Tumor Angiogenesis Targeted Radiosensitization Therapy Using Gold

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Tumor Angiogenesis Targeted Radiosensitization Therapy using Gold Nanoprobes Guided by MRI/SPECT Imaging Yi Yang, Lu Zhang, Jiali Cai, Xiao Li, Dengfeng Cheng, Huilan Su, Jianping Zhang, Shiyuan Liu, Hongcheng Shi, Yingjian Zhang, and Chunfu Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09274 • Publication Date (Web): 05 Jan 2016 Downloaded from http://pubs.acs.org on January 10, 2016

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ACS Applied Materials & Interfaces

Tumor Angiogenesis Targeted Radiosensitization Therapy

using

Gold

Nanoprobes Guided

by

MRI/SPECT Imaging Yi Yang,ζ,

‡

Lu Zhang,ζ, ‡ Jiali Cai,ψ Xiao Li,$ Dengfeng Cheng, $ Huilan Su,∆ Jianping Zhang,Φ

Shiyuan Liu, ψ Hongcheng Shi, $ Yingjian Zhang, Φ Chunfu Zhang ζ, φ, * ζ State Key Laboratory of Oncogenes and Related Genes，Shanghai Cancer Institute, School of Biomedical Engineering，Shanghai Jiao Tong University, Shanghai 200030, China.

ψ Changzheng Hospital, Secondary Military Medical University, Shanghai 200003, China. $ Department of Nuclear Medicine, Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai 200032, China. ∆ State Key Lab of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai, China. Φ Department of Nuclear Medicine, Shanghai Cancer Center, Fudan University, Shanghai 200032, China φ Department of Nuclear Medicine, Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200025, P. R. China ‡ These authors contributed equally

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KEYWORDS Gold nanoparticles; radiotherapy; MRI/SPECT; αvβ3 integrin; theranostics

ABSTRACT Gold nanoparticles (AuNPs) have recently garnered great interest as potential radiosensitizers in tumor therapy. However, major challenges facing their application in this regard are further enhancement of tumor accumulation of the particles in addition to enhanced permeability retention (EPR) effect and an understanding of the optimal particle size and time for applying radiotherapy after the particle administration. In this study, we fabricated novel cyclic c(RGDyC) peptide conjugated-Gd and 99mTc labeled-AuNPs (RGD@AuNPs-Gd99mTc) probes with different sizes (29, 51 and 80 nm) and evaluated their potential as radiosensitization therapy both in vitro and in vivo. We found that these probes have a high specificity for αvβ3 integrin positive cells, which resulted in their high cellular uptake and thereby enhanced radiosensitization. Imaging in vivo with MRI and SPECT/CT directly showed that the RGD@AuNPs-Gd99mTc probes specifically target tumors and exhibit greater accumulation within tumors than the RAD@AuNPs-Gd99mTc probes. Interestingly, we found that the 80 nm RGD@AuNPs-Gd99mTc probes exhibit the greatest effects in vitro, however the 29 nm RGD@AuNPs-Gd99mTc probes were clearly most efficient in vivo. As a result, radiotherapy of tumors with the 29 nm probe was the most potent. Our study demonstrates that RGD@AuNPsGd99mTc probes are highly useful radiosensitizers capable of guiding and enhancing radiation therapy of tumors.

INTRODUCTION Gold nanoparticles (AuNPs) possess a range of unique physical and chemical properties that make them ideal platforms for a variety of biomedical applications, including imaging, drug


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delivery, and therapy.

1-3

With regards to the latter, there recently has been great interest in

utilizing AuNPs as radiosensitizers in cancer radiation therapy, owing to their strong absorption and high efficiency in generating secondary electrons under γ-ray or X-ray irradiation. 4 Indeed, AuNPs of various sizes and shapes have been shown to significantly enhance the effectiveness of radiation doses both in vitro and in vivo. 5-8 However, for this to become an established, reliable methodology, it is essential to determine the optimal conditions of their application in a physiological setting. For one, it is not immediately obvious what size of AuNPs should be employed for maximal effectiveness.

6

On

the one hand, the photoelectric absorption cross-section of a radiosensitive material, and thus also its effectiveness of radiation enhancement, depends on the size of the constituent particle, and so larger sizes should generally provide greater enhancement.

4, 9-11

However, on the other

hand, there are physical or chemical barriers of biological systems that limit the accumulation of AuNPs in tumors and tissues. For example, AuNPs greater than 50 nm in diameter do not pass through the outer barrier of the reticuloendothelial system, and instead form large aggregates in the blood, leading to a poor deposition of AuNPs in tumors.12-14 In contrast, AuNPs with a 12.1 nm diameter coated with polyethylene glycol (PEG) accumulate in tumors to a much greater degree than those of AuNPs with diameters of 27.6 or 46.6 nm.15, 16 Thus, with such opposing effects of AuNP size, it is critically important to experimentally determine the optimal AuNP size for maximal radiosensitization under physiologically relevant conditions. In addition, we also note that it is also essential to experimentally determine the optimal time point at which the particle accumulation within the tumor is maximal, as this is also expected to significantly influence the measured outcome.


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To enable evaluation of these properties in a physiological setting, advanced imaging techniques that provide noninvasive, quantitative, and reproducible monitoring of the particle accumulations in tumors are needed. In this regard, we note that many biomedical imaging techniques, such as magnetic resonance imaging (MRI), imaging

23, 24

17-19

nuclear,

20-22

and fluorescent

have recently been extensively employed in studies of the bio-distribution of

nanoparticles in vivo. Among these techniques, MRI exhibits a high spatial resolution in three dimensions and can also provide anatomical information, while nuclear imaging is highly sensitive and quantitative and can dynamically resolve the bio-distribution of nanoparticles in the whole body. 25 Therefore, combining these two imaging modalities is expected to take advantage of the strengths of both modalities simultaneously and thereby enable a better understanding of the in vivo behavior of AuNPs. 26, 27 In addition, it is well documented that the radiotherapy enhancement is proportional to the amount of AuNPs accumulated in tumor region.28, 29 Currently, tumor accumulation of AuNPs delivered intravenously is achieved mainly by enhanced permeability retention (EPR) effect, which is less efficient than that by active targeting. 30, 31 To further enhance tumor accumulation, and thus the therapeutic effect, active targeting is plausible. 32 In this study, we developed cyclic c(RGDyC) peptide (abbreviated RGD) conjugated-Gd and 99mTc labeled-AuNPs probes (RGD@AuNPs-Gd99mTc) to enable specific localization to tumors of AuNPs that could be simultaneously monitored with both MRI and nuclear imaging methodologies. Further, we produced three different sizes (particles with diameters of 29, 51, or 80 nm) and evaluated their effectiveness as radiosensitizers both in vitro and in vivo. We found that all three probes exhibit high specificity for αvβ3 integrin positive cells and tumor angiogenic vessels. The 80 nm RGD@AuNPs-Gd99mTc were internalized by tumor cells most


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efficiently, and thus exhibited the greatest degree of radiosensitization in vitro. However, in vivo, the 29 nm RGD@AuNPs-Gd99mTc exhibited greater tumor accumulation and thus were ultimately found to be most effective for tumor radiosensitization therapy.

MATERIALS AND METHODS Synthesis of Gold Nanoparticles All the chemicals were purchased from Sigma-Aldrich ( St. Louis, MO.) unless indicated otherwise. Gold nanoparticles were synthesized with seed-growth method according to previous report.

33

Briefly, gold nanoseeds were first synthesized. To this

end, trisodium citrate dihydrate (5 mM, 1 mL) and HAuCl4 (5 mM, 1 mL) were mixed into 18 mL deionized H2O, and then freshly made NaBH4 solution (0.1 M, 0.6 mL) was quickly injected into the mixture under vigorous stirring. After stirring for 4 h, the solution was collected as the seed solution for subsequent seeded growth. Next, a certain amount of the seed solution was quickly injected into a growth solution, which consisted of 0.5 mL of polyvinylpyrrolidone (PVP, Mw 29,000) (5 wt%), 0.25 mL of L-ascorbic acid (0.1 M), 0.2 mL of KI (0.2 M), 0.06 mL of HAuCl4 (0.25M) and 2 mL of H2O, under vigorous stirring. The AuNPs were allowed to grow for 10 min and then collected by centrifugation and redispersed in water. For synthesis of 29 nm, 51 nm and 80 nm AuNPs, 200 µL, 20 µL and 5 µL seed solutions were added, respectively. Fabrication of RGD Peptide Conjugated-, Gd and 99mTc labeled-AuNPs Probe (RGD@AuNPs-Gd99mTc) For fabrication of the probe, AuNPs were first modified with amino-poly(ethylene glycol)-thiol (NH2-PEG-SH, MW 3400, Laysan Bio Inc, Alabama). To this end, twelve milligram AuNPs synthesized above was dispersed into 1mL ethanol, sonicated for 10 min, collected and then washed with water. Subsequently, the particles were dispersed into


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the PEG aqueous solution (3 mL, 4 mg/mL), sonicated for 30 min and then stirred at 4 oC overnight. Next, PEG-modified AuNPs (1mg) were dispersed into 200 µL phosphate buffer (PBS, pH 8), then sulfosuccinimidyl-4-[N -maleimidomethyl]-cyclohexane-1-carboxylate (sulfo-SMCC, 50 µL, 1mM. Pierce, Tianjin, China), 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (NHS-DOTA, 50 µL, 1mM. Macrocyclics Inc, Dallas) and N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycinate (NHS-MAG3, 50 µL, 1mM. Pierce, Tianjin, China) were added simultaneously and stirred for 30 min. The final AuNPs were retrieved by ultrafiltration with a Millipore centrifugal filter unit (Amicon Ultra-0.5, MWCO 100,000), washed three times and re-suspended into 0.5 mL PBS (pH 7.4). RGD peptide, Gd and 99mTc were then conjugated and labeled onto the particles successively. For RGD peptide conjugation, 100 µg of the peptide was added into above AuNPs suspension and vibrated overnight to produce a thiol-maleimide linkage between peptide and Sulfo-SMCC on the particle surface. The peptide conjugated AuNPs were retrieved by ultrafiltration (Amicon Ultra-0.5, MWCO 100,000), washed with PBS (pH 7.4) three times and then dispersed into GdCl3 aqueous solution (40 mg/mL, 1mL) for Gd labeling. The pH of the solution was adjusted to 5.0 ∼ 5.5 with dilute sodium hydroxide and the labeling was carried out for 24 hours under stirring at room temperature. Afterward, the RGD peptide-conjugated, Gd-labeled AuNPs were collected by ultrafiltration (Amicon Ultra-0.5, MWCO 100,000) and washed with PBS (pH7.4) three times. For 99mTc labeling, as-prepared particles were dispersed into a mixture of ammonium acetate (90 µL, 0.25 M) and tartrate buffer (30 µL, 50 mM).

Subsequently, 99mTc-pertechnetate

generator eluate (10 µL, 1mCi ∼ 5mCi) was added. After vortexing for a while, a freshly prepared stannous chloride dihydrate solution (10 µL, 4 mg/mL in tartrate buffer) was added and, after gently vortexing for a while, the solution was incubated at room temperature for 1 h.


34

To

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evaluate the labeling efficiency, radio-thin-layer chromatography (RTLC; AR2000, Bioscan, Washington, DC) was performed, using acetone as the mobile phase. In this system, 99mTclabeled AuNPs remain at the origin, while 99mTc-pertechnetate migrates to retardation factor (Rf) = 0.7-0.9. The labeling efficiency was calculated by dividing the radioactivity retained at the origin to the total radioactivity added. 35 The final product, RGD@AuNPs-Gd99mTc probe, was purified using size exclusion filters with a 10 kDa molecular weight cutoff (Millipore, Amicon Ultra-0.5) and size exclusion chromatography with disposable columns containing Sephadex G25 medium using saline as an eluent. The rose color eluate containing the probe was collected. A centrifugal concentrator was then used to concentrate the samples to the desired volume. In addition, RGD peptide-conjugated, gadolinium and technetium (Tc)-labeled AuNPs (RGD@AuNPs-GdTc) were also prepared as a “cold” probe using NaTcO4 (10 µL, 0.1M) as a precursor under the same conditions for 99mTc labeling. At the same time, RAD peptide (c(RADyC))-coupled, Gd and 99mTc or Tc-labeled AuNPs were also prepared as control probes (RAD@AuNPs-Gd99mTc, RAD@AuNPs-GdTc). Characterizations of the Probes Zeta potentials of AuNPs at each step of the probe preparation were measured. The probe (RGD@AuNPs-GdTc) was characterized by the following methods. The hydrodynamic sizes were measured using a Malvern Instruments Zetasizer Nano Series Nano-ZS. The size and morphology were characterized by transmission electron microscopy (TEM, JEOL 2010) at an accelerating voltage of 200 kV. The particle size distribution was calculated using an ImageJ analysis software by measuring the diameter of more than 100 individual particles. The longitudinal relaxation times were measured at 1.41 T (60 MHz) and 37 oC on a Bruker mq60 nuclear magnetic resonance analyzer. An inversion recovery (IR) pulse sequence was used to measure the longitudinal relaxation time (T1). The gadolinium,


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technetium and gold concentrations were determined using inductively coupled plasma optical emission spectrometry (ICP-OES, ICAP-6300, Thermo Fisher, Portsmouth, New Hampshire). The T1 relaxivity (r1) was deduced by fitting inverse relaxation times (1/T1) as a function of the gadolinium concentrations. The number of AuNPs per milliliter was calculated using equation (1) and (2) assuming the ideal spherical shape of the particles. They are expressed as: N = m/vs

(1);

v = 4 × πr3/3 (2)

in which N is the amount of AuNPs per milliliter, m is the mass of gold per milliliter in the substance [g mL-1], v is the volume of an AuNP, r is the radius of an AuNP, and s is the specific gravity of colloidal gold [19.3 g cm-3]. The number of gadolinium and technetium per milliliter was calculated using equation (3), in which m is the mass of gadolinium or technetium in the solution [g mL-1], M is the mass of gadolinium or technetium atom. n = m/M (3) Moreover, the conjugation efficiency of RGD peptide was also evaluated with Ellman method by measuring the free sulfhydryl groups in the peptide in the reaction media before and after conjugation spectrophotometrically.

36, 37

Accordingly, the number of peptide conjugated

onto a particle could be determined. The possible release of Gd from the probes was studied in triplicate by co-incubating 1 mg of the probes in 0.3 mL of fresh mouse plasma at 37oC for different periods of time. After


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incubation, the probes were collected by ultrafiltration, and the free gadolinium in the serum was measured by ICP-OES. Stability of gadolinium was expressed as a percentage of gadolinium retained on AuNPs to the total amount of gadolinium on the probes. For the radioactive probes, the stability of the 99mTc was assessed in triplicate by coincubating 5 µL of the sample in 200 µL of fresh mouse plasma at 37oC for different periods of time. After incubation, the probes were collected by centrifugation, and the radioactivity retained on the particles was counted in a gamma well counter. Stability of 99mTc was expressed as a percentage of radioactivity retained on AuNPs to the radioactivity of the probes. Cytotoxicity of the Probes Cytotoxicity of the probes was evaluated by the typical 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT)

reduction

assays

using

RGD@AuNPs-GdTc probe. For this purpose, H1299 cells, a non-small-lung-cancer cell line, were seeded in a 96-well plate with 1×104 cells per well and incubated with the probe at different concentrations (10, 50, 100 and 150 µg Au/mL) for 24 h. After incubation, the culture media were removed, and the cells were washed with PBS (pH 7.4) three times. Subsequently, 100 µL aliquots of MTT solution were added. After co-incubation for another 4 h, the media were replaced with 100 µL of dimethyl sulfoxide per well, and the absorbance was monitored by a microplate reader at a wavelength of 490 nm. The cell viability was expressed as the percentage of absorbance of the cells incubated with the nanoparticles to that of the cells maintained in a normal culture medium. Specificity of the Probes Specificity of the probes for αvβ3 integrin was examined by cell silver staining and ICP-OES quantification of intracellular gold contents. For silver staining, H1299 cells were grown on cover slides and co-cultured with RGD@AuNPs-GdTc,


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RAD@AuNPs-GdTc or RGD@AuNPs-GdTc plus free RGD peptide (c(RGDyC), 10 µM)

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38, 39

at the concentration of 50 µg Au/mL for 1h. After incubation, the culture media were removed and cells were fixed with 4% paraformaldehyde. Silver staining was performed using a silver enhancement kit (ProteoSilver Silver Solution, Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, the cells were rinsed with deionized water, an equal volume of the kit’s two reagents were mixed in a 1:1 ratio in a microcentrifuge tube, and 50 uL was deposited onto the slides. Samples were stained for 20-30 minutes and the reaction was stopped by rinsing with water. To quantify the intracellular gold contents, the cells were treated with the probes at the same conditions described above. After incubation, cells were digested with aqua regia. The intracellular gold contents were determined by using ICP-OES, which were expressed in picogram (pg) of iron per cell. Subcellular Localizations of the Probes To identify the subcellular locations of the probes, TEM examination of H1299 cells were performed. For this purpose, cells were cultured with media containing RGD@AuNPs-GdTc, RAD@AuNPs-GdTc or RGD@AuNPs-GdTc plus free RGD peptide (10 µM) at the concentration of 50 µg Au/mL for 1 h. After incubation, the cells were washed with PBS three times, fixed with ice-cold 2.5% glutaraldehyde in 0.05 M sodium cacodylate (pH 7.2) for 40 minutes and then embedded in 2% agarose. Subsequently, the embedded cells were stained with 2% osmium tetroxide and 0.5% uranyl acetate successively, and processed for ultrathin sectioning. Micrographs were taken with TEM operating at an acceleration voltage of 80 kV (Philip CM-120, Eindhoven, Netherlands).


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Radiotherapy In Vitro H1299 cells were seeded into several six-well plates at a density of 105 cells/well

and

then

incubated

with

RGD@AuNPs-GdTc,

RAD@AuNPs-GdTc

or

RGD@AuNPs-GdTc plus free RGD peptide (10 µM) at the concentration of 50 µg Au/mL for 1h. After incubation, the cells were exposed to 4Gy of γ-ray radiation using cesium-137 (Cs-137, 662 keV) beam radiator and then continuously cultured. All the treatments were done only once. During the next 5 days, the live cells in each group were counted and the survival rate was calculated by dividing the number of live cells in each treated group by the number in the control group without any treatment. 40 MR Imaging of Tumors Animal procedure was in agreement with the guidelines of the Animal Welfare Committee of Shanghai Jiao Tong University. Balb/c nude mice (Slaccas, Shanghai, China) with average weight of 20 g were used. H1299 cells (5×106 cell/site) were implanted subcutaneously into nude mice, which were ready for use when the tumor size reached about 7-8 mm in diameter. Mice were injected with RGD@AuNPs-GdTc, RAD@AuNPs-GdTc or RGD@AuNPs-GdTc plus free RGD peptide (c(RGDyC), 100 µL, 0.15 mM)

38, 39

intravenously at the dose of 0.1mmol Gd/kg b. w. with five mice in each group. MR imaging was conducted using a 7 T MRI scanner (Biospec System 70/20, Brucker, Ettlingen, Germany) using a T1-weighted spin-echo sequence (TR = 1500 ms，TE = 6 ms，FOV =35×35mm，Matrix = 256×256，slice thickness = 1 mm) and T1-mapping sequence (TR = 3000 ms, TE = 15 ms and inversion delays of 500, 1000, 1500, 2000, 2500, 3000, and 3500 ms). Pre- and post-contrast MR images were analyzed using ParaVision 6 software provided by the manufacturer. The average signal intensity inside a region of interest (ROI) drawn around the tumor was computed for each image. The relative signal intensity enhancement (rSIE) was defined as the ratio of average intensity inside the tumor post probe injection to that of pre injection.


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SPECT/CT Imaging Mice bearing H1299 tumors were intravenously injected with 0.1 mL (7.4 MBq, 10 mM in gold) of RGD@AuNPs-Gd99mTc, RAD@AuNPs-Gd99mTc, or RGD@AuNPs-Gd99mTc plus free RGD peptide (100 µL, 0.15 mM) with five mice in each group. SPECT/CT scans were obtained using a small-animal imaging system (Bioscan, Washington, DC). The CT images were used to provide anatomical references to the tumor location. The SPECT images were obtained at 32 projections over 360 oC (radius of rotation = 7.6 cm, 30 s/projection). Reconstructed data from SPECT and CT were visualized and coregistered using InVivoScope (Bioscan, Washington, DC). Bio-distribution Study of the Probes After SPECT/CT imaging, the mice were sacrificed and dissected. The entire tumor, kidney, spleen, and heart, and samples of the liver, intestines, stomach, lung, muscle and urine were weighed in plastic test tubes. The radioactivity was determined in a well-type scintillation detector along with 3 × 0.5 mL aliquots of the diluted standard representing 100% of the injected dose. The mean activities were used to obtain the percentage of injected dose per gram of tissue (% ID/g). 38 In addition, the probe distributions were also evaluated by examining gold contents in the organs described above and in urine. For this purpose, tumor bearing mice were grouped and injected with the probes at the doses exactly same as those for SPECT imaging. Six hours after injection, the mice were sacrificed and dissected. The organ and urine samples were weighed, and digested with nitric acid in a microwave digestion system (EXCEL, Preekem Scientific Instruments Co., Shanghai, China) and aqua regia successively. The digested solutions were diluted with Milli-Q water and the gold contents were quantified with ICP-OES. The percentage of injected dose per gram tissue (% ID g-1) of gold in a specific tissue was calculated.


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Determination of Blood Half-life of RGD@AuNPs-Gd99mTc Probes Five mice each received 7.4 MBq (0.1 mL) of RGD@AuNPs-Gd99mTc and blood samples were collected from a tail vein before and 2, 5, 10 and 30 min, 1, 2, 4 and 24 h after intravenous injection. For each blood sample (5 µL), radioactivity was countered and percentage of injection dose per gram of blood (%ID/g) was determined. Half-life of the temporal course of the %ID/g was fitted to a 2compartment bolus intravenous injection model. 12, 41 Radiotherapy In Vivo Mice bearing H1299 tumors with size about 7-8 mm were divided into five groups: control, radiation only, RGD@AuNPs-GdTc + radiation, RAD@AuNPs-GdTc + radiation, and competition (RGD@AuNPs-GdTc + free RGD peptide) + radiation. Each group included seven mice. Control groups received pure PBS (pH = 7.4). The probes were injected intravenously with dose of 2.5 mmol Au /kg b. w. Four hours post injection, the tumors were exposed to 10 Gy of γ-ray radiations (Cs-137, 662 keV). All the treatments were given only once. During the next 3 weeks, the tumor size of each mouse was measured by vernier caliper. The tumor volume was computed as V = (π/6) × A × B × C, for A, B, and C, the three tumor diameters.

42

Tumor growth was calculated by formula: 100% × (the calculated volume – initial

volume)/initial volume. Histological Studies To identify the expression of αvβ3 integrin and verify RGD@AuNPsGd99mTc targeting tumor angiogenic vessels, co-staining of αvβ3 integrin and AuNPs was performed. For this purpose, tumors were removed and imbedded in OCT glue (Miles, Elkhart, Indiana), frozen with nitrogen, and then sectioned into 5-µm slices using a cryostat (CM 1900, Leica, Nussloch, Germany). The sections were first fixed in acetone for 10 min at 4 oC and then air dried for 30 min. Silver staining was first performed using a silver enhancement kit


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(ProteoSilver Silver Solution, Sigma-Aldrich) and the staining procedure was same as that for cell staining. For αvβ3 integrin staining, the sections were treated with a primary rat anti-mouse CD61 monoclonal antibody (1:50 dilution, BD Biosciences, San Diego, CA) and a biotinylated goat anti-rat IgG (BD Bioscience, San Diego, CA) in combination with streptavidin-horseradish peroxidase (HRP) and the DAB detection system. The tumor sections were counterstained with hematoxylin and returned to blue by using an ammonia solution. Immunohistochemical staining of tumor tissues against CD31 and CD61 from control mice (mice without probe treatment) was also performed. To verify and compare the radiosensitization effects of the probes, γ-H2AX staining of tumor tissues was conducted. Tumors were fixed with paraformaldehyde and imbedded in paraffin. Representative 5µm thick tumor cross sections were cut. The sections were dewaxed in xylene, dehydrated in ethanol, and then incubated in 3% hydrogen peroxide for 10 min at room temperature. After the antigen was retrieved by microwave treatment, the sections were incubated overnight at room temperature with monoclonal anti-phosphohistone γ-H2AX primary antibody (2 µg/mL, Millipore, Billerica, MA) containing with 1% BSA at 4 oC overnight. After washing with PBS (pH=7.4) three times, the slices were incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (1:300 dilution, Molecular Probes, Eugene, Oregon) for 1h at room temperature. Cell nuclei were counter-stained with DAPI. Quantitative analysis of γ-H2AX positive area was conducted by a fluorescent microscope (Leica DM2500, Houston, Texas) using the Image-Pro Plus software. To this end, at least three randomly selected vision fields for each sample and total three samples were analyzed.


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The histological study and function examination of kidney were also performed 6 h after intravenous injection of RGD@AuNPs-GdTc probes (0.1 mL, 7.4 MBq, 10 mM in gold). For these purposes, kidney samples were fixed in 4% formalin and then processed for H&E staining. Kidney function was evaluated by examining urea nitrogen, serum creatinine and uric acid levels in blood samples. Statistical Analyses All data presented are the average ± SD of experiments repeated three or more times. Where appropriate, a Student’s t-test was used to determine if differences were statistically significant. A P value of < 0.05 was considered to indicate significant differences between groups.

Results Preparation and Characterization of the RGD@AuNPs-Gd99mTc Probes AuNPs with well-defined sizes were synthesized by a one-step seed growth method, by which Au seeds grow in a high concentration of gold precursor, where self-nucleation is avoided.33 As-prepared AuNPs possessed a uniform spherical morphology with narrow size distributions with diameters of 29 ± 2, 51 ± 2, and 80 ± 1 nm for the three different particles prepared here (Figure 1A, B, and C).

To improve the in vivo stability of these particles and provide a means of surface

functionalization, the AuNPs were modified with amino-poly(ethylene glycol)-thiol (NH2-PEGSH, MW 3400). The sulfhydryl group has a high affinity for gold atoms, 43 and the resulting AuS bond, which can be formed under mild conditions, thereby anchors the NH2-PEG-SH moiety on the surface of AuNPs with the amino group readily exposed to solution. To fabricate the RGD peptide conjugated-Gd and 99mTc labeled-AuNPs probe (RGD@AuNPs-Gd99mTc), the PEG-


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modified AuNPs were first incubated with sulfo-SMCC, NHS-DOTA, and NHS-MAG3 simultaneously. Each of these chemicals forms a covalent bond with the amino groups in the PEG via their succimide groups. After this reaction, the zeta potential of the particles was found

Figure 1. Characterizations and cytotoxicity of the probes. (A), (B), and (C) TEM images of the probes. The sizes of the probes are 29 ± 2 (A), 51 ± 2 (B) and 80 ± 1 nm (C). (D) Stability of the association of 99mTc and Gd on the probes in mouse serum. (E) Stability of the RGD@AuNPsGdTc probes in PBS (pH=7.4). (F) Viability of H1299 cells after incubation with the RGD@AuNPs-GdTc probes at different concentrations for 24 h. to be around -37 mV. The RGD peptides were then covalently conjugated to the particles through thiol-maleimide linkages.3,

39

The Gd and 99mTc were subsequently attached to the

particles by successively complexing Gd and 99mTc to DOTA and MAG3.34, 44 The overall procedure for the preparation of these probes is shown in Scheme 1. After the RGD peptide


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conjugation and Gd labeling, the zeta potentials of the particles were measured to be about -32 and -26 mV, respectively. The attachment of 99mTc on the particles was verified by radio-thinlayer chromatography (RTLC), from which we also measured a labeling efficiency of 87%.

Scheme 1. Schematic diagram of the procedure for fabrication of the RGD@AuNPs-Gd99mTc probes. Once fully labeled, the RGD@AuNPs-Gd99mTc were purified using size exclusion filters and size exclusion chromatography with disposable columns containing Sephadex G-25 medium, using saline as the eluent. The purified RGD@AuNPs-Gd99mTc were highly stable in mouse


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plasma, retaining 95% of the initial 99mTc content after 24 h incubation at 37 oC (Figure 1D), similar to previous findings.21 We also prepared particles without 99mTc for use as “cold” probes, labeling instead with technetium (RGD@AuNPs-GdTc) using NaTcO4 as a precursor. The zeta potential of these probes was found to be around -19 mV. The extent of Gd released from these probes in physiologically relevant solutions was investigated by incubating the probes in mouse serum at 37 oC, and as shown in Figure 1D, following a slight burst of Gd released during the first hour in the serum, the rate of Gd detachment slowed considerably. Overall, twenty fours after incubation, we found that 91.5 ± 0.6% Gd remained on the probes. The hydrodynamic sizes of the RGD@AuNPs-GdTc probes in phosphate buffer solution (PBS, pH 7.4) were 52, 94 and 118 nm for the 29, 51, and 80 nm AuNPs, respectively (Figure 1E), and increased slightly once dispersed in mouse serum (Figure S1). However, the probes were stable in both media. By measuring the Gd, Tc, and gold contents by ICP-OES, we determined the number of Gd3+ ions per nanoparticle to be 9800 ± 400, 27500 ± 900,

and 69000 ± 2000 and the number of Tc ions per

nanoparticle to be 1200 ± 200, 6400 ±500, and 11000 ± 400 for the 29, 51, and 80 nm AuNPs, respectively. Also, using the Ellman assay, we determined the number of c(RGDyC) conjugated onto each particle to be 350 ±90, 960 ± 400, and 1500± 200 for the 29, 51, and 80 nm AuNPs, respectively. The longitudinal (r1) relaxivities of these probes at 1.41 T and 37 oC were 10.1, 12.3 and 9.2 s−1 mM−1 for the 29, 51, and 80 nm AuNPs, respectively, which is much higher than that of conventional Magnevist (Gd-DTPA, r1 = 3.6 s-1 mM − 1 at 1.41 T).45 Such relaxivity enhancements of gadolinium complexes tethered to AuNPs have also been observed by others. For example, Coughlin et al. conjugated OPSS-PEG-Gd(DOTA) to a gold nanoshell and


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achieved an r1 relaxivity of 37 mM −1 s −1 at 1.41 T and 37 °C. 46 Similarly, Song et al. coupled Gd-chelates to 30 nm gold nanoparticles via thiol-terminated DNA and observed an r1 value of 20 mM

−1

s −1 under the same measurement conditions.

47

These observed enhancements in r1

relaxivity are likely a result of the restricted molecular tumbling and therefore increased rotational correlation times (τR) of the Gd-chelates once immobilized to nanocarriers. 48 Cytotoxicity of the RGD@AuNPs-Gd99mTc Probes The cytotoxicity of the probes in vitro was evaluated with the cold probes using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) reduction assay. H1299 cells were incubated with the RGD@AuNPs-GdTc probes at different concentrations of particles (10, 50, 100 or 200 µg Au /mL) and the cell viability was assessed after 24 h incubation (Figure 1F). At relatively lower concentrations (10 or 50 µg/mL), the cytotoxicity of the probes was not significant, with cell viability still greater than 85%. However, the cell viability decreased to about 73%, 82%, and 90% at a probe concentration of 100 µg/mL for the 29, 51, and 80 nm probes, respectively, and decreased even further at probe concentrations of 200 µg/mL. These data are in agreement with previous investigations showing that the cytotoxicity of AuNPs is dose-dependent 49, with larger AuNPs exhibiting less cytotoxicity.15, 50 Specificity of the Probes The specificity of the probes for αvβ3 integrin was evaluated using the cold probes. H1299 cells, which are αvβ3 positive,

51

were cultured with media containing

the probes, RGD@AuNPs-GdTc, RAD@AuNPs-GdTc or RGD@AuNPs-GdTc plus free RGD peptide (10 µM), at a concentration of 50 µg Au/mL for 1 h, followed by silver staining. We found that, regardless of the probe size, the cellular uptake of the RGD@AuNPs-GdTc probes was greater than that of the RAD@AuNPs-GdTc probes and that the uptake could be inhibited


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by free RGD peptide (Figure 2A). Further, by quantifying the intracellular gold contents using ICP-OES, we also found that the cellular uptake of the RGD@AuNPs-GdTc probes was indeed more than that of RAD@AuNPs-GdTc probes (p < 0.01) and free RGD peptide could inhibit the uptake (Figure 2B and 2C). Together, these observations demonstrate that the RGD@AuNPsGdTc probes could specifically target αvβ3-positive cells, and the cellular uptake of the probes is

．Specificity of the probes for αvβ3 integrin. (A) Silver staining of the H1299 cells Figure 2． treated with the probes, RGD@AuNPs-GdTc (RGD), RAD@AuNPs-GdTc (RAD) or RGD@AuNPs-GdTc plus free RGD peptide (10 µM) (COM), at a concentration of 50 µg Au/mL


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for 1h. Bar: 10 µm. (B) Intracellular Au contents quantified by ICP-OES. (C) Number of AuNPs per cell. * p < 0.05, ** p < 0.01. mediated at least partially by the αvβ3 integrin.39 However, we also found that the extent of cellular uptake depended on the particle size, with the uptake of the 80 nm probe most efficient (51.9 ± 0.9 pg/cell), followed by the 51 nm (44.6 ± 1.2 pg/cell) and 29 nm (25.8 ± 4.4 pg/cell) probes.


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Figure 3．．Intracellular localizations of the probes and radiotherapy of tumor cells. (A) TEM micrographs of H1299 cells treated with the different sized probes, RGD@AuNPs-GdTc (RGD), RAD@AuNPs-GdTc (RAD) or RGD@AuNPs-GdTc plus free RGD peptide (10 µM) (COM), at a concentration of 50 µg Au/mL for 1h. Arrow: probes internalized by the cells. Bar: 1 µm. (B) Plots of H1299 cell viability after treatment with the 80 nm probes. (C) Comparison of the radiosensitization effects of the RGD@AuNPs-GdTc probes with different sizes. Subcellular Locations of the Probes Previous work has demonstrated that the subcellular location of the AuNPs influences the efficiency of radiosensitization therapy, with particles closer to the cell nucleus exhibiting a higher efficiency.52, 53 To identify the subcellular locations of the probes, H1299 cells were treated with the probes, RGD@AuNPs-GdTc, RAD@AuNPsGdTc or RGD@AuNPs-GdTc plus free RGD peptide (10 µM), at 50 µg Au/mL for 1h and then imaged with TEM. In this way, we found that the RGD@AuNPs-GdTc probes were internalized by the cells and accumulated in cytoplasmic vesicles unevenly distributed near the cell nuclei (Figure 3A, arrow head). Seldom particles were observed at the cell surface. Moreover, consistent with the silver staining and ICP-OES measurements, the cellular uptake of the RGD@AuNPs-GdTc probes was greater than that of the RAD@AuNPs-GdTc probes and this uptake was reduced by the presence of free RGD peptide. Radiotherapy In Vitro To demonstrate the radiosensitization effects in vitro, H1299 cells were treated with the probes, RGD@AuNPs-GdTc, RAD@AuNPs-GdTc or RGD@AuNPsGdTc plus free RGD peptide (10 µM), at 50 µg Au/mL for 1h, and then exposed to 4Gy of γ-ray radiation (Cs-137, 662 keV). The cells were continuously cultured and the cell survival rates, calculated by comparison to a control group without radiation treatment,

40

were measured each

day after exposure. As shown in Figure 3B and Figure S2, for each of the three differently sized probes, both RGD@AuNPs-GdTc and RAD@AuNPs-GdTc probes dramatically decreased the


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survival rates of the H1299 cells. However, the RGD@AuNPs-GdTc probes were found to exhibit greater cytotoxicity and stronger radiosensitization effects than those of the RAD@AuNPs-GdTc probes, and competition with free RGD peptide reduced the effects markedly. Among the RGD@AuNPs-GdTc probes, the 80 nm diameter particles had the strongest radiosensitization effect, followed by the 51 and 29 nm probes (Figure 3C). These results are consistent with a previous report by Chithrani et al., which demonstrated that the efficiency of radiosensitization was dependent on the amount of AuNPs internalized by the cells and the particles with the greatest degree of internalization exhibited the greatest degree of radiosensitization enhancement.28 MR Imaging To optimize the incubation time and probe size for radiation therapy, tumor accumulations of the probes were evaluated in mice using MRI with a 7T MRI scanner. MR imaging was performed after the mice were injected intravenously with the probes, RGD@AuNPs-GdTc, RAD@AuNPs-GdTc or

RGD@AuNPs-GdTc plus free RGD peptide

(100 µL, 0.15 mM), at a dose of 0.1mmol Gd/kg b. w. The T1-weighted MRI images revealed that 30 min after the injection of the RGD@AuNPs-GdTc probes, there are hyper-intense MR signals in tumor regions that are much more pronounced than those of control mice receiving the RAD@AuNPs-GdTc probes (Figure 4A). After this burst period, the signal intensity increased more gradually with time and plateaued 2 h post injection (Figure 4A and 4B). However, for mice co-injected with the free RGD peptide, the signal intensity was dramatically lower. For control mice treated with the RAD@AuNPs-GdTc probes, the increase in the MR signal intensity in the tumor regions was only marginal. To quantify these observations, we determined the relative signal intensity enhancement (rSIE), which is the ratio of the average signal intensity in the tumor regions after probe injection to that prior to injection (Figure 4, left column).


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Figure 4．．T1-weighted MR imaging (left) and relative signal intensity enhancement (right) of the H1299 tumors. The mice were injected intravenously with the probes, RGD@AuNPs-GdTc (RGD), RAD@AuNPs-GdTc (RAD) or RGD@AuNPs-GdTc plus free RGD peptide (100 µL, 0.15 mM) (COM), with a probe size of 29 nm (A), 51 nm (B) or 80 nm(C) at the dose of 0.1mmol Gd/kg b. w.


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Consistent with previous reports, these results showed that the RGD@AuNPs-GdTc probes specifically targeted αvβ3 integrin in tumors. 19, 39, 54 Clearly, since this accumulation was greater than that which can be attributed to the EPR effect, tumor accumulations of the RGD-containing probes were mainly mediated by αvβ3 integrin. Moreover, tumor accumulations of the 29 nm RGD@AuNPs-GdTc probes were greatest, followed by the 51 and 80 nm probes. SPECT/CT Imaging To confirm the MRI observations and measurements and directly quantify probe accumulations in the tumors, we examined mice treated with the particles with SPECT imaging. In particular, mice were injected intravenously with the probes, RGD@AuNPsGd99mTc, RAD@AuNPs-Gd99mTc, or RGD@AuNPs-Gd99mTc plus free RGD peptide, and then radiation dosages of 7.4 MBq were applied. In line with MRI observations, half an hour post injection, SPECT/CT imaging revealed that tumor accumulations of the RGD@AuNPsGd99mTc probes were significant (Figure 5). The radiative signals from these particles in the tumor regions were augmented with time (Figure 5A). Also, as found with MRI, the particle accumulations were reduced in the presence of competing free RGD peptide. Tumor accumulations of the RAD@AuNPs-Gd99mTc probes were marginal (Figure 5A, Figure S3, S4). These observations further confirm that the RGD@AuNPs-Gd99mTc probes can specifically target αvβ3 integrin in vivo.

55

Also, we note that, these images also revealed that in addition to

tumors, both RGD@AuNPs-Gd99mTc and RAD@AuNPs-Gd99mTc probes were also present in other parts of body. After SPECT imaging, the mice were sacrificed and the bio-distributions of the probes, as measured by the percentage of injected dose per gram of tissue (%ID/g), was determined. For both the RGD@AuNPs-Gd99mTc and RAD@AuNPs-Gd99mTc probes, high amounts of


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Figure 5．．SPECT/CT imaging of mice bearing H1299 tumors and the bio-distribution of the probes in vivo. (A) SPECT/CT imaging of mice after intravenous injection with the probes, RGD@AuNPs-Gd99mTc (RGD), RAD@AuNPs-Gd99mTc (RAD) or RGD@AuNPs-Gd99mTc


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plus free RGD peptide (100 µL, 0.15 mM) (COM), at the dose of 7.4 MBq. (B) Bio-distribution of the probes 6 h after injection. ** p < 0.01. radioactivity were found in the spleen and liver, suggesting that these probes are cleared mainly through the reticuloendothelial system (RES), which is slower than renal clearance.56 High accumulations and long term retention of the probes in the RES may induce damage to the normal tissues, thus probes with renal clearance are more desirable.

57

Tumor accumulations of

the RGD@AuNPs-Gd99mTc probes were 14.6 ± 2.1%ID/g, 9.4 ± 0.4%ID/g, and 8.4 ± 1.5%ID/g for the 29, 51, and 80 nm probes, respectively, and decreased to 6.2±1.4 (p < 0.01), 5.1±0.6 (p < 0.01) and 4.9±0.7 %ID/g (p < 0.01), respectively, when also added with free RGD peptide. For the RAD@AuNPs-Gd99mTc probes, the measurements were 4.0±1.4%ID/g, (p < 0.01), 4.4±0.3 %ID/g (p < 0.01), and 3.3±0.6 %ID/g (p < 0.01), respectively, significantly lower than those of RGD probes (Figure 5B, Figure S3, S4). These data suggest that tumor accumulation of the 29 nm RGD@AuNPs-Gd99mTc probes is most efficient and the accumulation culminated 4 h post injection (Figure 5A). The bio-distributions of the probes were also evaluated by examining the gold content (%ID/g) in different organs and in the urine. We found that the gold distributions were similar to those determined by γ-ray counting (99mTc) (Figure 5B, Figure S3, S4). However, a lower amount of gold (%ID/g) was found in urine, compared to the 99mTc measurement. This discrepancy may be due to the detachment of 99mTc from the probes. The findings of gold nanoparticles in the urine may be due to the temporary damage of the kidney by the probes (Figure S5, Table S1). We evaluated the pharmacokinetic properties of the probes using the radiolabeled nanoparticles. By applying a two-compartment model,41 the blood clearance half-lives of the


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RGD@AuNPs-Gd99mTc probes were determined to be 34.7, 26.7, and 17 minutes for the 29, 51, and 80 nm particles, respectively.

Figure 6．．Evaluation of the in vivo efficacy of radiotherapy in mice bearing H1299 tumors. (A) Tumor growth curves following the different treatment modes. Mice were injected intravenously with the 29 nm probe at a dose of 2.5 mmol Au/kg. RGD: RGD@AuNPs-GdTc. RAD: RAD@AuNPs-GdTc. COM: RGD@AuNPs-GdTc plus free RGD peptide (100 µL, 0.15 mM). Radiotherapy was performed 4 h post injection with 10 Gy of γ-ray radiation. Control groups received PBS. (B) Tumor weights three weeks after the treatments. * p < 0.05, ** p < 0.01. (C) Comparison of probe size dependence of the radiosensitization therapy with the RGD@AuNPsGdTc probes.

Radiotherapy In Vivo Due to the high tumor accumulation of the 29 nm RGD@AuNPsGd99mTc probes, we next evaluated the potential of this probe for radiosensitization therapy of tumors. Mice with H1299 tumors were divided into five groups with seven mice in each group


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(control, radiation only, RGD@AuNPs-GdTc + radiation, RAD@AuNPs-GdTc + radiation, and competition + radiation (RGD@AuNPs-GdTc + free RGD peptide)). Four hours post-injection, the tumors were irradiated under 137Cs gamma radiation (662 keV) at a 10 Gy dose, and then the tumor volumes were measured. Three weeks after radiation, the mice were sacrificed and the tumor weights were also measured. Compared to the control and radiation-only groups (which exhibited a tumor volume increase of ≈ 472 % and ≈ 310%, respectively), the tumor growth was significantly suppressed, with tumor volumes increases of only ~ 43% (P < 0.01 vs radiation), ~ 220% (P < 0.05 vs radiation) and ~ 65% (P < 0.01 vs radiation) for the mice treated with the probes, RGD@AuNPs-GdTc, RAD@AuNPs-GdTc and RGD@AuNPs-GdTc plus free RGD peptide, respectively (Figure 6A). In agreement with these measurements, the tumor weights of the control and radiation-only groups were 1.3 ± 0.4 and 1.0 ± 0.3 g, whereas for the mice treated with the probes, RGD@AuNPs-GdTc, RAD@AuNPs-GdTc and RGD@AuNPs-GdTc plus free RGD peptide, the tumor weights decreased to 0.3 ± 0.1 (P < 0.01 vs radiation), 0.6 ± 0.2 (P < 0.05 vs radiation) and 0.6 ± 0.1 g (P < 0.05 vs radiation), respectively (Figure 6B). These in vivo data further confirm the strong radiation enhancement from the RGD@AuNPs-GdTc probes in tumor radiotherapy. We also evaluated the effectiveness of the 51 nm and 80 nm probes as tumor therapy in a similar way and the tumor volumes and weights are presented in Figure S6. Overall, the 29 nm RGD@AuNPs-GdTc probes showed stronger enhancement of radiosensitization than the other two sizes of RGD@AuNPs-GdTc probes (Figure 6C), which is probably attributable to the greater extent of accumulation of the 29 nm probes in the tumors.


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Histological Studies To evaluate the expression of αvβ3 integrin in tumors and confirm the probes targeted the angiogenic vessels of the tumors, we analyzed the tumors from the

Figure 7．．IHC studies of tumor tissues. (A) γ-H2AX immunostaining of tumor tissues from mice treated with the 29 nm probe at a dose of 0.5g Au/kg b. w. in combination with 10 Gy of γray radiation. RGD: RGD@AuNPs-GdTc. RAD: RAD@AuNPs-GdTc. COM: RGD@AuNPs-


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GdTc plus free RGD peptide (100 µL, 0.15 mM). (B) Comparison of DNA damage in tumor tissues induced by the different probes. * p < 0.05, ** p < 0.01. AuNP-treated mice with immunohistochemistry (IHC), using antibodies against αvβ3 integrin and silver-staining to detect AuNPs. As shown in Figure S7, there was indeed high expression of

αvβ3 integrin (CD61) in the tumor angiogenic vessels and, further, these vessels were welltargeted by the RGD@AuNPs-GdTc probes. Co-incubation of the particles with free RGD peptide reduced the probe targeting efficiency, consistent with the aforementioned results. We note that the RAD@AuNPs-GdTc probes were also found in tumors, but to a lesser degree. Finally, to verify and compare the effectiveness of radiosensitization by the three differently sized probes, we examined these tumor tissues with IHC using antibodies against γ-H2AX, which is a generally accepted marker of dsDNA damage.

58

We found that exposure to γ-ray

radiation induced DNA damage (Figure 7A) and that the damage was more pronounced in the presence of the RGD@AuNPs-GdTc probes than that of the RAD@AuNPs-GdTc probes (Figure 7, Figure S8, S9). The fractions of γ-H2AX positive areas were 0.36 ± 0.02, 0.28 ± 0.04 and 0.17 ± 0.05, for the 29, 51, and 80 nm RGD@AuNPs-GdTc probes (Figure 7B), respectively.

Discussion AuNPs have great potential in tumor radiosensitization therapy. However, there are challenges facing the therapy, including how to enhance tumor accumulation of the particles beyond what occurs owing to the EPR effect, while avoiding accumulation in non-target organs (e.g. liver and spleen). Further, it is critical to understand at what time point should the radiotherapy be performed following administration of AuNPs, and the optimal size of AuNPs for therapy. In this


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work, we developed RGD peptide conjugated-Gd and 99mTc labeled-AuNPs probes (RGD@AuNPs-Gd99mTc) with different sizes (29, 51 and 80 nm) for radiosensitization therapy of tumors. We found that the RGD peptide enhanced both cell and tumor accumulation of AuNPs dramatically. We also found that the cellular uptake of the 80 nm RGD@AuNPsGd99mTc probes was most efficient in vitro, whereas in vivo, tumor accumulation of the 29 nm RGD@AuNPs-Gd99mTc probes was most efficient, leading to significant suppression of tumor growth after radiotherapy. Overall, the RGD@AuNPs-Gd99mTc probes thus represent a very promising formulation for guiding and enhancing tumor radiation therapy. To clearly determine the probe size dependence on the effectiveness of radiosensitization, it was essential to synthesize AuNPs with well-defined sizes. Conventionally, AuNPs are synthesized by the reduction of HAuCl4 with sodium citrate,59 which usually leads to size distributions in a wide range due to the secondary nucleation of HAuCl4 during the AuNPs growth. Multi-step seeded growth can produce AuNPs with improved size distribution and monodispersity, but it is time and labor consuming, with low AuNPs yield. 60 In the current study, a one-step seeded growth method was employed, which suppressed self-nucleation by stabilizing a concentrated growth solution with strongly coordinating ligands, leading to precise size control and convenient, scalable fabrication of AuNPs. 33 Targeting tumor angiogenesis through αvβ3 integrin may represent a potential strategy to complement radiotherapy. Angiogenesis is an essential step for the growth and spread of malignant tumors.

61, 62

Tumor cells secrete pro-angiogenic factors to induce and protect

vascular endothelial cells, which in turn supply tumor tissue with oxygen and nutrients.63,

64

AuNPs targeting tumor angiogenic vessels localize radiation damage to tumor-associated endothelial cells, which could break this tumor protective cycle. Thus, with such damage, the


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vasculature would fail to deliver nutrients to the tumor tissue, leading to ischemic necrosis and a reduced production of pro-angiogenic factors. Damage to the tumor microvasculature during radiation therapy may thus be a more effective mechanism for tumor suppression than clonogenic cell death itself. 65 In addition, some kinds of tumor cells, such as H1299 cells used in this study, are also αvβ3 positive. αvβ3-targeted AuNPs that have extravasated from vessels can also target these tumor cells and potently sensitize the cells to radiation therapy. In our study, the RGD@AuNPs-Gd99mTc probes demonstrate high specificity for αvβ3 integrin, which improved the internalization of AuNPs by tumor cells and the accumulation within tumors, leading to higher cell toxicity and more significant inhibition of tumor growth after radiotherapy compared to the scramble peptide conjugated AuNPs. Timing in radiation therapy may be of utmost clinical significance.66 Previous theoretical studies indicate that higher circulating AuNPs levels yield greater vasculature targeted radiotherapy damage;

67, 68

thus, radiotherapy may need to be applied immediately following an

AuNPs administration. By contrast, for intravenously injected AuNPs directly targeting tumor cells, more time would be needed so that the particles can extravasate into tumor stroma via the EPR effect. Therefore, the timing for most effective radiation therapy varies from several minutes to several days after AuNPs administration.

29

We labeled AuNPs with Gd and 99mTc,

which enabled a direct determination of the timing for maximal tumor accumulations of the particles by MRI/SPECT imaging. Tumor accumulations of the probes were apparent 30 min after injection, increased gradually, and largely plateaued 2 h post injection. Therefore, we applied radiation therapy 4 h post probe injection.


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Previous studies have shown that for radiosensitization therapy with AuNPs, the radiation dose enhancement is proportional to the extent of accumulation of the particles in the tumors,29, 31, 69, 70

which is mainly governed by the particle size and surface properties. 15, 21, 71, 72 Hence, in

recent years, AuNPs with various sizes and surface coatings have been explored in tumor radiosensitization therapy, some showing significantly enhanced therapeutic effects. For example, Zhang et al. developed glutathione-coated ultra-small AuNPs (around 2 nm in diameter) that were efficiently accumulated within tumors via the EPR effect and demonstrated enhanced radiosensitization of tumors.

71, 72

In addition, Zhang et al. prepared PEG-coated AuNPs with

relatively larger sizes (4.8, 12.1, 27.3 and 46.6 nm), finding that the 12.1 nm AuNPs exhibited greatest tumor accumulation and thereby produced the greatest radiosensitization effect. 15 However, we note that both ultra-small and larger sized AuNPs have their assets and limitations in radiosensitization therapy. Ultra-small gold nanoparticles are highly effective for radiation dose enhancement, 73, 74 and, due to its small size, the particles readily clear through the renal system, which appears to be the most appropriate way non-degradable nanoparticle are removed from the body since it is the most rapid. Previous studies have also found that ultrasmall gold nanoparticles efficiently accumulate within tumors through the EPR effect.

70-72

However, the accumulation efficiency may vary with different tumor types since the renal clearance minimizes the residential time of the nanoparticles in the blood. Large particles are also effective as radiosensitizers, mainly owing to the low-energy electrons produced from a shell a few nanometers in depth.

74

However the particles are not removed by the renal system

and are readily sequestered by the reticuloendothelial system after intravenous injection, leading to a short blood circulation time. However, the blood half-lives of the particles can be rationally changed from several minutes to more than ten hours, with appropriate surface modification. 1, 75


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Longer blood half-lives promote greater tumor uptake and enhanced tumor-associated vasculature radiotherapy,

66

which should result in greater damage to the tumor than tumor cell-

targeted therapy as discussed above. Unlike previous studies, we functionalized AuNPs with the RGD peptide to specifically target αvβ3 integrin-positive cells and enable higher cellular uptake and tumor accumulations. Among the three different sizes of probes studied here, cellular uptake of the 80 nm probe was found to be most efficient, although tumor accumulation of the 29 nm probe was the greatest. This disparity may be owing to easier sequestration of larger size probes by the reticuloendothelial system, as indicated by their shorter blood half-lives. Therefore, tumor accumulation of active-targeting AuNPs may be a balance between the target specificity and particle size.

Conclusions In summary, novel RGD peptide-conjugated-Gd and 99mTc-labeled AuNPs probes (RGD@AuNPs-Gd99mTc) with three different sizes were fabricated for radiosensitization therapy of tumors. These probes exhibit high specificity for cells expressing αvβ3 integrin, enabling higher accumulation of AuNPs by αvβ3 positive tumor cells in vitro and tumors in vivo. We found that the cellular uptake of the 80 nm RGD@AuNPs-Gd99mTc probes is greater than that of the 51 or 29 nm probes in vitro, whereas tumor accumulation of the 29 nm probe is most efficient in vivo. Further, tumor accumulations of these RGD@AuNPs-Gd99mTc probes can be imaged by MRI/SPECT in real time, which makes it possible to determine the optimal time after administration of the probes at which to apply the radiation. Thus, the RGD@AuNPs-Gd99mTc probes represent a powerful formulation for both guiding and enhancing radiation therapy of tumors.


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AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Tel:+86-21-62933323. Author Contributions: Yi Yang and Lu Zhang contributed equally to this work. Notes: The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (81230030, 81571729), the Grants from the State Key Laboratory of Oncogenes and Related Genes (90-1503) and the Med-Engineering Crossing Foundation of SJTU (YG2012MS15). We thank Professor Weihai Yin and Dr. Ban Wang for their helps in performing immunohistochemical staining of tumor tissues. We appreciate Professor Daniel M. Czajkowsky for improvement in English writing.

Supporting Information Available:

Stability of the probe in mouse serum, in vitro cell

radiotherapy, SPECT/CT imaging of tumors and bio-distribution of 51 and 80 nm probes, kidney function studies, in vivo radiotherapy with 51 and 80 nm probes and histological studies. This material is available free of charge via the Internet at http://pubs.acs.org.


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Table of Contents Graphic


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Tumor Angiogenesis Targeted Radiosensitization Therapy Using Gold

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