Research Article www.acsami.org
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 2016.8:1718-1732. Downloaded from pubs.acs.org by OPEN UNIV OF HONG KONG on 01/23/19. For personal use only.
†
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China ‡ 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 Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, China ⊥ Department of Nuclear Medicine, Shanghai Cancer Center, Fudan University, Shanghai 200032, China # Department of Nuclear Medicine, Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China S Supporting Information *
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 99 mTc-labeled AuNPs (RGD@AuNPs-Gd99 mTc) 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-Gd99 mTc probes specifically target tumors and exhibit greater accumulation within tumors than the RAD@AuNPsGd99 mTc probes. Interestingly, we found that the 80 nm RGD@AuNPs-Gd99 mTc probes exhibit the greatest effects in vitro; however, the 29 nm RGD@AuNPs-Gd99 mTc 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@AuNPs-Gd99 mTc probes are highly useful radiosensitizers capable of guiding and enhancing radiation therapy of tumors. KEYWORDS: gold nanoparticles, radiotherapy, MRI/SPECT, αvβ3 integrin, theranostics
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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, 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
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 delivery, and therapy.1−3 With regard 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 © 2016 American Chemical Society
Received: September 30, 2015 Accepted: January 5, 2016 Published: January 5, 2016 1718
DOI: 10.1021/acsami.5b09274 ACS Appl. Mater. Interfaces 2016, 8, 1718−1732
Research Article
ACS Applied Materials & Interfaces
allowed to grow for 10 min, then collected by centrifugation, and redispersed in water. For synthesis of 29, 51, and 80 nm AuNPs, 200, 20, and 5 μL seed solutions were added, respectively. Fabrication of RGD-Peptide-Conjugated, Gd- and 99 mTcLabeled AuNPs Probe (RGD@AuNPs-Gd99 mTc). For fabrication of the probe, AuNPs were first modified with aminopoly(ethylene glycol)thiol (NH2−PEG−SH; MW 3400; Laysan Bio Inc., Arab, AL, USA). To this end, 12 mg of the AuNPs previously synthesized was dispersed into 1 mL of ethanol, sonicated for 10 min, collected, and then washed with water. Subsequently, the particles were dispersed into the PEG aqueous solution (3 mL, 4 mg/mL), sonicated for 30 min, and then stirred at 4 °C overnight. Next, PEG-modified AuNPs (1 mg) were dispersed into 200 μL of phosphate buffer (PBS, pH 8), then sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC; 50 μL, 1 mM; Pierce, Tianjin, China), 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (NHS-DOTA; 50 μL, 1 mM; Macrocyclics Inc., Dallas, TX, USA), and N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycinate (NHS-MAG3; 50 μL, 1 mM; Pierce) were added simultaneously, and stirred for 30 min. The final AuNPs were retrieved by ultrafiltration with a Millipore centrifugal filter unit (Amicon Ultra0.5; MWCO 100,000), washed three times, and resuspended into 0.5 mL of PBS (pH 7.4). RGD peptide, Gd ,and 99 mTc were then conjugated and labeled onto the particles successively. For RGD peptide conjugation, 100 μg of the peptide was added into the aforementioned 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, 1 mL) 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 h 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 99 mTc 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, 99 mTc− pertechnetate generator eluate (10 μL, 1−5 mCi) 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 evaluate the labeling efficiency, radio-thinlayer chromatography (RTLC; AR2000, Bioscan, Washington, DC, USA) was performed, using acetone as the mobile phase. In this system, 99 mTc-labeled AuNPs remain at the origin, while 99 mTc− 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-Gd99 mTc probe, was purified using size exclusion filters with a 10 kDa molecular weight cutoff (Millipore, Amicon Ultra0.5) and size exclusion chromatography with disposable columns containing Sephadex G-25 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 as those for 99 mTc labeling. At the same time, RADpeptide (c(RADyC))-coupled, Gd- and 99 mTc- or Tc-labeled AuNPs were also prepared as control probes (RAD@AuNPs-Gd99 mTc, RAD@AuNPs-GdTc). Characterizations of the Probes. ζ 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
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. 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)17−19 and nuclear20−22 and fluorescent imaging23,24 have recently been extensively employed in studies of the biodistribution 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 biodistribution 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 the 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 99 mTc-labeled AuNPs probes (RGD@AuNPs-Gd99 mTc) 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 a diameter 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@AuNPsGd99 mTc were internalized by tumor cells most efficiently and thus exhibited the greatest degree of radiosensitization in vitro. However, in vivo, the 29 nm RGD@AuNPs-Gd99 mTc exhibited greater tumor accumulation and thus were ultimately found to be most effective for tumor radiosensitization therapy.
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MATERIALS AND METHODS
Synthesis of Gold Nanoparticles. All of the chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless indicated otherwise. Gold nanoparticles were synthesized with seed-growth method according to a 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 of 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 Lascorbic 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 1719
DOI: 10.1021/acsami.5b09274 ACS Appl. Mater. Interfaces 2016, 8, 1718−1732
Research Article
ACS Applied Materials & Interfaces
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 min, 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 previously. 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 was 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 of 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 min, 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, The Netherlands). Radiotherapy in Vitro. H1299 cells were seeded into several sixwell 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 of Au/ mL for 1 h. After incubation, the cells were exposed to 4 Gy of γ-ray radiation using cesium-137 (Cs-137, 662 keV) beam radiator and then continuously cultured. All of 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
distribution was calculated using 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 °C 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, technetium, and gold concentrations were determined using inductively coupled plasma optical emission spectrometry (ICP-OES; ICAP-6300, Thermo Fisher, Portsmouth, NH, USA). 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 eqs 1 and 2 assuming the ideal spherical shape of the particles. They are expressed as
N = m/vs
(1)
v = 4πr 3/3
(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 eq 3, in which m is the mass of gadolinium or technetium in the solution (g mL−1) and M is the mass of the gadolinium or technetium atom.
n = m/M
(3)
Moreover, the conjugation efficiency of RGD peptide was also evaluated with the 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 peptides 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 37 °C for different periods of time. After 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 99 mTc was assessed in triplicate by co-incubating 5 μL of the sample in 200 μL of fresh mouse plasma at 37 °C for different periods of time. After incubation, the probes were collected by centrifugation and the radioactivity retained on the particles was counted in a γ well counter. Stability of 99 mTc 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-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays using RGD@AuNPsGdTc probe. For this purpose, H1299 cells, a nonsmall-cell-lungcancer cell line, were seeded in a 96-well plate with 1 × 104 cells/well and incubated with the probe at different concentrations (10, 50, 100, and 150 μg of 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/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 cocultured with RGD@AuNPs-GdTc, RAD@AuNPs-GdTc, or RGD@AuNPs-GdTc plus free RGD peptide (c(RGDyC); 10 μM)38,39 at the concentration of 50 μg of Au/mL for 1 h. After incubation, the culture media were removed and cells were fixed with 4% paraformaldehyde. Silver
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MR IMAGING OF TUMORS The 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 c(RGDyC) (100 μL, 0.15 mM)38,39 intravenously at the dose of 0.1 mmol of Gd/(kg of body weight (b.w.)) with five mice in each group. MR imaging was conducted using a 7 T MRI scanner (Biospec System 70/20, Bruker, Ettlingen, Germany) using a T1weighted spin−echo sequence (repeat time (TR) = 1500 ms, echo time (TE) = 6 ms, field of view (FOV) = 35 × 35 mm, matrix = 256 × 256, and slice thickness = 1 mm) and T1mapping sequence (TR = 3000 ms, TE = 15 ms, and inversion delays of 500, 1000, 1500, 2000, 2500, 3000, and 3500 ms). Pre- and postcontrast 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 preinjection. SPECT/CT Imaging. Mice bearing H1299 tumors were intravenously injected with 0.1 mL (7.4 MBq, 10 mM in gold) of RGD@AuNPs-Gd99 mTc, RAD@AuNPs-Gd99 mTc, or RGD@AuNPs-Gd99 mTc plus free RGD peptide (100 μL, 1720
DOI: 10.1021/acsami.5b09274 ACS Appl. Mater. Interfaces 2016, 8, 1718−1732
Research Article
ACS Applied Materials & Interfaces
Figure 1. Characterizations and cytotoxicity of the probes. (A−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 99 mTc and Gd on the probes in mouse serum. (E) Stability of the RGD@AuNPs-GdTc 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.
injected dose per gram of tissue (% ID g−1) of gold in a specific tissue was calculated. Determination of Blood Half-life of RGD@AuNPsGd99 mTc Probes. Five mice each received 7.4 MBq (0.1 mL) of RGD@AuNPs-Gd99 mTc, and blood samples were collected from a tail vein before and 2, 5, 10, and 30 min and 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 was determined. The half-life of the temporal course of the %ID/g was fitted to a twocompartment 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@AuNPsGdTc + free RGD peptide) + radiation. Each group included seven mice. Control groups received pure PBS (pH = 7.4). The probes were injected intravenously with a dose of 2.5 mmol of Au/(kg of b.w.). Four hours postinjection, the tumors were exposed to 10 Gy of γ-ray radiations (Cs-137, 662 keV). All of 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)ABC, 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@AuNPs-Gd99 mTc targeting tumor
0.15 mM) with five mice in each group. SPECT/CT scans were obtained using a small-animal imaging system (Bioscan). The CT images were used to provide anatomical references to the tumor location. The SPECT images were obtained at 32 projections over 360 °C (radius of rotation = 7.6 cm, 30 s/ projection). Reconstructed data from SPECT and CT were visualized and co-registered using InVivoScope (Bioscan). Biodistribution 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 welltype 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 previously and in urine. For this purpose, tumor bearing mice were grouped and injected with the probes at the doses exactly the 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 1721
DOI: 10.1021/acsami.5b09274 ACS Appl. Mater. Interfaces 2016, 8, 1718−1732
Research Article
ACS Applied Materials & Interfaces angiogenic vessels, co-staining of αvβ3 integrin and AuNPs was performed. For this purpose, tumors were removed and imbedded in OCT glue (Miles, Elkhart, IN, USA), 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 °C and then air-dried for 30 min. Silver staining was first performed using a silver enhancement kit (ProteoSilver Silver Solution, Sigma-Aldrich), and the staining procedure was the same as that for cell staining. For αvβ3 integrin staining, the sections were treated with a primary rat antimouse CD61 monoclonal antibody (1:50 dilution; BD Biosciences, San Diego, CA, USA) and a biotinylated goat antirat IgG (BD Bioscience) 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. Immuno-histochemical 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 5 μm 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 antiphosphohistone γH2AX primary antibody (2 μg/mL; Millipore, Billerica, MA, USA) containing 1% BSA at 4 °C overnight. After washing with PBS (pH = 7.4) three times, the slices were incubated with Alexa Fluor 488 goat antirabbit secondary antibody (1:300 dilution; Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. Cell nuclei were counterstained with DAPI. Quantitative analysis of the γ-H2AX positive area was conducted by a fluorescent microscope (Leica DM2500, Houston, TX, USA) using the Image-Pro Plus software. To this end, at least three randomly selected vision fields for each sample and a total of three samples were analyzed. The histological study and function examination of kidney were also performed 6 h after intravenous injection of RGD@ AuNPs-Gd99 mTc 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 hematoxylin and eosin (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 ± standard deviation (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
