- Email: [email protected]
Multifunctional liposomal drug delivery with dual probes of magnetic resonance and fluorescence imaging
Author’s Accepted Manuscript Multifunctional liposomal drug delivery with dual probes of magnetic resonance and fluorescence imaging Chih-Ling Huang, Wan-Ru Hsieh, Che-Wei Lin, Hung-Wei Yang, Chih-Kuang Wang www.elsevier.com/locate/ceri
PII: DOI: Reference:
S0272-8842(18)30889-7 https://doi.org/10.1016/j.ceramint.2018.04.034 CERI17951
To appear in: Ceramics International Received date: 14 January 2018 Revised date: 29 March 2018 Accepted date: 5 April 2018 Cite this article as: Chih-Ling Huang, Wan-Ru Hsieh, Che-Wei Lin, Hung-Wei Yang and Chih-Kuang Wang, Multifunctional liposomal drug delivery with dual probes of magnetic resonance and fluorescence imaging, Ceramics International, https://doi.org/10.1016/j.ceramint.2018.04.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Multifunctional liposomal drug delivery with dual probes of magnetic resonance and fluorescence imaging Chih-Ling Huang 1,4, +, Wan-Ru Hsieh 2, +, Che-Wei Lin 2, Hung-Wei Yang 3, and Chih-Kuang Wang 2,4 *
1
Center for Fundamental Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan
2
Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical
University, Kaohsiung 807, Taiwan 3
Institute of Medical Science and Technology, National Sun Yat-sen University 804, Taiwan
4
Orthopaedic Research Center, College of Medicine, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
Correspondence: [Chih-Kuang Wang]
100, Shih-Chuan 1st Road,Kaohsiung,80708,Taiwan
Tel [+886- 7-3121101 #2677]
Fax [+886-7-3125339]
Email: [email protected]
1
Abstract: Many liposomal drug carriers have shown great promise in the clinic. To ensure the efficient
preclinical development of drug-loaded liposomes, the drug retention and circulation properties
of these systems should be characterized. Iron oxide (Fe3O4) magnetic nanoparticles (MNPs)
are used as T2 contrast agents in magnetic resonance imaging (MRI). Gold nanoclusters
(GNCs) contain tens of atoms with subnanometer dimensions; they have very low cytotoxicity
and possess superb red emitting fluorescent properties, which prevents in vivo background
autofluorescence. The aim of this study was to develop dual imaging, nanocomposite,
multifunctional liposome drug carriers (Fe3O4-GNCs) comprising MNPs of iron oxide and
GNCs. First, MNPs of iron oxide were synthesized by co-precipitation. The MNP surfaces
were modified with amine groups using 3-aminopropyltriethoxysilane (APTES). Second, GNCs
were synthesized by reducing HAuCl4·3H2O with NaBH4 in the presence of lipoic acid (as a
stabilizer and nanosynthetic template). The GNCs were grown by adsorption onto particles to
control the size and stability of the resultant colloids. Subsequently, dual Fe3O4-GNCs imaging
probes were fabricated by conjugating the iron oxide MNPs with the GNCs via amide bonds.
Finally, liposome nanocarriers were used to enclose the Fe3O4-GNCs in an inner phase
(liposome@Fe3O4-GNCs) by reverse phase evaporation. These nanocarriers were
characterized by dynamic light scattering (DLS), fluorescence spectroscopy, X-ray diffraction
(XRD), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR)
2
spectrophotometry, superconducting quantum interference device (SQUID), nuclear magnetic
resonance (NMR) imaging and in vivo imaging systems (IVIS). These multifunctional liposomal
drug delivery systems with dual probes are expected to prove useful in preclinical trials for
cancer diagnosis and therapy.
Keywords: dual probes; fluorescence imaging; iron oxide; magnetic resonance imaging
3
1. Introduction In recent years, a large number of therapeutic drugs have been developed. However, the
process of developing drugs from preclinical animal experiments to therapeutic applications
takes many years. Furthermore, the development time averages 6.5 years (ranging from
1.6-19.4 years) from when the Food and Drug Administration (FDA) authorizes initial testing in humans to market approval and clinical trials 1. To reduce the time between preclinical animal
experiments and to shorten the overall drug development phase, a drug delivery system with
imaging capabilities was proposed. This system can be used to track in vivo metabolic
pathways and can be combined with one or more traditional drug delivery methods to provide
controlled release capabilities.
Magnetic resonance imaging (MRI) is a powerful non-invasive imaging method offering high spatial resolution. MRI can be significantly improved with contrast agents 2. MRI signal
intensity is correlated with the relaxation times (T1, spin–lattice relaxation; and T2, spin–spin relaxation) of in vivo water protons 3. T2 agents, such as iron oxide nanoparticles, provide
negative signals and have been used to visualize organs, which have high signal intensity
(e.g., kidneys or lymphoid tissues). Moreover, iron oxide nanoparticles have been
functionalized with bioactive materials and used for targeted imaging via site-specific accumulation 4, 5. For example, superparamagnetic iron oxide nanoparticles co-coated with
polyethylene glycol (PEG) and polyetherimide (PEI) polymers were fabricated to improve drug
4
delivery
and
MRI
in
cancer
6
therapy
.
Similarly,
chondroitin-4-sulfate-capped
super-paramagnetic iron oxide nanoparticles have been used for doxorubicin hydrochloride delivery in cancer targeting 7.
Fluorescent gold nanoclusters (GNCs) have been considered as contrast agents for
diagnosis strategies. Due to the high biocompatibility and non-toxic property of gold, studies
have aimed at reducing the size and increasing the surface area of GNCs. Furthermore,
because of the strong quantum confinement effect of free electrons in GNC particles, these
particles are characterized by discrete energy levels. These properties result in
size-dependent fluorescence and other attractive molecule-like properties, such as a facile surface tailoring ability and color tenability 8. When combined with near-infrared fluorescent imaging, these GNCs can provide information for effective therapies 9. GNCs have generally
been used as fluorescent probes or as pro-drugs in tumor diagnosis and therapy. Fluorescent,
GNC-based nanoprobes with low cytotoxicity can specifically target carcinomas in vitro and in vivo 10.
In this study, we proposed a multi-functional drug delivery system for therapy with dual
magnetic resonance and fluorescence imaging capabilities. A liposome-based system was
selected as the delivery system because liposomes have hydrophilic-lipophilic characteristics
and have shown demonstrable efficacy in drug delivery
5
11
. The dual imaging functions were
prepared by combining superparamagnetic iron oxide nanoparticles and gold nanoclusters as MRI imaging contrast agents and fluorescent probes 12, respectively.
To evaluate the in vitro affinity of this system and its ability to trace metabolic pathways,
an in vivo assessment was performed using fluorescent imaging in Sprague–Dawley (SD) rats.
Each synthesis step in the creation of the multifunctional liposomal drug delivery system was
systematically identified, and the physicochemical properties, such as average size
distribution,
morphology,
chemical
functional
groups,
crystal
structures,
saturation
magnetization, and cell toxicity, were characterized. This multifunctional, liposomal drug
delivery system could be the foundation for dual magnetic resonance and fluorescence
imaging probes. Additionally, this system could aid in developing promising liposomal
drug delivery systems for cancer therapy by furthering the understanding of various types of
targeting moieties and biomarkers.
6
2. Materials and methods 2.1 Materials (±)-α-Lipoic acid (C8H13O2S2, ≥99%, LA), cholesterol (C27H46O, Chol), ethanol (CH3CH2OH, ≥99.8%, EtOH), 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (C40H80NO8P, DPPC), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (C8H17N3, EDC), iron (ш) chloride hexahydrate (FeCl3·6H2O, ≥98%), and Trypan blue solution (0.4%) were purchased from Sigma-Aldrich Co. (U.S.A.); 3-Aminopropyltriethoxysilane (C9H23NO3Si, ≥98%, APTES), citric acid (C6H8O7, ≥99%, CA), ferrous chloride tetrahydrate (FeCl2·4H2O, ≥99%), methanol
(CH3OH, MeOH), and trichloromethane (CHCl3, 99.8%) were purchased from Merck Co.
(Germany). Cell Titer 96® Aqueous One Solution Cell Proliferation Assay was purchased from Promega Co. (U.S.A.). Dulbecco’s Modified Eagle’s Medium (DMEM, low glucose), and
Trypsin-EDTA were purchased from GIBCO. Fetal bovine serum (FBS) was purchased from Biological Industries. Gold (ш) chloride trihydrate (HAuCl4·3H2O, 99%) and sodium
borohydride (NaBH4, 98%) were purchased from Alfa Aesar Co. (Germany). Phosphate buffer
solution (PBS, without calcium and magnesium) was purchased from MP Biomedical Co.
(U.S.A.). Methoxy-poly (ethylene glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N
(DSPE-mPEG2000) was purchased from Laysan Bio, Inc. Sodium hydroxide (NaOH, 98.9%)
was purchased from J. T. Baker Co. (U.S.A.).
2.2 Synthesis of dual probes nanocomposites (Fe3O4-GNCs)-loaded liposomes 7
In this study, a multifunctional liposomal drug delivery system with dual imaging
nanocomposite (Fe3O4-GNC) probes was fabricated. A schematic synthesis of the Fe3O4-GNC
nanocomposite-loaded liposomes is shown in Fig. 1. First, superparamagnetic Fe3O4
nanoparticles were provided as MRI contrast media. Second, GNCs with near IR fluorescence
capabilities at excitation wavelengths of 700–800 nm were provided to prevent in vivo
background autofluorescence. Third, the dual Fe3O4-GNC nanocomposite probes were
prepared by combining the Fe3O4 nanoparticles and GNCs via an amidation reaction. Finally,
the Fe3O4-GNC nanocomposites were loaded into the inner phase of the liposomes
(liposome@Fe3O4-GNCs), which consisted of 1,2-dipalmitoyl-rac-glycero-3-phosphocholine
(DPPC),
cholesterol
and
methoxy-poly(ethylene
glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N (DSPE-PEG2000). The following
sub-items describe the process method and related analysis method for each step.
2.2.1 Preparation of Fe3O4 nanoparticles and Fe3O4-APTES
The superparamagnetic Fe3O4 nanoparticles were fabricated by chemical co-precipitation 13,14
. The typical synthesis method is shown below, wherein 1.6218 g (6 millimole) iron (ш)
chloride hexahydrate (FeCl3·6H2O) and 0.5964 g (3 millimole) ferrous chloride tetrahydrate
(FeCl2·4H2O) were dissolved in 100 ml de-ionized (DI water) at a mole ratio of 2:1. The mixing
solution was put in a flask equipped with a mechanical stirrer at 85°C and purged with
nitrogen. Sodium hydroxide (NaOH) was used as a co-precipitation agent. Then, 10 ml 8
aqueous 25% NaOH solution was added to the flask and stirred for 60 minutes. Finally, the
resulting black Fe3O4 nanoparticles precipitates were collected and washed with DI water by
magnet until pH 7.
While the Fe3O4 nanoparticle cores served as MRI contrast agents, silica coatings, such
as 3-aminopropyltriethoxysilane (APTES), on the Fe3O4 NPs provided modification sites
15
.
APTES molecules were covalently bound to or adsorbed onto the MNPs (MNPs-APTES). The
active amino groups in the APTES molecules were able to combine with GNCs. Thus,
APTES-coated MNPs were prepared following a method similar to that described by Yamaura et al. 16. Briefly, APTES was hydrolyzed by dissolving 5 ml APTES in 2 ml HCl solution (pH=4),
stirring for 6 hours and adding 20 ml DI water. Fe3O4 nanoparticles were soaked in 100 ml
ethanol solution (50%) and well dispersed by ultrasonic mixing for 30 minutes. The APTES
hydrolysis solution was added to the Fe3O4 solution at 40°C overnight to modify the surfaces of
the Fe3O4 nanoparticles. The resulting functionalized MNPs-APTES with amino groups were
collected by magnet and dispersed in ethanol by ultrasonic mixing for 30 minutes three times
and washed with DI water.
2.2.2 Preparation of gold nanoclusters The preparation of gold nanoclusters (GNCs) was modified from a previous study 17. In this
process, 0.012 g lipoic acid was dissolved in a 2-ml NaOH aqueous solution (25%) in a ring
opening reaction. Then, 2 ml HAuCl4 solution (0.01 M) and 2 ml NaBH4 solution (0.02 M) were
9
then sequentially added and stirred in an ice bath for 30 minutes. The resultant GNCs were
isolated by methanol-induced agglomeration and re-dissolved in toluene. The solution was
vacuum dried, 6 ml NaOH aqueous solution (pH = 8) was added, and the dispersion was well
mixed. Finally, the GNCs were collected by centrifugal filter (10 kDa) at 7500 rpm for 15
minutes at 25°C and were maintained in NaOH solution (pH = 8).
2.2.3 Preparation of Fe3O4-GNC nanocomposites
Fe3O4-GNCs were prepared by combining the Fe3O4 magnetic nanoparticles and the
fluorescent GNCs via amidation reaction
18
. As described earlier, Fe3O4 magnetic
nanoparticles (MNPs) were modified with 3-aminopropyltriethoxysilane (APTES) to
functionalize the MNP surfaces with amino groups (-NH2). To prepare the corresponding
carboxyl groups (-COOH) on the fluorescent GNCs, 20 mg EDC were added to 10 ml GNC
solution. To these activated GNCs, 0.01 g Fe3O4-APTES was added for one day at room
temperature. Conjugated Fe3O4-GNCs were collected by magnet and washed with DI water
five times to remove unreacted GNCs and EDC.
2.2.4 Preparation of liposome@Fe3O4-GNCs
The multifunctional liposome@Fe3O4-GNC liposomal drug delivery system was prepared
according to a modified method described in a previous study
19
. Reverse-phase evaporation
was used to construct the functional liposome (DPPC:Chol:DSPE-PEG2000 = 80:20:5 molar
ratio) to enclose the Fe3O4-GNC nanocomposites. Unloaded Fe3O4-GNC nanocomposites
10
were collected by magnet. Supernatant liquid was centrifugally purified at 4000 rpm for 15
minutes at 25°C to obtain the liposome@Fe3O4-GNC drug nanocarriers, which have dual
probes for magnetic resonance and fluorescence imaging.
2.2.5 Characterization of as-synthesis materials
The chemical structures and functional groups of the prepared samples were analyzed
using a Fourier transform infrared spectrometer (System 2000 FT-IR, Perkin Elmer) with a
germanium crystal and a Harrick KBr prism. The spectra were obtained by averaging 64 scans at a resolution of 2 cm−1 over a range of 400–4000 cm−1. Peak intensity variations of amine
groups (-NH) and Si-O-Si bonds were examined to confirm chemical modification by APTES.
Intensity variations in Fe-O bonds were used to confirm the Fe3O4.
X-ray diffraction (XRD, D/MAX, Rigaku, Japan) was used to identify the crystal structure
and to analyze the phase orientation of Fe3O4. The scanning mode was set at continuous and the scanning rate was 2 °/min. A CuKα radiation source (λ = 1.5405981 Ǻ) was used and
operated at 40 kV and 30 mA. The incident beam angle on the surface was 1 °.
The particle size and polydispersity index (PDI) of the nanoparticle samples were
measured using a transmission electron microscope (TEM; HT7700, Hitachi) and a
nanoparticle analyzer (SZ-100, Horiba). The TEM samples prepared as follows: a drop of
nanoparticle suspension was placed on a carbon-coated copper grid, and the grid was
vacuum dried at room temperature for 24 hours. The concentrations of Fe and Au were 11
determined by inductively coupled plasma mass spectrometry (XSERIES II ICP-MS, Thermo
Scientific).
A superconducting quantum interference device (SQUID, MPMS XL-7, Quantum Design)
was used to measure the saturation magnetization of the samples. The applied field
magnitude ranged from -15,000 to +15,000 Oe. Residual magnetization was measured to
confirm super-paramagnetism. The precise effect of transverse relaxation was represented by
transverse relativity and samples were measured using a 7.0 T Fast field-cycling nuclear
magnetic resonance spectrometer (NMR, Stelar). The samples were diluted to six
concentrations, i.e., 1.7, 3.5, 7.0, 13.9, 27.8, and 55.7 μM Fe, placed in microtubes, and tested
at repetition times (TR) and multiple echo times (TE) of 7 s and 10-150 ms, respectively.
2.3 Cell viability assay of liposome@Fe3O4-GNCs
Liposome@Fe3O4-GNCs colloid nanocomposites were injected into the tail vein of rats.
The types of cells that may be encountered include blood cells, epithelial cells, bone marrow
stem cells (BMSCs) and other targeting cells. Therefore, bone marrow mesenchymal stem
cells are one of the options to evaluate liposome@Fe3O4-GNCs colloid nanocomposites cell
toxicity. Changes in the viability of D1 cells (BALB/c mouse bone marrow mesenchymal stem
cells, BMSCs) were quantitatively assessed using a tetrazolium compound (MTS; Sigma, USA)
for
one
24-h
culture
period.
This
compound,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium 12
inner salt (MTS) produces a water-soluble formazan product that has an absorbance
maximum at 490 nm in phosphate-buffered saline in the presence of phenazine methosulfate
(PMS). The amount of colored product formed is proportional to the number of cells and their
time of incubation with MTS/PMS. To evaluate cell viability while in direct contact with the cells, a concentration of 2 x 104 cells per well was seeded on a 96-well plate and cultured at 37°C for
1 day in a humidified incubator containing 5% CO2. Liposome@Fe3O4-GNCs with different Fe concentrations of 3.5, 7.0, 13.9, 27.8, 55.7 μM, or different Au concentrations of 13, 26, 52,
105, 211 mM, were prepared by adding culture medium previously incubated at 37°C
overnight and sterilized by UV light. Culture plates without any samples were used as normal
controls. After 24 h of cultivation, MTS solution (5 mg/ml in PBS) was added to each well, and
the plates were incubated for 2 h (37°C). The MTS solution and medium were mixed well at a
1:9 ratio. The optical density of the water-soluble formazan produced in the solution was
measured using an ELISA reader (SLT, Crailheim, Germany). Data were collected and
averaged from three different wells per condition.
2.4 Fluorescence intensity measurement of liposome@Fe3O4-GNCs in vivo
In Vivo Imaging Spectrum (IVIS, IVIS® Spectrum, Perkin Elmer) was used to measure the
fluorescent intensity of the liposome@Fe3O4-GNCs. The samples were prepared at five
concentrations, i.e., 13, 26, 52, 105, and 211 mM GNCs, and placed in a 96-well plate. The
13
excitation wavelength was 640 nm, and the emission wavelength was 740 nm. IVIS images
were captured, and fluorescence intensities were calculated using ImageJ software.
Liposome@Fe3O4-GNCs were evaluated in vivo using Sprague Dawley (SD) rats.
Experimental protocols followed the national guidelines for the care and use of laboratory
animals and the study was approved by the Animal Experimental Ethics Committee of KMU.
Three 6-month-old female SD rats per group were used. In each rat, 0.7 ml of the
liposome@Fe3O4-GNCs solution comprising an Au concentration of 211 mM (or Fe concentration of 55.7 μM) were injected into the tail vein each time. This is the safest dose for
the rats. However, the risk of death was higher when each injection was more than 1 ml
volume for the rat. After the injection, all animals were provided free access to food and water.
After 1 and 6 hours, the animals were sacrificed, and the major organs (i.e., heart, liver, spleen,
lung, and kidney) were collected. The fluorescence signals (640–740 nm) in these organs from
three rats in each group were detected using the IVIS imaging system, and all the organs of
the three rats in each group were quantified using IVIS 200 software. Mean ± SD values were
used to express the data, unless otherwise noted. Statistical analyses of the data were performed using Student’s t test. Differences characterized by p < 0.05 were considered to be
statistically significant.
14
3 Results and Discussion 3.1 Characterization of the liposome@Fe3O4-GNCs
Fig. 2(a) shows the FT-IR spectra of the Fe3O4 and Fe3O4-APTES nanoparticles from 400 – 4000 cm-1. Characteristic peaks of the Fe-O metal-oxygen bond were observed at 587 cm-1. This corresponded with stretching vibrations of the metal at tetrahedral sites
20
. APTES
modification of the Fe3O4 nanoparticle surfaces was confirmed by the presence of peaks indicative of Si-O-Si stretching and N-H bending at 1110 cm-1 and 1540 cm-1, respectively 21.
The phase structures of the Fe3O4 and Fe3O4-APTES nanoparticles were examined by
XRD. The diffraction peaks could be characteristic of the face-centered cubic structure of
magnetite according to ICDD card No. 19-0629. Fig. 2(b) shows the XRD patterns of Fe3O4
and Fe3O4-APTES. Crystalline diffraction peaks at 30.1°, 35.6°, 43.1°, 53.4°, 57.1°, and 62.7° 2θ are correlated with Miller Indices of (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) typical
of cubic spinel Fe3O4, respectively. No significant changes were observed in the XRD pattern
of the Fe3O4 core upon APTES functionalization. The obtained Fe3O4-APTES particles
presented similar crystal structures to those of pure Fe3O4 nanoparticles
22
. The size
distributions of the resultant Fe3O4 and Fe3O4-APTES nanoparticles were analyzed by TEM,
and their images are shown in the Supporting Information. As indicated, the Fe3O4 and
Fe3O4-APTES nanoparticles exhibited average sizes of 10.351.6 nm and 12.210.9 nm,
respectively. 15
A fluorescent spectrometer was used to characterize gold nanoclusters (GNCs)
synthesized with (±)-α-diaphorase. GNC colloidal solutions were excited at 350 nm, and
emission signals between 730-740 nm were collected as shown in Fig. 3(A). This wave band
region not only reduced the background noise from the biomatrix but also actively enabled the
imaging and tracking of cell fluorescence, meeting the experimental needs of cellular
observation or image tracking in vivo. The average GNC diameter was approximately 3 nm, as
measured by the nanoparticle analyzer. Under UV light, the Fe3O4-GNC nanocomposites
emitted near red light at 740-760 nm, as shown in Fig. 3(B). Through a simple fluorescence
test, the Fe3O4-GNC nanocomposites were shown to be capable for fluorescent tracking.
The particle size and polydisperse index (PDI) values of liposomes, liposome@Fe3O4
and liposome@Fe3O4-GNCs were measured using a nanoparticle analyzer; the results are
presented in Table 1. The particle sizes of liposomes, liposome@ Fe3O4 and liposome@
Fe3O4-GNCs were 112.7, 195.6 and 186.0 nm, respectively. The size of these three particles
was all greater than 100 nm but less than 200 nm. However, this size range of nanoparticles
can be absorbed by cells and enter the circulatory system. Furthermore, these nanoparticles
can be absorbed by lymphatic tissue and intestinal cells
23
. The size of the liposome@
Fe3O4-GNCs (186.0 nm) would be large enough to prevent their rapid leakage into blood
capillaries (normal blood vessel pores are approximately 2–6 nm in size) and small enough to
prevent capture by fixed macrophages lodged in the reticuloendothelial system, i.e., the sizes 16
of sinusoids in the spleen and the fenestra of Kupffer cells in the liver varies from 150 to 200 nm 24. Thus, the liposome@ Fe3O4-GNCs could be used in biomedical applications.
The PDI values of liposomes, liposome@Fe3O4 and liposome@Fe3O4-GNCs were 0.155,
0.417, and 0.083, respectively. PDI values range from 0 (monodispersed) to 1 (very broad
distribution) and are calculated to determine the degree of dispersion of polymer micelles or
particles. The PDI values of the liposomes, liposome@Fe3O4 and liposome@Fe3O4-GNCs
were less than 0.5, which suggested that the particles had narrow size distributions and were largely homogenous 25.
The
morphologies
and
sizes
of
the
liposomes,
liposome@Fe3O4
and
liposome@Fe3O4-GNCs were observed using TEM, as shown in Fig. 4. The results from the
TEM analyses agreed with those from the nanoparticle analyzer. We also observed that all
nanoparticles
had
spherical
vesicle
structures.
The
liposome
nanoparticles
were
sphere-shaped vesicles consisting of one or more phospholipid bilayers and were smaller than
the liposome@Fe3O4 and liposome@Fe3O4-GNC nanoparticles. The liposome@Fe3O4 and
liposome@Fe3O4-GNC nanoparticles were nanosized and enclosed Fe3O4 nanoparticles and
Fe3O4-GNC nanocomposites in their respective inner phases.
The hysteresis loops of the Fe3O4 nanoparticles and Fe3O4-APTES particles (a) and
liposome@Fe3O4-GNCs (b) are presented in Fig. 5. Magnetizations reached saturation after
an applied magnetic field of 15,000 Oe. The saturation magnetization (Ms) values of Fe3O4, 17
Fe3O4-APTES, and liposome@Fe3O4-GNCs were 55.98, 48,72, and 0.65 emu/g, respectively.
The Ms of Fe3O4-APTES was slightly decreased due to the reduced magnetic moment of the
Fe3O4 particles after APTES modification. This effect was likely due to the lower Fe3O4 weight
fraction of the Fe3O4-APTES relative to that of the Fe3O4 nanoparticles only. Alternatively,
Fe3O4-APTES could have created a magnetically dead layer on the Fe3O4 surface and thus
reduced the surface moments in the Fe3O4 particles. Piñeiro et al.
26
reported that the
magnetic quality strongly depended on the degree of order of crystalline lattices, oxidation
states
of
iron
ions
and
lattice
distortions
at
NP
surfaces.
Additionally,
the
liposome@Fe3O4-GNCs had a magnetic content of 1.2 wt.% based on the amount of pure
Fe3O4 particles due to the additional weight of the liposomes and GNCs. Although the Ms of
the liposome@Fe3O4-GNCs was smaller than that of the pure Fe3O4 particles, the curves of
Fig. 5 show typical superparamagnetic characteristics. No coercivity (Hc) was observed, and
remnant magnetism was nearly zero. This finding indicated that the Fe3O4, Fe3O4-APTES, and
liposome@Fe3O4-GNCs particles were nearly superparamagnetic
27
. The superparamagnetic
behavior of the liposome@Fe3O4-GNCs was due to the nanosize of the Fe3O4 particles
(approximately 10.4 nm) inside the liposome vesicles.
To confirm the magnetic resonance imaging effects of liposome@Fe3O4-GNCs as
contrast agents, the transverse relaxation time T2 was measured using fast field-cycling
nuclear magnetic resonance (NMR, Stelar). Because superparamagnetic materials induce
18
decreases in the transverse relaxation time T2, this would lead to increased contrast in MRI
images. Fig. 6 shows the MR signal intensity for different Fe concentrations. As shown in Fig.
6(a), the increased Fe concentration resulted in a reduced transverse relaxation time T2,
leading to improved MR signals. As shown in Fig. 6(a), the MR signal intensity decreased with
increasing concentrations of Fe
28
. In Fig. 6(b), the transverse relaxation rate (1/T2) of the
liposome@Fe3O4-GNCs was a function of Fe concentration in DI water. The transverse
relaxivity (r2) of the liposome@Fe3O4-GNCs was determined from the slope of the linear
equation: 1/T2 = 1/T02 + r2 [Fe]
where
1/T2 is
the
observed
transverse
relaxation
rate
in
the
presence
of
liposome@Fe3O4-GNCs, 1/T02 is the transverse relaxation rate in the absence of
liposome@Fe3O4-GNCs, r2 is the transverse relaxivity, and [Fe] is the concentration of Fe ions. Based on the slope of the regression line (R2 = 0.9981), the transverse relaxivity of the liposome@Fe3O4-GNCs was 872.73 mM−1 s−1. According to a previous study, the relaxivity
rate, r2, of hybrid nanospheres (chitosan@superparamagnetic iron oxide nanoparticles) showed an approximately 8-fold increment and reached the maximum of 533 mM−1 s−1
29
.
Moreover, the common magnetic fields of a clinical MRI system were 1-3 T, but the higher magnetic fields could increase the relaxivity rate. For example, the value of r2 was 10.64 mM–1 s–1 at 212 μT and increased to 152.47 mM–1 s–1 at 14.1 T for the ultrasmall superparamagnetic
19
iron oxide nanoparticles
30
. In this study, the magnetic field was 7 T and displayed the higher
relaxivity rate than the typical system. Compared to free iron oxide nanoparticles, this relaxivity
of our proposed material indicated that liposome@Fe3O4-GNCs are a promising material for molecular imaging applications31.
3.2 Cell viability assay of liposome@Fe3O4-GNCs
The results of the MTS assay are presented as absorbance levels to reflect altered
cellular mitochondrial activity in response to different Fe concentrations from 3.5 to 55.7 μM, or
Au concentrations from 211 to 13 mM, within liposome@Fe3O4-GNCs. Therefore, we also
present a direct cytotoxicity test for liposome@Fe3O4-GNCs and after incubating for 24 hours, liposome@Fe3O4-GNCs with different Fe concentrations of 3.5, 7.0, 13.9, 27.8, and 55.7 μM
did not exhibit cytotoxicity
(Fig. 7). According to results of the MTS assay and the
absorbance of the water-soluble formazan product value, cell viability values were all above
98.72%. The results of the MTS assay demonstrated that the liposome@Fe3O4-GNCs could
be used for in vitro cell-material interactions as a multifunctional liposomal drug delivery
system.
3. 3 Fluorescence intensity measurement of liposome@Fe3O4-GNCs in vivo
Fig. 8 shows images of the liposomes, liposome@Fe3O4 and liposome@Fe3O4-GNC
particles in daylight and under UV illumination. The liposome solution is a white and turbid
aqueous solution under daylight. The liposome@Fe3O4 and liposome@Fe3O4-GNCs were 20
yellow and brown in color due to the Fe3O4 particles. The liposome and liposome@Fe3O4
particles had no fluorescent properties, while the liposome@Fe3O4-GNCs emitted red fluorescent light. The wavelength of the emitted light ranged from 700 – 760 nm. This range of wavelength minimizes in vivo background autofluorescence 32.
Fig. 9 shows the fluorescence intensities of liposome@Fe3O4-GNCs for different GNC
concentrations as measured by IVIS imaging. Based on the slope of the regression line (R2 = 0.9996), the fluorescence intensity showed a highly linear relation to the GNC
concentration. This demonstrated that the liposome@Fe3O4-GNCs could function as a
fluorescent probe.
An in vivo biodistribution assay in SD rats was performed to further examine the ability of
liposome@Fe3O4-GNCs as fluorescent imaging probes. Fig. 10 shows the photos and
fluorescent images taken using IVIS imaging at 0, 1, 3, and 6 hours after the
liposome@Fe3O4-GNC transport into the SD rats. Organs such as the heart, spleen, lungs,
kidneys,
and
liver
were
taken
to
observe
the
metabolic
pathway
of
the
liposome@Fe3O4-GNCs. The fluorescence signals were barely detectable in the hearts of the
rats and were marginally detectable in the liver, spleen, lungs, and especially detectable in the
kidneys. After the liposome@Fe3O4-GNCs had been introduced into the SD rats for 6 hours,
fluorescence signals were primarily observed in the kidneys. This finding showed that the
liposome@Fe3O4-GNCs were hydrophilic carriers and primarily metabolized in the kidneys.
21
The fluorescence intensities of the liposome@Fe3O4-GNCs that reached the organs (i.e., heart, liver, spleen, lungs, and kidneys) were evaluated after vein administration via the rat’s
tails
for
1
and
6
hours.
Fig.
11
shows
the
fluorescence
intensities
of
the
liposome@Fe3O4-GNCs in the heart, liver, spleen, lungs, and kidneys after 1 and 6 hours. The
fluorescence intensities of the liposome@Fe3O4-GNCs in the heart and kidneys were the
lowest and highest, respectively. Moreover, the fluorescence intensity increased with time.
The fluorescence intensity of the liposome@Fe3O4-GNCs remained high and showed a
primary metabolic pathway through the kidneys.
4 Conclusion We successfully synthesized a multifunctional drug delivery system comprising of the
liposome@Fe3O4-GNCs. This dual MRI and near IR fluorescent imaging capability of the
Fe3O4-GNC nanocomposite provided a tracking tool for preclinical animal and human clinical
trials. The size of the liposome@Fe3O4-GNCs was approximately 186.0 nm, which was large
enough to prevent rapid leakage into the blood capillaries but small enough to escape capture
by fixed macrophages lodged in the reticuloendothelial system. The saturation magnetization
of the liposome@Fe3O4-GNCs was 0.65 emu/g; however, the nanoparticles maintained their
superparamagnetic property as an MRI contrast agent. This multi-functional drug delivery
system composited of nanosized gold clusters (GNCs) also had fluorescent properties. The
22
GNCs had an emission wavelength range of 700 – 760 nm, which minimized in vivo
background
autofluorescence.
The
in
vitro
affinity
tests
in
SD
rats
on
the
liposome@Fe3O4-GNC system demonstrated the non-toxic and in vivo use of these
nanoclusters in tracking their metabolic pathways. This promising liposome@Fe3O4-GNCs
with dual probes of magnetic resonance and fluorescence imaging show its potential.
However, there is still a need to verify the accuracy of target molecules conjugated with
liposome@Fe3O4-GNCs in animal clinical study stages in the future.
5 Acknowledgments The authors gratefully acknowledge the financial support provided to this study by the Ministry of Science and Technology (MOST) in Taiwan under Grant No. MOST- 105-2218-E-037-002. This study is also supported partially by Aim for the Top Universities Grant of Kaohsiung Medical University (KMU-TP105B03) and Kaohsiung Medical University Research Foundation (KMU-Q106007).
6 References 1.
Moore TJ, Furberg CD. Development times, clinical testing, postmarket follow-up, and
safety risks for the new drugs approved by the us food and drug administration: The
class of 2008. JAMA Internal Medicine 2014;174:90-95.
23
2.
Liu Q, Song L, Chen S, Gao J, Zhao P, Du J. A superparamagnetic polymersome with
extremely high T2 relaxivity for MRI and cancer-targeted drug delivery. Biomaterials
2017;114:23-33.
3.
Estelrich J, Sánchez-Martín MJ, Busquets MA. Nanoparticles in magnetic resonance
imaging: from simple to dual contrast agents. International Journal of Nanomedicine
2015;10:1727-1741.
4.
Xu P, Shen Z, Zhang B, Wang J, Wu R. Synthesis and characterization of
superparamagnetic iron oxide nanoparticles as calcium-responsive MRI contrast
agents. Applied Surface Science 2016;389:560-566.
5.
Weinstein JS, Varallyay CG, Dosa E, Gahramanov S, Hamilton B, Rooney WD,
Muldoon LL, Neuwelt EA. Superparamagnetic iron oxide nanoparticles: diagnostic
magnetic resonance imaging and potential therapeutic applications in neurooncology
and central nervous system inflammatory pathologies, a review. Journal of Cerebral
Blood Flow and Metabolism: Official Journal of the International Society of Cerebral
Blood Flow and Metabolism 2010;30:15-35.
6.
Huang Y, Mao K, Zhang B, Zhao Y. Superparamagnetic iron oxide nanoparticles
conjugated with folic acid for dual target-specific drug delivery and MRI in cancer
theranostics. Materials Science and Engineering: C 2017;70, Part 1:763-771.
24
7.
Mallick N, Anwar M, Asfer M, Mehdi SH, Rizvi MMA, Panda AK, Talegaonkar S,
Ahmad FJ. Chondroitin sulfate-capped super-paramagnetic iron oxide nanoparticles
as
potential
carriers
of
doxorubicin
hydrochloride.
Carbohydrate
Polymers
2016;151:546-556.
8.
Qu X, Li Y, Li L, Wang Y, Liang J, Liang J. Fluorescent Gold Nanoclusters: Synthesis
and Recent Biological Application. Journal of Nanomaterials 2015;2015:1-23.
9.
Zhang X, Zhao N, Wang B, Tian Z, Dai Y, Ning P, Chen D. Structure-inherent
near-infrared fluorescent probe mediates apoptosis imaging and targeted drug
delivery in vivo. Dyes and Pigments 2017;138:204-212.
10.
Chen H, Li B, Ren X, Li S, Ma Y, Cui S, Gu Y. Multifunctional near-infrared-emitting
nano-conjugates based on gold clusters for tumor imaging and therapy. Biomaterials
2012;33:8461-8476.
11.
Akhtar N, Khan RA. Liposomal systems as viable drug delivery technology for skin
cancer sites with an outlook on lipid-based delivery vehicles and diagnostic imaging
inputs for skin conditions'. Progress in Lipid Research 2016;64:192-230.
12.
Wang C, Yao Y, Song Q. Gold nanoclusters decorated with magnetic iron oxide
nanoparticles for potential multimodal optical/magnetic resonance imaging. Journal of
Materials Chemistry C 2015;3:5910-5917.
25
13.
Fauconnier N, Bée A, Roger J, Pons JN. Synthesis of aqueous magnetic liquids by
surface complexation of maghemite nanoparticles. Journal of Molecular Liquids
1999;83:233-242.
14.
Massart R. Preparation of aqueous magnetic liquids in alkaline and acidic media.
IEEE Transactions on Magnetics 1981;17:1247-1248.
15.
Asenath Smith E, Chen W. How To Prevent the Loss of Surface Functionality Derived
from Aminosilanes. Langmuir 2008;24:12405-12409.
16.
Yamaura M, Camilo RL, Sampaio LC, Macêdo MA, Nakamura M, Toma HE.
Preparation and characterization of (3-aminopropyl)triethoxysilane-coated magnetite
nanoparticles. Journal of Magnetism and Magnetic Materials 2004;279:210-217.
17.
Lin C-AJ, Yang T-Y, Lee C-H, Huang SH, Sperling RA, Zanella M, Li JK, Shen J-L,
Wang H-H, Yeh H-I and others. Synthesis, Characterization, and Bioconjugation of
Fluorescent Gold Nanoclusters toward Biological Labeling Applications. ACS Nano
2009;3:395-401.
18.
Feng B, Hong RY, Wang LS, Guo L, Li HZ, Ding J, Zheng Y, Wei DG. Synthesis of
Fe3O4/APTES/PEG diacid functionalized magnetic nanoparticles for MR imaging.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 2008;328:52-59.
26
19.
Wang L, Zhang P, Shi J, Hao Y, Meng D, Zhao Y, Yanyan Y, Li D, Chang J, Zhang Z.
Radiofrequency-Triggered Tumor-Targeting
Delivery System
for
Theranostics
Application. ACS Applied Materials & Interfaces 2015;7:5736-5747.
20.
Kurtan
U,
Baykal
A.
Fabrication
and
characterization
of
Fe3O4@APTES@PAMAM-Ag highly active and recyclable magnetic nanocatalyst:
Catalytic reduction of 4-nitrophenol. Materials Research Bulletin 2014;60:79-87.
21.
Mirzabe GH, Keshtkar AR. Application of response surface methodology for thorium
adsorption on PVA/Fe3O4/SiO2/APTES nanohybrid adsorbent. Journal of Industrial
and Engineering Chemistry 2015;26:277-285.
22.
Jafarzadeh M, Soleimani E, Norouzi P, Adnan R, Sepahvand H. Preparation of
trifluoroacetic acid-immobilized Fe3O4@SiO2–APTES nanocatalyst for synthesis of
quinolines. Journal of Fluorine Chemistry 2015;178:219-224.
23.
Ai J, Biazar E, Jafarpour M, Montazeri M, Majdi A, Aminifard S, Zafari M, Akbari HR,
Rad HG. Nanotoxicology and nanoparticle safety in biomedical designs. International
Journal of Nanomedicine 2011;6:1117-1127.
24.
Wisse E, Braet F, Dianzhong L, De Zanger R, Jans D, Crabbe E, Vermoesen AN.
Structure and Function of Sinusoidal Lining Cells in the Liver. Toxicologic Pathology
1996;24:100-111.
27
25.
Long J, Xu E, Li X, Wu Z, Wang F, Xu X, Jin Z, Jiao A, Zhan X. Effect of chitosan
molecular weight on the formation of chitosan–pullulanase soluble complexes and
their application in the immobilization of pullulanase onto Fe3O4–κ-carrageenan
nanoparticles. Food Chemistry 2016;202:49-58.
26.
Piñeiro Y, Vargas Z, Rivas J, López-Quintela MA. Iron Oxide Based Nanoparticles for
Magnetic Hyperthermia Strategies in Biological Applications. European Journal of
Inorganic Chemistry 2015;2015:4495-4509.
27.
Luo Y, Zhou Z, Yue T. Synthesis and characterization of nontoxic chitosan-coated
Fe3O4 particles for patulin adsorption in a juice-pH simulation aqueous. Food
Chemistry 2017;221:317-323.
28.
Rezayan AH, Mousavi M, Kheirjou S, Amoabediny G, Ardestani MS, Mohammadnejad
J. Monodisperse magnetite (Fe3O4) nanoparticles modified with water soluble
polymers for the diagnosis of breast cancer by MRI method. Journal of Magnetism and
Magnetic Materials 2016;420:210-217.
29.
Lin Y, Wang S, Zhang Y, Gao J, Hong L, Wang X, Wu W, Jiang X. Ultra-high relaxivity
iron oxide nanoparticles confined in polymer nanospheres for tumor MR imaging.
Journal of Materials Chemistry B 2015;3:5702-5710.
30.
Wang W, Dong H, Pacheco V, Willbold D, Zhang Y, Offenhaeusser A, Hartmann R,
Weirich TE, Ma P, Krause H-J and others. Relaxation Behavior Study of Ultrasmall
28
Superparamagnetic Iron Oxide Nanoparticles at Ultralow and Ultrahigh Magnetic
Fields. The Journal of Physical Chemistry B 2011;115:14789-14793.
31.
Sitthichai S, Pilapong C, Thongtem T, Thongtem S. CMC-coated Fe3O4 nanoparticles
as new MRI probes for hepatocellular carcinoma. Applied Surface Science
2015;356:972-977.
32.
Lin MZ, McKeown MR, Ng H-L, Aguilera TA, Shaner NC, Campbell RE, Adams SR,
Gross LA, Ma W, Alber T and others. Autofluorescent Proteins with Excitation in the
Optical Window for Intravital Imaging in Mammals. Chemistry & Biology
2009;16:1169-1179.
29
Table 1 Average sizes and polydisepersity index (PDI) of liposome, liposome@Fe3O4 and
liposome@Fe3O4-GNCs. Material
Average size(nm)
PDI
liposome
112.7
0.155
liposome@ Fe3O4
195.9
0.417
liposome@ Fe3O4-GNCs
186.0
0.083
Fig. 1 Schematic diagram of synthesis Fe3O4-GNC nanocomposite-loaded liposomes.
30
Fig. 2 (a) FT-IR spectra and (b) XRD patterns of Fe3O4 and Fe3O4-APTES.
31
Fig. 3 (a) GNCs of (i) UV-vis absorption spectrum, (ii) fluorescence emission (excitation at 350
nm) spectra. (b) Fluorescence emission (excitation at 350 nm) spectra of Fe3O4-GNC
nanocomposites.
32
Fig. 4 TEM images and the sketch of nanoparticles from (a) liposomes, (b) liposome@Fe3O4
and (c) liposome@Fe3O4-GNCs.
33
Fig.
5
Hysteresis
loops
of
(a)
Fe3O4
liposome@Fe3O4-GNCs.
34
and
Fe3O4-APTES
particles
and
(b)
Fig. 6 (a) T2-weighted MRI of liposome@Fe3O4-GNCs at various Fe concentrations: 0, 1.8, 3.5,
7.0,
13.9,
27.8,
55.7
μM
(b)
The
transverse
(1/T2)
liposome@Fe3O4-GNCs as a function of Fe concentrations in DI water.
35
relaxation
rates
of
Fig. 7 The cell viability of liposome@Fe3O4-GNCs with different Fe/Au concentrations (μM/mM) were analyzed by the MTS assay after incubating for 24 hours. These values are
shown as the mean ± standard error of the mean, n=3.
36
Fig. 8 Photos of nanoparticles from liposomes (a; d), liposome@Fe3O4 (b; e) and
liposome@Fe3O4-GNCs (c; f) in daylight and under UV illumination.
37
Fig. 9 Fluorescence intensity of liposome@Fe3O4-GNCs with different GNC concentrations for
IVIS imaging.
38
Fig. 10 Photos and fluorescence images taken using IVIS imaging at (a) 0, (b) 1, (c) 3, and (d)
6 hours after liposome@Fe3O4-GNCs transport into SD rats.
39
Fig. 11 The fluorescence intensity after liposome@Fe3O4-GNCs reached the heart, liver, spleen, lung, and kidney transport into SD rats for 1 and 6 hours (n =3). ** denote p < 0.05 were statistically significant.
40
PII: DOI: Reference:
S0272-8842(18)30889-7 https://doi.org/10.1016/j.ceramint.2018.04.034 CERI17951
To appear in: Ceramics International Received date: 14 January 2018 Revised date: 29 March 2018 Accepted date: 5 April 2018 Cite this article as: Chih-Ling Huang, Wan-Ru Hsieh, Che-Wei Lin, Hung-Wei Yang and Chih-Kuang Wang, Multifunctional liposomal drug delivery with dual probes of magnetic resonance and fluorescence imaging, Ceramics International, https://doi.org/10.1016/j.ceramint.2018.04.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Multifunctional liposomal drug delivery with dual probes of magnetic resonance and fluorescence imaging Chih-Ling Huang 1,4, +, Wan-Ru Hsieh 2, +, Che-Wei Lin 2, Hung-Wei Yang 3, and Chih-Kuang Wang 2,4 *
1
Center for Fundamental Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan
2
Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical
University, Kaohsiung 807, Taiwan 3
Institute of Medical Science and Technology, National Sun Yat-sen University 804, Taiwan
4
Orthopaedic Research Center, College of Medicine, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
Correspondence: [Chih-Kuang Wang]
100, Shih-Chuan 1st Road,Kaohsiung,80708,Taiwan
Tel [+886- 7-3121101 #2677]
Fax [+886-7-3125339]
Email: [email protected]
1
Abstract: Many liposomal drug carriers have shown great promise in the clinic. To ensure the efficient
preclinical development of drug-loaded liposomes, the drug retention and circulation properties
of these systems should be characterized. Iron oxide (Fe3O4) magnetic nanoparticles (MNPs)
are used as T2 contrast agents in magnetic resonance imaging (MRI). Gold nanoclusters
(GNCs) contain tens of atoms with subnanometer dimensions; they have very low cytotoxicity
and possess superb red emitting fluorescent properties, which prevents in vivo background
autofluorescence. The aim of this study was to develop dual imaging, nanocomposite,
multifunctional liposome drug carriers (Fe3O4-GNCs) comprising MNPs of iron oxide and
GNCs. First, MNPs of iron oxide were synthesized by co-precipitation. The MNP surfaces
were modified with amine groups using 3-aminopropyltriethoxysilane (APTES). Second, GNCs
were synthesized by reducing HAuCl4·3H2O with NaBH4 in the presence of lipoic acid (as a
stabilizer and nanosynthetic template). The GNCs were grown by adsorption onto particles to
control the size and stability of the resultant colloids. Subsequently, dual Fe3O4-GNCs imaging
probes were fabricated by conjugating the iron oxide MNPs with the GNCs via amide bonds.
Finally, liposome nanocarriers were used to enclose the Fe3O4-GNCs in an inner phase
(liposome@Fe3O4-GNCs) by reverse phase evaporation. These nanocarriers were
characterized by dynamic light scattering (DLS), fluorescence spectroscopy, X-ray diffraction
(XRD), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR)
2
spectrophotometry, superconducting quantum interference device (SQUID), nuclear magnetic
resonance (NMR) imaging and in vivo imaging systems (IVIS). These multifunctional liposomal
drug delivery systems with dual probes are expected to prove useful in preclinical trials for
cancer diagnosis and therapy.
Keywords: dual probes; fluorescence imaging; iron oxide; magnetic resonance imaging
3
1. Introduction In recent years, a large number of therapeutic drugs have been developed. However, the
process of developing drugs from preclinical animal experiments to therapeutic applications
takes many years. Furthermore, the development time averages 6.5 years (ranging from
1.6-19.4 years) from when the Food and Drug Administration (FDA) authorizes initial testing in humans to market approval and clinical trials 1. To reduce the time between preclinical animal
experiments and to shorten the overall drug development phase, a drug delivery system with
imaging capabilities was proposed. This system can be used to track in vivo metabolic
pathways and can be combined with one or more traditional drug delivery methods to provide
controlled release capabilities.
Magnetic resonance imaging (MRI) is a powerful non-invasive imaging method offering high spatial resolution. MRI can be significantly improved with contrast agents 2. MRI signal
intensity is correlated with the relaxation times (T1, spin–lattice relaxation; and T2, spin–spin relaxation) of in vivo water protons 3. T2 agents, such as iron oxide nanoparticles, provide
negative signals and have been used to visualize organs, which have high signal intensity
(e.g., kidneys or lymphoid tissues). Moreover, iron oxide nanoparticles have been
functionalized with bioactive materials and used for targeted imaging via site-specific accumulation 4, 5. For example, superparamagnetic iron oxide nanoparticles co-coated with
polyethylene glycol (PEG) and polyetherimide (PEI) polymers were fabricated to improve drug
4
delivery
and
MRI
in
cancer
6
therapy
.
Similarly,
chondroitin-4-sulfate-capped
super-paramagnetic iron oxide nanoparticles have been used for doxorubicin hydrochloride delivery in cancer targeting 7.
Fluorescent gold nanoclusters (GNCs) have been considered as contrast agents for
diagnosis strategies. Due to the high biocompatibility and non-toxic property of gold, studies
have aimed at reducing the size and increasing the surface area of GNCs. Furthermore,
because of the strong quantum confinement effect of free electrons in GNC particles, these
particles are characterized by discrete energy levels. These properties result in
size-dependent fluorescence and other attractive molecule-like properties, such as a facile surface tailoring ability and color tenability 8. When combined with near-infrared fluorescent imaging, these GNCs can provide information for effective therapies 9. GNCs have generally
been used as fluorescent probes or as pro-drugs in tumor diagnosis and therapy. Fluorescent,
GNC-based nanoprobes with low cytotoxicity can specifically target carcinomas in vitro and in vivo 10.
In this study, we proposed a multi-functional drug delivery system for therapy with dual
magnetic resonance and fluorescence imaging capabilities. A liposome-based system was
selected as the delivery system because liposomes have hydrophilic-lipophilic characteristics
and have shown demonstrable efficacy in drug delivery
5
11
. The dual imaging functions were
prepared by combining superparamagnetic iron oxide nanoparticles and gold nanoclusters as MRI imaging contrast agents and fluorescent probes 12, respectively.
To evaluate the in vitro affinity of this system and its ability to trace metabolic pathways,
an in vivo assessment was performed using fluorescent imaging in Sprague–Dawley (SD) rats.
Each synthesis step in the creation of the multifunctional liposomal drug delivery system was
systematically identified, and the physicochemical properties, such as average size
distribution,
morphology,
chemical
functional
groups,
crystal
structures,
saturation
magnetization, and cell toxicity, were characterized. This multifunctional, liposomal drug
delivery system could be the foundation for dual magnetic resonance and fluorescence
imaging probes. Additionally, this system could aid in developing promising liposomal
drug delivery systems for cancer therapy by furthering the understanding of various types of
targeting moieties and biomarkers.
6
2. Materials and methods 2.1 Materials (±)-α-Lipoic acid (C8H13O2S2, ≥99%, LA), cholesterol (C27H46O, Chol), ethanol (CH3CH2OH, ≥99.8%, EtOH), 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (C40H80NO8P, DPPC), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (C8H17N3, EDC), iron (ш) chloride hexahydrate (FeCl3·6H2O, ≥98%), and Trypan blue solution (0.4%) were purchased from Sigma-Aldrich Co. (U.S.A.); 3-Aminopropyltriethoxysilane (C9H23NO3Si, ≥98%, APTES), citric acid (C6H8O7, ≥99%, CA), ferrous chloride tetrahydrate (FeCl2·4H2O, ≥99%), methanol
(CH3OH, MeOH), and trichloromethane (CHCl3, 99.8%) were purchased from Merck Co.
(Germany). Cell Titer 96® Aqueous One Solution Cell Proliferation Assay was purchased from Promega Co. (U.S.A.). Dulbecco’s Modified Eagle’s Medium (DMEM, low glucose), and
Trypsin-EDTA were purchased from GIBCO. Fetal bovine serum (FBS) was purchased from Biological Industries. Gold (ш) chloride trihydrate (HAuCl4·3H2O, 99%) and sodium
borohydride (NaBH4, 98%) were purchased from Alfa Aesar Co. (Germany). Phosphate buffer
solution (PBS, without calcium and magnesium) was purchased from MP Biomedical Co.
(U.S.A.). Methoxy-poly (ethylene glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N
(DSPE-mPEG2000) was purchased from Laysan Bio, Inc. Sodium hydroxide (NaOH, 98.9%)
was purchased from J. T. Baker Co. (U.S.A.).
2.2 Synthesis of dual probes nanocomposites (Fe3O4-GNCs)-loaded liposomes 7
In this study, a multifunctional liposomal drug delivery system with dual imaging
nanocomposite (Fe3O4-GNC) probes was fabricated. A schematic synthesis of the Fe3O4-GNC
nanocomposite-loaded liposomes is shown in Fig. 1. First, superparamagnetic Fe3O4
nanoparticles were provided as MRI contrast media. Second, GNCs with near IR fluorescence
capabilities at excitation wavelengths of 700–800 nm were provided to prevent in vivo
background autofluorescence. Third, the dual Fe3O4-GNC nanocomposite probes were
prepared by combining the Fe3O4 nanoparticles and GNCs via an amidation reaction. Finally,
the Fe3O4-GNC nanocomposites were loaded into the inner phase of the liposomes
(liposome@Fe3O4-GNCs), which consisted of 1,2-dipalmitoyl-rac-glycero-3-phosphocholine
(DPPC),
cholesterol
and
methoxy-poly(ethylene
glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N (DSPE-PEG2000). The following
sub-items describe the process method and related analysis method for each step.
2.2.1 Preparation of Fe3O4 nanoparticles and Fe3O4-APTES
The superparamagnetic Fe3O4 nanoparticles were fabricated by chemical co-precipitation 13,14
. The typical synthesis method is shown below, wherein 1.6218 g (6 millimole) iron (ш)
chloride hexahydrate (FeCl3·6H2O) and 0.5964 g (3 millimole) ferrous chloride tetrahydrate
(FeCl2·4H2O) were dissolved in 100 ml de-ionized (DI water) at a mole ratio of 2:1. The mixing
solution was put in a flask equipped with a mechanical stirrer at 85°C and purged with
nitrogen. Sodium hydroxide (NaOH) was used as a co-precipitation agent. Then, 10 ml 8
aqueous 25% NaOH solution was added to the flask and stirred for 60 minutes. Finally, the
resulting black Fe3O4 nanoparticles precipitates were collected and washed with DI water by
magnet until pH 7.
While the Fe3O4 nanoparticle cores served as MRI contrast agents, silica coatings, such
as 3-aminopropyltriethoxysilane (APTES), on the Fe3O4 NPs provided modification sites
15
.
APTES molecules were covalently bound to or adsorbed onto the MNPs (MNPs-APTES). The
active amino groups in the APTES molecules were able to combine with GNCs. Thus,
APTES-coated MNPs were prepared following a method similar to that described by Yamaura et al. 16. Briefly, APTES was hydrolyzed by dissolving 5 ml APTES in 2 ml HCl solution (pH=4),
stirring for 6 hours and adding 20 ml DI water. Fe3O4 nanoparticles were soaked in 100 ml
ethanol solution (50%) and well dispersed by ultrasonic mixing for 30 minutes. The APTES
hydrolysis solution was added to the Fe3O4 solution at 40°C overnight to modify the surfaces of
the Fe3O4 nanoparticles. The resulting functionalized MNPs-APTES with amino groups were
collected by magnet and dispersed in ethanol by ultrasonic mixing for 30 minutes three times
and washed with DI water.
2.2.2 Preparation of gold nanoclusters The preparation of gold nanoclusters (GNCs) was modified from a previous study 17. In this
process, 0.012 g lipoic acid was dissolved in a 2-ml NaOH aqueous solution (25%) in a ring
opening reaction. Then, 2 ml HAuCl4 solution (0.01 M) and 2 ml NaBH4 solution (0.02 M) were
9
then sequentially added and stirred in an ice bath for 30 minutes. The resultant GNCs were
isolated by methanol-induced agglomeration and re-dissolved in toluene. The solution was
vacuum dried, 6 ml NaOH aqueous solution (pH = 8) was added, and the dispersion was well
mixed. Finally, the GNCs were collected by centrifugal filter (10 kDa) at 7500 rpm for 15
minutes at 25°C and were maintained in NaOH solution (pH = 8).
2.2.3 Preparation of Fe3O4-GNC nanocomposites
Fe3O4-GNCs were prepared by combining the Fe3O4 magnetic nanoparticles and the
fluorescent GNCs via amidation reaction
18
. As described earlier, Fe3O4 magnetic
nanoparticles (MNPs) were modified with 3-aminopropyltriethoxysilane (APTES) to
functionalize the MNP surfaces with amino groups (-NH2). To prepare the corresponding
carboxyl groups (-COOH) on the fluorescent GNCs, 20 mg EDC were added to 10 ml GNC
solution. To these activated GNCs, 0.01 g Fe3O4-APTES was added for one day at room
temperature. Conjugated Fe3O4-GNCs were collected by magnet and washed with DI water
five times to remove unreacted GNCs and EDC.
2.2.4 Preparation of liposome@Fe3O4-GNCs
The multifunctional liposome@Fe3O4-GNC liposomal drug delivery system was prepared
according to a modified method described in a previous study
19
. Reverse-phase evaporation
was used to construct the functional liposome (DPPC:Chol:DSPE-PEG2000 = 80:20:5 molar
ratio) to enclose the Fe3O4-GNC nanocomposites. Unloaded Fe3O4-GNC nanocomposites
10
were collected by magnet. Supernatant liquid was centrifugally purified at 4000 rpm for 15
minutes at 25°C to obtain the liposome@Fe3O4-GNC drug nanocarriers, which have dual
probes for magnetic resonance and fluorescence imaging.
2.2.5 Characterization of as-synthesis materials
The chemical structures and functional groups of the prepared samples were analyzed
using a Fourier transform infrared spectrometer (System 2000 FT-IR, Perkin Elmer) with a
germanium crystal and a Harrick KBr prism. The spectra were obtained by averaging 64 scans at a resolution of 2 cm−1 over a range of 400–4000 cm−1. Peak intensity variations of amine
groups (-NH) and Si-O-Si bonds were examined to confirm chemical modification by APTES.
Intensity variations in Fe-O bonds were used to confirm the Fe3O4.
X-ray diffraction (XRD, D/MAX, Rigaku, Japan) was used to identify the crystal structure
and to analyze the phase orientation of Fe3O4. The scanning mode was set at continuous and the scanning rate was 2 °/min. A CuKα radiation source (λ = 1.5405981 Ǻ) was used and
operated at 40 kV and 30 mA. The incident beam angle on the surface was 1 °.
The particle size and polydispersity index (PDI) of the nanoparticle samples were
measured using a transmission electron microscope (TEM; HT7700, Hitachi) and a
nanoparticle analyzer (SZ-100, Horiba). The TEM samples prepared as follows: a drop of
nanoparticle suspension was placed on a carbon-coated copper grid, and the grid was
vacuum dried at room temperature for 24 hours. The concentrations of Fe and Au were 11
determined by inductively coupled plasma mass spectrometry (XSERIES II ICP-MS, Thermo
Scientific).
A superconducting quantum interference device (SQUID, MPMS XL-7, Quantum Design)
was used to measure the saturation magnetization of the samples. The applied field
magnitude ranged from -15,000 to +15,000 Oe. Residual magnetization was measured to
confirm super-paramagnetism. The precise effect of transverse relaxation was represented by
transverse relativity and samples were measured using a 7.0 T Fast field-cycling nuclear
magnetic resonance spectrometer (NMR, Stelar). The samples were diluted to six
concentrations, i.e., 1.7, 3.5, 7.0, 13.9, 27.8, and 55.7 μM Fe, placed in microtubes, and tested
at repetition times (TR) and multiple echo times (TE) of 7 s and 10-150 ms, respectively.
2.3 Cell viability assay of liposome@Fe3O4-GNCs
Liposome@Fe3O4-GNCs colloid nanocomposites were injected into the tail vein of rats.
The types of cells that may be encountered include blood cells, epithelial cells, bone marrow
stem cells (BMSCs) and other targeting cells. Therefore, bone marrow mesenchymal stem
cells are one of the options to evaluate liposome@Fe3O4-GNCs colloid nanocomposites cell
toxicity. Changes in the viability of D1 cells (BALB/c mouse bone marrow mesenchymal stem
cells, BMSCs) were quantitatively assessed using a tetrazolium compound (MTS; Sigma, USA)
for
one
24-h
culture
period.
This
compound,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium 12
inner salt (MTS) produces a water-soluble formazan product that has an absorbance
maximum at 490 nm in phosphate-buffered saline in the presence of phenazine methosulfate
(PMS). The amount of colored product formed is proportional to the number of cells and their
time of incubation with MTS/PMS. To evaluate cell viability while in direct contact with the cells, a concentration of 2 x 104 cells per well was seeded on a 96-well plate and cultured at 37°C for
1 day in a humidified incubator containing 5% CO2. Liposome@Fe3O4-GNCs with different Fe concentrations of 3.5, 7.0, 13.9, 27.8, 55.7 μM, or different Au concentrations of 13, 26, 52,
105, 211 mM, were prepared by adding culture medium previously incubated at 37°C
overnight and sterilized by UV light. Culture plates without any samples were used as normal
controls. After 24 h of cultivation, MTS solution (5 mg/ml in PBS) was added to each well, and
the plates were incubated for 2 h (37°C). The MTS solution and medium were mixed well at a
1:9 ratio. The optical density of the water-soluble formazan produced in the solution was
measured using an ELISA reader (SLT, Crailheim, Germany). Data were collected and
averaged from three different wells per condition.
2.4 Fluorescence intensity measurement of liposome@Fe3O4-GNCs in vivo
In Vivo Imaging Spectrum (IVIS, IVIS® Spectrum, Perkin Elmer) was used to measure the
fluorescent intensity of the liposome@Fe3O4-GNCs. The samples were prepared at five
concentrations, i.e., 13, 26, 52, 105, and 211 mM GNCs, and placed in a 96-well plate. The
13
excitation wavelength was 640 nm, and the emission wavelength was 740 nm. IVIS images
were captured, and fluorescence intensities were calculated using ImageJ software.
Liposome@Fe3O4-GNCs were evaluated in vivo using Sprague Dawley (SD) rats.
Experimental protocols followed the national guidelines for the care and use of laboratory
animals and the study was approved by the Animal Experimental Ethics Committee of KMU.
Three 6-month-old female SD rats per group were used. In each rat, 0.7 ml of the
liposome@Fe3O4-GNCs solution comprising an Au concentration of 211 mM (or Fe concentration of 55.7 μM) were injected into the tail vein each time. This is the safest dose for
the rats. However, the risk of death was higher when each injection was more than 1 ml
volume for the rat. After the injection, all animals were provided free access to food and water.
After 1 and 6 hours, the animals were sacrificed, and the major organs (i.e., heart, liver, spleen,
lung, and kidney) were collected. The fluorescence signals (640–740 nm) in these organs from
three rats in each group were detected using the IVIS imaging system, and all the organs of
the three rats in each group were quantified using IVIS 200 software. Mean ± SD values were
used to express the data, unless otherwise noted. Statistical analyses of the data were performed using Student’s t test. Differences characterized by p < 0.05 were considered to be
statistically significant.
14
3 Results and Discussion 3.1 Characterization of the liposome@Fe3O4-GNCs
Fig. 2(a) shows the FT-IR spectra of the Fe3O4 and Fe3O4-APTES nanoparticles from 400 – 4000 cm-1. Characteristic peaks of the Fe-O metal-oxygen bond were observed at 587 cm-1. This corresponded with stretching vibrations of the metal at tetrahedral sites
20
. APTES
modification of the Fe3O4 nanoparticle surfaces was confirmed by the presence of peaks indicative of Si-O-Si stretching and N-H bending at 1110 cm-1 and 1540 cm-1, respectively 21.
The phase structures of the Fe3O4 and Fe3O4-APTES nanoparticles were examined by
XRD. The diffraction peaks could be characteristic of the face-centered cubic structure of
magnetite according to ICDD card No. 19-0629. Fig. 2(b) shows the XRD patterns of Fe3O4
and Fe3O4-APTES. Crystalline diffraction peaks at 30.1°, 35.6°, 43.1°, 53.4°, 57.1°, and 62.7° 2θ are correlated with Miller Indices of (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) typical
of cubic spinel Fe3O4, respectively. No significant changes were observed in the XRD pattern
of the Fe3O4 core upon APTES functionalization. The obtained Fe3O4-APTES particles
presented similar crystal structures to those of pure Fe3O4 nanoparticles
22
. The size
distributions of the resultant Fe3O4 and Fe3O4-APTES nanoparticles were analyzed by TEM,
and their images are shown in the Supporting Information. As indicated, the Fe3O4 and
Fe3O4-APTES nanoparticles exhibited average sizes of 10.351.6 nm and 12.210.9 nm,
respectively. 15
A fluorescent spectrometer was used to characterize gold nanoclusters (GNCs)
synthesized with (±)-α-diaphorase. GNC colloidal solutions were excited at 350 nm, and
emission signals between 730-740 nm were collected as shown in Fig. 3(A). This wave band
region not only reduced the background noise from the biomatrix but also actively enabled the
imaging and tracking of cell fluorescence, meeting the experimental needs of cellular
observation or image tracking in vivo. The average GNC diameter was approximately 3 nm, as
measured by the nanoparticle analyzer. Under UV light, the Fe3O4-GNC nanocomposites
emitted near red light at 740-760 nm, as shown in Fig. 3(B). Through a simple fluorescence
test, the Fe3O4-GNC nanocomposites were shown to be capable for fluorescent tracking.
The particle size and polydisperse index (PDI) values of liposomes, liposome@Fe3O4
and liposome@Fe3O4-GNCs were measured using a nanoparticle analyzer; the results are
presented in Table 1. The particle sizes of liposomes, liposome@ Fe3O4 and liposome@
Fe3O4-GNCs were 112.7, 195.6 and 186.0 nm, respectively. The size of these three particles
was all greater than 100 nm but less than 200 nm. However, this size range of nanoparticles
can be absorbed by cells and enter the circulatory system. Furthermore, these nanoparticles
can be absorbed by lymphatic tissue and intestinal cells
23
. The size of the liposome@
Fe3O4-GNCs (186.0 nm) would be large enough to prevent their rapid leakage into blood
capillaries (normal blood vessel pores are approximately 2–6 nm in size) and small enough to
prevent capture by fixed macrophages lodged in the reticuloendothelial system, i.e., the sizes 16
of sinusoids in the spleen and the fenestra of Kupffer cells in the liver varies from 150 to 200 nm 24. Thus, the liposome@ Fe3O4-GNCs could be used in biomedical applications.
The PDI values of liposomes, liposome@Fe3O4 and liposome@Fe3O4-GNCs were 0.155,
0.417, and 0.083, respectively. PDI values range from 0 (monodispersed) to 1 (very broad
distribution) and are calculated to determine the degree of dispersion of polymer micelles or
particles. The PDI values of the liposomes, liposome@Fe3O4 and liposome@Fe3O4-GNCs
were less than 0.5, which suggested that the particles had narrow size distributions and were largely homogenous 25.
The
morphologies
and
sizes
of
the
liposomes,
liposome@Fe3O4
and
liposome@Fe3O4-GNCs were observed using TEM, as shown in Fig. 4. The results from the
TEM analyses agreed with those from the nanoparticle analyzer. We also observed that all
nanoparticles
had
spherical
vesicle
structures.
The
liposome
nanoparticles
were
sphere-shaped vesicles consisting of one or more phospholipid bilayers and were smaller than
the liposome@Fe3O4 and liposome@Fe3O4-GNC nanoparticles. The liposome@Fe3O4 and
liposome@Fe3O4-GNC nanoparticles were nanosized and enclosed Fe3O4 nanoparticles and
Fe3O4-GNC nanocomposites in their respective inner phases.
The hysteresis loops of the Fe3O4 nanoparticles and Fe3O4-APTES particles (a) and
liposome@Fe3O4-GNCs (b) are presented in Fig. 5. Magnetizations reached saturation after
an applied magnetic field of 15,000 Oe. The saturation magnetization (Ms) values of Fe3O4, 17
Fe3O4-APTES, and liposome@Fe3O4-GNCs were 55.98, 48,72, and 0.65 emu/g, respectively.
The Ms of Fe3O4-APTES was slightly decreased due to the reduced magnetic moment of the
Fe3O4 particles after APTES modification. This effect was likely due to the lower Fe3O4 weight
fraction of the Fe3O4-APTES relative to that of the Fe3O4 nanoparticles only. Alternatively,
Fe3O4-APTES could have created a magnetically dead layer on the Fe3O4 surface and thus
reduced the surface moments in the Fe3O4 particles. Piñeiro et al.
26
reported that the
magnetic quality strongly depended on the degree of order of crystalline lattices, oxidation
states
of
iron
ions
and
lattice
distortions
at
NP
surfaces.
Additionally,
the
liposome@Fe3O4-GNCs had a magnetic content of 1.2 wt.% based on the amount of pure
Fe3O4 particles due to the additional weight of the liposomes and GNCs. Although the Ms of
the liposome@Fe3O4-GNCs was smaller than that of the pure Fe3O4 particles, the curves of
Fig. 5 show typical superparamagnetic characteristics. No coercivity (Hc) was observed, and
remnant magnetism was nearly zero. This finding indicated that the Fe3O4, Fe3O4-APTES, and
liposome@Fe3O4-GNCs particles were nearly superparamagnetic
27
. The superparamagnetic
behavior of the liposome@Fe3O4-GNCs was due to the nanosize of the Fe3O4 particles
(approximately 10.4 nm) inside the liposome vesicles.
To confirm the magnetic resonance imaging effects of liposome@Fe3O4-GNCs as
contrast agents, the transverse relaxation time T2 was measured using fast field-cycling
nuclear magnetic resonance (NMR, Stelar). Because superparamagnetic materials induce
18
decreases in the transverse relaxation time T2, this would lead to increased contrast in MRI
images. Fig. 6 shows the MR signal intensity for different Fe concentrations. As shown in Fig.
6(a), the increased Fe concentration resulted in a reduced transverse relaxation time T2,
leading to improved MR signals. As shown in Fig. 6(a), the MR signal intensity decreased with
increasing concentrations of Fe
28
. In Fig. 6(b), the transverse relaxation rate (1/T2) of the
liposome@Fe3O4-GNCs was a function of Fe concentration in DI water. The transverse
relaxivity (r2) of the liposome@Fe3O4-GNCs was determined from the slope of the linear
equation: 1/T2 = 1/T02 + r2 [Fe]
where
1/T2 is
the
observed
transverse
relaxation
rate
in
the
presence
of
liposome@Fe3O4-GNCs, 1/T02 is the transverse relaxation rate in the absence of
liposome@Fe3O4-GNCs, r2 is the transverse relaxivity, and [Fe] is the concentration of Fe ions. Based on the slope of the regression line (R2 = 0.9981), the transverse relaxivity of the liposome@Fe3O4-GNCs was 872.73 mM−1 s−1. According to a previous study, the relaxivity
rate, r2, of hybrid nanospheres (chitosan@superparamagnetic iron oxide nanoparticles) showed an approximately 8-fold increment and reached the maximum of 533 mM−1 s−1
29
.
Moreover, the common magnetic fields of a clinical MRI system were 1-3 T, but the higher magnetic fields could increase the relaxivity rate. For example, the value of r2 was 10.64 mM–1 s–1 at 212 μT and increased to 152.47 mM–1 s–1 at 14.1 T for the ultrasmall superparamagnetic
19
iron oxide nanoparticles
30
. In this study, the magnetic field was 7 T and displayed the higher
relaxivity rate than the typical system. Compared to free iron oxide nanoparticles, this relaxivity
of our proposed material indicated that liposome@Fe3O4-GNCs are a promising material for molecular imaging applications31.
3.2 Cell viability assay of liposome@Fe3O4-GNCs
The results of the MTS assay are presented as absorbance levels to reflect altered
cellular mitochondrial activity in response to different Fe concentrations from 3.5 to 55.7 μM, or
Au concentrations from 211 to 13 mM, within liposome@Fe3O4-GNCs. Therefore, we also
present a direct cytotoxicity test for liposome@Fe3O4-GNCs and after incubating for 24 hours, liposome@Fe3O4-GNCs with different Fe concentrations of 3.5, 7.0, 13.9, 27.8, and 55.7 μM
did not exhibit cytotoxicity
(Fig. 7). According to results of the MTS assay and the
absorbance of the water-soluble formazan product value, cell viability values were all above
98.72%. The results of the MTS assay demonstrated that the liposome@Fe3O4-GNCs could
be used for in vitro cell-material interactions as a multifunctional liposomal drug delivery
system.
3. 3 Fluorescence intensity measurement of liposome@Fe3O4-GNCs in vivo
Fig. 8 shows images of the liposomes, liposome@Fe3O4 and liposome@Fe3O4-GNC
particles in daylight and under UV illumination. The liposome solution is a white and turbid
aqueous solution under daylight. The liposome@Fe3O4 and liposome@Fe3O4-GNCs were 20
yellow and brown in color due to the Fe3O4 particles. The liposome and liposome@Fe3O4
particles had no fluorescent properties, while the liposome@Fe3O4-GNCs emitted red fluorescent light. The wavelength of the emitted light ranged from 700 – 760 nm. This range of wavelength minimizes in vivo background autofluorescence 32.
Fig. 9 shows the fluorescence intensities of liposome@Fe3O4-GNCs for different GNC
concentrations as measured by IVIS imaging. Based on the slope of the regression line (R2 = 0.9996), the fluorescence intensity showed a highly linear relation to the GNC
concentration. This demonstrated that the liposome@Fe3O4-GNCs could function as a
fluorescent probe.
An in vivo biodistribution assay in SD rats was performed to further examine the ability of
liposome@Fe3O4-GNCs as fluorescent imaging probes. Fig. 10 shows the photos and
fluorescent images taken using IVIS imaging at 0, 1, 3, and 6 hours after the
liposome@Fe3O4-GNC transport into the SD rats. Organs such as the heart, spleen, lungs,
kidneys,
and
liver
were
taken
to
observe
the
metabolic
pathway
of
the
liposome@Fe3O4-GNCs. The fluorescence signals were barely detectable in the hearts of the
rats and were marginally detectable in the liver, spleen, lungs, and especially detectable in the
kidneys. After the liposome@Fe3O4-GNCs had been introduced into the SD rats for 6 hours,
fluorescence signals were primarily observed in the kidneys. This finding showed that the
liposome@Fe3O4-GNCs were hydrophilic carriers and primarily metabolized in the kidneys.
21
The fluorescence intensities of the liposome@Fe3O4-GNCs that reached the organs (i.e., heart, liver, spleen, lungs, and kidneys) were evaluated after vein administration via the rat’s
tails
for
1
and
6
hours.
Fig.
11
shows
the
fluorescence
intensities
of
the
liposome@Fe3O4-GNCs in the heart, liver, spleen, lungs, and kidneys after 1 and 6 hours. The
fluorescence intensities of the liposome@Fe3O4-GNCs in the heart and kidneys were the
lowest and highest, respectively. Moreover, the fluorescence intensity increased with time.
The fluorescence intensity of the liposome@Fe3O4-GNCs remained high and showed a
primary metabolic pathway through the kidneys.
4 Conclusion We successfully synthesized a multifunctional drug delivery system comprising of the
liposome@Fe3O4-GNCs. This dual MRI and near IR fluorescent imaging capability of the
Fe3O4-GNC nanocomposite provided a tracking tool for preclinical animal and human clinical
trials. The size of the liposome@Fe3O4-GNCs was approximately 186.0 nm, which was large
enough to prevent rapid leakage into the blood capillaries but small enough to escape capture
by fixed macrophages lodged in the reticuloendothelial system. The saturation magnetization
of the liposome@Fe3O4-GNCs was 0.65 emu/g; however, the nanoparticles maintained their
superparamagnetic property as an MRI contrast agent. This multi-functional drug delivery
system composited of nanosized gold clusters (GNCs) also had fluorescent properties. The
22
GNCs had an emission wavelength range of 700 – 760 nm, which minimized in vivo
background
autofluorescence.
The
in
vitro
affinity
tests
in
SD
rats
on
the
liposome@Fe3O4-GNC system demonstrated the non-toxic and in vivo use of these
nanoclusters in tracking their metabolic pathways. This promising liposome@Fe3O4-GNCs
with dual probes of magnetic resonance and fluorescence imaging show its potential.
However, there is still a need to verify the accuracy of target molecules conjugated with
liposome@Fe3O4-GNCs in animal clinical study stages in the future.
5 Acknowledgments The authors gratefully acknowledge the financial support provided to this study by the Ministry of Science and Technology (MOST) in Taiwan under Grant No. MOST- 105-2218-E-037-002. This study is also supported partially by Aim for the Top Universities Grant of Kaohsiung Medical University (KMU-TP105B03) and Kaohsiung Medical University Research Foundation (KMU-Q106007).
6 References 1.
Moore TJ, Furberg CD. Development times, clinical testing, postmarket follow-up, and
safety risks for the new drugs approved by the us food and drug administration: The
class of 2008. JAMA Internal Medicine 2014;174:90-95.
23
2.
Liu Q, Song L, Chen S, Gao J, Zhao P, Du J. A superparamagnetic polymersome with
extremely high T2 relaxivity for MRI and cancer-targeted drug delivery. Biomaterials
2017;114:23-33.
3.
Estelrich J, Sánchez-Martín MJ, Busquets MA. Nanoparticles in magnetic resonance
imaging: from simple to dual contrast agents. International Journal of Nanomedicine
2015;10:1727-1741.
4.
Xu P, Shen Z, Zhang B, Wang J, Wu R. Synthesis and characterization of
superparamagnetic iron oxide nanoparticles as calcium-responsive MRI contrast
agents. Applied Surface Science 2016;389:560-566.
5.
Weinstein JS, Varallyay CG, Dosa E, Gahramanov S, Hamilton B, Rooney WD,
Muldoon LL, Neuwelt EA. Superparamagnetic iron oxide nanoparticles: diagnostic
magnetic resonance imaging and potential therapeutic applications in neurooncology
and central nervous system inflammatory pathologies, a review. Journal of Cerebral
Blood Flow and Metabolism: Official Journal of the International Society of Cerebral
Blood Flow and Metabolism 2010;30:15-35.
6.
Huang Y, Mao K, Zhang B, Zhao Y. Superparamagnetic iron oxide nanoparticles
conjugated with folic acid for dual target-specific drug delivery and MRI in cancer
theranostics. Materials Science and Engineering: C 2017;70, Part 1:763-771.
24
7.
Mallick N, Anwar M, Asfer M, Mehdi SH, Rizvi MMA, Panda AK, Talegaonkar S,
Ahmad FJ. Chondroitin sulfate-capped super-paramagnetic iron oxide nanoparticles
as
potential
carriers
of
doxorubicin
hydrochloride.
Carbohydrate
Polymers
2016;151:546-556.
8.
Qu X, Li Y, Li L, Wang Y, Liang J, Liang J. Fluorescent Gold Nanoclusters: Synthesis
and Recent Biological Application. Journal of Nanomaterials 2015;2015:1-23.
9.
Zhang X, Zhao N, Wang B, Tian Z, Dai Y, Ning P, Chen D. Structure-inherent
near-infrared fluorescent probe mediates apoptosis imaging and targeted drug
delivery in vivo. Dyes and Pigments 2017;138:204-212.
10.
Chen H, Li B, Ren X, Li S, Ma Y, Cui S, Gu Y. Multifunctional near-infrared-emitting
nano-conjugates based on gold clusters for tumor imaging and therapy. Biomaterials
2012;33:8461-8476.
11.
Akhtar N, Khan RA. Liposomal systems as viable drug delivery technology for skin
cancer sites with an outlook on lipid-based delivery vehicles and diagnostic imaging
inputs for skin conditions'. Progress in Lipid Research 2016;64:192-230.
12.
Wang C, Yao Y, Song Q. Gold nanoclusters decorated with magnetic iron oxide
nanoparticles for potential multimodal optical/magnetic resonance imaging. Journal of
Materials Chemistry C 2015;3:5910-5917.
25
13.
Fauconnier N, Bée A, Roger J, Pons JN. Synthesis of aqueous magnetic liquids by
surface complexation of maghemite nanoparticles. Journal of Molecular Liquids
1999;83:233-242.
14.
Massart R. Preparation of aqueous magnetic liquids in alkaline and acidic media.
IEEE Transactions on Magnetics 1981;17:1247-1248.
15.
Asenath Smith E, Chen W. How To Prevent the Loss of Surface Functionality Derived
from Aminosilanes. Langmuir 2008;24:12405-12409.
16.
Yamaura M, Camilo RL, Sampaio LC, Macêdo MA, Nakamura M, Toma HE.
Preparation and characterization of (3-aminopropyl)triethoxysilane-coated magnetite
nanoparticles. Journal of Magnetism and Magnetic Materials 2004;279:210-217.
17.
Lin C-AJ, Yang T-Y, Lee C-H, Huang SH, Sperling RA, Zanella M, Li JK, Shen J-L,
Wang H-H, Yeh H-I and others. Synthesis, Characterization, and Bioconjugation of
Fluorescent Gold Nanoclusters toward Biological Labeling Applications. ACS Nano
2009;3:395-401.
18.
Feng B, Hong RY, Wang LS, Guo L, Li HZ, Ding J, Zheng Y, Wei DG. Synthesis of
Fe3O4/APTES/PEG diacid functionalized magnetic nanoparticles for MR imaging.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 2008;328:52-59.
26
19.
Wang L, Zhang P, Shi J, Hao Y, Meng D, Zhao Y, Yanyan Y, Li D, Chang J, Zhang Z.
Radiofrequency-Triggered Tumor-Targeting
Delivery System
for
Theranostics
Application. ACS Applied Materials & Interfaces 2015;7:5736-5747.
20.
Kurtan
U,
Baykal
A.
Fabrication
and
characterization
of
Fe3O4@APTES@PAMAM-Ag highly active and recyclable magnetic nanocatalyst:
Catalytic reduction of 4-nitrophenol. Materials Research Bulletin 2014;60:79-87.
21.
Mirzabe GH, Keshtkar AR. Application of response surface methodology for thorium
adsorption on PVA/Fe3O4/SiO2/APTES nanohybrid adsorbent. Journal of Industrial
and Engineering Chemistry 2015;26:277-285.
22.
Jafarzadeh M, Soleimani E, Norouzi P, Adnan R, Sepahvand H. Preparation of
trifluoroacetic acid-immobilized Fe3O4@SiO2–APTES nanocatalyst for synthesis of
quinolines. Journal of Fluorine Chemistry 2015;178:219-224.
23.
Ai J, Biazar E, Jafarpour M, Montazeri M, Majdi A, Aminifard S, Zafari M, Akbari HR,
Rad HG. Nanotoxicology and nanoparticle safety in biomedical designs. International
Journal of Nanomedicine 2011;6:1117-1127.
24.
Wisse E, Braet F, Dianzhong L, De Zanger R, Jans D, Crabbe E, Vermoesen AN.
Structure and Function of Sinusoidal Lining Cells in the Liver. Toxicologic Pathology
1996;24:100-111.
27
25.
Long J, Xu E, Li X, Wu Z, Wang F, Xu X, Jin Z, Jiao A, Zhan X. Effect of chitosan
molecular weight on the formation of chitosan–pullulanase soluble complexes and
their application in the immobilization of pullulanase onto Fe3O4–κ-carrageenan
nanoparticles. Food Chemistry 2016;202:49-58.
26.
Piñeiro Y, Vargas Z, Rivas J, López-Quintela MA. Iron Oxide Based Nanoparticles for
Magnetic Hyperthermia Strategies in Biological Applications. European Journal of
Inorganic Chemistry 2015;2015:4495-4509.
27.
Luo Y, Zhou Z, Yue T. Synthesis and characterization of nontoxic chitosan-coated
Fe3O4 particles for patulin adsorption in a juice-pH simulation aqueous. Food
Chemistry 2017;221:317-323.
28.
Rezayan AH, Mousavi M, Kheirjou S, Amoabediny G, Ardestani MS, Mohammadnejad
J. Monodisperse magnetite (Fe3O4) nanoparticles modified with water soluble
polymers for the diagnosis of breast cancer by MRI method. Journal of Magnetism and
Magnetic Materials 2016;420:210-217.
29.
Lin Y, Wang S, Zhang Y, Gao J, Hong L, Wang X, Wu W, Jiang X. Ultra-high relaxivity
iron oxide nanoparticles confined in polymer nanospheres for tumor MR imaging.
Journal of Materials Chemistry B 2015;3:5702-5710.
30.
Wang W, Dong H, Pacheco V, Willbold D, Zhang Y, Offenhaeusser A, Hartmann R,
Weirich TE, Ma P, Krause H-J and others. Relaxation Behavior Study of Ultrasmall
28
Superparamagnetic Iron Oxide Nanoparticles at Ultralow and Ultrahigh Magnetic
Fields. The Journal of Physical Chemistry B 2011;115:14789-14793.
31.
Sitthichai S, Pilapong C, Thongtem T, Thongtem S. CMC-coated Fe3O4 nanoparticles
as new MRI probes for hepatocellular carcinoma. Applied Surface Science
2015;356:972-977.
32.
Lin MZ, McKeown MR, Ng H-L, Aguilera TA, Shaner NC, Campbell RE, Adams SR,
Gross LA, Ma W, Alber T and others. Autofluorescent Proteins with Excitation in the
Optical Window for Intravital Imaging in Mammals. Chemistry & Biology
2009;16:1169-1179.
29
Table 1 Average sizes and polydisepersity index (PDI) of liposome, liposome@Fe3O4 and
liposome@Fe3O4-GNCs. Material
Average size(nm)
PDI
liposome
112.7
0.155
liposome@ Fe3O4
195.9
0.417
liposome@ Fe3O4-GNCs
186.0
0.083
Fig. 1 Schematic diagram of synthesis Fe3O4-GNC nanocomposite-loaded liposomes.
30
Fig. 2 (a) FT-IR spectra and (b) XRD patterns of Fe3O4 and Fe3O4-APTES.
31
Fig. 3 (a) GNCs of (i) UV-vis absorption spectrum, (ii) fluorescence emission (excitation at 350
nm) spectra. (b) Fluorescence emission (excitation at 350 nm) spectra of Fe3O4-GNC
nanocomposites.
32
Fig. 4 TEM images and the sketch of nanoparticles from (a) liposomes, (b) liposome@Fe3O4
and (c) liposome@Fe3O4-GNCs.
33
Fig.
5
Hysteresis
loops
of
(a)
Fe3O4
liposome@Fe3O4-GNCs.
34
and
Fe3O4-APTES
particles
and
(b)
Fig. 6 (a) T2-weighted MRI of liposome@Fe3O4-GNCs at various Fe concentrations: 0, 1.8, 3.5,
7.0,
13.9,
27.8,
55.7
μM
(b)
The
transverse
(1/T2)
liposome@Fe3O4-GNCs as a function of Fe concentrations in DI water.
35
relaxation
rates
of
Fig. 7 The cell viability of liposome@Fe3O4-GNCs with different Fe/Au concentrations (μM/mM) were analyzed by the MTS assay after incubating for 24 hours. These values are
shown as the mean ± standard error of the mean, n=3.
36
Fig. 8 Photos of nanoparticles from liposomes (a; d), liposome@Fe3O4 (b; e) and
liposome@Fe3O4-GNCs (c; f) in daylight and under UV illumination.
37
Fig. 9 Fluorescence intensity of liposome@Fe3O4-GNCs with different GNC concentrations for
IVIS imaging.
38
Fig. 10 Photos and fluorescence images taken using IVIS imaging at (a) 0, (b) 1, (c) 3, and (d)
6 hours after liposome@Fe3O4-GNCs transport into SD rats.
39
Fig. 11 The fluorescence intensity after liposome@Fe3O4-GNCs reached the heart, liver, spleen, lung, and kidney transport into SD rats for 1 and 6 hours (n =3). ** denote p < 0.05 were statistically significant.
40