). An alternative to the use of antibodies as targeting groups is the use of peptides; these smaller molecules more readily allow structural engineering, giving improved binding regiocontrol, and a corresponding retention of receptor affinity, as well as reduced blood residence time which allows for more rapid imaging.
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SO3H
N HO3S
N
OH
N
O
64Cu
N
NO2 SO3H 60
Figure 27 Metalloporphyrin structure 60, with the N-benzyl functionalization offering enhanced chelate stability and more rapid incorporation of 64Cu.
Shi et al. demonstrated the synthesis and metallation of the folate receptor-targeted peptide GDEVDGSGK-folate chlorin conjugate (61), with radiolabeling occurring in >99.9% RCY to produce a conjugate with high specific activity (2.66 GBq μmol1). Stability of 61 was confirmed both in vitro and in vivo over 24 h. Tumor uptake was evaluated in a FR-positive mouse model; approximately 3% of the injected dose accumulated in the tumor with good tumor:muscle selectivity (8.8:1), allowing clear delineation of the tumor during imaging. Additionally, this uptake was shown to be blocked by administration of excess folic acid, confirming the folatemediated uptake process (Figure 28) (83). A bombesin analogue was utilized as a targeting peptide for the endogenous porphyrin protoporphyrin IX (PPIX), with the radiochemistry and localization compared to an untargeted PPIX control. Radiolabeling required moderate heating (50 °C) and long reaction times (2 h), with the addition of ethanol as a stabilizer, with poorer RCY obtained for PPIX than conjugate 62 (25% and 40% decay corrected, respectively). In vitro analysis was carried out in a GRP receptor expressing human prostate cell line, with targeted 62 demonstrating significantly higher uptake than PPIX. Pharmacokinetic evaluation demonstrated similar excretion profiles for both PPIX and 62; however, 62 showed significantly lower accumulation in normal skin tissue. Despite this, no difference in tumor uptake was observed between 62 and the untargeted control, attributed to the in vivo
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N
N 64Cu
N
NH
N
N
N
NH2
N
O
HN O
O NH GDEVDGSGK NH
COOH NH O
O 61
Figure 28 Structure of the folate receptor-targeted metallochlorin conjugate 61.
O
NH
O
O
O
O
O
d-Tyr Gln Trp Ala Val β-Ala His Thi Nle
O
N
N 64
N
Cu
62
N O OH
Figure 29 Cu-radiolabeled protoporphyrin IX derivative 62, with conjugated bombesin analogue to improve tumor uptake. 64
interactions between 62 and serum proteins dominating the uptake profile (Figure 29) (84).
5.2 Gallium 68 While the dominance of 18F-FDG as a radiotracer means that 18F is currently the most widely used clinical radioisotope in PET imaging, the positronemitting isotope of gallium, 68Ga, is gaining popularity. While its 68 min half-life is significantly shorter than that of 18F, it is sufficiently long to allow chemical manipulation, and this combined with its high positron abundance (89%) and relatively low maximum positron energy (1.92 MeV) means that 68 Ga is well suited to the synthesis of clinically relevant radiotracers. Furthermore, its production from Ge/Ga generators makes it an attractive proposition for clinical use, as it can be produced without the need for an on-site cyclotron.
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In addition, gallium is an attractive option for radiolabeling porphyrins; unlike 99mTc, the small size of the metal means that it can easily be chelated by porphyrins to produce stable metalloporphyrins which have been shown to demonstrate minimal toxicity (85), and the diamagnetic nature of the metal means that, unlike 64Cu, the photophysical properties of the porphyrin are largely retained. The first example of 68Ga radiolabeling of a porphyrin was carried out by Benham Azad et al., utilizing an RGD peptide-functionalized protoporphyrin IX derivative targeting the αvβ3 integrin. Radiolabeling was carried out using microwave heating, with irradiation at 120 °C for 45 min demonstrating the best yields (RCY: 33%, decay corrected, RCP: 97%), with higher temperatures leading to instability of peptide and hydrolysis of free gallium. In vitro fluorescence microscopy in comparison to an untargeted control showed cell uptake observed for 63 only, with blocking experiments utilizing the cyclic peptide cRGDfV showing displacement of 63, suggested that cellular uptake of this compound is dominated by active binding to the integrin receptors (Figure 30) (86). Use of untargeted gallium porphyrins as radiotracers for PET has also been demonstrated to be an effective strategy. Radiolabeling of four examples of endogenous tetrapyrrolic structures, and the commercially available tetraphenylporphyrin (66) was carried out using high temperatures under microwave irradiation for between 1 and 15 min. RCY obtained varied between 22% and 73%, with the best results obtained for 66. RCP were also varied (between 85% and 98%) with 65 and 68 showing insufficient purity for biological studies. 64, 66, and 67 showed good stability to transchelation from DTPA and apo-transferrin and transmetallation with iron; however, only 66 and 67 were found to be stable in serum, with 64 showing a limited
N
N 68
N
Ga
63
N
HN
NH2 O
NH O HO
O
O
NH
O
O
H N
NH O
OH
O N H
NH2 O
Figure 30 RGD-peptide functionalized gallium protoporphyrin IX derivative 63.
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half-life of 72 min. Preliminary in vivo PET imaging was carried out in a mouse sarcoma model using 64. While high renal uptake was observed, around 0.5% of the injected dose localized in the tumor, with a modest tumor: tissue ratio of around 2 (Figure 31) (87). Use of untargeted lipophilic porphyrins has also been explored by Fazaeli et al. Radiolabeling of tetra(pentafluorophenyl)porphyrin with 68Ga was shown to require lower temperatures (100 °C) but long reaction times (1 h); however, no RCY was recorded. The porphyrin demonstrated excellent stability in human serum, and in vivo imaging showed selective tumor uptake, with 5:1 tumor:muscle ratios achieved (88). Despite these successes, more recent papers have utilized water-soluble exogenous porphyrins as radiotracers; the hydrophilicity of these porphyrins makes them more applicable to in vivo use, and the use of nonnative porphyrins allows for increased synthetic flexibility and facile tuning of porphyrin properties. The preferential tumor uptake (89) and cellular localization (90) of the cationic tetra(N-methyl-4-pyridyl)porphyrin have previously been demonstrated in its use as a therapeutic agent, with Bhadwal et al. publishing the first example of this porphyrin utilized in a radiotracer. 68Ga chelation again required elevated temperatures (100 °C) for extended periods of time, producing the desired product with an RCY of 90%, and excellent radiochemical purity (99%). The conjugate demonstrated excellent serum stability and showed good tumor uptake into a fibrosarcoma mouse model despite rapid renal clearance, with maximum tumor:blood ratios of 2.8:1 reported (91). The limited tumor selectivity of many radiolabeled metalloporphyrins has led to the development of hydrophilic, conjugatable metalloporphyrin radiotracers, allowing both ease of administration and facile bioconjugation to targeting biomolecules. Bryden et al. demonstrated the first example of targeted exogenous gallium porphyrins; radiolabeling was shown to be extremely rapid, with microwave irradiation achieving an RCY of >95% in 5 min. Subsequent click conjugation of the porphyrin to an integrin-targeting peptide was carried out at room temperature, and evaluation of the conjugate 69 as a targeted theranostic agent was carried out in vitro, demonstrating good selectivity for target integrin upregulating cell lines only (Figure 32) (92).
5.3 Other Isotopes In addition to these commonly utilized isotopes, there is a growing interest in novel isotopes for PET imaging. 62Zn offers many favorable properties for
OH
N
N
N
68Ga
N
N
HO
O
N
OH
N
64
O
OH
HO
O
N
N
N
65
N
68Ga
68Ga
O
N
OH 66
HO
O
N
H3COOC
N
N
H3CO
O
N
N
N
67
N 68Ga
68Ga
O
OCH3
H3CO
O
N
68
O
OCH3
Figure 31 A range of both endogenous (64–65, 67–68) and exogenous (66) tetrapyrroles labeled with 68Ga.
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Francesca Bryden and Ross W. Boyle
O N+
H N O
N N 68Ga N Cl N N+ 69
O 3Cl–
N
N
N
TWYKIAFQRNRK
N+
Figure 32 Structure of the integrin-targeting
NH2 O
68
Ga porphyrin–peptide conjugate 69.
incorporation into metalloporphyrin radiotracers, with a half-life of 9.3 h, 100% positron emission, and the diamagnetic nature of the metal meaning that, as for gallium, the photophysical properties of porphyrin are largely retained. 62ZnCl2 was utilized by Tamura et al. to produce three examples of glycosylated porphyrin radiotracers, with only 10 min of heating required for zinc insertion. Cytotoxicity and cellular localization were evaluated in control and target cell lines, with the chelated zinc showing minimal impact on porphyrin localization. PET imaging in an animal model showed a large proportion of all three porphyrins accumulated rapidly in the liver; while 71 and 72 showed improved tumor localization in comparison to 70, uptake was still relatively low, with future adjustment of hydrophilic/lipophilic properties suggested by the authors to minimize liver uptake (Figure 33) (93). Despite its popularity in MRI, use of manganese isotopes in PET imaging is rare, with 51Mn (t1/2 ¼ 46.2 min, positron energy ¼ 2.5 MeV) being the only isotope to date which has been incorporated into a metalloporphyrin radiotracer, with widespread use hindered by its limited availability and short half-life. Radiolabeling of tetra(4-sulfonatophenyl)-porphyrin (TPPS) with 51 Mn was carried out to produce an alternative to the widely used MnTPPS MRI contrast agent; the problems associated with the high effective dose required for MRI can be avoided by using PET instead, as the quantity of imaging agent required is orders of magnitude lower. Radiolabeling was achieved after 20 min heating at 100 °C, with an RCY of 32% (decay corrected) and 99.9% incorporation; however, no biological evaluation was conducted (94).
6. MAGNETIC RESONANCE IMAGING The application of magnetic resonance to imaging of living systems was first demonstrated Damadian (95), with NMR utilized to differentiate
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F
N
N
F
62Zn
N
N H
NaOOC
N
F
F
R N
N
F
F
F
COONa F
F R 71
O OH
F
N
62 Zn
70
OH HO HO
F
F
NH
R=
F F
F
COONa
O
F
F
H NaOOC
F
S F
F
F
F
F
F
F
N
F
N
F
62
R
R
Zn
F
N
N
F
F
F
F
F F
F F 72
Figure 33
62
Zn-labeled glycosylated porphyrin structures 70–72.
between malignant tumors and normal tissue. Unlike PET and SPECT, MRI does not utilize ionizing radiation, instead using a strong magnetic field and an oscillating radiofrequency to excite nuclei containing odd numbers of protons and/or neutrons, with the subsequent differences in relaxation time producing contrast in the image. Images can be generated utilizing either T1 or T2 weighting, measuring longitudinal and transverse relaxation, respectively, with the change in weighting highlighting different pathologies and anatomical features. Generation of MRI images in native tissue is possible through excitation of the hydrogen nuclei in water present naturally in body tissues, with
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contrast in the image resulting from differences in tissue density; however, this is often insufficient to allow identification of tumor tissue from normal tissue. Addition of exogenous contrast agents reduces the relaxation time of nearby protons, increasing contrast and improving tumor delineation. As a result, MRI is capable of producing clinically relevant images without the need for exposure to ionizing radiation, with superior spatial resolution and contrast in comparison to radio-imaging techniques; however, sensitivity is low and the cost can be high. While there are numerous clinically approved exogenous contrast agents, none have yet been commercially produced which offer any appreciable specificity for neoplastic diseases. As a result, there is a growing interest in the development of alternative contrast agents, including the use of porphyrins and other tetrapyrroles. While there has been some interest in porphyrins as platforms for attachment of 19F (96,97), these structures require high levels of symmetry and multiple loading of fluorine atoms to create appreciable reduction in relaxation times. In contrast, porphyrins chelating paramagnetic metals have been extensively explored, with many showing good stability, high relaxivities, and minimal toxicity. Good selectivity for tumor tissue has also been observed for many porphyrins, although the exact reason for this localization is poorly understood, and in some cases has been attributed to nonspecific accumulation in necrotic tissues (98, 99).
6.1 Gadolinium As the only ion containing seven unpaired electrons, Gd3+ is a highly paramagnetic metal and an obvious choice for use in MRI agents, despite the high toxicity associated with free gadolinium (100). As a result, a plethora of gadolinium chelators are described within the literature, and the clinical use of a number of approved gadolinium-containing MRI contrast agents is well established, with Gd-DTPA being the most commonly used for clinical applications (101). However to date, use of porphyrins as clinical gadolinium chelators is limited, which can be attributed to the fact that the large size of the gadolinium ion is a poor fit with the relatively small porphyrin cavity, resulting in instability and potential leaching of toxic-free gadolinium. Despite this, examples of gadolinium porphyrins synthesized and evaluated as MRI agents have been published. Despite the loss of gadolinium from TPPS in vivo noted as early as 1987 (102), the high relaxivity of Gd-TPPS in solution makes it an attractive target for clinical use, and promising results have been obtained from both T1 and T2 brain imaging (103).
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Metalloporphyrins for Medical Imaging Applications
Examples of gadolinium endogenous porphyrins have also been synthesized including gadolinium hematoporphyrin, which showed improved in vitro uptake and higher relaxivity values in comparison to control compounds GaCl3 and Ga-DTPA (104). Subsequent evaluation of gadolinium hematoporphyrin and an exogenous boronated porphyrin 73 was carried out, with 73 showing R1 and R2 relaxivity values 5 and 10 times higher than Gd-DTPA, respectively. Tumor uptake of Gd hematoporphyrin and 73 was evaluated in a mouse melanoma model with the tumor showing 28% and 21% uptake of the injected dose, respectively, compared to 10% of Gd-DTPA. MRI showed good contrast enhancement in both cases, with the highest tumor signal enhancement (120%) found for Gd hematoporphyrin (105). Good uptake of both contrast agents was also found in a mouse colorectal tumor model, with T1 relaxation times reduced by 15% for hematoporphyrin and 12% for 73, compared to 3.3% in DTPA. However, as seen previously (105), high levels of gadolinium were found in the liver, which was attributed to possible free gadolinium dissociating from the porphyrin (Figure 34) (106). The first example of a gadolinium chlorin contrast agent was synthesized by Kim et al. using Radachlorin. The species was shown to be stable with no loss of gadolinium, and evaluation in a tissue phantom showed 20–25% signal intensity enhancement, with minimal change with changing concentration. However, the poor water solubility of the species limits the clinical applicability of the conjugate (107). H10B10
O
O
B10H10
N N
Gd N N
H10B10
O
73
O
B10H10
Figure 34 Structure of the boronated gadolinium porphyrin 73.
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As a result of the poor size match between porphyrins and Gd3+, there has been a growing interest in the clinical use of texaphyrins as gadolinium chelators. These large, expanded porphyrin analogues are modified to contain five nitrogen atoms in the larger central cavity, allowing enhanced stability of gadolinium chelates. The most well-known example is motexafin gadolinium (74) which was first developed in 1996 (108) as a radiation sensitizer for the treatment of cancer, particularly brain metastasis (109,110). However, the gadolinium core means that it also has a potential use as an MRI contrast agent, either for monitoring the uptake of motexafin gadolinium (111) or as an MRI agent for both diagnostic MRI (112) and intraoperative MRI (Figure 35) (113). An alternative strategy to the use of expanded porphyrins is the use of porphyrins attached to macrocycles or linear chelators designed for incorporation of gadolinium, a strategy which has been used to great effect in the development of the commercial imaging agents Gadophrin 2 (75) and 3 (76). Both structures have two gadolinium ions per molecule chelated outside of the porphyrin core, with the addition of the copper ion into the porphyrin of Gadophrin 3 significantly improving the stability of the contrast agent without affecting pharmacological properties. As a result, the stability of the gadolinium chelation is significantly improved, in addition to a significantly improved safety profile in comparison to other porphyrin contrast agents (114). Both Gadophrin 2 and 3 are of interest due to their intracellular targeting of necrotic tissue, and subsequent slow clearance as a result of poor OH O O– N N
O
N
Gd N
N
O O
O O
O O
O O
–
O 74 OH
Figure 35 The expanded porphyrin-like structure of texaphyrin motexafin gadolinium 74 allows chelation of gadolinium with improved stability due to its larger cavity size.
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Metalloporphyrins for Medical Imaging Applications
lymphatic drainage (114). This uptake allows for specific targeting of necrotic tissue in pathological conditions, particularly in the diagnosis of myocardial necrosis and ischemia following heart infarction (115–117). Their use has also been evaluated in neoplastic conditions, with Gadophrin 3 showing promise in the determination of necrosis in breast cancer (118); however, it was found that the high affinity of both contrast agents for necrosis can impede distinguishing tumors from other necrotic tissues, such as abscesses (Figure 36) (119). Following the success of the Gadophrin structures, development of a number of other contrast agents employing this chelation strategy has been attempted. Conjugation of a zinc phthalocyanine to a DOTA chelator via click chemistry was carried out to produce 77, with the increased molecular weight of the phthalocyanine expected to enhance the molecular relaxivity of the structure. While the photophysical properties of the phthalocyanine were retained following conjugation, the R1 relaxivity of 77 was found to be less than half of that of unconjugated gadolinium DOTA, with this reduction attributed to the amide spacer arm blocking the gadolinium waterbinding site. In vitro assessment of the structure demonstrated some toxicity, with concentrations of above 50 μM decreasing cell viability to ca. 75%; however, no assessment of the imaging potential of 77 was carried out in a biological system (Figure 37) (120). In contrast, Trivedi et al. utilized this strategy with two examples of metalloporphyrazines, each conjugated to two DOTA chelators, to produce effective contrast agents. Cellular uptake of both 78 and 79 was evaluated in a breast cancer cell line in comparison to the unconjugated Gd-DOTA, with 78 showing twofold better uptake in comparison to 79 and the control. In vitro imaging confirmed this, with the clearest images obtained for 78 with COO–
O N
N H
N
H N O
M N
N
H N O
N
O N H
N
N Gd3+ COO– –OOC COO– –OOC Gd3+ N
N
COONa
N
COONa
COO–
75 M = 2H 76 M = Cu
Figure 36 Commercial imaging agents Gadophrin 2 (75) and 3 (76).
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Francesca Bryden and Ross W. Boyle
R
O
R
O
N N
N
N
R N R
Zn
S N
N
N
N N N
H N
N O
N
O
O
O
O
O
N 77 R=
R
N Gd
HN
S
O
O
O
R
Figure 37 Incorporation of a chelating macrocyclic unit into the structure of conjugate 77 allows for stable chelation of gadolinium for MRI while retaining the photophysical characteristics of the zinc phthalocyanine.
significantly higher contrast:noise ratios obtained. Subsequent in vivo evaluation of 78 showed the best contrast enhancement after 4 h, with reduction in T1 found to be slower but considerably more persistent than in comparison to Gd-DOTA alone (Figure 38) (121).
6.2 Manganese In contrast to gadolinium, manganese has only five unpaired electrons, with the corresponding reduction in paramagnetism leading to it being less optimal for use within MRI contrast agents. Despite this, manganese porphyrins are widely utilized in the literature as potential MRI agents, with the smaller size of the manganese ion allowing for more facile synthesis and greater stability of the resulting complexes in comparison to gadolinium. The first examples of manganese porphyrin MRI contrast agents were produced from the metallation of endogenous porphyrins. Both manganese protoporphyrin IX (122) and hematoporphyrin (123) demonstrated strongly paramagnetic character and good in vivo stability, with clear visualization of the liver in T1 weighted images, although toxicity at higher concentrations of hematoporphyrin (38 μmol kg1) was observed. Similarly, while manganese uroporphyrin demonstrated excellent chelate stability and clear enhancement of tumor contrast in an in vivo model, toxic effects were present at all effective doses (124). In contrast, evaluation of manganese(III) mesoporphyrin in comparison to the exogenous contrast agent manganese tetra-(4-sulfonatophenyl)porphyrin showed no reported toxicity. In addition, manganese
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Metalloporphyrins for Medical Imaging Applications
O
O MeO
O N
MeO
N
M
O
N
O OMe
N
N O OMe
N
O
N
N Gd
N
O
N
O
O O
N
N
N
N
O
N
O MeO
NH
MeO
O
N
N
O O O
N H N
N
N
Gd
O
N
O N
O
O 78 M = Cu 79 M = Zn
O
Figure 38 Structure of metalloporphyrazine–DOTA conjugates 78 and 79.
mesoporphyrin was found to be a promising contrast agent, displaying enhanced lesion-to-liver contrast in both hepatic liver abscesses and metastatic liver disease, while no contrast enhancement was observed for MnTPPS at the same dose (125). As a result of the limited success of endogenous porphyrin MRI contrast agents, there is a growing interest in the development of exogenous manganese porphyrins. The most widely exploited example of these is manganese tetra-(4-sulfonatophenyl)porphyrin (MnTPPS), which was first reported as a potential MRI contrast agent in 1984, showing the greatest enhancement in water relaxation rates in comparison to both other metalloporphyrins and free Mn (126). Subsequent studies have demonstrated both the superior image enhancement capabilities of MnTPPS in comparison to cationic manganese porphyrins, and its improved in vivo stability in comparison to analogous gadolinium porphyrins (127). In addition, evaluation of its use as an MRI contrast agent has been carried out in lymphoma (128), carcinoma and sarcoma (129), and glioma (130,131), with good tumor contrast enhancement observed in all cases. Despite these initial promising results, a number of papers noted serious issues with the use of MnTPPS; significant uptake and localization of the contrast agent was observed in the liver and kidneys, and the high doses required to provide sufficient contrast enhancement resulted in green
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pigmentation of the skin and eyes (130,132), as well as more serious side effects including a number of animal deaths associated with high toxicity (132). As a result, a number of attempts have been made to optimize and modify the MnTPPS core structure, with the aims of reducing associated toxicity and improving both tumor localization and contrast enhancement. Altering the degree of porphyrin sulfonation (2, 3, or 4 sulfonate groups) has been demonstrated to affect both toxicity and tumor localization, with the trisulfonated 80 presenting the most favorable combination of biodistribution, tumor uptake, and tumor relaxivity, with the higher relaxivities of 81 and 82 countered by higher liver uptake (133). Comparison of both MnTPPS and 80 as contrast agents in a breast tumor model demonstrated that 80 offered significantly improved contrast enhancement at low doses, although its toxicity was even higher than that of MnTPPS (Figure 39) (134). Manganese tetrasulfophthalocyanine was also proposed as an alternative to MnTPPS, with its potential as a contrast agent evaluated in a mouse mammary tumor model. Both the relaxation rate and the biodistribution profile
–O
SO3–
SO3–
3S
N N
Mn
N N
N
N
–O
3S
Mn
N
N
–O S 3
80 –O
81
3S
N N
Mn
N
N
–O
3S
82
Figure 39 Mn-TPPS analogues 80––82 displaying varying degrees of sulfonation.
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Metalloporphyrins for Medical Imaging Applications
were shown to be similar to those of MnTPPS, and good selective tumor uptake was also noted (tumor: muscle ratio of 9.2:1). While the phthalocyanine was found to be both less toxic and more stable to oxidation than MnTPPS, blue staining of skin following administration was noted, persisting for 3–5 days after injection (135). Modification of the MnTPPS structure for application in different MRI applications has also been carried out, with Lee et al. demonstrating the development of a zinc-sensing MRI agent from the functionalization of 83 with chelating arms to act both as axial ligands for the Mn core and as a zinc sensor. Addition of zinc to 83 was found to decrease the R1 relaxivity from 8.7 to 6.7 mM1 s1, while in contrast a zinc-induced enhancement of T2 relaxation was observed. The efficacy of 83 was also assessed in vitro in both zinc-treated and control cells, with significantly shorter T1 values found for the control (80 ms) in comparison to the treated cells (510 ms), while T2 values were shorter for zinc-treated samples than the control. In contrast, significantly higher cytotoxicity and no differentiation between zinc-treated and control cells were observed with MnTPPS (136). Subsequent work examined the efficacy of both 83 and MnTPPS in a rat brain model. Two days after intracranial administration, strong contrast was observed in the zinc-rich gray matter with 83 and persisted for 10 days, while little enhancement was observed with MnTPPS, although this difference was attributed to more efficient uptake of 83 into zinc-rich tissue rather than significantly enhanced contrast in the presence of zinc (Figure 40) (137). –O
SO3–
3S
N N
Mn
N HN
N
N N
HN –O
3S
N
N N N 83
Figure 40 Sulfonated manganese porphyrin 83 which demonstrates capabilities as a zinc-sensing MRI sensor due to functionalization with pyridyl pendant arms.
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Most recently, Nofiele et al. produced a dimer structure based on TPPS (85), with the aim of increasing both molecular weight and rotational correlation time to increase R1. Synthesis of a second alternative structure 84 was also carried out, with the aim producing a contrast agent with improved in vivo clearance. R1 relaxivity of both 84 and 85 was evaluated against MnTPPS and Gd-DTPA, with 85 exhibiting R1 higher than both (20.9 mM1 s1 per Mn at 1T), while R1 values for 84 were higher than for Gd-DTPA but lower than for MnTPPS. Biodistribution in rats showed significant contrast enhancements in both cases, with 84 showing renal clearance within 24 h, while 85 displayed slow hepatobiliary excretion over 3 days (138,139). Use of 85 as an alternative to the commercial blood pool agent Gadofosveset was subsequently demonstrated. 85 was found to form a 1:1 complex with HSA, with an associated decrease in R1 due to protein binding restricting accessibility of water. While R1 relaxivity of 85 was significantly lower than those of Gadofosveset at low field strengths (0.5–1 T), values were similar at a clinically applicable 3 T and 85 was found to remain in the blood for longer than 24 h, offering a significant advantage in comparison to the rapidly cleared Gd-DTPA (140). In vivo evaluation of 84 was also carried out in a rat breast tumor model, with good tumor visualization observed. Use of 84 gave larger contrast enhancement than Gd-DTPA at all time points measured, and while Gd-DTPA showed shorter accumulation and washout time, 84 demonstrated improved contrast against surrounding tissue and sustained contrast enhancement over a longer time period (Figure 41) (141). As a result of the limited clinical relevance of many contrast agents based on the structure of MnTPPS, development of a range of alternative exogenous porphyrin MRI contrast agents has been carried out, with the use of alternative water-solubilization and derivatization strategies employed to tune pharmacokinetic properties, reduce toxicity, and enhance utility as contrast agents. Two examples of manganese porphyrins were produced by Bradshaw et al., specifically engineered to incorporate highly biocompatible polyhydroxylamide substituents, with the aim of producing structures displaying the hydrophilicity of MnTPPS without the associated toxicity issues. Despite this, the minimum lethal dose of 86 was higher than that of MnTPPS and in vivo imaging showed limited image enhancement and long liver retention times (Figure 42) (142). Fujimori et al. synthesized the manganese porphyrin–chelator complex ATN-10 (88) for a range of biomedical imaging applications, first used as
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HO
HO
O
N
N
O
N
OH
Mn O
N
OH
O 84
SO3–
SO3–
N
N
N –O
3S
N
N SO3–
Mn
Mn N
N
N
85 SO3–
SO3–
Figure 41 Structures of small, hydrophilic manganese porphyrin 84 and porphyrin dimer 85.
R
R
O OH
86: R = NH
OH
N N Mn
X
O HO
N 87: R =
N
R
NH
OH
R
Figure 42 Manganese porphyrins 86 and 87 bearing polyhydroxylamide substituents to improve water solubility and reduce toxicity.
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an MRI agent in 1997 for brain tumors. Uptake in rat models of both brain tumors and pathological conditions which disrupt the blood–brain barrier was examined. 88 demonstrated good uptake and retention in the tumor model, with contrast enhancement still observed 24 h after uptake, while nontumor models showed limited uptake and clearance from the body in less than 2 h. In contrast, the control Gd-DTPA demonstrated no tumor-specific uptake, showing uptake in all examples of damaged blood–brain barrier (143). Subsequent work examined uptake in both normal brain and tumor tissue; while initial uptake showed poor differentiation, good tumor:brain accumulation ratios (10.4:1) were observed after 24 h. This work also confirmed the selectivity of uptake, with no contrast enhancement observed in necrotic tissue or peritumoral tissue; however, it was noted that this uptake selectivity could be due to known poor uptake of contrast agents into brain tissue rather than enhanced uptake into tumor tissue (Figure 43) (144). Chelation of gadolinium into the ATN-10 structure was also attempted; with 89 proposed for use in both MRI and neutron capture therapy. The relaxivity of 89 was found to be significantly higher than that of both Gd-DTPA and 88, with in vivo imaging of a brain tumor model showing peak contrast enhancement after 30 min. However, 89 displayed limited in vivo stability, with evidence of both Gd and Mn dissociation after 24 h present during imaging (Figure 44) (145). Modification of porphyrin structures to engender amphiphilicity has also been attempted, with the aim of optimizing tumor uptake. Synthesis of 90 was carried out and evaluated in a squamous cell carcinoma mouse model in comparison to Gd-DTPA. While the relaxivity of both compounds was comparable, 90 showed sustained tumor enhancement after injection, with slow tumor clearance and excellent accumulation, attributed to the
H2O O N
N Mn
N
O
O
HOOC N H
COOH N
N
COOH
COOH
N 88
HOOC
COOH
Figure 43 Porphyrin–chelator complex ATN-10 88, with manganese chelation carried out by the porphyrin core rather than the peripheral chelator.
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H2O
OOC
O O
N
N N
N
O
Mn
COO
N
COO N Gd
N
COO
89
HOOC
COOH
Figure 44 Incorporation of both manganese into the porphyrin core and gadolinium into the peripheral chelator of the porphyrin–chelator complex ATN-10 89.
O O N
N Mn
N
AspOC
N
COAsp 90
Figure 45 Structure of manganese porphyrin 90.
amphiphilicity of the structure, while Gd-DTPA showed good initial tumor enhancement, followed by complete clearance after 2 h (Figure 45) (146). Modification of the structure and amphiphilicity of two cationic porphyrins has also been carried out in order to alter bioavailability. Both structures showed high T1 relaxation rates in a tissue phantom in comparison to commercial contrast agents, with 92 demonstrating superior T1 alternation in an animal model. However, 91 showed improved selective accumulation and a good toxicity profile and was suggested as a more suitable imaging agent despite the smaller effect on T1 relaxation (Figure 46) (147). 6.2.1 Macromolecular Structures There has been a growing interest in macromolecular manganese porphyrin structures, with a number of these now evaluated as MRI contrast agents.
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N+
N+
N+
N+
N N N+
Mn
N N
N
N
Mn
N
N
N+ N+
N+ 92
91
Figure 46 Cationic manganese porphyrins 91 and 92, with the altered amphiphilicity of the structures allowing modification of in vivo uptake.
O
O NH R
HO
N N
Mn
Cl N
R = chitosan (20 kDa)
N
HO
OH
O
93
O
Figure 47 Manganese porphyrin–chitosan conjugate 93, with the conjugation carried out through peptide coupling to a single carboxylic acid functionality.
Yu et al. demonstrated the synthesis of a chitosan-functionalized porphyrin, with evaluation of 93 demonstrating higher R1 values and superior image quality in comparison to the commercially available Gd-DTPA (Figure 47) (148). Subsequent work demonstrated the PEGylation of a manganese metalloporphyrin, followed by encapsulation of 94 into mono- and bis(permethyl-β-cyclodextran) structures, with the aim of producing stabilized Mn(II) contrast agents with enhanced paramagnetic character. Conjugates with 1:1 and 1:2 ratios were produced, with the minimal cytotoxicity of these structures in comparison to free 94 demonstrated. In situ reduction of the Mn(III) core to Mn(II) with sodium ascorbate was monitored by UV–vis
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spectrophotometry, with both conjugates shown to stabilize the Mn(II) species to autoxidation giving a Mn(II) half-life of 24 min. Assessment of in vitro relaxivity showed similar values for both Mn(II) and Mn(III) species, and minimal differences between the mono- and bis-cyclodextran structures, with all having higher values than commercial imaging agents (149). Further enhancement of relaxivity was attempted through the development of a branched tris-cyclodextran structure. As for the bis structure, limited cytotoxicity and improved Mn(II) stability were observed, with the tris structure having a longer Mn(II) half-life (ca. 30 min) and 7% higher longitudinal relaxivity in comparison to the bis structure, which was attributed to the enhanced rigidity of the structure (Figure 48) (150). Exploitation of the ability of cyclodextran structures to encapsulate multiple metalloporphyrins was also exploited by Aime et al. in the development of contrast agents sensitive to the partial pressure of oxygen, allowing applications in functional MRI. Incorporation of MnTPPS into a commercial cyclodextran structure bearing six to nine cyclodextran units was carried out, with large numbers of chelated MnTPPS engendering significant differences between the relaxivity of the Mn(II) and Mn(III) species (40.8 and 15.2 mM1 s1, respectively) (151). PEGylation of manganese porphyrins in order to improve relaxivity and reduce cytotoxicity has also been demonstrated, with 95 demonstrating R1 values ca. 7.5 times higher than MnTPPS, with the high R1 attributed to the slow tumbling rate of 95 rather than increased viscosity due to mPEG550 O
O – mPEG550
N
N Mn
N
N
O mPEG550
94
O mPEG550
Figure 48 PEGylated metalloporphyrin 94, which was subsequently incorporated into mono- and bis-cyclodextran structures.
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O O
O
O n
O
O
O
O
n
N N
Mn
N
N
O
O n
O
95
O
n
Figure 49 PEGylated metalloporphyrin 95, which was subsequently incorporated into an oligocyclodextran structure.
PEGylation. Cytotoxicity of 95 was also evaluated, with the IC50 shown to be significantly higher than MnTPPS (4.1 and 0.25 mM, respectively) (Figure 49) (152). Exploitation of nanoparticle delivery vehicles for porphyrin contrast agents has been demonstrated within the literature, with this approach favored due to allowing delivery of increased quantities of contrast agents to the tumor site, enhanced R1 values and alteration of both toxicity and pharmacokinetic properties. However, conjugation strategies must be carefully considered; although encapsulation of structures to incorporate them into nanoparticles is a popular and facile methodology, it also limits water access to the contrast agent, leading to deterioration of R1 values. Polylactic acid nanoparticles were utilized by Jing et al., with the synthesis of a theranostic delivery platform through encapsulation of therapeutic doxorubicin and covalent surface functionalization with 96. R1 relaxivity of the nanoparticles was found to be significantly higher than that of unconjugated 96 (27.8 and 6.7 mM1 s1, respectively), with this enhanced relaxivity attributed to reduction of the contrast agent tumbling rate following attachment to the solid support. In vivo evaluation in a mouse tumor model allowed clear visualization of tumor, including areas of inhomogeneity not observed without contrast agent, with signal enhancement increased from 30 min to 24 h (153). Subsequent work from this group utilized doxorubicin-loaded polylactic acid coated with a gold nanoshell, with surface functionalization with 96 carried out via a PEG spacer group. In contrast to nonPEGylated systems, the synthesized system demonstrated reduced aggregation, significantly longer blood circulation time, and good colloidal stability over 1 week. While
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the R1 relaxivity of the system (22.18 mM1 s1) was higher than that of free 96, it was also lower than that of previously synthesized nanoparticle systems (153). Despite this, in vivo imaging showed good visualization of the tumor, as well as therapeutic effects through both release of doxorubicin and photothermal therapy (154). Conjugation of 96 to oxidized dextran has also been carried out by Zhang et al., with the resulting high molecular weight conjugate showing reduced toxicity in comparison to free 96. While both the dextran conjugate and 96 showed enhanced R1 values in comparison to the control Gd-DTPA, relaxivity showed only minimal enhancement upon dextran conjugation (8.9 and 7.4 mM1 s1, respectively), as a result of the increasing molecular weight. In vivo evaluation of the dextran conjugate in comparison to Gd-DTPA showed tumor enhancement in both cases; however, higher signal enhancement of the tumor over a longer time period was observed for the conjugate (155). Covalent surface attachment of 96 has also been demonstrated on organic nanoparticles synthesized through self-assembly of tetraphenylporphyringrafted lipids. R1 relaxivity was found to be about four times that of the free 96 (20.6 and 5.2 mM1 s1, respectively) but lower than that of previously synthesized nanoparticle contrast agents. However, in vivo imaging showed good diagnostic and therapeutic capabilities, with clear tumor signal enhancement and effective treatment both in vitro and in vivo utilizing the photodynamic therapy capabilities of the free-base porphyrin–lipid (Figure 50) (156). SO3–
N –
N Mn
O3S N
NH2
N
SO3– 96
Figure 50 Sulfonated manganese porphyrin 96, with the amine functional group allowing conjugation to a number of nanosized delivery vehicles.
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MacDonald et al. also demonstrated the synthesis of porphyrin–lipid nanostructures to create porphysomes; organic, biodegradable liposome-like structures formed from self-assembly of a pyropheophorbide–lipid conjugates in aqueous media. Use of manganese-chelated 97 allows for labeling of all 80,000 porphyrins in the porphysome to created high payload contrast agents, with no disruption of the porphysome structure by the axial ligand observed. In addition, manganese chelation was found to improve photostability, and reduce phototoxicity, with the structure showing only limited cytotoxicity in vitro. Relaxivity values were determined for both intact and detergent disrupted Mn porphysomes; while both R1 and R2 values for intact porphysomes were low (1.2 and 7.0 mM1 s1, respectively), disrupted relaxivity was comparable to that of Gd-DTPA (R1 ¼ 4.0 mM1 s1 and R2 ¼ 12.9 mM1 s1). The increase in relaxivity upon disruption of Mn porphysomes was attributed to the result of increased water access to the inner coordination shell of the Mn ions, with the authors suggesting that optimization of the porphysome structure could allow enhancements to relaxivity (Figure 51) (157). Recently, Qazi et al. demonstrated an alternative strategy utilizing novel nanoparticles constructed from a hybrid of synthetic polymer and virus-like capsid particles. The porphyrin contrast agent was encapsulated into the particle through reaction between the activated porphyrin 98 and the amine-functionalized interior surface of the particle, to achieve loading ratios ranging from 121 to 3646 molecules of 98 molecules per capsid. T1 relaxivity remained constant per Mn ion, with the highest whole particle values of 7100 mM1 s1 achieved. Despite this, relaxivity of the porphyrin O
O O O
13
O O
P O O–
N+
O
N
N Mn N
97 N
Figure 51 Manganese pyropheophorbide–lipid monomer 97 which spontaneously self-assembles in aqueous environments to produce porphysome contrast agents.
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did not increase upon capsid conjugation, which can most likely be attributed to the poor water access inside the capsid. R2 relaxivity was also evaluated for this system and displayed an inverse relationship with increasing porphyrin loading ratios, allowing the system to be modified for use as either a T1 or T2 contrast agent (Figure 52) (158). Inorganic nanoparticles have also been utilized as solid supports, with Hitomi et al. demonstrating the synthesis of thiol-functionalized 99, allowing direct attachment to gold nanoparticles. Alterations to porphyrin: nanoparticle ratios were found to vary size, optical, and MRI properties, while relaxivity was found to vary with nanoparticle size. In all cases, R1 per Mn center was up to 4.3 times that of free 99 (Figure 53) (159). Proteins have also been explored as an alternative to synthetically produced nanoparticles for the targeted delivery of metalloporphyrin contrast agents. Initially, Shapiro et al. demonstrated the use of the heme domain of Bacillus megaterium cytochrome P450 BM3 (BM3h) to detect dopamine OH O N
N Mn
N
N O O O N
98
O
Figure 52 Contrast agent 98 bearing an NHS ester modality for facile conjugation to semisynthetic nanoparticle structures.
NH
S O O S
N N Mn
NH
N
S
NH O O
N NH
S
99
Figure 53 Structure of manganese porphyrin 99 functionalized with peripheral thiol groups to facilitate attachment to gold nanoparticles.
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release in living rat brains, with the native iron porphyrin acting as the contrast agent. However, these experiments were relatively insensitive due to the low R1 relaxivity of the unmodified proteins (ca. 1 mM1 s1) (160). Substitution of the iron heme for Mn protoporphyrin IX was then attempted, with the similar structure of the porphyrin allowing improved R1 values without affecting protein folding. The synthesized Mn-BM3h showed >95% manganese incorporation, increased heat stability, and twofold increased T1 relaxivity (2.6 mM1 s1) over the native variant. The relaxivity of a number of other Mn-PPIX-derived proteins was also measured, with the Mn-C1634 showing the highest R1 and R2 relaxivities (3.3 and 7.4 mM1 s1, respectively) (161). A similar analysis was carried out using the Thermoanaerobacter tengcongensis nitric oxide/oxygen-binding protein (NOX), with the native iron-containing heme form showing limited application as an MR imaging agent with T1 and T2 relaxivities of 1.6 and 2.9 mM1 s1, respectively. Substitution of the heme for manganese protoporphyrin IX chelated with both Mn(II) and Mn(III) was shown to produce stable structures. While Mn(II) showed modest enhancement in relaxivities (R1 ¼ 3.6 mM1 s1 and R2 ¼ 3.8 mM1 s1), significant enhancements were observed for the Mn(III) structure (R1 ¼ 12.0 mM1 s1 and R2 ¼ 16.8 mM1 s1). In addition, incorporation of gadolinium mesoporphyrin IX was carried out to produce even higher relaxivity values, with R1 ¼ 28.7 mM1 s1 and R2 ¼ 39.9 mM1 s1 (162).
7. PHOTO-ACOUSTIC IMAGING Photo-acoustic imaging has recently emerged as one of the newest and most rapidly growing medical imaging techniques. This hybrid technique combines laser irradiation with ultrasound detection, with target chromophores excited with a near-infrared laser light to induce thermoelastic expansion within the tissue. As a result of this, acoustic pressure waves are generated, which can be detected by an ultrasound transducer to form a photo-acoustic image. Imaging in this way offers multiple advantages over both traditional optical and ultrasound imaging techniques, with high spatial resolution, applicability across both the macro- and microscale, greater specificity than traditional ultrasound imaging, and imaging at depths of up to 5–6 cm, far beyond the limitations of optical imaging (163). The first examples of photo-acoustic imaging utilized naturally occurring pigments including hemoglobin as contrast agents, allowing distinction of
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the blood vessels from surrounding tissue. However, early detection of tumors utilizing these endogenous chromophores was limited, with the poor vascularization of small tumors limited the quantity of hemoglobin present. As a result, addition of an exogenous contrast agent is required (164). To this end, a number of exogenous dyes have been evaluated as contrast agents, including nonporphyrin structures such as methylene blue, congo red, indocyanine green (163), and a limited number of exogenous tetrapyrroles, pyropheophorbide-α (165), bacteriochlorins (166), and modified porphyrin derivatives (167). While tetrapyrroles do offer high optical absorption which generates strong PA signals, their high fluorescence acts as a competing relaxation mechanism, reducing heat generation and consequently ultrasound wave generation. Recently, the first examples of metallophthalocyanine contrast agents have been developed, with the clinical relevance of the lipophilic zinc phthalocyanine 100 compared to four other photosensitizers; protoporphyrin IX, a squarine, Ce6, and methylene blue in a scattering phantom. 100 demonstrated the highest photo-acoustic quantum yield (0.47) and greatest light absorption between 700 and 900 nm of all evaluated contrast agents; however, relatively high cell toxicity was observed. While in vivo evaluation in a mouse tumor model showed evidence of photo-acoustic signals in the liver, spleen, and intestines, tumor selectivity was good, with maximum tumor:normal tissue ratios of 7:1 observed after 60 min, providing good contrast in all images (Figure 54) (168). As a result, synthesis of a range of hydrophilic metallophthalocyanines was then carried out, with these potential contrast agents examined in both a tissue phantom and a mouse oral squamous cell carcinoma model. In both cases, free-base porphyrin 101 exhibited the best photo-acoustic properties, with this effect attributed to the metallation promoting internal conversion to the triplet state rather than heat generation. In vivo imaging showed SO3H
HO3S N N
N N
Zn
N N
N N
M
N N
N N 100
N N
101 M = 2H 102 M = Zn 103 M = AI
N N
HO3S
SO3H
Figure 54 Metallophthalocyanine photoacoustic imaging agents 100–103.
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superior localization and imaging with 101, with 102 producing signals of three times lower intensity, and 103 providing no tumor delineation (169).
8. MULTIMODAL IMAGING Interest in the development of multimodal imaging has arisen as a natural consequence of the inherent limitations of each of the discussed imaging technologies, including spatial resolution, sensitivity, and speed of imaging. As a result of these shortcomings, all generated images provide only limited information, and there is a growing need to combine the generated information from two or more scans to provide both morphological and functional information. While simply fusing the information from two or more separate scans is widely used, difficulty in controlling patient movement and position means that confirmation of co-registration of signals is complex. An alternative is the combination of two scanners into a single unit to simultaneously provide two images, an approach which has been successfully applied to SPECT/CT and PET/CT for around 20 years (170). As well as the improved speed and the increased amount of information provided by combined scanning methodologies, combining two imaging techniques into a single multimodal imaging agent also offers numerous clinical advantages. These include allowing the study of processes and structures from differing resolution and depths, as well as permitting investigation of tumor morphology, growth, pharmacokinetics, and mechanism of drug action, while only requiring administration of a single compound. In particular, the structure of porphyrins makes them particularly appealing as multimodal imaging agents; as well as offering inherent fluorescent properties and facile chelation, they also offer multiple sites for functionalization, allowing development of a wide range of multimodal agents.
8.1 SPECT–Fluorescence SPECT and fluorescent imaging technologies were combined by El-Tamer et al. in 2003 to produce the first example of a multimodal metallophthalocyanine imaging agent. Synthesis of the 99mTc-radiolabeled tetrasulfophthalocyanine was carried out to produce an imaging agent suitable for the detection of breast cancer metastases in sentinel lymph nodes, with the aim of overcoming the wide discrepancies observed when SPECT and fluorescent imaging agents were administered separately. 99mTc radiolabeling was carried out in 30 min under mild reaction conditions, with the resulting conjugate allowing sentinel lymph node visualization in a
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rabbit model through both SPECT and fluorescent imaging, with no uptake into nontarget lymph nodes or accumulation associated with free 99mTc observed (171).
8.2 Photo-Acoustic/Fluorescence The combination of photo-acoustic imaging and fluorescence imaging into a multimodal agent is an obvious choice, with many endogenous porphyrin structures demonstrating efficacy as both fluorescent and photo-acoustic imaging agents. Despite this, only a single example of this strategy has been published to date, with Lovell et al. demonstrating the use of porphysomes containing a range of different metals. Metallation was shown to modulate both absorption and emission wavelength, allowing facile tuning to meet specific wavelengths. Although absorption of 104 was unchanged following porphysome formation, extensive fluorescence quenching was observed due to the close packing in the structure. As a result, following irradiation, the energy was released via thermal expansion, with significant temperature increases (35 °C) observed following laser irradiation. Assessment of both fluorescence and photo-acoustic imaging was carried out in vivo, with initial tumor localization of the porphysomes clearly detected using photo-acoustic imaging with no damage to surrounding tissue. While tumor fluorescence was unchanged after 15 min, after 2 days significant fluorescence was observed, which was attributed to porphysome degradation inside cells, and subsequent reduction in quenching (Figure 55) (172). O O O
O
O P–
O
N+
O
O
N
N M
N
104 M = Zn, Cu, or Pd
N
Figure 55 Pyropheophorbide–lipid monomer 104 which spontaneously self-assembles in aqueous environments to produce porphysomes. Metallation of this structure with zinc, copper, and palladium was carried out to modulate both absorption and emission wavelengths.
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8.3 PET–Fluorescence PET–fluorescence combines two popular imaging modalities to allow imaging on both the micro- and macroscale and, as a result, is a popular option for metalloporphyrin imaging despite the difficulty in designing structures which retain the fluorescent properties of the porphyrins while allowing functionalization with paramagnetic radioisotopes. This problem was encountered by Liu et al., who utilized a targeted 64Cu pyropheophorbide 105 to carry out ovarian cancer imaging in primary cell line-derived tumors. While 105 showed good tumor:muscle ratios (4.97 at 4 h), fluorescence imaging could not be carried out due to quenching of the excited state by the copper core. As a result, fluorescent imaging was carried out with a mixture of 105 and the free-base derivative, with PET imaging showing clear tumor delineation, while fluorescence imaging showed excellent uptake into metastases, with 3.5-fold higher fluorescence in comparison with adjacent normal tissue (Figure 56) (173). Alternatively, use of radioisotopes other than 64Cu allows retention of fluorescence after radiolabeling. Labeling of metalloporphyrins and phthalocyanines with 18F was carried out by Ranyuk et al. for PET/fluorescence imaging, with relatively poor yields (18%) of the cold analogues obtained even under forcing conditions (80 °C for 20 min). Yields obtained using 18 F were also poor, with 107 obtained in RCY of 10%, while synthesis of 18F-labeled 106 was not attempted (Figure 57) (174). An alternative labeling methodology offering improved yields and retention of fluorescence is the incorporation of the radioisotope into chelators attached to the tetrapyrrole structure. Ranyuk et al. attached both DOTA
N
N
NH2
64Cu
N
N N
N
NH N
O
HN O
O NH GDEVDGSGK–NH
COOH 105
NH O
O
Figure 56 Targeted 64Cu pyropheophorbide 105, which was combined with the unmetallated analogue to produce a multimodal fluorescence-PET imaging agent.
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R
R
F
R
N
N Zn
N
R
N
R = tBu
R
R 106 t
C4H9
N tC
4H9
N N
N
N
N
Zn N
tC
N
4H9
18F
107
Figure 57 Metalloporphyrin 106 and metallophthalocyanine 107 labeled with positron-emitting 18F.
and NOTA chelators via varying lengths of linker chain to a zinc phthalocyanine to create multimodal imaging agents, with radiolabeling with 64Cu and 68Ga carried out in good RCY (ca. 70% and 60% decay-corrected yields, respectively). The 64Cu chelates showed superior serum stability, with no loss of copper observed, and therefore, 109a and 109c were selected for in vivo evaluation. Biodistribution studies showed rapid kidney uptake and excretion of 109c, while 109a demonstrated 10-fold larger retention. Despite this, in vivo imaging showed high surface distribution and limited tumor uptake, with the tumor only poorly distinguished from the surrounding tissue using both fluorescence and PET imaging (Figure 58) (175). Incorporation of 64Cu directly into a metalloporphyrin structure can also be an effective method of producing multimodal imaging agents through the
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H n N
NaO3S N N
N Zn
N
O
N
O
N N
N
O
N
68
Ga
N
O N
108a n = 1 108b n = 3 108c n = 5
O O
N
O
SO3Na
NaO3S
N N
N Zn
N
O
N
N
O
O
H n N
NaO3S
N
64
Cu N
109a n = 1 109b n = 3 109c n = 5
N
O
N O
N NaO3S
SO3Na
Figure 58 Structures of imaging agents 108–109, with the conjugated DOTA and NOTA macrocycles allowing chelation of paramagnetic metals without loss of the zinc phthalocyanine fluorescence.
use of multiply-loaded nanosized delivery vehicles; this allows a proportion of the porphyrin to be radiolabeled, allowing retention of some of the fluorescent characteristic. Liu et al. demonstrated this technique through the use of porphysomes, with radiolabeling carried out on 110, and subsequent self-assembly producing a porphysome with high specific activity (103,600 GBq mmol1). Despite this, only 5% of porphyrins present in each porphysome were labeled, leaving porphysome size and photophysical properties unaffected. Evaluation as an imaging agent in a prostate cancer model demonstrated excellent tumor uptake after 24 h, with very little background uptake and high tumor:muscle ratios (12.7:1) (176). The utility of 110 was also demonstrated in both large and small prostate tumor models, with clear delineation of tumors of all sizes, as well as metastases present in the spine and bones, via both fluorescence and radio-imaging (Figure 59) (177).
8.4 MRI/Fluorescence In considering the combination of PET and fluorescent imaging, multimodal MRI/fluorescence agents are an attractive option for imaging, but
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O O O
13
O
O P O –O O
N+
O 110 N
N 64
Cu N
N
Figure 59 Pyropheophorbide–lipid monomer 110 which spontaneously self-assembles in aqueous environments to produce porphysomes, allowing the nanostructure to be partly radiolabeled with 64Cu to produce a multimodal imaging agent.
O O
O N H
O
N
H N
N O
N N
N
N
N Zn
N 111
N N
N
Figure 60 Zinc phthalocyanine 111 conjugated to super paramagnetic iron oxide nanoparticles via a click chemistry methodology.
the synthesis of suitable metalloporphyrin agents is limited by the paramagnetic properties of metals suitable for MRI. The challenge is further complicated by the lower limits of detection required for MRI compared to PET, making partial-metallation methodologies impractical. As a result, attachment of tetrapyrrole structures to an external MRI contrast agent is the only practicable method to produce such multimodal agents. Boudon et al. employed this strategy using super paramagnetic iron oxide (SPIO) nanoparticles, which were silanized to allow functionalization with zinc phthalocyanine 111 in ratios of ca. 690 per nanoparticle. The excitation and emission properties of 111 were unaffected by conjugation, with measured R2 and R1 found to be similar to commercial contrast agents (73 and 3 mMFe1 s1, respectively), with the low R2 value attributed to the highly hydrophobic phthalocyanine coating limiting access of water molecules to the SPIO core (Figure 60) (178).
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Most recently, attachment of zinc porpholactol to two chelating agents has been carried out to produce two multimodal imaging agents. In both cases, fluorescence was not affected by conjugation, and T1 relaxivities (ca. 18 mM1 s1 at 0.5 T and 37 °C) were found to be significantly higher than that of Gd-DTPA. In vitro imaging via both MRI and fluorescence was carried out successfully, with good images and no dark toxicity found for 112 or 113 (Figure 61) (179). O O
F
F
F
F
F
FO
F
N
F N
N O O
O
F
O
F
Zn F
N Gd N
O F
N
N
N=N N
N F
F F
F
F
F
112
F F O O N
F
F
N=N N
F
F
F
FO
F
N
F F
N
N O O
O F
Zn N
N Gd O
O F
N
H N
O F
N F
F F
F
F
F
113
F F
Figure 61 Combining zinc porpholactols with gadolinium chelating macrocycles allows retention of the porpholactol photophysical properties to produce MRI/fluorescence imaging agents 112 and 113.
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8.5 PET/MRI The development of combined PET/MRI imaging agents is a novel combination of two powerful imaging modalities, which was restricted for many years due to the technical challenges involved in the combination of both technologies into a single scanner. The first example of a metalloporphyrin-based system was produced by Gros et al., utilizing a structure consisting of two chelators, a porphyrin and a tetraazamacrocycle. Metal chelation was performed in a two-step process, with gadolinium metallation carried out at 50 °C for 12 h, followed by copper insertion at 50 °C for 2 h. 114 demonstrated good stability in water at physiological pH, with no transmetallation or release of gadolinium observed. T1 relaxivity of the structure at 300 MHz was found to be similar to that of unconjugated gadolinium-DOTA, with the attached metalloporphyrin having little effect, although the increased molecular weight resulted in a medium field relaxivity higher than that of commercially available contrast agents. Metallation of the porphyrin with 64Cu was not attempted, with no evaluation of the conjugate as a PET imaging agent carried out (Figure 62) (180). Subsequently, the same group also developed two examples of PET/MRI agents, with 115 functionalized with an amine functional group for attachment to targeting biomolecules, and 116 functionalized with two gadolinium chelators for improved contrast enhancement. Both structures were shown to exhibit no cytotoxicity; however, some cell growth inhibition was observed in both cases. T1 relaxivity values were found to be typical of gadolinium complexes of this type, with again no influence from the copper porphyrin observed. Despite this, preliminary MRI in mouse tumor models showed rapid blood clearance and no enhanced tumor uptake was observed (Figure 63) (181). H2O O
N O
O
O N Gd
N
N O
O NH
N
N Cu
O 114
N
Figure 62 Structure of bifunctional PET/MRI chelator 114.
N
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H2O O
N O
O
O N Gd
N
O
N
N
O
N Cu
HN
O
N
NH2 N 115
H2O O
O
N O
O
N Gd
N
N O
O
N
O
N
O
Cu
HN N
NH N
O N
O
N Gd
N
O N
O
116 O
O
H 2O
Figure 63 Single and double gadolinium-chelator functionalization of copper porphyrins 115 and 116.
9. CONCLUSIONS While exploitation of porphyrins and other tetrapyrrolic photosensitizers as therapeutic modalities has been of interest for over 100 years, the application of these discussed structures as clinical treatments for neoplastic conditions is still a relatively new field, with the clinical photosensitizer Photofrin receiving regulatory approval as recently as 1993. Similarly, although the utility of metalloporphyrins as imaging agents has been recognized for over 60 years, none of the discussed structures have yet been approved for clinical imaging applications.
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This lack of clinically approved metalloporphyrin imaging agents cannot be attributed to a lack of clinical relevance of these structures; as demonstrated throughout this review, metalloporphyrins can offer both selective tumor uptake and excellent imaging potential across a plethora of imaging technologies. Despite this, the nature of metalloporphyrins presents innate challenges in the development of imaging agents; the complex synthesis and functionalization of many described structures can limit scalability of production and purification, while their highly lipophilic nature results in high nontarget organ uptake in liver, spleen, and kidneys. As a result, there is a pressing need for future work in this area to focus on addressing these issues. In particular, strategies for imparting full water solubility on structures without increasing structural complexity or engendering toxicity are needed. Development of structures with both accessible and flexible bioconjugation handles is also required in order to meet the growing appreciation of the efficacy of biomolecule-targeted structures to improve tumor uptake and reduce nontarget organ uptake, rather than relying on passive uptake alone. In addition, it is becoming apparent that the greatest strength of metalloporphyrins is their multifunctionality, simultaneously allowing conjugation and metal chelation while in many cases retaining both innate fluorescent and therapeutic properties. This property aligns well with the growing interest in the area of cancer research in both multimodal imaging and theranostics. These multifaceted techniques offer the most promising step forward to date toward overcoming disease heterogeneity. They offer increased accuracy in disease staging and determination of receptor expression in both metastatic lesions and primary tumors, in addition to allowing exploitation of emerging technologies such as MRI/PET, providing a drive toward the eventual goal of personalized medicine.
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Metalloporphyrins for Medical Imaging Applications Francesca Bryden1, Ross W. Boyle Department of Chemistry, University of Hull, Hull, United Kingdom 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Fluorescence Imaging 2.1 Phthalocyanine Dyes 2.2 Nanoscale Structures 2.3 FRET Systems 3. Raman Imaging 4. Gamma Imaging 4.1 Indium-111 4.2 Technecium 99m 4.3 Gallium 67 4.4 Other Radionuclides 5. Positron Imaging (PET) 5.1 Copper 64 5.2 Gallium 68 5.3 Other Isotopes 6. Magnetic Resonance Imaging 6.1 Gadolinium 6.2 Manganese 7. Photo-Acoustic Imaging 8. Multimodal Imaging 8.1 SPECT–Fluorescence 8.2 Photo-Acoustic/Fluorescence 8.3 PET–Fluorescence 8.4 MRI/Fluorescence 8.5 PET/MRI 9. Conclusions References
Advances in Inorganic Chemistry, Volume 68 ISSN 0898-8838 http://dx.doi.org/10.1016/bs.adioch.2015.09.003
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Abstract In this account, the synthesis and biological evaluation of metalloporphyrins and related structures for use in biomedical imaging applications are outlined. The structural design of these tetrapyrrolic metal chelators is discussed, with evaluation of strategies designed to facilitate more rapid chelation under milder conditions, improve water solubility, and allow for conjugation to targeting groups and macromolecular structures. Particular emphasis is given to the application of these metalloporphyrin structures to clinically relevant imaging techniques including magnetic resonance imaging, fluorescence imaging, radio-imaging, and Raman and photo-acoustic imaging, with a broad overview of the progress made in these areas. The future of research in this area is also examined, with the emerging research interest in more modern multimodal imaging set to dominate the future of biomedical imaging.
1. INTRODUCTION Porphyrins are a class of macrocycles comprised of four pyrrole units conjugated through methine bridges, with this highly conjugated structure giving intense absorption in both the UV and visible regions of the electromagnetic spectrum. This in turn leads to both the characteristic purple color of these structures and their name; the word porphyrin is derived from the Greek porphyra, meaning purple. Modifications to the porphyrin structure including alterations to the degree of conjugation and number of heteroatoms can be carried out to produce a host of tetrapyrrole structures, including the naturally occurring chlorins and bacteriochlorins (Figure 1).
Porphyrin
Chlorin
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N
N
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N NH
N
N
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NH N
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N
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N
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N
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N HN
N
N
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Phthalocyanine
Porphyrazine
NH HN
Corrole
Figure 1 Skeleton structures of porphyrin and a range of other tetrapyrrolic molecules.
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Porphyrins are most well known for their essential roles within a number of biological processes, most notably as the iron-containing heme which permits hemoglobin oxygen transportation in the blood of all vertebrates. It is therefore unsurprising that the first examples of porphyrins described in the literature were produced from endogenous sources, with Nencki and Zeleski first describing the isolation of hematoporphyrin from hemoglobin in 1900 (1). Subsequently, a range of both endogenous and synthetic exogenous porphyrins have been described, in addition to numerous alternative tetrapyrrolic structures; while these structures are of interest due to their enhanced photophysical characteristics and altered reactivity, their challenging synthesis and functionalization make them less commonly utilized in comparison to porphyrins. Although porphyrins and related structures have been exploited in many applications including light harvesting (2) and the development of supramolecular structures (3), they are best known for their use in medical applications, in particular as photosensitizers in photodynamic therapy. These photosensitizers are essentially nontoxic in the absence of light; however, upon irradiation these structures can undergo a cascade of photochemical processes to produce cytotoxic reactive oxygen species, capable of triggering cell death and leading to tumor eradication. However, there is also a growing interest in porphyrins and other tetrapyrroles for use in diagnostic medical applications, with the inherent fluorescence, facile derivatization, low cytotoxicity in the absence of light, and preferential tumor uptake of these structures, making them highly attractive as diagnostic modalities as well as therapeutics. To date, the vast majority of research into porphyrins for medical applications has exploited the use of free-base porphyrins; these unmetallated structures are generally easier to synthesize, and chelation of paramagnetic metals into the porphyrin core is incompatible with many uses, causing quenching of the porphyrin excited state and associated loss of both fluorescence and therapeutic activity. Despite this, metalloporphyrins also offer significant research interest; porphyrins are highly efficient metal chelators, with the planar porphyrin ring distorting to allow chelation to metals which are either too small or too large to fit well into the porphyrin cavity, and to date complexes hosting a range of at least 50 metals have been described in the literature (4). In particular, the utility of metalloporphyrins as imaging agents is a relatively unappreciated but growing field, with the metallation of porphyrins offering application of the natural tumor affinity of porphyrins to imaging techniques including fluorescence, magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT).
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2. FLUORESCENCE IMAGING Fluorescence imaging was one of the earliest medical imaging techniques to be adopted, exploiting the innate property of some chromophores to absorb light at a specific wavelength and subsequently reemit light at a longer wavelength. Autofluorescence of hematoporphyrin in a rat tumor upon irradiation with UV light was first observed in 1921 (5), while the first deliberate application of fluorescence to tumor imaging was carried out in 1942, with administered hematoporphyrin showing localization in both tumor tissue and lymph nodes (6). Despite this long history, fluorescence imaging is still highly relevant to clinical imaging applications today, allowing both in vivo and in vitro imaging on the micro- and macroscale. In addition, fluorescence imaging offers high sensitivity, rapid imaging, and continuous real-time monitoring without high cost or the need for ionizing radiation, and as a result, it is still one of the most popular imaging techniques. Despite this, the use of light to both activate and detect the imaging agent presents challenges; both resolution (2–3 mm) and imaging depth (<1 cm) are limited, and obtaining quantitative data is difficult (7). Due to the well-understood photophysical properties of porphyrins, much of the early literature focusses on the use of free-base porphyrins as fluorescence imaging agents. However, metalloporphyrins and derivatives, in particular metallophthalocyanines, are also of significant interest for optical imaging. Although metallation can affect the wavelength and intensity of both the UV–vis and fluorescence spectra of tetrapyrrolic structures, it allows greater flexibility in the construction of synthetic reaction schemes as well as optimization of photophysical characteristics of the fluorescence imaging agent.
2.1 Phthalocyanine Dyes Phthalocyanines show particular promise in the area of fluorescence imaging due to their favorable optical properties, with their high molar absorptivity (ε > 200,000 M1 cm1) (8) at wavelengths greater than 680 nm allowing for good quality fluorescent images to be produced using exciting light in the red and near-infrared portion of the electromagnetic spectrum. As a result of this shift in wavelength, both sensitivity of the technique and light penetration in human tissue increase significantly due to a simultaneous reduction in both autofluorescence from endogenous fluorophores, and light scattering in the near-infrared region. Metallophthalocyanines
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incorporating diamagnetic metals such as aluminum, zinc, and gallium are of particular interest in this application due to their increased fluorescence quantum yield and increased stability, while no significant quenching of fluorescence is observed, in contrast to paramagnetic metals such as copper. Chloroaluminum sulfophthalocyanine was the first metallophthalocyanine utilized for optical imaging, with its excellent photostability and long emission lifetimes (ca. 7.5 ns), making it an ideal chromophore for time-delayed fluorescence imaging. Although its use in vivo was not explored, in vitro tests in a model tissue phantom demonstrated excellent potential as a fluorescence imaging agent, operating well over a range of concentrations (9). However, despite these early promising results and the commercial availability of the sulfonated aluminum phthalocyanine derivative Photosens® as a therapeutic agent in Russia, no attempt to utilize this phthalocyanine in fluorescence imaging has been published. In contrast, zinc phthalocyanine derivatives have been widely explored as optical imaging agents, particularly in the imaging of neoplastic conditions. The first example of this was carried out by Mantavera et al., who utilized a range of cationic zinc phthalocyanines to image B16 melanoma; a highly pigmented cancer which cannot be imaged easily with traditional porphyrin imaging agents due to the strong visible absorption of the highly pigmented melanoma. Functionalization of cationic phthalocyanines with varying lengths of alkyl chain produced a range of phthalocyanines with varying amphiphilicity which retained photophysical properties of the parent phthalocyanine, demonstrating excellent light absorption outside the absorption range of melanin. In vitro uptake of all three dyes into melanoma cells over time was monitored; with 2 demonstrating the best uptake profile, attributed to the highly amphiphilic nature of the molecule. In vivo imaging also demonstrated promising results, with a mouse model showing good (2.4–3.8 higher fluorescence) differentiation between normal skin and tumors measuring 3–5 mm, although fluorescence of smaller tumors was minimal (Figure 2) (10). Imparting water solubility on zinc phthalocyanines has also been demonstrated through peripheral functionalization with a range of mono- and oligosaccharides. In addition to improving hydrophilicity, these saccharides may also demonstrate a tumor-targeting effect, with the overexpression of glucose transporting and galactose receptors reported on the cell surface of some neoplastic tissues (11). This strategy was first utilized in the synthesis of fluorescent probes by Liu et al., who utilized a click chemistry to conjugate a zinc phthalocyanine to four azide-functionalized glucose groups (4).
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R N+ 1
N+ R
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O
O N 2
R=
N N
4 3
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N 10
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Figure 2 Cationic zinc phthalocyanines 1–3, with differing aliphatic chain lengths allowing modification of amphiphilicity.
Analysis of 4 in DMSO demonstrated retention of clinically relevant photophysical properties: absorbance at 618 and 682 nm, fluorescence at 690 nm with a high fluorescence quantum yield (0.48), and no significant photobleaching on timescales relevant to imaging. Despite the observation of significant aggregation of the photosensitizer and corresponding complete fluorescence quenching during in vitro imaging, imaging in an animal model showed no fluorescence quenching, with the difference between in vivo and in vitro results attributed to disaggregation of 4 following administration. While 4 demonstrated clear liver and kidney uptake and no associated organ toxicity, tumor uptake was found to be negligible, with fluorescence shown to be no higher than in control animals (Figure 3) (12). As a result of the demonstrated limited tumor affinity of glucose moieties, the same group subsequently utilized an alternative saccharide, galactose, for targeting liver cancer. Synthesis of a series of phthalocyanines bearing between 1 and 4 galactose groups was carried out (5–8), with only 7 and 8 exhibiting complete water solubility. As for 4, while aggregation in aqueous media led to weak fluorescence in vitro, all phthalocyanines demonstrated good in vivo fluorescence. Optical imaging in a mouse model demonstrated clear fluorescence of the tumor in comparison to surrounding tissue when utilizing phthalocyanines 7 and 8, with the best images obtained between 12 and 24 h after administration. In contrast, imaging agents 5 and 6 showed no significant increase in tumor fluorescence compared to the
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OH HO
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Figure 3 Structure of glucose-functionalized zinc phthalocyanine 4.
R1
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5: R1 = R, R2 = H, R3 = H, R4 = H 6: R1 = R, R2 = H, R3 = R, R4 = H 7: R1 = R, R2 = R, R3 = R, R4 = H 8: R1 = R, R2 = R, R3 = R, R4 = R
Figure 4 Zinc phthalocyanines 5–8, with the differing levels of saccharide functionalization shown to have significant effects on both water solubility and pharmacokinetic character of the imaging agents.
control, with this lack of fluorescence attributed to poor solubility and an associated enhanced potential for aggregation (Figure 4) (13). An alternative to galactose for the targeting of liver cancer is the disaccharide lactose; similar to galactose, it allows active targeting due to the hepatic asialoglycoprotein receptors overexpressed on the surface of many liver tumors, while also offering improved water solubility. A click conjugation strategy was again used by Lv et al. to produce the saccharidesubstituted phthalocyanine 9, with four lactose units attached per phthalocyanine. While photophysical properties were found to be similar to previously synthesized optical imaging agents 4–8, in contrast 9 also showed limited cytotoxicity and improved photostability. In vivo evaluation in a mouse model demonstrated good selectivity, with a clearly visible signal
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O
O N N N
HO OH O
R= HO
OH
HO O
O
OH
N Zn
N
N R N N
OH
N N
N R N N N
O
O
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9
Figure 5 Saccharide-substituted zinc phthalocyanine 9. The use of the disaccharide lactose was shown to allow improved potential for active targeting of ASGP receptor overexpression on some tumors.
from the tumor and limited fluorescence in nontarget organs, while nontarget tumors, including lung and melanoma models, showed no uptake over a 12-h period (Figure 5) (14). The utility of this lactose-functionalized phthalocyanine has also been demonstrated in sentinel lymph node mapping. In vivo imaging utilizing both lactose-functionalized 9 and glucose-functionalized 4 was carried out in mice, with 9 showing strong sentinel lymph node fluorescence after just 10 min with visible fluorescence persisting for over 30 min, while only background levels of fluorescence were observed in nontarget muscle and fat. In contrast, 4 was found to show no accumulation in the lymph nodes, with fluorescence no higher than the control (15).
2.2 Nanoscale Structures An alternative solution to the problem of imaging agent solubility is the use of nanoscale delivery vehicles, with the literature describing a plethora of recently developed nanoparticles including biological macromolecules, inorganic and organic nanotubes and nanoparticles, and liposome-like structures. All of the described structures allow for incorporation of fluorescent imaging agents via either encapsulation or conjugation, in both cases aiding solubility and allowing delivery of imaging agents directly to tumor tissue. In addition to aiding solubility, these macromolecular structures can also reduce aggregation and allow for greater concentrations of fluorophores through multiple loading onto the delivery platform, allowing for increased fluorescence signal and improved contrast ratios. The use of serum components, such as cholesterol as delivery vehicles, has been demonstrated, exploiting the increased catabolism and
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receptor-mediated uptake of cholesterol in some tumor cells (16). Wu et al. demonstrated the use of LDL cholesterol as a delivery platform for transportation of NIR-absorbing oligoporphyrin chromophores, for the treatment of B16 melanoma. Four examples of fluorescent probes were synthesized, all showing broad-spectrum light absorption and highly lipophilic character, with the aim of preventing leakage from the cholesterol in vivo. However, only 10 and 11 were successfully incorporated into LDL, with an average of 30 chromophores per LDL unit. In vitro studies demonstrated no toxicity even after 24 h, with both 10 and 11 showing rapid internalization into target cells, while the corresponding untargeted chromophores showed no uptake or fluorescence. Fluorescent imaging compared to a glucose-targeted pyropheophorbide control showed excellent fluorescent image quality for both 10 and 11, even at low dye concentrations (ca. 60 nM dye concentration), while the control required around 20 times higher dye concentrations to achieve the same image quality (Figure 6) (17). While highly conjugated oligoporphyrin species represent a novel methodology to allow increased NIR absorbance of optical imaging agents, use of the biomolecule LDL as a delivery vehicle limits structural flexibility as functionalization and derivatization of this natural biomolecule are complex processes. An alternative is the use of polymersomes, synthetic liposome-like structures which utilize amphiphilic block copolymers to form a vesicle membrane, allowing development of delivery vehicles with excellent synthetic flexibility and biocompatibility. Polymersomes allow transportation of the imaging agent via incorporation into the polymer shell, making delivery of large numbers of fluorophores to each cell possible, while fluorophore spacing within the polymersome can be carefully controlled, limiting fluorescence quenching. Four examples of NIR-emitting zinc oligoporphyrin structures were synthesized (14–17), with varying peripheral chain composition and length, and all structures were incorporated into diblock copolymers comprised of polyethylene-oxide and polybutadiene. Self-assembly of the copolymers in aqueous solution results in the formation of the polymersome structures. All synthesized polymersomes showed excellent stability under physiological conditions, with no dye leakage and minimal photobleaching observed in aqueous conditions. In vivo imaging efficacy was examined in a rat tumor model, with ex vivo imaging of excised tumors carried out to confirm localization of the polymersomes. In all cases, the polymersomes demonstrated excellent potential as imaging agents, showing signal:noise ratios of at least 10:1 and retention in tumor tissue following direct injection (18).
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R1
N
R2
N
N
N
N
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R1
N
N
N
R1
N Zn
Zn N
N
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R1 =
12
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O
COOEt R2 =
3
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O
O 3
11
13 R1 =
R1, R2 = O
O R2 =
O
O NH
O O
Figure 6 Oligoporphyrin optical imaging agents 10–13 showing the diversity of peripheral functionalization produced on these structures.
Subsequent work in this area has demonstrated the synthetic flexibility of the polymersome system, demonstrating modification of both the polymersomes and zinc porphyrin fluorophores to allow optimization of both biological and photophysical properties. Modulating the amphiphilicity, substitution, and size of the oligoporphyrin imaging agents was shown to alter both absorption and fluorescence maxima (19). Changing porphyrin loading ratios was shown to affect significantly polymersome stability (20), and the
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alteration of porphyrin and polymer functionalization allowed alterations to bulk optical properties (21) and excited-state dynamics (22) of the imaging modality (Figure 7). The formation of synthetic porphyrin-based micelles has also been demonstrated without the use of block copolymers. Nakamura et al. exploited the highly hydrophobic nature of zinc protoporphyrin IX to form a conjugate with the hydrophilic HPMA; the resulting amphiphilic structure 18 spontaneously formed micelles of 30–80 nm in aqueous solution. Micelles were synthesized containing ca. 20% zinc porphyrin by weight and, unlike the parent porphyrin, demonstrated excellent water solubility. While the intact micellar form demonstrated minimal fluorescence and singlet oxygen generation, in vitro fluorescence was observed following internalization of the micelles, suggesting that intracellular disintegration of the micelles allowed reduction of the observed fluorescence quenching. The efficacy of these micelles as imaging agents was demonstrated in vivo, with good tumor uptake and minimal fluorescence of surrounding tissues observed. Micelles of 18 demonstrated tumor:normal tissue contrast ratios of 5:1 and greater, while the control zinc protoporphyrin IX showed no tumor uptake and rapid clearance from the body (Figure 8) (23). As well as facilitating transportation of lipophilic porphyrins, porphyrin nanogels also allow containment of porphyrins, reducing damage caused by generation of reactive oxygen species during irradiation. The first example of metalloporphyrin hydrogel nanoparticles was obtained through the reaction of the bis azide PEG chain (20) and the gallium tetraalkyne porphyrin 19 to produce a cross-linked structure, with the size of the nanoparticles formed controlled by the quantity of emulsifier added to produce particles between 30 and 110 nm. UV–vis analysis was conducted on both the gallium porphyrin nanogels and the demetallated free-base porphyrin derivative. In all cases, the gallium porphyrin showed improved photophysical characteristics, with a significant red shift in both the absorption band (682 nm) and the fluorescence emission (725 nm) (24). Synthesis of tumor-targeted nanogels was also demonstrated utilizing a folate-functionalized bis azide PEG (21), producing uniform nanoparticles of 120 nm diameter. While photophysical evaluation showed minimal change in both the fluorescence and absorption spectra in comparison to the hydroxyl-functionalized nanogels, the targeting ability of the folate functionalization was not evaluated in a tumor model (Figure 9) (24). The use of nanoscale structures also allows the incorporation of multiple fluorescent imaging agents into a single structure, allowing exploitation of a
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Figure 7 Oligoporphyrin optical imaging agents 14–17, with structures containing between 3 and 5 zinc porphyrin units to allow for improved NIR imaging efficacy.
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O
N
N
Zn N
N
O
O
16
Figure 7—Cont'd
O
O
N Zn
N
N
O
O
N
O
N
O
N
N
O
O
O
3
N
N
N
N
N
O
O
O
3
N
N
O
N
Zn N
N
O
O
O
O
Zn
O 17
Figure 7—Cont'd
3
3
N
Zn
3
Zn
O
O
O
O
N Zn
N
N
O
3
N
O
O
3
N
O
O
O
3
O
O
O
3
O
3
3
3
O
O
O
O
N
O
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Metalloporphyrins for Medical Imaging Applications
NH O
N
O
S O
CN HN
O
N
N Zn
N
N
O O
HO
O N
N Zn
N
N O OH
18
Figure 8 Amphiphilic diporphyrin monomer 18, which in aqueous solutions spontaneously forms micelles with improved tumor uptake and optical imaging potential.
OH O
O
N3
O
O OH 20
N N
Ga
N3
45
N
N FA O
O
O
O
N3
N3
45
FA 21
19
Figure 9 The synthesis of both tumor-targeted and untargeted nanogels for optical imaging application was demonstrated utilizing precursors zinc tetraalkyne porphyrin 19 and diazide chains 20 and 21 via a click chemistry methodology.
greater proportion of visible light, as well as development of theranostic agents. Incorporation of both zinc tetrasulfophthalocyanine and an adamantyl-nitroaniline fluorescent imaging agent into an epichlorohydrinβ-cyclodextrin demonstrated both red and green fluorescence, as well as therapeutic singlet oxygen and NO generation. No interaction between
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the two chromophores was observed following encapsulation, with all photophysical properties of the single fluorophores retained. The suitability of the nanoconstructs as biocompatible vehicles with dual-color imaging capacity was demonstrated in melanoma cells, with fluorescence observed at both 457 and 640 nm (25). Imaging in human squamous carcinoma cells and skin tissue ex vivo was also carried out, with the nanostructure demonstrating cellular accumulation and visible fluorescence in human tissue (26).
2.3 FRET Systems The development of dual-chromophore fluorescence resonance energy transfer (FRET) systems as optical imaging probes is an area of growing research interest. FRET systems are comprised of two conjugated chromophores: a donor and an acceptor. When light excites the donor chromophore, energy is then transferred to the acceptor chromophore, which emits light through fluorescence. The transfer of energy in this way has numerous advantages, principally the ability to allow a very large Stokes shift between exciting and emitted light, reducing interference, and improving image quality. Chlorin–bacteriochlorin dyads were synthesized and explored as FRET fluorescence imaging agents, with either a zinc (22) or free-base chlorin (23) as the donor chromophore and the bacteriochlorin as the acceptor. While a large Stokes shift was observed for both structures, chelation of zinc led to a significant enhancement (110 nm) in comparison to 23 (85 nm), with good fluorescence quantum yields (0.19) and fluorescent lifetimes (5.3 ns) observed in both cases. Evaluation of the dyads in a tissue phantom in comparison to commercial cyanine dyes showed brighter images for the commercial dyes and significant quenching of the dyad fluorescence in polar solvents; however, both 22 and 23 demonstrated improved excitation and detection selectivity and longer fluorescence lifetimes, suggesting that they could be developed as promising alternatives to current commercial dyes (Figure 10) (27). Alternatively, conjugation of the chlorin donor to NIR-emitting lanthanide–DOTA acceptors has been demonstrated by Laakso et al., utilizing Nd- and Yb-chelated DOTA and both zinc and free-base chlorins. Photophysical properties of all synthesized dyads were evaluated, with shorter emission lifetimes observed for Nd than for Yb, and comparable results obtained for both zinc and free-base chlorins; however, no in vitro evaluation of these imaging agents was carried out (Figure 11) (28).
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N
N
N
N
NH
HN
M N
22 23
O N
M = Zn M = 2H
Figure 10 FRET imaging agents 22 and 23, with the combination of both chlorin and bacteriochlorin structures into a single agent allowing for significant enhancement of Stokes shift and fluorescence lifetimes in both structures.
Subsequently, the same group carried out a systematic analysis of a range of similar chlorin-functionalized DOTA–lanthanide dyads, evaluating the effect of altering both linker properties and metallation of the chlorin and cyclam units. Examination of photophysical properties of the synthesized metallochlorins demonstrated increasing absorption coefficients in the order 2H Ni < Pd < Zn < Cu, with a hypsochromic shift (30–50 nm) of absorption observed in all cases. Photophysical evaluation of the zinc chlorin dyads was also carried out, with the absorption spectra of the chlorin varying significantly based on the lanthanide attached; however, the very weak lanthanide emission could not be detected. In addition, the solubility of all complexes was found to be an issue, with all poorly soluble in water and insoluble in methanol (Figure 12) (29). Cyanine dyes have also been utilized as acceptor chromophores for metallochlorin donors; Ethirajan et al. demonstrated the synthesis of this FRET pair, conjugating a cyanine dye to nickel purpurinimide. Photophysical evaluation of the dyad suggested that FRET was taking place, with fluorescence emission observed from the cyanine dye following irradiation of the chlorin. Nickel metallation was also found to have a significant impact on the dyad, quenching singlet oxygen production and significantly increasing the photostability of the imaging agent in comparison to the free-base
O NH
N O
N
NH
NH
O
N O
HN N O
24
O
N Ln
N
N
NH
HN N
O
25
N
O
O O
O N
O
O
O
N O
Zn N
Ln N
O
O Ln = Nd, Yb
N
O
N
NH
N N 26
O
O
N Nd
N
O N
O O
O
Figure 11 DOTA–lanthanide/ chlorin FRET imaging agents 24–26, with varying metallation of both macrocycles shown to alter photophysical properties of the structures.
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Metalloporphyrins for Medical Imaging Applications
N
N O
Zn
N N N
N
N
N 27
O
N Yb
O
O
N
N
O O N
N
O
N O
Zn N
O
N 28
N
N N
N O
Ln
O N
N O
Ln = Yb, Gd, Nd
O
O
Figure 12 Structures of DOTA–lanthanide/zinc chlorin NIR FRET imaging agents 27 and 28. −
O3S N+
29 30
M = 2H M = Ni
O S
N
HN
N M
N
N
N+ −
O3S
O MeOOC
N
O
hexyl
Figure 13 Metallochlorin-cyanine FRET structures 29 and 30. Nickel metallation of 30 allowed for improved photostability of the structure and improved pharmacokinetic profile during imaging.
derivative 29. Fluorescence imaging was carried out in a mouse tumor model, with 30 showing good tumor specificity and uptake, allowing clear visualization of the tumor. In comparison, 29 showed a significantly higher uptake in the liver, kidneys, and spleen, and poor tumor fluorescence, confirming the importance of the metalloporphyrin unit (Figure 13) (30).
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3. RAMAN IMAGING The application of Raman spectroscopy in medical imaging is a novel technique of growing interest and is currently showing promise in both cellular and in vivo models. Surface-enhanced Raman scattering (SERS) techniques utilize highly conjugated chromophores as imaging agents, with the numerous highly polarizable double bonds making them Raman active. In addition, conjugation of these chromophores to metallic nanoparticles increases the intensity of the inelastically scattered photons by orders of magnitude, allowing improved limits of detection. Metalloporphyrins in many ways represent ideal optimal imaging agents for SERS, with the highly conjugated porphyrin being strongly Raman active, while the use of an appropriate chelated metal allows for quenching of any interfering fluorescent signals. The first example of the synthesis of a metalloporphyrin system for Raman imaging was developed by Tam et al., utilizing a manganese pyropheophorbide-a–lipid conjugate 31 as a surface coating for gold nanoparticles, allowing stabilization of the nanoparticle while acting as a Raman reporter. Surface coating of the nanoparticles was shown to be facile, producing a coating of 4–7 nm, with the Raman reporting properties of the metalloporphyrin retained without requiring direct contact with the nanoparticle. The nanoparticles were shown to have excellent serum stability, minimal cytotoxicity, and excellent potential as imaging agents, with in vitro studies demonstrating clear visualization of the nanoparticles in cells utilizing Raman microscopy (Figure 14) (31). Subsequently, the same group utilized the same porphyrin–lipid structure, but exchanged the manganese core for palladium (32); this change in the metallation allowed for the same reduction in porphyrin fluorescence, while preserving singlet oxygen generation and potential as a therapeutic agent. In vitro imaging demonstrated retention of the Raman reporter properties of the structure, as well as the potential of the structure to act as a theranostic agent, suggesting a possible use in the monitoring of the effects of photodynamic therapy in vivo (32).
4. GAMMA IMAGING While the use of gamma-emitting isotopes to study biological processes in living systems was first demonstrated in 1923 by de Hevesy, the
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Metalloporphyrins for Medical Imaging Applications
O
O O O
O O
P O O−
N+
O
31 32
M = Mn M = Pd
N
N M
N
N
Figure 14 Metalloporphyrin-lipid structures chelating either manganese (31) or palladium (32). Coating of gold nanoparticles with these structures was shown to produce effective Raman imaging agents, while the palladium metallation of 32 also allows for potential tumor treatment via photodynamic therapy.
major breakthrough in the use of radioisotopes in medical imaging came in 1948 with the discovery of the NaI(Tl) scintillation detector. The enhanced efficiency with which this technology could detect gamma rays allowed practical clinical detection of radiation inside the body for the first time (33). Early planar gamma imaging operated on the principle of the administration of a gamma-emitting radioisotope followed by detection by scintigraphy to produce a 2D image. Subsequent improvements in imaging technology allowed for the development of SPECT, which uses a gamma camera which rotates around the patient, taking images from multiple angles to produce a true 3D image. These 3D images can be utilized to provide functional medical imaging and can be combined with SPECT/CT imaging to provide co-registered images, which allow overlay of the functional information obtained from SPECT over the gross anatomical CT image. Although SPECT shows poorer image quality and more limited resolution in comparison to more modern imaging techniques, it has not been superseded largely due to its use of cheaper, more long-lived isotopes than its counterpart PET, and subsequent lower cost. Modern gamma imaging is dominated by the use of the generatorproduced 99m-technetium, with over 80% of clinical SPECT imaging carried out utilizing this isotope (34). This popularity is due to the ease of its generation from 99Mo/99mTc generators, allowing the isotope to be produced effectively and reliably on-site in hospitals at low cost. While 99mTc is largely utilized in the form of ionic species such as the pertechnetate anion [99mTcO4], porphyrins offer an attractive option as chelators for 99mTc
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and other gamma-emitting metals, reducing undesirable uptake resulting from free ions and offering increased control over tumor localization.
4.1 Indium-111 111
In was the first metal used to label porphyrins for gamma imaging, decaying primarily by electron capture to emit two gamma photons of 173 and 247 keV. The stability of indium porphyrins makes them an attractive synthetic target, with the first examples of 111In porphyrins synthesized in 1972, with radiolabeling carried out to produce radiolabeled indium derivatives of tetraphenylporphyrin (TPP), tetratolylporphyrin (TTP), and tetra(4-methoxyphenyl)porphyrin (35). Subsequent publications also demonstrated the potential of 111In radiolabeling endogenous porphyrins, with indium-labeled structures including mesoprotoporphyrin IX (36) and hematoporphyrin derivative (HPD) synthesized (37). The first synthesis of an 111In porphyrin for biological use was carried out in 1975; Merrick et al. examined the biodistribution of indium TPP in rats, with the porphyrin displaying high liver uptake in comparison to other indium chelators, which was attributed to the high lipophilicity of the structure (38). The lipophilicity of this structure was also found to limit biological applicability in its use in imaging platelets in vivo; while platelet labeling in greater than 30% RCY was achieved and conjugates showed good plasma stability, during human trials after 24 h a 50% redistribution of 111In to erythrocytes was observed (39). Despite limited success of imaging with 111In-TPP, synthesis and biological evaluation of a number of alternative 111In porphyrins with improved biological relevance have been carried out. 111In-labeled HPD has been trialed successfully in animal breast cancer models, demonstrating clear visualization of malignant tumors by nuclear scintigraphy, with limited radiotracer uptake observed in either benign tumors or abdominal cysts. Good selectivity in uptake was also observed, with around 2.5% of the injected dose localizing in malignant tumors, and tumor to blood, heart, and lung ratios averaging 50:1, 10:1, and 3:1, respectively (40). A number of hydrophilic exogenous porphyrins have also been developed for biological use. 111In labeling of tetra(methyl-4-pyridyl)porphyrin was first carried out by Vaum et al. for the development of lymph node imaging agents. Imaging in a rat model demonstrated excellent lymph node selectivity compared to both muscle and bone (ratios of 72:1 and 16:1, respectively), despite the challenging nature of selective lymph node
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localization (41). Following the successful use of 33 in lymph node imaging, its efficacy was compared to two alternative water-soluble 111In porphyrins 34 and 35. Initial biodistribution studies in a rat model demonstrated slowest blood clearance for 34, with superior lymph node-to-muscle uptake ratios observed for 35. In vivo imaging in a rabbit model was carried out utilizing a scintillation camera, with the image quality varying significantly dependent on the radiotracer utilized. While porphyrin 35 demonstrated clear delineation of the popliteal nodes from 4 h after administration, the nodes were only faintly visualized after 24 h utilizing 33, with significant skeletal uptake also observed, and 34 showing no node uptake (Figure 15) (42). In addition to lymph node imaging, 33 has also been demonstrated to show promise as an imaging agent for melanoma. In vivo imaging in a hamster melanoma model demonstrated high uptake into tumor tissue (3.3% of ID); however, interestingly only poor localization in the necrotic core of the tumor was observed. Despite this, clear images of both the primary tumor and kidney metastases were obtained after 6 h, with the viable periphery
N+
−O
N+
SO3−
3S
N
N N
111
In
111
In
N
N
N
N
N
N+
N+
−O
33
3S
34
N+
N+
N N
111In
N
N
N+
Figure 15 Charged hydrophilic
35 111
N+
In porphyrins 33–35.
SO3−
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clearly delineated (43). Subsequently, this porphyrin was also utilized for imaging of human melanoma cells in a mouse model, with uptake and imaging potential compared to both free gallium citrate and a 67Ga-radiolabeled porphyrin. Rapid tumor uptake of 33 was observed, showing higher tumor activity than the 67Ga porphyrin at all times. While absolute tumor uptake of 33 was substantially lower than that of 67Ga citrate, rapid blood clearance of the porphyrin resulted in substantially higher tumor-to-blood ratios, with imaging after 6 h showing good contrast with the tumor clearly visible (44). In contrast, uncharged hydrophilic porphyrins have only recently been evaluated as radiotracers, with their pharmacokinetics being largely dominated by noncovalent interactions with serum proteins. 111In radiolabeling was carried out on three examples of hydroxyl-functionalized porphyrins by heating at 80 °C for 60 min, with the resulting structures demonstrating excellent RCP (>99%) and good serum stability over 2 days. Biodistribution in rats differed significantly in comparison to free indium; all porphyrins accumulated in both the liver and kidneys to some extent, although the rapid renal clearance and reduced liver accumulation observed for 38 made it the most suitable for further biological use. This pattern was confirmed by SPECT imaging in rats, with all three porphyrins showing clear visualization of spleen, liver, and kidneys, although uptake was not evaluated in a tumor model (Figure 16) (45). Alternatively, synthesis of bioconjugatable 111In porphyrins allows for attachment to targeting groups such as tumor-associated antibodies, allowing improved uptake into tumor tissue and reducing uptake into nontarget organs. The first example of this strategy was carried out by Bedel-Cloutour et al.; chelation of 111In to produce porphyrin 39 was carried out in 97% RCY, with subsequent peptide coupling of the unpurified mixture to both IgG and F(ab0 )2 antibody fragments. Conjugates were produced bearing 9–9.5 porphyrins per Mab and 1.5 porphyrins per F(ab0 )2, with 90–95% RCY and full retention of immunoreactivity in all cases (Figure 17) (46). Synthesis and conjugation of a range of water-soluble 111In porphyrins were then demonstrated, with metallation carried out using a mixture of both 111In and 115In to produce carrier-added derivatives in RCYs of between 53% and 90%. Subsequent bioconjugation was carried out to BSA and an anti-CEA MAb, with specific activities varying considerably between 7.4 and 25.9 MBq mg1 of protein. Synthesis of conjugates with improved specific activity was also achieved through removal of the isotopic dilution step, to obtain conjugates with 9.5–10 porphyrins per antibody, and average specific activity of 73 MBq mg1. All porphyrin–antibody
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OH
OH
HO
HO
HO
OH N
N N
111In
N
N
111In
N
N
N
HO HO
OH
OH
36
OH MeO
MeO
37
HO
OMe
MeO N N
111In
N
N
OMe MeO
OMe
OMe
38
Figure 16 Uncharged synthetic hydrophilic
111
In porphyrins 36–38.
R1
R2
N+
N N
111
In
N
Cl N
N
N
O
111In
N
N N+
39
N+
O 40: R1 = NO2
R2 = F
41: R1 = H
R2 = F
42: R1 = H
R2 = OCH2COOH
43: R1 = H
R2 = OCH2COO(C6H4)SCH3
Figure 17 Structures of bioconjugatable In porphyrins 39–43. Conjugation to both antibody fragments and full monoclonal antibodies was demonstrated via peptide coupling to allow for targeted gamma imaging. 111
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conjugates demonstrated good stability in aqueous solution at room temperature over 48 h; however, no biological evaluation of these conjugates was carried out (47). Although promising results have been obtained by 111In-chelating porphyrin radiotracers, synthesis of 111In radiotracers can also be carried out through the conjugation of metalloporphyrins to alternative 111In chelators; this allows exploitation of the tumor localization of porphyrins while allowing RCY and reaction times to be optimized through the use of nonmacrocyclic chelators. Nakajima et al. first demonstrated this methodology utilizing a gallium porphyrin attached to the commercially available chelator DTPA for 111In chelation. Biodistribution was evaluated in pancreatic cancer and lung cancer models, with 44 showing significantly higher tumor: blood ratios than the gallium citrate control (84:1 and 4:1, respectively), with good tumor:organ ratios also observed. Low levels of accumulation of 44 were seen in a control inflammation animal model, while uptake of gallium citrate remained high. Phototoxicity of 44 was also low in contrast to the commercial photosensitizer Photofrin, minimizing the risk of skin sensitivity after administration. Tumor imaging also showed improved results with 44; imaging after 72 h showed visible tumor tissue, while tumor tissue could not be distinguished using the gallium citrate control (48). In subsequent work by the same group an alternative metalloporphyrin conjugated to DTPA was utilized, with the latter structure allowing very rapid chelation of 111In at room temperature. Whole-body autoradiography was carried out in hamsters utilizing gallium citrate as a control. While both 45 and gallium citrate showed recticuloendothelial uptake, bone accumulation was observed only with gallium citrate. Uptake was also more selective with 45, showing equal or better tumor:organ ratios in all cases, and tumor: blood ratios up to 17 times higher than those observed for the control. In addition, while both radiotracers produced clear images with good contrast between tumor and normal tissue, 45 accumulated in viable tumor tissue only, while gallium citrate accumulated in both necrotic and viable tumor tissue, thus demonstrating that 45 shows specific affinity for tumor tissue rather than for all inflammation (49). While 45 demonstrated excellent affinity for tumor tissue, the phototoxic effects associated with the gallium porphyrin are undesirable for an imaging agent. As a result, modification of the porphyrin to produce manganese derivative 46 was carried out, which demonstrated low triplet lifetimes and no phototoxicity. In vivo imaging was carried out in three animal tumor models, with 46 giving significantly clearer images of tumor after
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O O N
HO
O
O N
N
O
111ln
N
O
O
O
O
O
O
O
N Ga
N
HOOC
N
44
COOH
H2O
OOC
O N
N M N
N
O
O
N COO
COO N
111ln
N OOC
45 M = Ga 46 M = Mn HOOC
COOH
Figure 18 Metalloporphyrin structures 44–46, with the use of 111In chelators external to the porphyrin structure allowing for improved radiochemical yields and reduced reaction times in comparison to porphyrin chelators.
72 h than the control gallium citrate. Selective uptake was also improved for 46, with all tumors having tumor:blood ratios of >50:1 and tumor:muscle ratios of >4:1, and two of the three tumor models showing higher tumor uptake and lower organ uptake than gallium citrate (Figure 18) (50).
4.2 Technecium 99m The metastable isotope of technetium, 99mTc, is one of the most widely utilized isotopes in gamma imaging, with its facile production from 99Mo generators, and relatively short half-life allowing for low patient radiation exposure and high clinical applicability. As for 111In, the larger size of 99m Tc means that chelation into porphyrins can be difficult, with longer reaction times and poorer chelate stability, and as a result porphyrin structure must be carefully controlled to optimize radiotracer parameters.
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The first example of the synthesis of a 99mTc porphyrin radiotracer was carried out in 1982, with radiolabeling of HPD carried out at ambient temperature for 30 min with 97.3% RCY. However, stability of the complex was limited, with storage for longer than 3 h at room temperature showing significant loss of 99mTc (51). Despite this, in vivo evaluation of the 99mTcHPD was carried out in an adenocarcinoma animal model, with selective uptake into primary tumors as well as in metastatic lesions present in the lungs and liver observed. In contrast, uptake into a benign tumor model was low and could only be visualized by fluorescence (52). Radiolabeling has also been demonstrated on Photosan, a commercial variant of HPD. While radiolabeling was found to proceed well under mild reaction conditions, stability was again found to be limited, with loss of chelated 99mTc after 2 h in vitro. In contrast, the conjugate displayed good in vivo stability for up to 24 h and scintigraphy imaging demonstrated good tumor uptake, particularly into small, vascularized tumors, and maximum tumor: muscle ratios of 7.9:1 (53). In contrast, labeling of tetra(4-sulfonatophenyl)porphyrin (TPPS) with 99m Tc has been shown to generate stable complexes. While radiolabeling required more forcing conditions, with heating to 100 °C for 30 min producing a 90% RCY, the generated complex was found to be stable in ambient conditions for 14 h, with this improved stability attributed to conformational changes in the porphyrin following chelation. While in vitro evaluation showed rapid uptake into Hep2 tumor cells, SPECT imaging in mice showed no uptake into the tumor and rapid clearance of the porphyrin from the body (54). 99m Tc-TPPS has also been exploited in the imaging of nonneoplastic conditions, including the imaging of inflammatory response. The first example of this was demonstrated by Zanelli et al., with 99mTc-TPPS showing accumulation at the site of injections of histamine, a well-known mediator of the inflammatory response (55). Subsequent work demonstrated the use of this inflammatory uptake for imaging of osteomyelitis, with 99mTc-TPPS compared to the commercially available 111InCl and 111In-WBC in a rabbit model. While all radiotracers demonstrated increased uptake in inflamed tissue, 99mTc-TPPS showed the best clinical relevance, with more selective uptake, improved image quality and reduced time between administration and scanning (56). More recently, the radiolabeling of a pheophorbide-a derivative with 99m Tc was carried out by Ocakoglu et al. with the aim of exploiting the antimicrobial effect of pheophorbide to produce an infection imaging agent.
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Heating to 60–70 °C for 25 min produced the 99mTc derivative 47 in 87% RCY; however, again in vitro serum stability was shown to be limited to 2 h or less. Uptake was evaluated in both a bacterially infected muscle model and a sterile inflamed control; while both showed uptake of 47, maximum target: nontarget muscle uptake varied significantly between the models, with maximum ratios of 1.29:1 and 5.61:1 for the control and infection models, respectively, allowing the potential for distinguishing infection from inflammation (Figure 19) (57). Due to the instability of 99mTc chelates, attempts have also been made to increase stability utilizing an alternative chelation methodology, chelation of the radioisotope by peripheral functionalities of the porphyrin rather than the central core. This was first demonstrated using UV–vis analysis by Shetty et al., with the radiolabeling of novel porphyrins 48 and 49 carried out in 30 min at room temperature. Radiolabeling of porphyrin derivatives containing Mn in the central core confirmed 99mTc binding to the peripheral carboxylic acid functionalities rather than the porphyrin core. In vivo imaging using 48 was carried out in sarcoma-bearing mice, with good visualization of both the primary tumor and brain metastases observed (58). Subsequent work confirmed improved in vitro stability of 48, with the porphyrin showing good stability at room temperature for up to 4 h. Evaluation in an animal model was carried out in comparison to the commercial radiotracers 99mTc(V)-DMSA, 99mTc-citrate, and 201TlCl, with 48 showing the highest tumor:muscle ratios (5.87), but tumor:liver and tumor:kidney ratios of below 1, due to both rapid blood and muscle clearance, and slow renal and hepatobiliary excretion (Figure 20) (59). Evaluation of the chlorin analogue 50 was also carried out by the same group, with this analogue offering identical chelation properties but enhanced potential to be utilized as a theranostic for photodynamic therapy.
N
N
99mTc
N
O
Figure 19 Structure of
99m
O N
O O
47
Tc-labeled pheophorbide-a derivative 47.
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Francesca Bryden and Ross W. Boyle
COOH
HOOC O
O
COOH
O O
COOH
N HN
NH N O
HOOC
O
HOOC
O
O 48
COOH
HOOC
HOOC O
O
COOH
N HN
NH N
HOOC
O
O 49
COOH
Figure 20 Chelation of 99mTc utilizing the peripheral carboxylic acid functionalization present on 48 and 49 allows for the development of gamma imaging agents with improved chelate stability in comparison to conventional 99mTc porphyrin structures.
Radiolabeling was carried out in 15 min at room temperature, with the resulting complex showing in vitro stability for up to 4 h at room temperature. Pharmacokinetics in a rat model were similar to 48, showing rapid blood clearance and high liver and kidney uptake, while in vivo imaging demonstrated higher tumor:muscle uptake ratios in comparison to 99mTc(V)-DMSA and 201TlCl in all tumor models, although uptake did not differ significantly between 50 and 99mTc-citrate (Figure 21) (60). The carboxylic acid-functionalized tetra-(4-carbomethoxyphenyl)porphyrin was also used for noncore chelation of 99mTc, with UV analysis confirming chelation by the peripheral functionalities only. Biodistribution
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HOOC O
COOH O
O O
COOH COOH
N NH
HN N
HOOC HOOC
O O
50
O HOOC
O COOH
Figure 21 The structure of chlorin 50 allows for chelation of 99mTc via the peripheral carboxylic acid moieties rather than the core of the tetrapyrrole structure, while also offering enhanced absorption at clinically relevant wavelengths in comparison to porphyrin analogues.
studies demonstrated limited uptake in most organs; however, very high liver accumulation was observed, with associated short-term liver damage also observed (61). Most recently, development of the externally chelating porphyrin 51 was carried out by Santos et al. with the aim of producing a radiotracer with high structural similarities to natural porphyrins, but with a simple structure to allow for rapid clearance. 99mTc incorporation was carried out with 92.5% RCY, with UV–vis analysis confirming chelation of the metal outside of the porphyrin core. 51 demonstrated significantly improved serum stability, with no loss of 99mTc after 6 h at 37 °C. In vivo biodistribution in a mouse model demonstrated rapid clearance from the body, with low activity in all organs after 6 h; however, initial high uptake in the spleen, kidney, and liver was observed, possibly limiting the use for imaging colorectal tumors. Imaging by a gamma camera in tumor-bearing mice showed some selective tumor uptake, with tumor:muscle ratios of 3:1 and higher obtained in two tumor lines (Figure 22) (62).
4.3 Gallium 67 Use of 67Ga in gamma imaging radiotracers is an attractive prospect due to both its two gamma photopeaks (185 and 300 keV) and its long half-life (t1/2 ¼ 78 h), allowing increased manipulation and complex purification
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O NH
HOOC
N
O N
O
HN
O O O Tc 99m O O O
HOOC 51
Figure 22 Structure of 51 showing
99m
Tc chelation via peripheral functionalization.
steps without significant loss of activity. Despite this, its use has been largely superseded by the use of the positron-emitting 68Ga isotope for PET, which allows improved resolution and reduced patient exposure to radiation due to its short half-life. 67 Ga radiolabeling of a tetrapyrrole structure was first demonstrated by Rousseau et al. in 1985, utilizing both 67Ga and 99mTc to label tetrasulfophthalocyanine. Radiolabeling was shown to be significantly more efficient for 67Ga, with a maximum RCY of 63% obtained in comparison to an RCY of 19% utilizing 99mTc. In vivo distribution of both phthalocyanines was evaluated, with the 99mTc phthalocyanine showing high kidney accumulation and no significant tumor accumulation within the 24-h period of study. In contrast, the 67Ga phthalocyanine showed a significantly different distribution, with lower kidney uptake and higher tumor:blood and tumor: muscle ratios, although overall tumor uptake was lower than the gallium citrate control (63). Low overall tumor uptake was also observed with 67Ga-radiolabeled tetra(methyl-4-pyridyl)porphyrin. While radiolabeling was shown to proceed in high RCY (94.7%) after 30 min at 120 °C, absolute tumor uptake of the 67G-labeled porphyrin was substantially lower than that of both gallium citrate and an analogous 111In-labeled porphyrin at all time points. Despite this, rapid blood clearance of the 67Ga-labeled porphyrin over a period of 48 h resulted in tumor:blood ratios which were substantially higher than that of 67Ga citrate (44). Most recently, radiolabeling of a porphyrin with 67Ga was carried out by Paknafas et al., with 52 produced with an excellent radiochemical purity after heating to 100 °C for 60 min. While 52 demonstrated good in vitro stability over 24 h, imaging in a rat model showed skeletal and myocardial uptake after 2 h, attributed to release of gallium in vivo (Figure 23) (64).
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MeO
OMe
OMe OMe N
N
67Ga
N
N MeO MeO
Figure 23 Structure of
67
52
MeO
OMe
Ga-radiolabeled porphyrin 52.
4.4 Other Radionuclides Although dwarfed in popularity by the positron-emitting isotope 64Cu, 67 Cu has found use as both a therapeutic beta emitter and a diagnostic gamma emitter. The long half-life of 67Cu makes it particularly appropriate for imaging in combination with antibodies due to their long biological half-life, a strategy demonstrated by Bhalgat et al. utilizing n-benzyl functionalized porphyrins, which demonstrate improved metal chelate stability and more facile incorporation than porphyrins with unfunctionalized cores. Two examples of hydrophilic porphyrins were synthesized, and attachment to an antirenal cell carcinoma antibody was carried out via peptide coupling in 25–30% yield, with two to three porphyrins attached per antibody. Subsequent radiometallation was carried out in 41% RCY, yielding conjugates with a specific activity of 18.5 MBq mg1 protein, with all products having greater than 70% stability serum over 7 days. In vivo biodistribution in a mouse tumor model showed significant differences between targeted and untargeted porphyrins; free porphyrins showed high liver and kidney uptake, and modest tumor:blood selectivity (1.8–2:1). In contrast, the antibody conjugate demonstrated excellent tumor selectivity, with maximum tumor:blood ratios of 16.4:1 after 45 h (Figure 24) (65). Despite the prevalence of endogenous iron porphyrins, only a single example of the development of iron porphyrins for gamma imaging is known in the literature. Thaller et al. carried out radiolabeling on a number of both endogenous and exogenous porphyrins with 59Fe and 52Fe, evaluating tumor tissue uptake in an ex vivo model. Highest tumor tissue uptake was observed for endogenous deuteroporphyrins, while good uptake was
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HO O
O
O
S
S
O
O
OH
OH
OH
O
N N
67Cu
N N
N
HO O
53
N
N
N
O2N
67Cu
O
S
O OH
HO
O O
54
HO
Figure 24 Cu-labeled metalloporphyrins 53 and 54, with attachment to targeting antibodies carried out via peptide-coupling reactions with the peripheral carboxylic acid functionalities of the porphyrins. 67
also observed for exogenous porphyrins tetra(methyl-4-pyridyl)porphyrin and tetra-(4-carbomethoxyphenyl)porphyrin. Despite this, in vivo tumor imaging in rat models with 52Fe porphyrins demonstrated no tumor uptake at either 3 or 24 h time points (66). Similarly, while paramagnetic manganese has found favor in porphyrin imaging agents for MRI, gamma-emitting 54Mn has only been utilized once for the labeling of HPD, with heating to 150 °C for 60 min producing an RCY of 86.7%. In vivo biodistribution in mice showed no significant difference from free-base porphyrins, with limited selective accumulation in tumor tissue and high liver uptake (67). While 201Tl has largely been superseded by 99mTc due to its ease of generation, recently radiolabeling of tetrakis(pentafluorophenyl)porphyrin with 201 Tl was carried out by Fazaeli et al., producing the complex with greater than 99% purity and good specific activity (13–14 GBq mmol1). Despite this, clinical relevance of the conjugate was limited, with poor in vivo stability, limited tumor uptake, and rapid clearance observed (68). Although a gamma-emitting isotope of cobalt does exist, the extremely long half-life (271 days) of 57Co means that the use in gamma imaging is of little clinical relevance, with the only clinically developed variant being the radiolabeled vitamin B12 mimic 57Co-cyanocobalamin. The only example of the development of 57Co metalloporphyrins for medical imaging was the synthesis of 57Co hematoporphyrin by Angileri; however, in vivo studies showed limited applicability as an imaging agent, with normal and tumor-bearing mice showing no significant differences in uptake (69).
Metalloporphyrins for Medical Imaging Applications
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5. POSITRON IMAGING (PET) While PET also exploits radioisotopes for imaging purposes, it differs from SPECT in that the detected emissions are not produced directly from the radioisotope decay. Isotopes which decay to emit positrons are utilized, with the generated positrons undergoing an annihilation event with an electron to produce two coincident 511 keV gamma rays. These gamma rays are then detected by a ring of scintillation detectors around the patient, with only gamma photons originating from a single coincident event detected, removing the need for the collimators required by SPECT. As for SPECT, almost all clinical PET scanning today is carried out utilizing combined PET/CT scanners, which provide both functional and morphological imaging to aid in image registration. PET offers much higher sensitivity than SPECT, as well as improved spatial resolution, and resulting improved image quality. In addition, the shorter half-life of many clinically relevant positron-emitting isotopes means that radiation doses to patients are reduced; however, this also means that many isotopes require expensive on-site cyclotron generation, while even isotopes with comparatively longer half-lives (i.e., 18F) require rapid automated synthesis equipment in order to produce radiotracers with clinically relevant specific activities following transportation. Radiolabeling of biologically relevant structures with positron-emitting radioisotopes allows for functional imaging of disease states, with most well-known example being the current clinical gold-standard, 18F-FDG (fluorodeoxyglucose). Enhanced glucose uptake of many tumor cells as a result of the Warburg effect leads to increased uptake of this 18F-labeled glucose analogue, with subsequent phosphorylation preventing cellular egress and causing accumulation of 18F-FDG in target tissue. Since clinical approval, 18F-FDG has been utilized for diagnosis and staging of a plethora of malignant diseases, including digestive, lung, and breast cancers (70). Despite this, uptake of 18F-FDG occurs only as a result of enhanced metabolic activity and does not provide information about receptor expression in these cancers. As a result, other radioligands have been developed which allow assessment of tumor hypoxia, apoptosis, and expression of receptors including somatostatin, steroid hormone receptors, and epidermal growth factor receptors (71). Radiolabeling of porphyrins has also been widely explored, and while there is some interest in labeling via covalent attachment of nonmetal isotopes including 18F (72) and 124I (73), the real utility of
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porphyrin radiotracers is in their ability to efficiently chelate metallic radioisotopes to produce stable metal complexes.
5.1 Copper 64 Despite the popularity of 18F in the development of radiotracers, the first examples of PET imaging carried out with metalloporphyrin radiotracers utilized 64Cu and were first used in preclinical in vivo studies in 1951 (74). The popularity of 64Cu in radiotracers can be attributed to both the long half-life and favorable decay characteristics of this isotope (t1/2 ¼ 12.7 h, decay: β+ (61%) β (39%), energy β+max ¼ 656 keV), as well as the ready cyclotron preparation from proton bombardment of the stable 64 Ni target. Copper also offers good compatibility with porphyrins, with several studies showing that the pharmacokinetics of metalloporphyrin radiotracers are dominated by the chelating porphyrin rather than the coordinated metal (75, 76) although the paramagnetic nature of the copper alters the photophysical properties of the porphyrin, quenching both fluorescence and singlet oxygen generation. Early imaging using copper porphyrin radiotracers utilized endogenous porphyrins including hematoporphyrin, uroporphyrin, and coproporphyrin and showed mixed results; while tumor uptake was observed in mice, localization in human tumors was insufficient to allow imaging, with high liver uptake observed in all cases suggesting hepatobiliary metabolism (77). Use of commercially available derivatives of endogenous porphyrins including HPD was found to be more effective, showing a threefold reduction in liver uptake; however, addition of the copper was found to alter the composition of the HPD, and uptake ratios varied considerably between animals (78). As a result, subsequent work has utilized a range of exogenous porphyrins and phthalocyanines, with these synthetic structures allowing facile tuning of the porphyrin properties to improve chelation, improve tumor localization, and limit risk of in vivo degradation. Radiolabeling of tetra(pentafluorophenyl)porphyrin with 64Cu has been carried out by Fazaeli et al., with the aim of producing porphyrin radiotracers with an improved pharmacokinetic profile. Forcing radiolabeling conditions and long reaction times (100 °C for 60 min) were required, with the radiotracer showing good biostability over a 48-h period. In vivo pharmacokinetic studies showing minimal liver uptake, with most excreted via the kidneys, however, no imaging or tumor uptake studies were carried out (79).
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Sulfonated phthalocyanines also offer improved solubility profiles as well as improved biological stability in comparison to endogenous porphyrins. Radiolabeling of a mixture of sulfonated phthalocyanines bearing between one and four sulfonate groups was carried out by Soucy-Faulkner. While comparison of the pharmacokinetics of 55 and free copper acetate suggested no in vivo phthalocyanine demetallation, significant uptake into both liver and kidneys was observed. Additionally, while the tumor could be distinguished during imaging, tumor activity was low, with these poor results attributed to the complexity of the radiolabeled mixture (Figure 25) (80). For this reason, subsequent work by this group utilized purified fractions of this mixture, bearing 2, 3, or 4 sulfonate groups, as well as phthalocyanine 59, designed to show improved amphiphilic character. Radiolabeling of all phthalocyanines was rapid, with microwave irradiation of 150 W for 1 min producing the desired 64Cu metallophthalocyanines. In vivo studies demonstrated marked differences in the pharmacokinetics, with significant kidney and bladder uptake, and no tumor activity observed for the highly hydrophilic phthalocyanines 56 and 57. In contrast, the more lipophilic 58 and 59 showed liver uptake in addition to good tumor localization, with the best PET images, showing clear tumor delineation, obtained for phthalocyanine 59 (Figure 26) (81). The targeting ability of a range of biomolecules has also been exploited in the synthesis of porphyrin radiotracers, with the aim of improving tumor uptake and enhancing PET image quality. 64Cu-radiolabeled porphyrins in particular offer good compatibility with large biomolecules such as proteins and antibodies, with the long half-life of 64Cu corresponding well with the slow tumor accumulation and blood clearance of these biomacromolecules. NaO3S
R N N N
N Cu64
N
R = H or SO3Na
N
N N R
R 55
Figure 25 Representative structure of 55, which comprises a mixture of sulfonated phthalocyanines bearing between one and four of these peripheral water-solubilizing functionalities.
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NaO3S
SO3Na
NaO3S
N N N
N
N Cu64
N
N
N Cu64
N
N
N
N
N N
N
N SO3Na
NaO3S
NaO3S
SO3Na
56
57 NaO3S
C6H11
N N N
N
N Cu64
N
N N 58
N Cu64
N
N
N
N SO3Na
NaO3S
N N
N
NaO3S
SO3Na 59
(mixture of cis and trans isomers)
Figure 26 64Cu-radiolabeled phthalocyanines 56–59, with varying levels of peripheral sulfonation.
Roberts et al. synthesized a range of antibody-targeted radiotracers utilizing N-benzyl porphyrin 60, which allows the incorporation of 64Cu more rapidly and at temperatures more appropriate for antibody radiolabeling. Radiolabeling was shown to proceed well under mild reaction conditions (40 °C for 30–60 min), with the macrocyclic structure of 60 showing significantly enhanced stability in comparison to the linear chelator DTPA. Conjugation of 60 to antibodies was achieved via peptide coupling, with subsequent 64Cu insertion carried out in approximately 60% RCY. An average of two porphyrins was attached per antibody with specific activity of ca. 18 MBq mg1, with all conjugates showing good biostability (70% after 7 days). However, poor regiocontrol of porphyrin conjugation was observed, and antibody immunoreactivity was reduced to around 60% of that of the unmodified antibody (Figure 27) (82). An alternative to the use of antibodies as targeting groups is the use of peptides; these smaller molecules more readily allow structural engineering, giving improved binding regiocontrol, and a corresponding retention of receptor affinity, as well as reduced blood residence time which allows for more rapid imaging.
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SO3H
N HO3S
N
OH
N
O
64Cu
N
NO2 SO3H 60
Figure 27 Metalloporphyrin structure 60, with the N-benzyl functionalization offering enhanced chelate stability and more rapid incorporation of 64Cu.
Shi et al. demonstrated the synthesis and metallation of the folate receptor-targeted peptide GDEVDGSGK-folate chlorin conjugate (61), with radiolabeling occurring in >99.9% RCY to produce a conjugate with high specific activity (2.66 GBq μmol1). Stability of 61 was confirmed both in vitro and in vivo over 24 h. Tumor uptake was evaluated in a FR-positive mouse model; approximately 3% of the injected dose accumulated in the tumor with good tumor:muscle selectivity (8.8:1), allowing clear delineation of the tumor during imaging. Additionally, this uptake was shown to be blocked by administration of excess folic acid, confirming the folatemediated uptake process (Figure 28) (83). A bombesin analogue was utilized as a targeting peptide for the endogenous porphyrin protoporphyrin IX (PPIX), with the radiochemistry and localization compared to an untargeted PPIX control. Radiolabeling required moderate heating (50 °C) and long reaction times (2 h), with the addition of ethanol as a stabilizer, with poorer RCY obtained for PPIX than conjugate 62 (25% and 40% decay corrected, respectively). In vitro analysis was carried out in a GRP receptor expressing human prostate cell line, with targeted 62 demonstrating significantly higher uptake than PPIX. Pharmacokinetic evaluation demonstrated similar excretion profiles for both PPIX and 62; however, 62 showed significantly lower accumulation in normal skin tissue. Despite this, no difference in tumor uptake was observed between 62 and the untargeted control, attributed to the in vivo
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N
N 64Cu
N
NH
N
N
N
NH2
N
O
HN O
O NH GDEVDGSGK NH
COOH NH O
O 61
Figure 28 Structure of the folate receptor-targeted metallochlorin conjugate 61.
O
NH
O
O
O
O
O
d-Tyr Gln Trp Ala Val β-Ala His Thi Nle
O
N
N 64
N
Cu
62
N O OH
Figure 29 Cu-radiolabeled protoporphyrin IX derivative 62, with conjugated bombesin analogue to improve tumor uptake. 64
interactions between 62 and serum proteins dominating the uptake profile (Figure 29) (84).
5.2 Gallium 68 While the dominance of 18F-FDG as a radiotracer means that 18F is currently the most widely used clinical radioisotope in PET imaging, the positronemitting isotope of gallium, 68Ga, is gaining popularity. While its 68 min half-life is significantly shorter than that of 18F, it is sufficiently long to allow chemical manipulation, and this combined with its high positron abundance (89%) and relatively low maximum positron energy (1.92 MeV) means that 68 Ga is well suited to the synthesis of clinically relevant radiotracers. Furthermore, its production from Ge/Ga generators makes it an attractive proposition for clinical use, as it can be produced without the need for an on-site cyclotron.
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In addition, gallium is an attractive option for radiolabeling porphyrins; unlike 99mTc, the small size of the metal means that it can easily be chelated by porphyrins to produce stable metalloporphyrins which have been shown to demonstrate minimal toxicity (85), and the diamagnetic nature of the metal means that, unlike 64Cu, the photophysical properties of the porphyrin are largely retained. The first example of 68Ga radiolabeling of a porphyrin was carried out by Benham Azad et al., utilizing an RGD peptide-functionalized protoporphyrin IX derivative targeting the αvβ3 integrin. Radiolabeling was carried out using microwave heating, with irradiation at 120 °C for 45 min demonstrating the best yields (RCY: 33%, decay corrected, RCP: 97%), with higher temperatures leading to instability of peptide and hydrolysis of free gallium. In vitro fluorescence microscopy in comparison to an untargeted control showed cell uptake observed for 63 only, with blocking experiments utilizing the cyclic peptide cRGDfV showing displacement of 63, suggested that cellular uptake of this compound is dominated by active binding to the integrin receptors (Figure 30) (86). Use of untargeted gallium porphyrins as radiotracers for PET has also been demonstrated to be an effective strategy. Radiolabeling of four examples of endogenous tetrapyrrolic structures, and the commercially available tetraphenylporphyrin (66) was carried out using high temperatures under microwave irradiation for between 1 and 15 min. RCY obtained varied between 22% and 73%, with the best results obtained for 66. RCP were also varied (between 85% and 98%) with 65 and 68 showing insufficient purity for biological studies. 64, 66, and 67 showed good stability to transchelation from DTPA and apo-transferrin and transmetallation with iron; however, only 66 and 67 were found to be stable in serum, with 64 showing a limited
N
N 68
N
Ga
63
N
HN
NH2 O
NH O HO
O
O
NH
O
O
H N
NH O
OH
O N H
NH2 O
Figure 30 RGD-peptide functionalized gallium protoporphyrin IX derivative 63.
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Francesca Bryden and Ross W. Boyle
half-life of 72 min. Preliminary in vivo PET imaging was carried out in a mouse sarcoma model using 64. While high renal uptake was observed, around 0.5% of the injected dose localized in the tumor, with a modest tumor: tissue ratio of around 2 (Figure 31) (87). Use of untargeted lipophilic porphyrins has also been explored by Fazaeli et al. Radiolabeling of tetra(pentafluorophenyl)porphyrin with 68Ga was shown to require lower temperatures (100 °C) but long reaction times (1 h); however, no RCY was recorded. The porphyrin demonstrated excellent stability in human serum, and in vivo imaging showed selective tumor uptake, with 5:1 tumor:muscle ratios achieved (88). Despite these successes, more recent papers have utilized water-soluble exogenous porphyrins as radiotracers; the hydrophilicity of these porphyrins makes them more applicable to in vivo use, and the use of nonnative porphyrins allows for increased synthetic flexibility and facile tuning of porphyrin properties. The preferential tumor uptake (89) and cellular localization (90) of the cationic tetra(N-methyl-4-pyridyl)porphyrin have previously been demonstrated in its use as a therapeutic agent, with Bhadwal et al. publishing the first example of this porphyrin utilized in a radiotracer. 68Ga chelation again required elevated temperatures (100 °C) for extended periods of time, producing the desired product with an RCY of 90%, and excellent radiochemical purity (99%). The conjugate demonstrated excellent serum stability and showed good tumor uptake into a fibrosarcoma mouse model despite rapid renal clearance, with maximum tumor:blood ratios of 2.8:1 reported (91). The limited tumor selectivity of many radiolabeled metalloporphyrins has led to the development of hydrophilic, conjugatable metalloporphyrin radiotracers, allowing both ease of administration and facile bioconjugation to targeting biomolecules. Bryden et al. demonstrated the first example of targeted exogenous gallium porphyrins; radiolabeling was shown to be extremely rapid, with microwave irradiation achieving an RCY of >95% in 5 min. Subsequent click conjugation of the porphyrin to an integrin-targeting peptide was carried out at room temperature, and evaluation of the conjugate 69 as a targeted theranostic agent was carried out in vitro, demonstrating good selectivity for target integrin upregulating cell lines only (Figure 32) (92).
5.3 Other Isotopes In addition to these commonly utilized isotopes, there is a growing interest in novel isotopes for PET imaging. 62Zn offers many favorable properties for
OH
N
N
N
68Ga
N
N
HO
O
N
OH
N
64
O
OH
HO
O
N
N
N
65
N
68Ga
68Ga
O
N
OH 66
HO
O
N
H3COOC
N
N
H3CO
O
N
N
N
67
N 68Ga
68Ga
O
OCH3
H3CO
O
N
68
O
OCH3
Figure 31 A range of both endogenous (64–65, 67–68) and exogenous (66) tetrapyrroles labeled with 68Ga.
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Francesca Bryden and Ross W. Boyle
O N+
H N O
N N 68Ga N Cl N N+ 69
O 3Cl–
N
N
N
TWYKIAFQRNRK
N+
Figure 32 Structure of the integrin-targeting
NH2 O
68
Ga porphyrin–peptide conjugate 69.
incorporation into metalloporphyrin radiotracers, with a half-life of 9.3 h, 100% positron emission, and the diamagnetic nature of the metal meaning that, as for gallium, the photophysical properties of porphyrin are largely retained. 62ZnCl2 was utilized by Tamura et al. to produce three examples of glycosylated porphyrin radiotracers, with only 10 min of heating required for zinc insertion. Cytotoxicity and cellular localization were evaluated in control and target cell lines, with the chelated zinc showing minimal impact on porphyrin localization. PET imaging in an animal model showed a large proportion of all three porphyrins accumulated rapidly in the liver; while 71 and 72 showed improved tumor localization in comparison to 70, uptake was still relatively low, with future adjustment of hydrophilic/lipophilic properties suggested by the authors to minimize liver uptake (Figure 33) (93). Despite its popularity in MRI, use of manganese isotopes in PET imaging is rare, with 51Mn (t1/2 ¼ 46.2 min, positron energy ¼ 2.5 MeV) being the only isotope to date which has been incorporated into a metalloporphyrin radiotracer, with widespread use hindered by its limited availability and short half-life. Radiolabeling of tetra(4-sulfonatophenyl)-porphyrin (TPPS) with 51 Mn was carried out to produce an alternative to the widely used MnTPPS MRI contrast agent; the problems associated with the high effective dose required for MRI can be avoided by using PET instead, as the quantity of imaging agent required is orders of magnitude lower. Radiolabeling was achieved after 20 min heating at 100 °C, with an RCY of 32% (decay corrected) and 99.9% incorporation; however, no biological evaluation was conducted (94).
6. MAGNETIC RESONANCE IMAGING The application of magnetic resonance to imaging of living systems was first demonstrated Damadian (95), with NMR utilized to differentiate
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Metalloporphyrins for Medical Imaging Applications
F
N
N
F
62Zn
N
N H
NaOOC
N
F
F
R N
N
F
F
F
COONa F
F R 71
O OH
F
N
62 Zn
70
OH HO HO
F
F
NH
R=
F F
F
COONa
O
F
F
H NaOOC
F
S F
F
F
F
F
F
F
N
F
N
F
62
R
R
Zn
F
N
N
F
F
F
F
F F
F F 72
Figure 33
62
Zn-labeled glycosylated porphyrin structures 70–72.
between malignant tumors and normal tissue. Unlike PET and SPECT, MRI does not utilize ionizing radiation, instead using a strong magnetic field and an oscillating radiofrequency to excite nuclei containing odd numbers of protons and/or neutrons, with the subsequent differences in relaxation time producing contrast in the image. Images can be generated utilizing either T1 or T2 weighting, measuring longitudinal and transverse relaxation, respectively, with the change in weighting highlighting different pathologies and anatomical features. Generation of MRI images in native tissue is possible through excitation of the hydrogen nuclei in water present naturally in body tissues, with
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contrast in the image resulting from differences in tissue density; however, this is often insufficient to allow identification of tumor tissue from normal tissue. Addition of exogenous contrast agents reduces the relaxation time of nearby protons, increasing contrast and improving tumor delineation. As a result, MRI is capable of producing clinically relevant images without the need for exposure to ionizing radiation, with superior spatial resolution and contrast in comparison to radio-imaging techniques; however, sensitivity is low and the cost can be high. While there are numerous clinically approved exogenous contrast agents, none have yet been commercially produced which offer any appreciable specificity for neoplastic diseases. As a result, there is a growing interest in the development of alternative contrast agents, including the use of porphyrins and other tetrapyrroles. While there has been some interest in porphyrins as platforms for attachment of 19F (96,97), these structures require high levels of symmetry and multiple loading of fluorine atoms to create appreciable reduction in relaxation times. In contrast, porphyrins chelating paramagnetic metals have been extensively explored, with many showing good stability, high relaxivities, and minimal toxicity. Good selectivity for tumor tissue has also been observed for many porphyrins, although the exact reason for this localization is poorly understood, and in some cases has been attributed to nonspecific accumulation in necrotic tissues (98, 99).
6.1 Gadolinium As the only ion containing seven unpaired electrons, Gd3+ is a highly paramagnetic metal and an obvious choice for use in MRI agents, despite the high toxicity associated with free gadolinium (100). As a result, a plethora of gadolinium chelators are described within the literature, and the clinical use of a number of approved gadolinium-containing MRI contrast agents is well established, with Gd-DTPA being the most commonly used for clinical applications (101). However to date, use of porphyrins as clinical gadolinium chelators is limited, which can be attributed to the fact that the large size of the gadolinium ion is a poor fit with the relatively small porphyrin cavity, resulting in instability and potential leaching of toxic-free gadolinium. Despite this, examples of gadolinium porphyrins synthesized and evaluated as MRI agents have been published. Despite the loss of gadolinium from TPPS in vivo noted as early as 1987 (102), the high relaxivity of Gd-TPPS in solution makes it an attractive target for clinical use, and promising results have been obtained from both T1 and T2 brain imaging (103).
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Metalloporphyrins for Medical Imaging Applications
Examples of gadolinium endogenous porphyrins have also been synthesized including gadolinium hematoporphyrin, which showed improved in vitro uptake and higher relaxivity values in comparison to control compounds GaCl3 and Ga-DTPA (104). Subsequent evaluation of gadolinium hematoporphyrin and an exogenous boronated porphyrin 73 was carried out, with 73 showing R1 and R2 relaxivity values 5 and 10 times higher than Gd-DTPA, respectively. Tumor uptake of Gd hematoporphyrin and 73 was evaluated in a mouse melanoma model with the tumor showing 28% and 21% uptake of the injected dose, respectively, compared to 10% of Gd-DTPA. MRI showed good contrast enhancement in both cases, with the highest tumor signal enhancement (120%) found for Gd hematoporphyrin (105). Good uptake of both contrast agents was also found in a mouse colorectal tumor model, with T1 relaxation times reduced by 15% for hematoporphyrin and 12% for 73, compared to 3.3% in DTPA. However, as seen previously (105), high levels of gadolinium were found in the liver, which was attributed to possible free gadolinium dissociating from the porphyrin (Figure 34) (106). The first example of a gadolinium chlorin contrast agent was synthesized by Kim et al. using Radachlorin. The species was shown to be stable with no loss of gadolinium, and evaluation in a tissue phantom showed 20–25% signal intensity enhancement, with minimal change with changing concentration. However, the poor water solubility of the species limits the clinical applicability of the conjugate (107). H10B10
O
O
B10H10
N N
Gd N N
H10B10
O
73
O
B10H10
Figure 34 Structure of the boronated gadolinium porphyrin 73.
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Francesca Bryden and Ross W. Boyle
As a result of the poor size match between porphyrins and Gd3+, there has been a growing interest in the clinical use of texaphyrins as gadolinium chelators. These large, expanded porphyrin analogues are modified to contain five nitrogen atoms in the larger central cavity, allowing enhanced stability of gadolinium chelates. The most well-known example is motexafin gadolinium (74) which was first developed in 1996 (108) as a radiation sensitizer for the treatment of cancer, particularly brain metastasis (109,110). However, the gadolinium core means that it also has a potential use as an MRI contrast agent, either for monitoring the uptake of motexafin gadolinium (111) or as an MRI agent for both diagnostic MRI (112) and intraoperative MRI (Figure 35) (113). An alternative strategy to the use of expanded porphyrins is the use of porphyrins attached to macrocycles or linear chelators designed for incorporation of gadolinium, a strategy which has been used to great effect in the development of the commercial imaging agents Gadophrin 2 (75) and 3 (76). Both structures have two gadolinium ions per molecule chelated outside of the porphyrin core, with the addition of the copper ion into the porphyrin of Gadophrin 3 significantly improving the stability of the contrast agent without affecting pharmacological properties. As a result, the stability of the gadolinium chelation is significantly improved, in addition to a significantly improved safety profile in comparison to other porphyrin contrast agents (114). Both Gadophrin 2 and 3 are of interest due to their intracellular targeting of necrotic tissue, and subsequent slow clearance as a result of poor OH O O– N N
O
N
Gd N
N
O O
O O
O O
O O
–
O 74 OH
Figure 35 The expanded porphyrin-like structure of texaphyrin motexafin gadolinium 74 allows chelation of gadolinium with improved stability due to its larger cavity size.
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lymphatic drainage (114). This uptake allows for specific targeting of necrotic tissue in pathological conditions, particularly in the diagnosis of myocardial necrosis and ischemia following heart infarction (115–117). Their use has also been evaluated in neoplastic conditions, with Gadophrin 3 showing promise in the determination of necrosis in breast cancer (118); however, it was found that the high affinity of both contrast agents for necrosis can impede distinguishing tumors from other necrotic tissues, such as abscesses (Figure 36) (119). Following the success of the Gadophrin structures, development of a number of other contrast agents employing this chelation strategy has been attempted. Conjugation of a zinc phthalocyanine to a DOTA chelator via click chemistry was carried out to produce 77, with the increased molecular weight of the phthalocyanine expected to enhance the molecular relaxivity of the structure. While the photophysical properties of the phthalocyanine were retained following conjugation, the R1 relaxivity of 77 was found to be less than half of that of unconjugated gadolinium DOTA, with this reduction attributed to the amide spacer arm blocking the gadolinium waterbinding site. In vitro assessment of the structure demonstrated some toxicity, with concentrations of above 50 μM decreasing cell viability to ca. 75%; however, no assessment of the imaging potential of 77 was carried out in a biological system (Figure 37) (120). In contrast, Trivedi et al. utilized this strategy with two examples of metalloporphyrazines, each conjugated to two DOTA chelators, to produce effective contrast agents. Cellular uptake of both 78 and 79 was evaluated in a breast cancer cell line in comparison to the unconjugated Gd-DOTA, with 78 showing twofold better uptake in comparison to 79 and the control. In vitro imaging confirmed this, with the clearest images obtained for 78 with COO–
O N
N H
N
H N O
M N
N
H N O
N
O N H
N
N Gd3+ COO– –OOC COO– –OOC Gd3+ N
N
COONa
N
COONa
COO–
75 M = 2H 76 M = Cu
Figure 36 Commercial imaging agents Gadophrin 2 (75) and 3 (76).
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R
O
R
O
N N
N
N
R N R
Zn
S N
N
N
N N N
H N
N O
N
O
O
O
O
O
N 77 R=
R
N Gd
HN
S
O
O
O
R
Figure 37 Incorporation of a chelating macrocyclic unit into the structure of conjugate 77 allows for stable chelation of gadolinium for MRI while retaining the photophysical characteristics of the zinc phthalocyanine.
significantly higher contrast:noise ratios obtained. Subsequent in vivo evaluation of 78 showed the best contrast enhancement after 4 h, with reduction in T1 found to be slower but considerably more persistent than in comparison to Gd-DOTA alone (Figure 38) (121).
6.2 Manganese In contrast to gadolinium, manganese has only five unpaired electrons, with the corresponding reduction in paramagnetism leading to it being less optimal for use within MRI contrast agents. Despite this, manganese porphyrins are widely utilized in the literature as potential MRI agents, with the smaller size of the manganese ion allowing for more facile synthesis and greater stability of the resulting complexes in comparison to gadolinium. The first examples of manganese porphyrin MRI contrast agents were produced from the metallation of endogenous porphyrins. Both manganese protoporphyrin IX (122) and hematoporphyrin (123) demonstrated strongly paramagnetic character and good in vivo stability, with clear visualization of the liver in T1 weighted images, although toxicity at higher concentrations of hematoporphyrin (38 μmol kg1) was observed. Similarly, while manganese uroporphyrin demonstrated excellent chelate stability and clear enhancement of tumor contrast in an in vivo model, toxic effects were present at all effective doses (124). In contrast, evaluation of manganese(III) mesoporphyrin in comparison to the exogenous contrast agent manganese tetra-(4-sulfonatophenyl)porphyrin showed no reported toxicity. In addition, manganese
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O
O MeO
O N
MeO
N
M
O
N
O OMe
N
N O OMe
N
O
N
N Gd
N
O
N
O
O O
N
N
N
N
O
N
O MeO
NH
MeO
O
N
N
O O O
N H N
N
N
Gd
O
N
O N
O
O 78 M = Cu 79 M = Zn
O
Figure 38 Structure of metalloporphyrazine–DOTA conjugates 78 and 79.
mesoporphyrin was found to be a promising contrast agent, displaying enhanced lesion-to-liver contrast in both hepatic liver abscesses and metastatic liver disease, while no contrast enhancement was observed for MnTPPS at the same dose (125). As a result of the limited success of endogenous porphyrin MRI contrast agents, there is a growing interest in the development of exogenous manganese porphyrins. The most widely exploited example of these is manganese tetra-(4-sulfonatophenyl)porphyrin (MnTPPS), which was first reported as a potential MRI contrast agent in 1984, showing the greatest enhancement in water relaxation rates in comparison to both other metalloporphyrins and free Mn (126). Subsequent studies have demonstrated both the superior image enhancement capabilities of MnTPPS in comparison to cationic manganese porphyrins, and its improved in vivo stability in comparison to analogous gadolinium porphyrins (127). In addition, evaluation of its use as an MRI contrast agent has been carried out in lymphoma (128), carcinoma and sarcoma (129), and glioma (130,131), with good tumor contrast enhancement observed in all cases. Despite these initial promising results, a number of papers noted serious issues with the use of MnTPPS; significant uptake and localization of the contrast agent was observed in the liver and kidneys, and the high doses required to provide sufficient contrast enhancement resulted in green
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pigmentation of the skin and eyes (130,132), as well as more serious side effects including a number of animal deaths associated with high toxicity (132). As a result, a number of attempts have been made to optimize and modify the MnTPPS core structure, with the aims of reducing associated toxicity and improving both tumor localization and contrast enhancement. Altering the degree of porphyrin sulfonation (2, 3, or 4 sulfonate groups) has been demonstrated to affect both toxicity and tumor localization, with the trisulfonated 80 presenting the most favorable combination of biodistribution, tumor uptake, and tumor relaxivity, with the higher relaxivities of 81 and 82 countered by higher liver uptake (133). Comparison of both MnTPPS and 80 as contrast agents in a breast tumor model demonstrated that 80 offered significantly improved contrast enhancement at low doses, although its toxicity was even higher than that of MnTPPS (Figure 39) (134). Manganese tetrasulfophthalocyanine was also proposed as an alternative to MnTPPS, with its potential as a contrast agent evaluated in a mouse mammary tumor model. Both the relaxation rate and the biodistribution profile
–O
SO3–
SO3–
3S
N N
Mn
N N
N
N
–O
3S
Mn
N
N
–O S 3
80 –O
81
3S
N N
Mn
N
N
–O
3S
82
Figure 39 Mn-TPPS analogues 80––82 displaying varying degrees of sulfonation.
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were shown to be similar to those of MnTPPS, and good selective tumor uptake was also noted (tumor: muscle ratio of 9.2:1). While the phthalocyanine was found to be both less toxic and more stable to oxidation than MnTPPS, blue staining of skin following administration was noted, persisting for 3–5 days after injection (135). Modification of the MnTPPS structure for application in different MRI applications has also been carried out, with Lee et al. demonstrating the development of a zinc-sensing MRI agent from the functionalization of 83 with chelating arms to act both as axial ligands for the Mn core and as a zinc sensor. Addition of zinc to 83 was found to decrease the R1 relaxivity from 8.7 to 6.7 mM1 s1, while in contrast a zinc-induced enhancement of T2 relaxation was observed. The efficacy of 83 was also assessed in vitro in both zinc-treated and control cells, with significantly shorter T1 values found for the control (80 ms) in comparison to the treated cells (510 ms), while T2 values were shorter for zinc-treated samples than the control. In contrast, significantly higher cytotoxicity and no differentiation between zinc-treated and control cells were observed with MnTPPS (136). Subsequent work examined the efficacy of both 83 and MnTPPS in a rat brain model. Two days after intracranial administration, strong contrast was observed in the zinc-rich gray matter with 83 and persisted for 10 days, while little enhancement was observed with MnTPPS, although this difference was attributed to more efficient uptake of 83 into zinc-rich tissue rather than significantly enhanced contrast in the presence of zinc (Figure 40) (137). –O
SO3–
3S
N N
Mn
N HN
N
N N
HN –O
3S
N
N N N 83
Figure 40 Sulfonated manganese porphyrin 83 which demonstrates capabilities as a zinc-sensing MRI sensor due to functionalization with pyridyl pendant arms.
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Most recently, Nofiele et al. produced a dimer structure based on TPPS (85), with the aim of increasing both molecular weight and rotational correlation time to increase R1. Synthesis of a second alternative structure 84 was also carried out, with the aim producing a contrast agent with improved in vivo clearance. R1 relaxivity of both 84 and 85 was evaluated against MnTPPS and Gd-DTPA, with 85 exhibiting R1 higher than both (20.9 mM1 s1 per Mn at 1T), while R1 values for 84 were higher than for Gd-DTPA but lower than for MnTPPS. Biodistribution in rats showed significant contrast enhancements in both cases, with 84 showing renal clearance within 24 h, while 85 displayed slow hepatobiliary excretion over 3 days (138,139). Use of 85 as an alternative to the commercial blood pool agent Gadofosveset was subsequently demonstrated. 85 was found to form a 1:1 complex with HSA, with an associated decrease in R1 due to protein binding restricting accessibility of water. While R1 relaxivity of 85 was significantly lower than those of Gadofosveset at low field strengths (0.5–1 T), values were similar at a clinically applicable 3 T and 85 was found to remain in the blood for longer than 24 h, offering a significant advantage in comparison to the rapidly cleared Gd-DTPA (140). In vivo evaluation of 84 was also carried out in a rat breast tumor model, with good tumor visualization observed. Use of 84 gave larger contrast enhancement than Gd-DTPA at all time points measured, and while Gd-DTPA showed shorter accumulation and washout time, 84 demonstrated improved contrast against surrounding tissue and sustained contrast enhancement over a longer time period (Figure 41) (141). As a result of the limited clinical relevance of many contrast agents based on the structure of MnTPPS, development of a range of alternative exogenous porphyrin MRI contrast agents has been carried out, with the use of alternative water-solubilization and derivatization strategies employed to tune pharmacokinetic properties, reduce toxicity, and enhance utility as contrast agents. Two examples of manganese porphyrins were produced by Bradshaw et al., specifically engineered to incorporate highly biocompatible polyhydroxylamide substituents, with the aim of producing structures displaying the hydrophilicity of MnTPPS without the associated toxicity issues. Despite this, the minimum lethal dose of 86 was higher than that of MnTPPS and in vivo imaging showed limited image enhancement and long liver retention times (Figure 42) (142). Fujimori et al. synthesized the manganese porphyrin–chelator complex ATN-10 (88) for a range of biomedical imaging applications, first used as
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HO
HO
O
N
N
O
N
OH
Mn O
N
OH
O 84
SO3–
SO3–
N
N
N –O
3S
N
N SO3–
Mn
Mn N
N
N
85 SO3–
SO3–
Figure 41 Structures of small, hydrophilic manganese porphyrin 84 and porphyrin dimer 85.
R
R
O OH
86: R = NH
OH
N N Mn
X
O HO
N 87: R =
N
R
NH
OH
R
Figure 42 Manganese porphyrins 86 and 87 bearing polyhydroxylamide substituents to improve water solubility and reduce toxicity.
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an MRI agent in 1997 for brain tumors. Uptake in rat models of both brain tumors and pathological conditions which disrupt the blood–brain barrier was examined. 88 demonstrated good uptake and retention in the tumor model, with contrast enhancement still observed 24 h after uptake, while nontumor models showed limited uptake and clearance from the body in less than 2 h. In contrast, the control Gd-DTPA demonstrated no tumor-specific uptake, showing uptake in all examples of damaged blood–brain barrier (143). Subsequent work examined uptake in both normal brain and tumor tissue; while initial uptake showed poor differentiation, good tumor:brain accumulation ratios (10.4:1) were observed after 24 h. This work also confirmed the selectivity of uptake, with no contrast enhancement observed in necrotic tissue or peritumoral tissue; however, it was noted that this uptake selectivity could be due to known poor uptake of contrast agents into brain tissue rather than enhanced uptake into tumor tissue (Figure 43) (144). Chelation of gadolinium into the ATN-10 structure was also attempted; with 89 proposed for use in both MRI and neutron capture therapy. The relaxivity of 89 was found to be significantly higher than that of both Gd-DTPA and 88, with in vivo imaging of a brain tumor model showing peak contrast enhancement after 30 min. However, 89 displayed limited in vivo stability, with evidence of both Gd and Mn dissociation after 24 h present during imaging (Figure 44) (145). Modification of porphyrin structures to engender amphiphilicity has also been attempted, with the aim of optimizing tumor uptake. Synthesis of 90 was carried out and evaluated in a squamous cell carcinoma mouse model in comparison to Gd-DTPA. While the relaxivity of both compounds was comparable, 90 showed sustained tumor enhancement after injection, with slow tumor clearance and excellent accumulation, attributed to the
H2O O N
N Mn
N
O
O
HOOC N H
COOH N
N
COOH
COOH
N 88
HOOC
COOH
Figure 43 Porphyrin–chelator complex ATN-10 88, with manganese chelation carried out by the porphyrin core rather than the peripheral chelator.
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H2O
OOC
O O
N
N N
N
O
Mn
COO
N
COO N Gd
N
COO
89
HOOC
COOH
Figure 44 Incorporation of both manganese into the porphyrin core and gadolinium into the peripheral chelator of the porphyrin–chelator complex ATN-10 89.
O O N
N Mn
N
AspOC
N
COAsp 90
Figure 45 Structure of manganese porphyrin 90.
amphiphilicity of the structure, while Gd-DTPA showed good initial tumor enhancement, followed by complete clearance after 2 h (Figure 45) (146). Modification of the structure and amphiphilicity of two cationic porphyrins has also been carried out in order to alter bioavailability. Both structures showed high T1 relaxation rates in a tissue phantom in comparison to commercial contrast agents, with 92 demonstrating superior T1 alternation in an animal model. However, 91 showed improved selective accumulation and a good toxicity profile and was suggested as a more suitable imaging agent despite the smaller effect on T1 relaxation (Figure 46) (147). 6.2.1 Macromolecular Structures There has been a growing interest in macromolecular manganese porphyrin structures, with a number of these now evaluated as MRI contrast agents.
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N+
N+
N+
N+
N N N+
Mn
N N
N
N
Mn
N
N
N+ N+
N+ 92
91
Figure 46 Cationic manganese porphyrins 91 and 92, with the altered amphiphilicity of the structures allowing modification of in vivo uptake.
O
O NH R
HO
N N
Mn
Cl N
R = chitosan (20 kDa)
N
HO
OH
O
93
O
Figure 47 Manganese porphyrin–chitosan conjugate 93, with the conjugation carried out through peptide coupling to a single carboxylic acid functionality.
Yu et al. demonstrated the synthesis of a chitosan-functionalized porphyrin, with evaluation of 93 demonstrating higher R1 values and superior image quality in comparison to the commercially available Gd-DTPA (Figure 47) (148). Subsequent work demonstrated the PEGylation of a manganese metalloporphyrin, followed by encapsulation of 94 into mono- and bis(permethyl-β-cyclodextran) structures, with the aim of producing stabilized Mn(II) contrast agents with enhanced paramagnetic character. Conjugates with 1:1 and 1:2 ratios were produced, with the minimal cytotoxicity of these structures in comparison to free 94 demonstrated. In situ reduction of the Mn(III) core to Mn(II) with sodium ascorbate was monitored by UV–vis
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spectrophotometry, with both conjugates shown to stabilize the Mn(II) species to autoxidation giving a Mn(II) half-life of 24 min. Assessment of in vitro relaxivity showed similar values for both Mn(II) and Mn(III) species, and minimal differences between the mono- and bis-cyclodextran structures, with all having higher values than commercial imaging agents (149). Further enhancement of relaxivity was attempted through the development of a branched tris-cyclodextran structure. As for the bis structure, limited cytotoxicity and improved Mn(II) stability were observed, with the tris structure having a longer Mn(II) half-life (ca. 30 min) and 7% higher longitudinal relaxivity in comparison to the bis structure, which was attributed to the enhanced rigidity of the structure (Figure 48) (150). Exploitation of the ability of cyclodextran structures to encapsulate multiple metalloporphyrins was also exploited by Aime et al. in the development of contrast agents sensitive to the partial pressure of oxygen, allowing applications in functional MRI. Incorporation of MnTPPS into a commercial cyclodextran structure bearing six to nine cyclodextran units was carried out, with large numbers of chelated MnTPPS engendering significant differences between the relaxivity of the Mn(II) and Mn(III) species (40.8 and 15.2 mM1 s1, respectively) (151). PEGylation of manganese porphyrins in order to improve relaxivity and reduce cytotoxicity has also been demonstrated, with 95 demonstrating R1 values ca. 7.5 times higher than MnTPPS, with the high R1 attributed to the slow tumbling rate of 95 rather than increased viscosity due to mPEG550 O
O – mPEG550
N
N Mn
N
N
O mPEG550
94
O mPEG550
Figure 48 PEGylated metalloporphyrin 94, which was subsequently incorporated into mono- and bis-cyclodextran structures.
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O O
O
O n
O
O
O
O
n
N N
Mn
N
N
O
O n
O
95
O
n
Figure 49 PEGylated metalloporphyrin 95, which was subsequently incorporated into an oligocyclodextran structure.
PEGylation. Cytotoxicity of 95 was also evaluated, with the IC50 shown to be significantly higher than MnTPPS (4.1 and 0.25 mM, respectively) (Figure 49) (152). Exploitation of nanoparticle delivery vehicles for porphyrin contrast agents has been demonstrated within the literature, with this approach favored due to allowing delivery of increased quantities of contrast agents to the tumor site, enhanced R1 values and alteration of both toxicity and pharmacokinetic properties. However, conjugation strategies must be carefully considered; although encapsulation of structures to incorporate them into nanoparticles is a popular and facile methodology, it also limits water access to the contrast agent, leading to deterioration of R1 values. Polylactic acid nanoparticles were utilized by Jing et al., with the synthesis of a theranostic delivery platform through encapsulation of therapeutic doxorubicin and covalent surface functionalization with 96. R1 relaxivity of the nanoparticles was found to be significantly higher than that of unconjugated 96 (27.8 and 6.7 mM1 s1, respectively), with this enhanced relaxivity attributed to reduction of the contrast agent tumbling rate following attachment to the solid support. In vivo evaluation in a mouse tumor model allowed clear visualization of tumor, including areas of inhomogeneity not observed without contrast agent, with signal enhancement increased from 30 min to 24 h (153). Subsequent work from this group utilized doxorubicin-loaded polylactic acid coated with a gold nanoshell, with surface functionalization with 96 carried out via a PEG spacer group. In contrast to nonPEGylated systems, the synthesized system demonstrated reduced aggregation, significantly longer blood circulation time, and good colloidal stability over 1 week. While
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the R1 relaxivity of the system (22.18 mM1 s1) was higher than that of free 96, it was also lower than that of previously synthesized nanoparticle systems (153). Despite this, in vivo imaging showed good visualization of the tumor, as well as therapeutic effects through both release of doxorubicin and photothermal therapy (154). Conjugation of 96 to oxidized dextran has also been carried out by Zhang et al., with the resulting high molecular weight conjugate showing reduced toxicity in comparison to free 96. While both the dextran conjugate and 96 showed enhanced R1 values in comparison to the control Gd-DTPA, relaxivity showed only minimal enhancement upon dextran conjugation (8.9 and 7.4 mM1 s1, respectively), as a result of the increasing molecular weight. In vivo evaluation of the dextran conjugate in comparison to Gd-DTPA showed tumor enhancement in both cases; however, higher signal enhancement of the tumor over a longer time period was observed for the conjugate (155). Covalent surface attachment of 96 has also been demonstrated on organic nanoparticles synthesized through self-assembly of tetraphenylporphyringrafted lipids. R1 relaxivity was found to be about four times that of the free 96 (20.6 and 5.2 mM1 s1, respectively) but lower than that of previously synthesized nanoparticle contrast agents. However, in vivo imaging showed good diagnostic and therapeutic capabilities, with clear tumor signal enhancement and effective treatment both in vitro and in vivo utilizing the photodynamic therapy capabilities of the free-base porphyrin–lipid (Figure 50) (156). SO3–
N –
N Mn
O3S N
NH2
N
SO3– 96
Figure 50 Sulfonated manganese porphyrin 96, with the amine functional group allowing conjugation to a number of nanosized delivery vehicles.
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MacDonald et al. also demonstrated the synthesis of porphyrin–lipid nanostructures to create porphysomes; organic, biodegradable liposome-like structures formed from self-assembly of a pyropheophorbide–lipid conjugates in aqueous media. Use of manganese-chelated 97 allows for labeling of all 80,000 porphyrins in the porphysome to created high payload contrast agents, with no disruption of the porphysome structure by the axial ligand observed. In addition, manganese chelation was found to improve photostability, and reduce phototoxicity, with the structure showing only limited cytotoxicity in vitro. Relaxivity values were determined for both intact and detergent disrupted Mn porphysomes; while both R1 and R2 values for intact porphysomes were low (1.2 and 7.0 mM1 s1, respectively), disrupted relaxivity was comparable to that of Gd-DTPA (R1 ¼ 4.0 mM1 s1 and R2 ¼ 12.9 mM1 s1). The increase in relaxivity upon disruption of Mn porphysomes was attributed to the result of increased water access to the inner coordination shell of the Mn ions, with the authors suggesting that optimization of the porphysome structure could allow enhancements to relaxivity (Figure 51) (157). Recently, Qazi et al. demonstrated an alternative strategy utilizing novel nanoparticles constructed from a hybrid of synthetic polymer and virus-like capsid particles. The porphyrin contrast agent was encapsulated into the particle through reaction between the activated porphyrin 98 and the amine-functionalized interior surface of the particle, to achieve loading ratios ranging from 121 to 3646 molecules of 98 molecules per capsid. T1 relaxivity remained constant per Mn ion, with the highest whole particle values of 7100 mM1 s1 achieved. Despite this, relaxivity of the porphyrin O
O O O
13
O O
P O O–
N+
O
N
N Mn N
97 N
Figure 51 Manganese pyropheophorbide–lipid monomer 97 which spontaneously self-assembles in aqueous environments to produce porphysome contrast agents.
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did not increase upon capsid conjugation, which can most likely be attributed to the poor water access inside the capsid. R2 relaxivity was also evaluated for this system and displayed an inverse relationship with increasing porphyrin loading ratios, allowing the system to be modified for use as either a T1 or T2 contrast agent (Figure 52) (158). Inorganic nanoparticles have also been utilized as solid supports, with Hitomi et al. demonstrating the synthesis of thiol-functionalized 99, allowing direct attachment to gold nanoparticles. Alterations to porphyrin: nanoparticle ratios were found to vary size, optical, and MRI properties, while relaxivity was found to vary with nanoparticle size. In all cases, R1 per Mn center was up to 4.3 times that of free 99 (Figure 53) (159). Proteins have also been explored as an alternative to synthetically produced nanoparticles for the targeted delivery of metalloporphyrin contrast agents. Initially, Shapiro et al. demonstrated the use of the heme domain of Bacillus megaterium cytochrome P450 BM3 (BM3h) to detect dopamine OH O N
N Mn
N
N O O O N
98
O
Figure 52 Contrast agent 98 bearing an NHS ester modality for facile conjugation to semisynthetic nanoparticle structures.
NH
S O O S
N N Mn
NH
N
S
NH O O
N NH
S
99
Figure 53 Structure of manganese porphyrin 99 functionalized with peripheral thiol groups to facilitate attachment to gold nanoparticles.
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release in living rat brains, with the native iron porphyrin acting as the contrast agent. However, these experiments were relatively insensitive due to the low R1 relaxivity of the unmodified proteins (ca. 1 mM1 s1) (160). Substitution of the iron heme for Mn protoporphyrin IX was then attempted, with the similar structure of the porphyrin allowing improved R1 values without affecting protein folding. The synthesized Mn-BM3h showed >95% manganese incorporation, increased heat stability, and twofold increased T1 relaxivity (2.6 mM1 s1) over the native variant. The relaxivity of a number of other Mn-PPIX-derived proteins was also measured, with the Mn-C1634 showing the highest R1 and R2 relaxivities (3.3 and 7.4 mM1 s1, respectively) (161). A similar analysis was carried out using the Thermoanaerobacter tengcongensis nitric oxide/oxygen-binding protein (NOX), with the native iron-containing heme form showing limited application as an MR imaging agent with T1 and T2 relaxivities of 1.6 and 2.9 mM1 s1, respectively. Substitution of the heme for manganese protoporphyrin IX chelated with both Mn(II) and Mn(III) was shown to produce stable structures. While Mn(II) showed modest enhancement in relaxivities (R1 ¼ 3.6 mM1 s1 and R2 ¼ 3.8 mM1 s1), significant enhancements were observed for the Mn(III) structure (R1 ¼ 12.0 mM1 s1 and R2 ¼ 16.8 mM1 s1). In addition, incorporation of gadolinium mesoporphyrin IX was carried out to produce even higher relaxivity values, with R1 ¼ 28.7 mM1 s1 and R2 ¼ 39.9 mM1 s1 (162).
7. PHOTO-ACOUSTIC IMAGING Photo-acoustic imaging has recently emerged as one of the newest and most rapidly growing medical imaging techniques. This hybrid technique combines laser irradiation with ultrasound detection, with target chromophores excited with a near-infrared laser light to induce thermoelastic expansion within the tissue. As a result of this, acoustic pressure waves are generated, which can be detected by an ultrasound transducer to form a photo-acoustic image. Imaging in this way offers multiple advantages over both traditional optical and ultrasound imaging techniques, with high spatial resolution, applicability across both the macro- and microscale, greater specificity than traditional ultrasound imaging, and imaging at depths of up to 5–6 cm, far beyond the limitations of optical imaging (163). The first examples of photo-acoustic imaging utilized naturally occurring pigments including hemoglobin as contrast agents, allowing distinction of
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the blood vessels from surrounding tissue. However, early detection of tumors utilizing these endogenous chromophores was limited, with the poor vascularization of small tumors limited the quantity of hemoglobin present. As a result, addition of an exogenous contrast agent is required (164). To this end, a number of exogenous dyes have been evaluated as contrast agents, including nonporphyrin structures such as methylene blue, congo red, indocyanine green (163), and a limited number of exogenous tetrapyrroles, pyropheophorbide-α (165), bacteriochlorins (166), and modified porphyrin derivatives (167). While tetrapyrroles do offer high optical absorption which generates strong PA signals, their high fluorescence acts as a competing relaxation mechanism, reducing heat generation and consequently ultrasound wave generation. Recently, the first examples of metallophthalocyanine contrast agents have been developed, with the clinical relevance of the lipophilic zinc phthalocyanine 100 compared to four other photosensitizers; protoporphyrin IX, a squarine, Ce6, and methylene blue in a scattering phantom. 100 demonstrated the highest photo-acoustic quantum yield (0.47) and greatest light absorption between 700 and 900 nm of all evaluated contrast agents; however, relatively high cell toxicity was observed. While in vivo evaluation in a mouse tumor model showed evidence of photo-acoustic signals in the liver, spleen, and intestines, tumor selectivity was good, with maximum tumor:normal tissue ratios of 7:1 observed after 60 min, providing good contrast in all images (Figure 54) (168). As a result, synthesis of a range of hydrophilic metallophthalocyanines was then carried out, with these potential contrast agents examined in both a tissue phantom and a mouse oral squamous cell carcinoma model. In both cases, free-base porphyrin 101 exhibited the best photo-acoustic properties, with this effect attributed to the metallation promoting internal conversion to the triplet state rather than heat generation. In vivo imaging showed SO3H
HO3S N N
N N
Zn
N N
N N
M
N N
N N 100
N N
101 M = 2H 102 M = Zn 103 M = AI
N N
HO3S
SO3H
Figure 54 Metallophthalocyanine photoacoustic imaging agents 100–103.
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superior localization and imaging with 101, with 102 producing signals of three times lower intensity, and 103 providing no tumor delineation (169).
8. MULTIMODAL IMAGING Interest in the development of multimodal imaging has arisen as a natural consequence of the inherent limitations of each of the discussed imaging technologies, including spatial resolution, sensitivity, and speed of imaging. As a result of these shortcomings, all generated images provide only limited information, and there is a growing need to combine the generated information from two or more scans to provide both morphological and functional information. While simply fusing the information from two or more separate scans is widely used, difficulty in controlling patient movement and position means that confirmation of co-registration of signals is complex. An alternative is the combination of two scanners into a single unit to simultaneously provide two images, an approach which has been successfully applied to SPECT/CT and PET/CT for around 20 years (170). As well as the improved speed and the increased amount of information provided by combined scanning methodologies, combining two imaging techniques into a single multimodal imaging agent also offers numerous clinical advantages. These include allowing the study of processes and structures from differing resolution and depths, as well as permitting investigation of tumor morphology, growth, pharmacokinetics, and mechanism of drug action, while only requiring administration of a single compound. In particular, the structure of porphyrins makes them particularly appealing as multimodal imaging agents; as well as offering inherent fluorescent properties and facile chelation, they also offer multiple sites for functionalization, allowing development of a wide range of multimodal agents.
8.1 SPECT–Fluorescence SPECT and fluorescent imaging technologies were combined by El-Tamer et al. in 2003 to produce the first example of a multimodal metallophthalocyanine imaging agent. Synthesis of the 99mTc-radiolabeled tetrasulfophthalocyanine was carried out to produce an imaging agent suitable for the detection of breast cancer metastases in sentinel lymph nodes, with the aim of overcoming the wide discrepancies observed when SPECT and fluorescent imaging agents were administered separately. 99mTc radiolabeling was carried out in 30 min under mild reaction conditions, with the resulting conjugate allowing sentinel lymph node visualization in a
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rabbit model through both SPECT and fluorescent imaging, with no uptake into nontarget lymph nodes or accumulation associated with free 99mTc observed (171).
8.2 Photo-Acoustic/Fluorescence The combination of photo-acoustic imaging and fluorescence imaging into a multimodal agent is an obvious choice, with many endogenous porphyrin structures demonstrating efficacy as both fluorescent and photo-acoustic imaging agents. Despite this, only a single example of this strategy has been published to date, with Lovell et al. demonstrating the use of porphysomes containing a range of different metals. Metallation was shown to modulate both absorption and emission wavelength, allowing facile tuning to meet specific wavelengths. Although absorption of 104 was unchanged following porphysome formation, extensive fluorescence quenching was observed due to the close packing in the structure. As a result, following irradiation, the energy was released via thermal expansion, with significant temperature increases (35 °C) observed following laser irradiation. Assessment of both fluorescence and photo-acoustic imaging was carried out in vivo, with initial tumor localization of the porphysomes clearly detected using photo-acoustic imaging with no damage to surrounding tissue. While tumor fluorescence was unchanged after 15 min, after 2 days significant fluorescence was observed, which was attributed to porphysome degradation inside cells, and subsequent reduction in quenching (Figure 55) (172). O O O
O
O P–
O
N+
O
O
N
N M
N
104 M = Zn, Cu, or Pd
N
Figure 55 Pyropheophorbide–lipid monomer 104 which spontaneously self-assembles in aqueous environments to produce porphysomes. Metallation of this structure with zinc, copper, and palladium was carried out to modulate both absorption and emission wavelengths.
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8.3 PET–Fluorescence PET–fluorescence combines two popular imaging modalities to allow imaging on both the micro- and macroscale and, as a result, is a popular option for metalloporphyrin imaging despite the difficulty in designing structures which retain the fluorescent properties of the porphyrins while allowing functionalization with paramagnetic radioisotopes. This problem was encountered by Liu et al., who utilized a targeted 64Cu pyropheophorbide 105 to carry out ovarian cancer imaging in primary cell line-derived tumors. While 105 showed good tumor:muscle ratios (4.97 at 4 h), fluorescence imaging could not be carried out due to quenching of the excited state by the copper core. As a result, fluorescent imaging was carried out with a mixture of 105 and the free-base derivative, with PET imaging showing clear tumor delineation, while fluorescence imaging showed excellent uptake into metastases, with 3.5-fold higher fluorescence in comparison with adjacent normal tissue (Figure 56) (173). Alternatively, use of radioisotopes other than 64Cu allows retention of fluorescence after radiolabeling. Labeling of metalloporphyrins and phthalocyanines with 18F was carried out by Ranyuk et al. for PET/fluorescence imaging, with relatively poor yields (18%) of the cold analogues obtained even under forcing conditions (80 °C for 20 min). Yields obtained using 18 F were also poor, with 107 obtained in RCY of 10%, while synthesis of 18F-labeled 106 was not attempted (Figure 57) (174). An alternative labeling methodology offering improved yields and retention of fluorescence is the incorporation of the radioisotope into chelators attached to the tetrapyrrole structure. Ranyuk et al. attached both DOTA
N
N
NH2
64Cu
N
N N
N
NH N
O
HN O
O NH GDEVDGSGK–NH
COOH 105
NH O
O
Figure 56 Targeted 64Cu pyropheophorbide 105, which was combined with the unmetallated analogue to produce a multimodal fluorescence-PET imaging agent.
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R
R
F
R
N
N Zn
N
R
N
R = tBu
R
R 106 t
C4H9
N tC
4H9
N N
N
N
N
Zn N
tC
N
4H9
18F
107
Figure 57 Metalloporphyrin 106 and metallophthalocyanine 107 labeled with positron-emitting 18F.
and NOTA chelators via varying lengths of linker chain to a zinc phthalocyanine to create multimodal imaging agents, with radiolabeling with 64Cu and 68Ga carried out in good RCY (ca. 70% and 60% decay-corrected yields, respectively). The 64Cu chelates showed superior serum stability, with no loss of copper observed, and therefore, 109a and 109c were selected for in vivo evaluation. Biodistribution studies showed rapid kidney uptake and excretion of 109c, while 109a demonstrated 10-fold larger retention. Despite this, in vivo imaging showed high surface distribution and limited tumor uptake, with the tumor only poorly distinguished from the surrounding tissue using both fluorescence and PET imaging (Figure 58) (175). Incorporation of 64Cu directly into a metalloporphyrin structure can also be an effective method of producing multimodal imaging agents through the
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H n N
NaO3S N N
N Zn
N
O
N
O
N N
N
O
N
68
Ga
N
O N
108a n = 1 108b n = 3 108c n = 5
O O
N
O
SO3Na
NaO3S
N N
N Zn
N
O
N
N
O
O
H n N
NaO3S
N
64
Cu N
109a n = 1 109b n = 3 109c n = 5
N
O
N O
N NaO3S
SO3Na
Figure 58 Structures of imaging agents 108–109, with the conjugated DOTA and NOTA macrocycles allowing chelation of paramagnetic metals without loss of the zinc phthalocyanine fluorescence.
use of multiply-loaded nanosized delivery vehicles; this allows a proportion of the porphyrin to be radiolabeled, allowing retention of some of the fluorescent characteristic. Liu et al. demonstrated this technique through the use of porphysomes, with radiolabeling carried out on 110, and subsequent self-assembly producing a porphysome with high specific activity (103,600 GBq mmol1). Despite this, only 5% of porphyrins present in each porphysome were labeled, leaving porphysome size and photophysical properties unaffected. Evaluation as an imaging agent in a prostate cancer model demonstrated excellent tumor uptake after 24 h, with very little background uptake and high tumor:muscle ratios (12.7:1) (176). The utility of 110 was also demonstrated in both large and small prostate tumor models, with clear delineation of tumors of all sizes, as well as metastases present in the spine and bones, via both fluorescence and radio-imaging (Figure 59) (177).
8.4 MRI/Fluorescence In considering the combination of PET and fluorescent imaging, multimodal MRI/fluorescence agents are an attractive option for imaging, but
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O O O
13
O
O P O –O O
N+
O 110 N
N 64
Cu N
N
Figure 59 Pyropheophorbide–lipid monomer 110 which spontaneously self-assembles in aqueous environments to produce porphysomes, allowing the nanostructure to be partly radiolabeled with 64Cu to produce a multimodal imaging agent.
O O
O N H
O
N
H N
N O
N N
N
N
N Zn
N 111
N N
N
Figure 60 Zinc phthalocyanine 111 conjugated to super paramagnetic iron oxide nanoparticles via a click chemistry methodology.
the synthesis of suitable metalloporphyrin agents is limited by the paramagnetic properties of metals suitable for MRI. The challenge is further complicated by the lower limits of detection required for MRI compared to PET, making partial-metallation methodologies impractical. As a result, attachment of tetrapyrrole structures to an external MRI contrast agent is the only practicable method to produce such multimodal agents. Boudon et al. employed this strategy using super paramagnetic iron oxide (SPIO) nanoparticles, which were silanized to allow functionalization with zinc phthalocyanine 111 in ratios of ca. 690 per nanoparticle. The excitation and emission properties of 111 were unaffected by conjugation, with measured R2 and R1 found to be similar to commercial contrast agents (73 and 3 mMFe1 s1, respectively), with the low R2 value attributed to the highly hydrophobic phthalocyanine coating limiting access of water molecules to the SPIO core (Figure 60) (178).
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Most recently, attachment of zinc porpholactol to two chelating agents has been carried out to produce two multimodal imaging agents. In both cases, fluorescence was not affected by conjugation, and T1 relaxivities (ca. 18 mM1 s1 at 0.5 T and 37 °C) were found to be significantly higher than that of Gd-DTPA. In vitro imaging via both MRI and fluorescence was carried out successfully, with good images and no dark toxicity found for 112 or 113 (Figure 61) (179). O O
F
F
F
F
F
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F
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F N
N O O
O
F
O
F
Zn F
N Gd N
O F
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N
N=N N
N F
F F
F
F
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F F O O N
F
F
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F
F
F
FO
F
N
F F
N
N O O
O F
Zn N
N Gd O
O F
N
H N
O F
N F
F F
F
F
F
113
F F
Figure 61 Combining zinc porpholactols with gadolinium chelating macrocycles allows retention of the porpholactol photophysical properties to produce MRI/fluorescence imaging agents 112 and 113.
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8.5 PET/MRI The development of combined PET/MRI imaging agents is a novel combination of two powerful imaging modalities, which was restricted for many years due to the technical challenges involved in the combination of both technologies into a single scanner. The first example of a metalloporphyrin-based system was produced by Gros et al., utilizing a structure consisting of two chelators, a porphyrin and a tetraazamacrocycle. Metal chelation was performed in a two-step process, with gadolinium metallation carried out at 50 °C for 12 h, followed by copper insertion at 50 °C for 2 h. 114 demonstrated good stability in water at physiological pH, with no transmetallation or release of gadolinium observed. T1 relaxivity of the structure at 300 MHz was found to be similar to that of unconjugated gadolinium-DOTA, with the attached metalloporphyrin having little effect, although the increased molecular weight resulted in a medium field relaxivity higher than that of commercially available contrast agents. Metallation of the porphyrin with 64Cu was not attempted, with no evaluation of the conjugate as a PET imaging agent carried out (Figure 62) (180). Subsequently, the same group also developed two examples of PET/MRI agents, with 115 functionalized with an amine functional group for attachment to targeting biomolecules, and 116 functionalized with two gadolinium chelators for improved contrast enhancement. Both structures were shown to exhibit no cytotoxicity; however, some cell growth inhibition was observed in both cases. T1 relaxivity values were found to be typical of gadolinium complexes of this type, with again no influence from the copper porphyrin observed. Despite this, preliminary MRI in mouse tumor models showed rapid blood clearance and no enhanced tumor uptake was observed (Figure 63) (181). H2O O
N O
O
O N Gd
N
N O
O NH
N
N Cu
O 114
N
Figure 62 Structure of bifunctional PET/MRI chelator 114.
N
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H2O O
N O
O
O N Gd
N
O
N
N
O
N Cu
HN
O
N
NH2 N 115
H2O O
O
N O
O
N Gd
N
N O
O
N
O
N
O
Cu
HN N
NH N
O N
O
N Gd
N
O N
O
116 O
O
H 2O
Figure 63 Single and double gadolinium-chelator functionalization of copper porphyrins 115 and 116.
9. CONCLUSIONS While exploitation of porphyrins and other tetrapyrrolic photosensitizers as therapeutic modalities has been of interest for over 100 years, the application of these discussed structures as clinical treatments for neoplastic conditions is still a relatively new field, with the clinical photosensitizer Photofrin receiving regulatory approval as recently as 1993. Similarly, although the utility of metalloporphyrins as imaging agents has been recognized for over 60 years, none of the discussed structures have yet been approved for clinical imaging applications.
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This lack of clinically approved metalloporphyrin imaging agents cannot be attributed to a lack of clinical relevance of these structures; as demonstrated throughout this review, metalloporphyrins can offer both selective tumor uptake and excellent imaging potential across a plethora of imaging technologies. Despite this, the nature of metalloporphyrins presents innate challenges in the development of imaging agents; the complex synthesis and functionalization of many described structures can limit scalability of production and purification, while their highly lipophilic nature results in high nontarget organ uptake in liver, spleen, and kidneys. As a result, there is a pressing need for future work in this area to focus on addressing these issues. In particular, strategies for imparting full water solubility on structures without increasing structural complexity or engendering toxicity are needed. Development of structures with both accessible and flexible bioconjugation handles is also required in order to meet the growing appreciation of the efficacy of biomolecule-targeted structures to improve tumor uptake and reduce nontarget organ uptake, rather than relying on passive uptake alone. In addition, it is becoming apparent that the greatest strength of metalloporphyrins is their multifunctionality, simultaneously allowing conjugation and metal chelation while in many cases retaining both innate fluorescent and therapeutic properties. This property aligns well with the growing interest in the area of cancer research in both multimodal imaging and theranostics. These multifaceted techniques offer the most promising step forward to date toward overcoming disease heterogeneity. They offer increased accuracy in disease staging and determination of receptor expression in both metastatic lesions and primary tumors, in addition to allowing exploitation of emerging technologies such as MRI/PET, providing a drive toward the eventual goal of personalized medicine.
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