Tumor-targeting multi-functional nanoparticles for theragnosis: New paradigm for cancer therapy

Advanced Drug Delivery Reviews 64 (2012) 1447–1458

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Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr

Tumor-targeting multi-functional nanoparticles for theragnosis: New paradigm for cancer therapy☆ Ju Hee Ryu a, 1, Heebeom Koo a, 1, In-Cheol Sun a, Soon Hong Yuk b, Kuiwon Choi a, Kwangmeyung Kim a, Ick Chan Kwon a,⁎ a b

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 6, Seongbuk-gu, Seoul 136-791, Republic of Korea College of Pharmacy, Korea University, Jochiwon, Yeongi, Chungnam 339-700, Republic of Korea

a r t i c l e

i n f o

Article history: Received 5 April 2012 Accepted 28 June 2012 Available online 4 July 2012 Keywords: Theragnosis Chemotherapy Photodynamic therapy siRNA therapy Photothermal therapy Optical imaging Personalized medicine Molecular imaging Cancer Nanomedicine

a b s t r a c t Theragnostic nanoparticles (NPs) contain diagnostic and therapeutic functions in one integrated system, enabling diagnosis, therapy, and monitoring of therapeutic response at the same time. For diagnostic function, theragnostic NPs require the inclusion of noninvasive imaging modalities. Among them, optical imaging has various advantages including sensitivity, real-time and convenient use, and non-ionization safety, which make it the leading technique for theragnostic NPs. For therapeutic function, theragnostic NPs have been applied to chemotherapy, photodynamic therapy, siRNA therapy and photothermal therapy. In this review, we present a recent progress reported in the development and applications of theragnostic NPs for cancer therapy. More specifically, we will focus on theragnostic NPs related with optical imaging, highlighting promising strategies based on optical imaging techniques. © 2012 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . 2. Theragnostic nanotechnology 3. Theragnostic nanotechnology 4. Theragnostic nanotechnology 5. Theragnostic nanotechnology 6. Conclusions . . . . . . . . Acknowledgments . . . . . . . References . . . . . . . . . . .

. . . . . . . . . . . . . in chemotherapy . . . . in photodynamic therapy in siRNA therapy . . . . in photothermal therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Theragnosis is a newly emerging concept which involves simultaneous execution of diagnostic tests and targeted therapy [1,2]. Theragnostic agents contain both diagnostic and therapeutic functions

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Personalized nanomedicine”. ⁎ Corresponding author. E-mail address: [email protected] (I.C. Kwon). 1 Authors contributed equally. 0169-409X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2012.06.012

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in one integrated system, enabling diagnosis, therapy, and monitoring of therapeutic response at the same time (Fig. 1) [3]. Diagnosis by theragnostic agents allows the characterization of diverse cellular phenotypes in an individual patient's cancer, which further enables therapy. In addition, co-delivery of diagnostic and therapeutic functions in theragnostic agents enables real-time validation of therapy. Real-time, noninvasive monitoring of the theragnostic agents, can allow clinicians to identify positive or negative patient response to the current therapeutic regimen and to guide decisions on continuation or alteration of the regimen before evaluation of traditional treatment efficacy, such as a change in tumor size [4,5].

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Fig. 1. Schematic illustration of theragnostic nanoparticle for simultaneous diagnosis, therapy, and therapeutic monitoring.

Nanotechnology could be utilized as a fascinating tool for theragnosis [6–8]. Various nanoparticles (NPs) containing either diagnostic or therapeutic function separately have now evolved to include both functions in theragnostic NPs [9]. One motivating factor for the integration is that both diagnostic and therapeutic agents should be focused on the tumors at an effective concentration to achieve the desired diagnosis and therapy [10]. Theragnostic NPs can be selectively delivered to tumors by passive or active targeting [11]. Passive targeting means that NPs can be extravasated from leaky vessels and selectively accumulated at the tumor site via the enhanced permeability and retention (EPR) effect [12,13]. In addition, NPs can be conjugated with ligands with strong binding affinity to targeted tumor cells for active targeting [14,15]. These ligands include peptides, small organic molecules, oligosaccharides, and antibodies. One NP can be conjugated with multiple copies of ligands or several ligands with different targets for efficient cellular uptake and enhanced tumor targeting [16]. Theragnosis requires noninvasive imaging modalities including optical imaging, magnetic resonance (MR) imaging, computed tomography (CT) or positron emission tomography (PET) [17]. Table 1 shows the characteristics of these imaging modalities currently applied in theragnostic systems. Individual imaging modality has its own advantages and disadvantages. Theragnosis needs noninvasive imaging modalities because imaging technology may allow for early diagnosis, non-invasive characterization of target-specific delivery and monitoring of therapeutic efficacy in cancer therapy. In particular, optical imaging based on fluorescence or bioluminescence is a sensitive, cost-effective technology that uses light to probe molecular targets and gene reporters in the living subject. Fluorescence imaging detects the light emitted from a fluorophore at a particular wavelength after excitation light typically illuminates the area of interest. Fluorophores for optical fluorescence imaging generally utilize near-infrared (NIR) Table 1 Characteristics of imaging modalities applied in theragnostic systems. Imaging modalities Advantages

Disadvantages

Optical imaging

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MR imaging CT imaging PET imaging

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High sensitivity Broad range of probes High spatial resolution Superior soft tissue contrast High spatial resolution Unlimited depth penetration Very high sensitivity Unlimited depth penetration Broad range of probes

Limited clinical translation Poor depth penetration Relatively low sensitivity Long imaging time Radiation exposure Poor soft tissue contrast Radiation exposure High cost

fluorescence light (λ = 650–900 nm), especially in in vivo imaging. The use of fluorophores within this range allows for low levels of interfering autofluorescence and high tissue penetration (up to several centimeters deep), providing enhanced image contrast with high sensitivity [18,19]. In addition, bioluminescence imaging measures the light emitted by the chemical reaction between a luciferase enzyme and luciferin. Advantages of optical imaging include sensitivity, real-time and convenient use, and non-ionization safety, which make optical imaging the leading technique among several potential imaging modalities available for the development of new theragnostic NPs in cancer therapy [20,21]. Theragnostic NPs are still in their infancy with regard to clinical application. However, extensive ongoing developments in nanotechnology and the call for a more personalized approach to medical treatment have already made theragnostic NPs a hot topic of research. In this review, we present a recent progress reported in the development and applications of theragnostic NPs for cancer therapy including chemotherapy, photodynamic therapy, siRNA therapy and photothermal therapy. More specifically, we will focus on theragnostic NPs related with optical imaging, highlighting promising strategies based on optical imaging techniques. 2. Theragnostic nanotechnology in chemotherapy Conventional small molecule drugs often exhibit disadvantages including the lack of sufficient specificity to the tumor, severe and toxic side effects in healthy tissues, limited delivery of hydrophobic drugs to the tumor cells, and drug-resistance [22,23]. NPs can provide a potential solution to these problems in traditional chemotherapy [22,24,25]. Among them, theragnostic NPs incorporating imaging agents and drugs such as doxorubicin (DOX) or paclitaxel (PTX) have been developed for simultaneous imaging and targeted chemotherapy [26,27]. Recently, our group has demonstrated that theragnostic NPs can simultaneously provide cancer detection, drug delivery, and real-time evaluation of therapeutic efficacy [6]. Chitosan-based NPs (CNPs) were labeled with Cy5.5, a NIR fluorescence dye for imaging, and encapsulated with PTX for cancer therapy (PTX–Cy5.5–CNP) (Fig. 2A) [28–30]. PTX–Cy5.5–CNP was delivered dose-dependently to the tumor sites in SCC7 tumor-bearing mouse models, which was visualized by optical imaging technology (Fig. 2B). The therapeutic efficacy of PTX–Cy5.5–CNP was able to be modulated and increased by repeating injections at three-day intervals. The targeted tumors displayed strong NIR fluorescence signals up to the frequency of injections at three-day intervals. With this optimal protocol, repeated injections of PTX–CNPs at three-day intervals could greatly increase the drug concentration in targeted tumors, resulting in enhanced therapeutic efficacy to tumor tissues while minimizing observed toxicity to normal tissues (Fig. 2C– E). This example demonstrated that theragnostic NPs can contribute to the identification of the optimal dosage of the drug in question, increasing both effectiveness and safety of the drug. NIR fluorescence imaging was also utilized to directly monitor tumor growth in response to PTX–Cy5.5–CNP administration in SCC7 tumor-bearing mouse models. This study showed the possibility to determine the optimal dosage of the drug for the right person at the right time when theragnostic NPs are localized in an individual. Therefore, this strategy using theragnostic NPs holds promise in paving the way for personalized medicine. Wang et al. recently reported theragnostic NPs with peptides that bind to histone H1 exposed on the surface of apoptotic cells for simultaneous drug delivery and real-time monitoring of therapeutic response via active targeting [4]. When tumor cells respond to chemotherapy, they generally undergo apoptosis, which could increase the expression of apoptotic biomarkers such as histone H1 in the tumor. Liposomes with or without apoptotic cell-targeting peptides contained DOX for chemotherapeutic treatment and Cy7.5 for imaging. Liposomes with apoptotic cell-targeting peptides suppressed tumor growth more

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Fig. 2. Theragnostic chitosan-based nanoparticles (CNPs) for cancer imaging and chemotherapy. (A) Conceptual description of theragnostic nanoparticles labeled with Cy5.5 for imaging and encapsulated with PTX for cancer therapy (PTX–Cy5.5–CNP). (B) In vivo images of the tumor-bearing mice treated with PTX–Cy5.5–CNP of different drug concentrations (5 mg/kg, 10 mg/kg, 20 mg/kg) every third day. The black arrow indicates intravenous injection of NP–PTX–Cy5.5. (C) Representative images of excised tumors treated with saline, CNPs, free PTX, and PTX–CNPs for 18 days. (D) Comparative therapeutic efficacy studies of PTX–CNPs in tumor-bearing mice. (E) Survival curve.

efficiently in mice compared to liposomes without apoptotic celltargeting peptides, which was demonstrated by a difference in the NIR fluorescence intensities. Theragnostic NPs could be designed to monitor the behavior of drug release using unique optical properties such as Förster (fluorescence) resonance energy transfer (FRET) [8,31]. FRET describes the nonradiative energy transfer from an excited state fluorophore (donor) to another fluorophore (acceptor) when the two fluorophores are in close proximity (typically b10 nm apart) [32,33]. Chen et al. recently demonstrated pH-responsive NPs encapsulated with DOX as an anticancer agent to understand drug release behavior and intracellular localization [34]. pH-responsive polymers were prepared by conjugating a hydrophobic N-palmitoyl group to chitosan, followed by attachment with Cy5. pH-responsive polymers could self-assemble into NPs encapsulating DOX in aqueous media, resulting in DOX–Cy5–NP. DOX (donor) and Cy5 (acceptor) in NPs were sufficiently close for energy transfer to occur (FRET on) at pH ≥7.0, whereas DOX and Cy5 were not in close proximity (FRET off) in the protonated form of DOX–Cy5–NP at low pH. Once internalized to cells, no fluorescence of DOX was observed when DOX–Cy5–NP was in the cavelolae/ caveosomes (FRET on with high efficiency), weak fluorescence in the cytosol when DOX–Cy5–NP was in the slightly acidic early endosome (FRET on with low efficiency), and strong fluorescence in the cytosol when DOX–Cy5–NP was in the more acidic late endosomes/lysosomes

(FRET off) (Fig. 3). This study indicated that theragnostic NPs employing the FRET technique can allow intracellular monitoring of drug release. There has been a great deal of progress with an intravital microscope to examine cellular behavior and molecular signals under conditions simulating a natural environment in living animals at subcellular resolution [29,35,36]. Intravital microscopy is different from whole body imaging using fluorescence or bioluminescence which usually provides only macroscopic resolution. The intravital microscope which includes an in vivo confocal laser scanning microscope (CLSM) can be used to directly assess in vivo distribution of the drug carrier or drug across various cellular locations. Intravital microscopy could enable a thorough understanding about the delivery mechanism to the targeted tissue [37,38]. Drug-loaded micelles composed of poly(glutamic acid) and hydrophilic polyethylene glycol (PEG) block copolymer were fabricated by Cabral et al. and were used for intravital imaging [39]. In vivo biodistribution and antitumor activity of drug-loaded micelles having a range of diameters were observed in both highly permeable tumors (C26) and poorly permeable tumors (BxPC3) (Fig. 4). Micelles were labeled with Alexa dyes for in vivo CLSM imaging. The real-time imaging via in vivo CLSM in a living mouse revealed that various sized micelles (with diameters of 30, 50, 70 and 100 nm) could penetrate through highly permeable tumors, however, only the 30 nm micelles accumulated in poorly permeable

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Fig. 3. pH-responsive theragnostic NPs for intracellular monitoring of drug release. Dual-emission fluorescence images of HT1080 cells; the cells were incubated with DOX-loaded Cy5-attached NPs and fluorescence images were taken in DOX imaging channel (560–600 nm) and Cy5 imaging channel (660–700 nm) when irradiating NPs at 488 nm.

tumors to accomplish an antitumor outcome. These results indicated that in vivo CLSM can contribute to the analysis of cellular internalization, tissue penetration and the extravasation profile of NPs in living mice. A study by Runnels et al. demonstrated several optical imaging techniques to track multiple myeloma tumor cells in the bone marrow and circulation of the mouse xenograft model [40]. Multiple myeloma is a cancer that originates from a single area and circulates through the circulatory system to multiple areas in the bone marrow. At 6 weeks after tumor cell injection, mice were treated with drug twice weekly. Throughout the course of treatment, the tumor volume decreased, as measured by bioluminescence imaging, which provided measurements from the bioluminescent reporter-expressing cells. The total number of circulating tumor cells also decreased, as they were continuously detected through an ear arteriole by in vivo flow cytometry [41]. As the tumor size decreased with continued therapy, the tumor size became below the bioluminescence imaging detection limit. However, residual tumor cells were still clearly detected by in vivo CLSM imaging. These results indicated that intravital microscopy can be used to detect even a small number of remaining tumor cells after the tumor bulk is removed at the very late stage of treatment [42]. During chemotherapy, theragnostic NPs are highly useful for providing real-time information on its location, release or efficacy of the contained drug and detecting residual tumor cells with various optical imaging techniques as shown in the above-mentioned studies. In addition, they enable the identification of the optimal dosage of the drug in question for the right person at the right time.

3. Theragnostic nanotechnology in photodynamic therapy Photodynamic therapy (PDT) is a clinical treatment which employs photo-triggered chemical drugs as photosensitizers. Photosensitizers can absorb light of particular wavelength and generate fluorescence and singlet oxygen molecules [43]. This singlet oxygen can damage major cellular organelles like mitochondria, resulting in cell death. Therefore, high accumulation of photosensitizers in cells can be applied to tumor therapy through photo-triggered death of tumor cells. With the aid of laser irradiation, PDT has been used for therapy in prostate, lung, head and neck, or skin cancers [44]. The representative photosensitizers for PDT are porphyrins, bacteriochlorins, phthalocyanines, hypericins, and chlorins. To improve therapeutic efficacy and reduce the side effects in normal tissue, various nanoscale photosensitizer delivery systems have been developed and applied to PDT [45–49]. Because photosensitizers can generate both fluorescence and singlet oxygen upon laser irradiation, they can be applied to optical imaging and PDT at the same time [43]. Based on the intrinsic fluorescence of photosensitizers, their tumor-targeted delivery can mark the location and area of the tumor tissue for optical imaging and further clinical treatment. Koo et al. applied pH-responsive micelles as tumor-targeted carriers for photosensitizers, and enabled simultaneous imaging and PDT under in vivo conditions [50]. The extracellular pH of tumor tissue was generally lower than that of normal tissue due to up-regulated glycolysis, and this acidic pH can be targeted by delivery systems including pH-responsive micelles. These micelles are composed of block copolymers with a hydrophilic PEG block

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Fig. 4. Drug-loaded differently sized micelles for observing in vivo biodistribution using intravital microscopy. Plots of relative tumor volumes of (A) highly permeable tumors (C26) and (B) poorly permeable tumors (BxPC3). (C) In vivo real-time microdistribution of drug-loaded micelles with different diameters in tumors. 30 nm micelles were labeled with Alexa 488 and 70 nm micelles were labeled with Alexa 594. Z-stack three-dimensional volume image of (a) C26 and (b) BxPC3 tumors 1 h after co-injection of 30 nm and 70 nm micelles. (c) Magnification of the perivascular area (showed by a white trapezium) of the z-stack three-dimensional volume image of BxPC3 tumors.

and a pH-responsive poly(β-amino ester) block (Fig. 5A) [51]. The tertiary amine group in poly(β-amino ester) can be protonated and become hydrophilic in acidic pH like the environment of tumor tissue. After this, the amphiphilic structure of the micelle is broken, and the inner cargo such as drugs or photosensitizers can be released. In this study, hydrophobic photosensitizer protophorphyrin IX (PpIX) was loaded to this pH-responsive micelle and applied to PDT. In tumor-bearing mouse models, the intense fluorescence signal in the tumor site was observed by optical imaging with this system (Fig. 5B). The tumor tissue was significantly delineated from the surrounding normal tissue, and this result showed the potential of this system for in vivo tumor imaging based on pH changes [20,32]. Upon laser irradiation, the successful therapeutic results were observed in a time-dependent manner with regard to the tumor volume and histological images (Fig. 5C and D). Another advantage of PDT is the possibility of an activatable system with improved specificity through a simple design based on self-quenching or with FRET quenchers [52]. Na and colleagues developed an activatable PDT system with chlorin e6 (Ce6) and black hole quencher-3 (BHQ-3) as the photosensitizer and FRET quencher, respectively [14]. Ce6 was conjugated to cationic mPEG-bPEI, and BHQ-3 was conjugated to anionic chondroitin sulfate (CS). These two polymers were able to bind to each other by electronic interaction in the aqueous phase to form NPs. Fluorescence and singlet oxygen generation ability were quenched in this structure, while it was recovered when CS was cleaved by an intracellular enzyme like esterase [53]. The fluorescence intensity and singlet oxygen generation

was highly enhanced in the presence of esterase. When they were injected into the tumor region and normal region in tumor-bearing mouse models, the tumor-specific recovery of fluorescence could be observed by optical imaging. Such activatable systems described in this study are expected to provide enhanced target-specificity for PDT in tumors [54]. As mentioned above, the intrinsic fluorescence of photosensitizers can provide easy tracking under both in vitro and in vivo conditions [43]. This characteristic can enable their application as a model drug to test the efficiency of delivery systems by optical imaging. Recently, Lee et al. fabricated two types of nanocarriers and tested their potential in tumor-bearing mouse models [55]. They prepared glycol chitosan NPs containing Ce6 by physical loading or chemical conjugation (Fig. 6A). In both types of NPs, Ce6 was successfully administered to glycol chitosan NPs with similar weight contents. However, when these two NPs were injected into the tumor-bearing mice through the tail vein, the conjugated particle demonstrated increased duration in the circulation and higher accumulation in the tumor tissue by optical imaging (Fig. 6B). The tumor images and tumor size graph showed higher therapeutic efficacy in the case of chemical conjugation in accordance with the optical imaging data (Fig. 6C and D). The difference in tumor accumulation of Ce6 may originate from the unintended release of the loaded drugs from amphiphilic NPs during blood circulation. This result suggested that the stability of amphiphilic NPs should be considered for their in vivo application, and further trials are needed to improve their stability [56]. In addition, this study also demonstrates that in vivo optical imaging can

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Fig. 5. pH-responsive micelles for simultaneous tumor imaging and photodynamic therapy. (A) Schematic illustration of tumor-targeted delivery of photosensitizers using pH-responsive micelles. (B) Time-dependent optical images of tumor-bearing mouse models after intravenous injection of photosensitizer (PpIX)-loaded pH-responsive micelles. (C) Tumor volume changes and (D) histological analysis of tumor-bearing mice treated with PpIX-loaded pH-responsive micelles.

be successfully used for the development and optimization of the PDT system for tumor therapy [57]. 4. Theragnostic nanotechnology in siRNA therapy Gene therapy involves the usage of nucleotide drugs for clinical purpose instead of chemical drugs. Until now, researchers have used plasmid DNA, antisense oligonucleotide (ODN), and small interference RNA (siRNA) for gene therapy [58–61]. siRNA has recently garnered much attention for its clinical use due to the lowest required dosage for gene regulation among the three genetic drugs available [62]. The mechanism of siRNA is via silencing of the expression of the target protein which shares the homologous sequence with the administered siRNA. After cellular uptake, siRNAs are bound to an RNA-induced silencing complex (RISC) with silencing activity through cutting and degradation of the target mRNA sequence. This specific suppression of protein expression can be useful in therapy through the reduction of target proteins associated with the disease [63]. siRNA should be localized within the cytosol of target cells to bring about the gene silencing effect. However, free siRNA itself cannot enter the cells due to its anionic charge. To deliver siRNA to target cells and their cytosol, various delivery systems have been developed using lipids, polymers, and metals [59,64,65]. Similar to the mechanism of chemotherapy, the targeted delivery of siRNA by carriers is also essential for successful gene therapy [12]. For this purpose, optical imaging techniques have been applied to test siRNA delivery systems by many researchers. Recently, Kwon's group showed that optical imaging can be used to monitor both the biodistribution of their siRNA delivery system and gene silencing under in vivo conditions [66]. They developed self-assembled NPs with hydrophobically modified glycol chitosan (GC) and polyethylenimine (PEI) (Fig. 7A). PEI was shown to provide a sufficient cationic charge for siRNA binding, and the presence of GC was able to reduce the interaction with serum protein and potential

aggregation during blood circulation (Fig. 7B) [67]. Cy5.5 was conjugated to siRNA by an end-group modification, and this Cy5.5-labeled siRNA can be tracked by optical imaging in both cells and mouse models. In tumor-bearing mouse models, a significant increase in the accumulation of siRNA in tumor tissue was observed based on NIR fluorescence of Cy5.5 (Fig. 7C). In addition, Kwon's group used red fluorescence protein (RFP)-expressing B16F10 tumor cells and an RFP-target siRNA sequence. Therefore, the targeted gene silencing effect in the tumor tissue was also monitored by the changes in the level of red fluorescence showing the potential of their carriers as a tumor-targeted siRNA delivery system (Fig. 7D). Despite the superior sensitivity of optical imaging, their low spatial resolution in living systems has limited their broad application. In accordance with this point of view, recent trials combining optical imaging with other imaging modalities are expected to compensate the weak attributes of each imaging technique and result in a synergistic approach for improved imaging and diagnosis [68,69]. For example, Weissleder's group used fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) imaging to evaluate the biomedical potential of their siRNA delivery system [70]. Their siRNA carrier was an 80 nm lipid-like NP composed of C12-200 lipid, disteroylphosphatidyl choline, cholesterol, polyethylenglycol1,2-di-O-tetradecyl-sn-glyceride (PEG-DMG), and siRNA [71]. This system was applied to in vivo experiments on atherosclerosis, islet transplantation, and tumor suppression. CT imaging can demonstrate the three-dimensional images of bone and major organs, and optical signals in FMT were overlaid to the CT images of mouse models to show the localization of the administered siRNA (Fig. 8A). The time-dependent pharmacokinetic analysis of siRNA was also enabled by FMT. These data demonstrated a quick disappearance of the administered siRNA from blood and resulted in the accumulation in the spleen, liver, and bone marrow (Fig. 8B). In addition, the change in the size of the tumors could be non-invasively monitored by CT imaging after administration of the NPs containing CCR2 chemokine

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receptor-targeted siRNA (Fig. 8C). After treatment, the tumor volume was significantly reduced compared to the tumors treated with the control siRNA sequence (Fig. 8D). Optical imaging can be also used with MR imaging to accomplish high resolution and high sensitivity simultaneously and acquire more minute anatomical or biological information on the target site. Magnetic NPs can be integrated by attaching various diagnostic and therapeutic agents such as optical reporting dyes and therapeutic genes (siRNA). A variety of magnetic NPs have been researched to find a promising platform for theragnosis [72]. Medravora et al. used optical and MR imaging techniques to track the siRNA conjugated with their carriers. They developed an iron oxide NP-based siRNA delivery system for optical/MR imaging and gene therapy [73]. This system was fabricated by conjugation of siRNA and Cy5.5 to the surface of cross-linked iron oxide NPs. In addition, myristoylated polyarginine peptides (MPAP) were also conjugated to the NPs for efficient membrane translocation. The localization of siRNA in the mouse model was able to be monitored by T2 MR imaging due to the paramagnetic property of iron oxide NPs. The amount of accumulated siRNA in the tumor was analyzed by optical imaging. With siSurvivin targeting the antiapoptotic gene BIRC5, this system could suppress the relative expression of Survivin in the tumor tissue. Recently, Kumar et al. also developed a similar iron oxide NP-based siRNA delivery system [74]. They used EPPT peptide for binding to the tumor-specific antigen uMUC-1 which provided enhanced tumor-targeting. In particular, they exhibited improved suppression of tumor growth in tumor-bearing mouse models by intravenous injection of this system containing BIRC5 siRNA.

Park's group focused on the FRET system to analyze the intracellular localization and unpacking mechanism of their siRNA delivery system [75]. For this purpose, quantum dots (emission: 624 nm) were conjugated to PEI, and the resulting QD–PEIs were complexed with Cy5-labeled siRNA (Cy5-siRNA) by electrostatic interaction (Fig. 9A). Protein transduction domain (Hph-1) peptide was also conjugated to these complexes. When Cy5–siRNA were in contact with QD in the complexes, the QD (donor) fluorescence decreased and Cy5 (acceptor) fluorescence increased by FRET. This effect diminished after the release of Cy5–siRNA from the complexes, and the fluorescence recovered at 624 nm. The complexes conjugated with Hph-1 peptide showed a faster dissociation and release profile of Cy5–siRNA, compared to the complexes without peptide in optical cellular imaging and fluorescence activated cell sorter (FACS) analysis (Fig. 9B and C). This fast dissociation is significantly different from the delayed dissociation of the complexes without peptide which enter tumor cells through traditional endocytosis and endosomal escape. This means that Hph-1 peptide could facilitate cellular uptake of the complex via a transcytosis process, but not by endocytosis. This study showed that optical imaging techniques such as FRET can be significantly useful in the development of new siRNA delivery system and in the analysis of their mechanism. In particular, siRNA should be stably protected against nuclease digestion and delivered to the cytosol of target cells to achieve the desired therapeutic results. Consequently, observing their localization in cells or bodies by optical imaging is essential to test siRNA delivery systems. In addition, siRNA is a promising material for optical imaging because it can be easily labeled with other molecules by intercalation

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Fig. 7. Glycol chitosan (GC)–PEI NP for siRNA delivery. (A) Chemical structures of hydrophobically modified GC and PEI. (B) Illustration of self-assembled GC–PEI NPs and siRNA binding. (C) Whole-body distribution analysis of GC–PEI NPs containing Cy5.5-labled siRNA by optical imaging. (D) Gene silencing test with RFP-expressing tumors and GC–PEI NPs containing RFP-targeted siRNA by optical imaging.

Fig. 8. In vivo therapeutic use of lipid-like NPs as siRNA carriers. (A) FMT–CT images after intravenous injection of lipid-like NPs containing Cy5.5-labeled siRNA. (B) Time-dependent pharmacokinetic analysis of (A) in blood and organs. (C) CT images of tumor-bearing mouse models treated with lipid-like NPs containing CCR2-targeted siRNA. (D) Tumor volume analysis based on CT data in (C).

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Fig. 9. Quantum dot-based siRNA delivery system for FRET optical imaging. (A) Synthetic procedure of quantum dot-based siRNA delivery system (QD–PEI–Cy5–siRNA). (B) Confocal images of PC-3 tumor cells treated with QD–PEI–Cy5–siRNA. (C) FRET analysis of the dissociation and release profile of siRNA from QD–PEI–Cy5–siRNA after cellular uptake.

or chemical conjugation. Furthermore, their large size can exhibit relatively constant physicochemical characteristics compared to small molecules such as chemical drugs after labeling [76]. Therefore, a great deal of research is underway to develop and optimize various siRNA delivery systems using optical imaging techniques, and these trials are highly important for effective in vivo siRNA therapy [73]. 5. Theragnostic nanotechnology in photothermal therapy The potential use of gold nanorods (AuNRs) as a simultaneous imaging and therapeutic agent has been demonstrated by many researchers. The shape of AuNRs generates several interesting characteristics such as unique optical absorption and photothermal properties. AuNRs have been applied in the imaging field because they have unique optical properties including scattering [77–79] and quenching [80,81]. These properties have been used for cancer imaging via dark-field microscopy [82,83] or molecular optical imaging [84,85]. In addition, their photothermal properties [86] and tunable absorption wavelength [87] make them suitable materials for photothermal therapy. Yi et al. showed the in vivo application of AuNRs in cancer imaging and therapy [88]. With regard to cancer imaging, AuNR was modified with a matrix metalloprotease (MMP)-sensitive fluorescence probe. In this MMP-AuNR, Cy5.5, a fluorescence dye, was attached to the surface of AuNR through the MMP enzyme substrate peptide. Fluorescence was significantly lowered by the quenching effect of AuNR until Cy5.5 was detached from AuNR due to the cleavage of the MMP substrate (Fig. 10A). Because MMP enzymes are abundant in tumors, the quenched fluorescence of MMP-AuNRs can be recovered in tumor tissues. The imaging of cancer was possible when MMP-AuNRs was intra-tumorally injected in mouse models (Fig. 10B). In addition, AuNRs can also be used as a heat source for photothermal therapy because they can convert light into heat upon laser irradiation. In the same study, the absorption wavelength was adjusted to the NIR

region by tuning their aspect ratio to enhance the laser absorption (671 nm). The temperature of MMP-AuNR increased up to 50 °C in 4 min after laser irradiation, causing damage to the tumor tissue. Huang et al. also used AuNRs for the in vitro demonstration of both molecular imaging and photothermal cancer therapy [89]. They conjugated the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies to the surface of AuNRs to make them selectively accumulate on the malignant tumor cell membrane. Confirmation was verified through the comparison of dark-field microscopic images after incubation with nonmalignant (HaCat) and malignant cell lines (HOC 313 clone 8 and HSC 3), respectively. Binding of AuNRs to cells was visualized using AuNRs' strong scattering of red light, and a strong signal was only observed in the malignant cells. This result demonstrated that the anti-EGFR antibody-conjugated AuNRs bound to the malignant cells with high affinity due to the overexpression of EGFR on the cell membrane. In addition, attempts were made to destroy malignant cells through laser irradiation. While HaCat cells were not influenced by the 80 mW laser, the HOC and HOC malignant cells were severely damaged. Therefore, these results suggested the possibility of AuNRs as a novel agent for theragnosis. Theragnostic NPs have been applied for early detection and treatment of metastasis, an important issue in recent cancer therapy. A study by Kim et al. demonstrated the potential of gold-coated carbon nanotubes for the photoacoustic detection and photothermal ablation of cancer metastasis in mouse lymph nodes [90]. Photoacoustic imaging, which combines non-ionizing optical waves and ultrasonic waves, allows in vivo molecular and functional imaging [91]. Photoacoustic imaging has overcome the limitations of pure optical imaging by acquiring strong optical contrast and high ultrasonic spatial resolution for imaging of deep tissue. Besides photoacoustic imaging or photothermal therapy, theragnostic NPs including quantum dots or fluorophores and siRNAs have been recently researched for detection and treatment of metastatic cancer [92].

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A

B

Fig. 10. Matrix metaloprotease-sensitive gold nanorod (MMP-AuNR) for optical imaging and photothermal therapy. (A) Schematic illustration of MMP-AuNR for simultaneous imaging and photothermal therapy (B) NIR fluorescence tomographic images of SCC7 tumor-bearing mice after intratumoral injection of the MMP-AuNR probe (1) without and (2) with inhibitor.

6. Conclusions This review presents the development and applications of theragnostic NPs for cancer therapy, including chemotherapy, photodynamic therapy, siRNA therapy and photothermal therapy, highlighting promising strategies based on optical imaging techniques. Theragnostic NPs showed potential for providing real-time information on the administered drug inside the body, selecting the optimal dosage of the drug, and monitoring the therapeutic response. However, there are some hurdles to overcome for successful clinical application of theragnostic NPs based on optical imaging. Firstly, optical imaging is limited by their poor penetrating depth. Because of this limitation, they are not widely used in the clinical settings even though they showed remarkable data in small animal studies or for superficial lesions in animals. In fact, the maximal tissue penetration depth of an FDA-approved NIRF dye, indocyanine green, is several millimeters. Nevertheless, in clinical settings, optical imaging is still useful in many areas including complete tumor resection through intraoperative tumor margin detection and early cancer detection by a real-time endoscopic fluorescence imaging system. In addition, several emerging optical techniques including intravital microscopy, photoacoustic imaging and optical coherence tomography have gained interest for translation to the clinic, due to their high spatial resolution and good tissue penetration. Secondly, theragnostic NPs also need to reconcile the apparent dismatch between dosage requirements of therapeutic agents and imaging agents for potential clinical use. In general, the dosage requirement of NPs for desired therapeutic efficacy may be much higher than the dosage required for imaging. Lastly, theragnostic agents include multiple functions

within a single system, which can increase the complexity of the system. Development of simple and reproducible methods to synthesize theragnostic agents can help theragnostic agents become more applicable to clinical practice. As the capabilities of flexible and multifunctional theragnostic NPs continue to increase, theragnostic NPs can play a critical role in the new era of personalized medicine, which represents a new paradigm for cancer therapy by offering the potential to change treatment strategies. Acknowledgments This work is supported by the M.D.-Ph.D. Program (2010-0019863, 2010-0019864), the Global Research Laboratory Project of MEST, and the Intramural Research Program (Theragnosis) of KIST. References [1] K.Y. Choi, G. Liu, S. Lee, X. Chen, Theragnostic nanoplatforms for simultaneous cancer imaging and therapy: current approaches and future perspectives, Nanoscale 4 (2012). [2] X. Ma, Y. Zhao, X.J. Liang, Theragnostic nanoparticles engineered for clinic and pharmaceutics, Acc. Chem. Res. 44 (2011) 1114–1122. [3] T. Lammers, S. Aime, W.E. Hennink, G. Storm, F. Kiessling, Theragnostic nanomedicine, Acc. Chem. Res. 44 (2011) 1029–1038. [4] K. Wang, M.H. Na, A.S. Hoffman, G. Shim, S.E. Han, Y.K. Oh, I.C. Kwon, I.S. Kim, B.H. Lee, In situ dose amplification by apoptosis-targeted drug delivery, J. Control. Release 154 (2011) 214–217. [5] T. Lammers, F. Kiessling, W.E. Hennink, G. Storm, Nanotheragnostics and image-guided drug delivery: current concepts and future directions, Mol. Pharm. 7 (2010) 1899–1912. [6] K. Kim, J.H. Kim, H. Park, Y.S. Kim, K. Park, H. Nam, S. Lee, J.H. Park, R.W. Park, I.S. Kim, K. Choi, S.Y. Kim, K. Park, I.C. Kwon, Tumor-homing multifunctional

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