Research Article www.acsami.org
A Novel Strategy through Combining iRGD Peptide with TumorMicroenvironment-Responsive and Multistage Nanoparticles for Deep Tumor Penetration Xingli Cun, Jiantao Chen, Shaobo Ruan, Li Zhang, Jingyu Wan, Qin He, and Huile Gao* Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, China S Supporting Information *
ABSTRACT: Despite the great achievements that nanomedicines have obtained so far, deep penetration of nanomedicines into tumors is still a major challenge in tumor treatment. The enhanced permeability and retention (EPR) effect was the main theoretical foundation for using nanomedicines to treat solid tumor. However, the antitumor efficiency is modest because the tumor is heterogeneous, with dense collagen matrix, abnormal tumor vasculature, and lymphatic system. Nanomedicines could only passively accumulate near leaky site of tumor vessels, and they cannot reach the deep region of tumor. To enhance further the tumor penetration efficiency, we developed a novel strategy of coadministering cell-homing penetration peptide iRGD with size-shrinkable and tumor-microenvironment-responsive multistage system (DOX-AuNPs-GNPs) to overcome these barriers. First, iRGD could specifically increase the permeability of tumor vascular and tumor tissue, leading to more DOX-AuNPs-GNPs leaking out from tumor vasculature. Second, the multistage system passively accumulated in tumor tissue and shrank from 131.1 to 46.6 nm to reach the deep region of tumor. In vitro, coadministering iRGD with DOX-AuNPs-GNPs showed higher cellular uptake and apoptosis ratio. In vivo, coadministering iRGD with DOX-AuNPs-GNPs presented higher penetration and accumulation in tumor than giving DOX-AuNPs-GNPs alone, leading to the best antitumor efficiency in 4T1 tumor-bearing mouse model. KEYWORDS: iRGD, size-shrinkable, multistage, deep penetration, tumor microenvironment sensitive
1. INTRODUCTION In recent years, nanomedicines have been widely used to diagnose and treat many kinds of diseases, such as cancer and inflammation for their targeting drug delivery ability and low side effects.1,2 Many of them could passively accumulate in solid tumor tissue through the enhanced permeability and retention (EPR) effect. However, tumor is heterogeneous with dense matrix, pathological blood and lymphatic networks, abnormal vascular barrier, and interstitial barrier.3 This microenvironment hinders the effective delivery of nanomedicines into tumor, in particular into the deep region far from the vasculature.4,5 Generally, systemically delivering nanomedicines to solid tumors needs three steps. First, nanomedicines have to reach tumor tissue through blood circulation. Next, nanomedicines must pass through the blood vessel wall and eventually pass through the tumor stroma to kill tumor cells. Thus, nanomedicines must have long circulation time to effectively penetrate the tumor vessel walls and interstitial space to achieve efficient delivery and deep penetration.3,6 There are many strategies for reaching optimal diffusion efficiency through design, such as designing tumor-microenvironment-responsive size-shrinkable nanocarriers, coinjecting drugs to decrease tumor interstitial pressure, and conjugating tumor-homing © XXXX American Chemical Society
and penetration ligands to improve their attack to tumor cells.6−8 To improve further the tumor targeting and penetration of nanomedicines, we proposed a novel strategy of coadministering tumor homing and penetration peptideiRGD with tumor-microenvironment-sensitive multistage nanoparticles for deep tumor penetration. iRGD first targets αvβ3 integrin receptors in the tumor vasculature and tumor cells by the RGD fragment. By its binding, a new binding motif specific for neuropilin-1 (NRP-1) was exposed through proteolytic cleavage of iRGD. This series of processes specifically enhanced the permeability of tumor blood vessels and tumor cells.9−11 Systemic injection with iRGD could improve tumor penetration of various compositions,12,13 and it has been suggested that systemic injection of iRGD could specifically increase the permeability of tumor vascular and tumor tissue, through interactions with integrin and NRP-1, the peptide increased drug penetration and thus improved its efficiency.3,12,14 More importantly, simple coadministration of iRGD was more effective in delivering Received: October 4, 2015 Accepted: November 25, 2015
A
DOI: 10.1021/acsami.5b09391 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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formulations in tissues. Then, tumor treatment was carried out on 4T1 tumor-bearing mice.
therapeutic agents into tumor parenchyma than was conjugation with nanoparticles.15 Though the travel of nanoparticles across tumor vessels has improved, the travel through tumor interstitial space was still hindered. The diffusion of nanoparticles in solid tumor interstitial space was affected by many factors, and particle size was considered as the most important one.16,17 Generally, compared with large-sized nanoparticles, small-sized nanoparticles could penetrate deeper in tumor tissue,3,18,19 but small-sized nanoparticles are easily rapidly cleared into other tissues because of fast migration rate, leading to poor accumulation and retention. On the contrary, the relatively low permeation rate of large-sized nanoparticles through the tumor matrix could contribute to better retention, but it cannot reach the deep location in tumor owing to dense tumor stroma and high interstitial fluid pressure in tumor.20 It is hard for nanoparticles with a fixed particle size to reach all areas of the tumor and obtain mass accumulation.7,21 Large-sized particles could not extravasate distant from the blood vessels, whereas small-sized particles could penetrate deep into the tumor but could not remain long.19 Therefore, the size of the particles should be relatively large to acquire a favored plasma half-life and selective penetration ability, but the particles need to be small enough for the effective deep tumor penetration so that they could enter the tumor interstitial space.22,23 It has been shown that the penetration depth and accumulation quantity could be controlled by changing the particle size of the nanocarriers’ to overcome the barriers in the tumor.16 Recently, the size-shrinkable multistage system DOX-AuNPs-GNPs was established by our lab. A pH-sensitive doxorubicin (DOX) fragment was anchored onto small-sized gold nanoparticles (AuNPs) to obtain pH-sensitive DOX-AuNPs; then, DOXAuNPs were modified on large-sized gelatin particles (GNPs) to obtain 131 nm size-shrinkable DOX-AuNPs-GNPs. The particle size of DOX-AuNPs-GNPs could shrink from over 130 nm to less than 50 nm through degradation by tumor matrix metalloproteinase-2 (MMP-2, an enzyme that is overexpressed in a variety of tumors).24,25 The 131 nm DOX-AuNPs-GNPs may benefit from their large size to accumulate around the tumor vasculature. Then, the local tumor MMP-2 degraded the large GNPs, releasing small-sized AuNPs to reach the deeper region of tumor.7,21 Thus, this system could satisfy the controversial requirement of tumor retention and penetration based on particle size, resulting both good tumor retention and high tumor penetration. Although the DOX-AuNPs-GNPs with size-shrinkable properties presented enhanced penetration and retention efficiency in tumor tissue, the penetration and accumulation of nanoparticles into deep region of tumor is still modest because of poor transport across the tumor vessel walls and lack of tumor-targeting ability. Thus, to enhance further the deep penetration in tumor tissues, we established a strategy through combining tumor-targeting and penetration peptide iRGD with tumor-microenvironment-sensitive multistage DOX-AuNPsGNPs for deep tumor penetration. This additional coadministration strategy greatly simplifies the path to clinical application, thereby providing a versatile way to enhance the antitumor efficiency.12 In this study, cellular uptake, cell toxicity, and cell apoptosis were tested on NRP-1 and αvβ3 receptors overexpressed 4T1 cells in vitro.12,26−28 In vivo, whole tissue imaging and slice confocal imaging were used to evaluate distribution of different
2. EXPERIMENTAL SECTION 2.1. Materials. iRGD was synthesized by Sango Biotech Co, Ltd. (Shanghai, China). Rabbit anti-integerin beta-3 was obtained from Abcam Ltd. (Hong Kong, China). Annexin V-FITC/PI apoptosis detection kit was obtained from Dojindo Laboratories (Shanghai, China). 4′-6-Diamidino-2-pheylindole (DAPI) and 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Beyotime Institute Biotechnology (Haimen, China). Gelatin type A was obtained from MP Biomedicals (Santa, USA). Rabbit anti-CD34 polyclonal antibody was obtained from Immunoway Biotechnology Co. (Newark, NJ, USA). Thiol-polyethylene glycol (PEG-SH, MW = 5000) was obtained from Laysan Bio, Inc. (Arab, AL, USA). Auric chloride acid was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Alexa Fluorescence 594-conjugated donkey anti-rabbit secondary antibody and Cy3-conjugated donkey anti-rabbit secondary antibody were purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA, USA). Doxorubicin hydrochloride was obtained from Beijing Huafeng United Technology Co., Ltd. (Beijing, China). Plastic cell culture dishes and plates were purchased from Wuxi NEST Biotechnology Co Ltd. (Wuxi, China). Rabbit antiNRP-1 antibody was purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). N-hydroxy-succinimide (NHS) and 1-[3(dimethylaminopropyl)]-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Keddia Reagent (Chengdu, China). The 4T1 cells were obtained from Chinese Academy of Sciences Cell Bank (Shanghai, China). 2.2. Animals. Female Balb/c mice (6 weeks old) were bought from Jiang’an Animal Center of Sichuan University (Chengdu, China). All animal experiments in this study were approved by the Laboratory Animal Management Committee of Sichuan University. 2.3. Synthesis and Characterization of pH-Sensitive-Fragment-Modified DOX. pH-sensitive-fragment-modified DOX was synthesized according to the method described previously.29 First, S(3-oxopropyl) ethanethioate was prepared. Second, the S-(3oxopropyl) ethanethioate was oxidized by sodium perchlorate to obtain 3-(acetylthio) propanoic acid. Third, 3-(acetylthio) propanoic acid reacted with hydrazine to get S-(3-hydrazinyl-3-oxopropyl) ethanethioate. Finally, the product was bonded on DOX via pHsensitive hydrazone. After a deacetylation reaction, pH-sensitive SH-RHyz-DOX was finally obtained. The product of each step was characterized by 1H NMR spectroscopy. 2.4. Preparation and Characterization of DOX-AuNPs and DOX-AuNPs-GNPs. AuNPs and GNPs were prepared according to the method described previously.29,30 The preparation of DOX-AuNPs was consistent with our previous studies.29 To prepare DOX-AuNPsGNPs, EDC and NHS were added to activate the carboxyl group of DOX-AuNPs. After centrifugation at 12 000 rpm for 10 min, free DOX and excessive EDC and NHS were removed. The precipitate was resuspended in 1 mL of GNPs and reacted at 37 °C for 8 h to obtain the DOX-AuNPs-GNPs. Data of particle size and zeta potential were obtained by a Zetasizer (Malvern, UK). The photos of AuNPs, AuNPs-GNPs, and AuNPsGNPs incubated with MMP-2 were recorded via transmission electron microscopy (TEM) (JEOL Ltd., Japan). 2.5. DOX Release Experiment. Spectrofluorophotometer (Shimadzu, Japan) was used to test the in vitro release of DOX from DOXAuNPs and DOX-AuNPs-GNPs. DOX-AuNPs and DOX-AuNPsGNPs were resuspended in PBS at pH 7.4 and 5.0 respectively. The fluorescent spectra of DOX from different formulations were detected at different time points in pH 7.4 and 5.0. Control was 4.9 μM free DOX. Light must be avoided during the whole experiment. 2.6. Cellular Uptake. 4T1 cells of ogarithmic growth phase were inoculated in 6-well plates at a density of 1 × 105 cells per well. After incubation for 24 h, DOX-AuNPs, DOX-AuNPs-GNPs, and iRGD + DOX-AuNPs-GNPs were added to the plates, respectively, with the same 10 μg/mL dose of DOX. The iRGD + DOX-AuNPs-GNPs B
DOI: 10.1021/acsami.5b09391 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 1. 1H NMR spectra of products of each step. (A) Spectra of S-(3-oxopropyl) ethanethioate. (B) Spectra of 3-(acetylthio) propanoic acid. (C) Spectra of S-(3-hydrazinyl-3-oxopropyl) ethanethioate. (D) Spectra of SH-R-Hyz-DOX. a−g represent the magnetic displacement of representative functional groups in each compound, respectively. group had iRGD added at a concentration of 43.3 μg/mL at the same time. After culture for 1 h, the cells were harvested. A flow cytometer (Beckman Coulter, USA) was used to measure the fluorescence intensity of different groups. For qualitative experiments, 4T1 cells were plated on a cover slide in 6-well plates at a density of 1 × 105 cells per well. After culture for 24 h, DOX-AuNPs, DOX-AuNPs-GNPs, and iRGD + DOX-AuNPsGNPs were added as described in previous steps. After incubation for 1 h, the cells were washed and fixed at room temperature. Then, DAPI (0.5 μg/mL) was added to stain the nuclei. Finally, the coverslips were placed on the slides for fluorescent imaging by a confocal microscope (Olympus, USA). 2.7. Apoptosis and Cell Toxicity Study in Vitro. To evaluate the cell apoptosis induced by DOX-AuNPs, DOX-AuNPs-GNPs, and iRGD + DOX-AuNPs-GNPs groups, 4T1 cells were plated as in section 2.6 and then treated with DOX-AuNPs, DOX-AuNPs-GNPs, and iRGD + DOX-AuNPs-GNPs. The concentration of DOX was 6.7 μg/mL, and the concentration of iRGD was 28.8 μg/mL. After incubation for 24 h, the cells were harvested and stained with Annexin V-FITC and PI.20 Finally, the stained cells were used to analyze the cell apoptosis in different groups by flow cytometer (Beckman Coulter, Brea, CA, USA). MTT assay was used to investigate the cytotoxicity of DOX-AuNPs, DOX-AuNPs-GNPs, and iRGD + DOX-AuNPs-GNPs. 4T1 cells were plated and cultured as in previous studies.31 DOX, DOX-AuNPs, and DOX-AuNPs-GNPs were added into each well. The final concentrations of DOX ranged from 0.082 to 20.0 μg/mL. The iRGD + DOX-AuNPs-GNPs group was coincubated with iRGD (0.363−88.6 μg/mL). After incubation for 24 h, the cytotoxicity of these formulations was measured by MTT.32 2.8. In Vivo and Ex Vivo Imaging. Tumor-bearing Balb/c mice were established as previously stated.33 Two weeks after implantation, mice were randomly divided into DOX, DOX-AuNPs, DOX-AuNPsGNPs, and iRGD + DOX-AuNPs-GNPs groups (3 mice per group). Mice were treated with the corresponding drug at the dose of DOX 3 mg/kg; the mice of iRGD + DOX-AuNPs-GNPs group were coinjected with iRGD at a final dose of 4 μM/kg. The mice in each group were photographed by IVI Spectrum system (Caliper, USA) 24
h after the injection. After being imaged, the mice were sacrificed. Tumors and all major tissues were collected and imaged as well. 2.9. In Vivo Tumor and Tissue Distribution. The major tissues of mice obtained in section 2.8 were collected and fixed with 4% (w/v) paraformaldehyde for immunofluorescence study. After dehydrating with 15% (w/v) glucose followed with 30% (w/v) glucose, all of these tissues were sectioned at a thickness of 16 μm using a freezing microtome (Leica, Germany). DAPI (0.5% μg/mL) was used to stain the nuclei, Cy3-labled donkey anti-rabbit secondary antibody and rabbit anti-mouse anti-CD34 antibody were used to stain tumor vasculature, rabbit anti-mouse anti-α v β3 antibody and Alexa Fluorescence 594-conjugated donkey anti-rabbit secondary antibody were used to stain αvβ3, and Cy3-labeled donkey anti-rabbit secondary antibody and rabbit anti-mouse anti-NRP-1 antibody were used to stain NRP-1. Then, the slices were observed by a confocal microscope. To evaluate further distribution of the formulations in vivo, the major tissues of 3 mice in each group were collected, dried, weighed, homogenized, ultrasonically extracted by chloroform/methanol = 4:1 for 1 h, and then centrifuged for 5 min at 2000 rpm. Next, a lower solution was obtained and dried in room temperature. The samples were tested on HPLC (Prominence, Japan). Subcutaneous 4T1 tumors were removed 24 h after injection to observe the distribution of DOX-AuNPs-GNPs and iRGD + DOXAuNPs-GNPs by TEM. The removed tumor tissues were diced, fixed, and sliced to observe.34 2.10. Antitumor Effect. Tumor-bearing mice were randomly divided into saline, DOX, DOX-AuNPs, DOX-AuNPs-GNPs, and iRGD + DOX-AuNPs-GNPs groups (n = 6). Saline group was treated with saline appropriately. Other groups treated with corresponding drugs at the dose of DOX 3 mg/kg; the mice of iRGD + DOX-AuNPsGNPs group were coinjected with iRGD at a final dose of 4 μM/kg every 2 days for a total of 7 doses. Mice were sacrificed immediately after the last injection. Then, tumors and hearts were sampled and hematoxylin and eosin (HE) staining and TUNEL staining were applied. 2.11. Statistical Analysis. All values were presented as mean ± SD. Two-tailed Student’s t test was used to measure the statistically significant. P < 0.05 was considered to be a statistically significant difference. C
DOI: 10.1021/acsami.5b09391 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 2. TEM images of (A) DOX-AuNPs-GNPs, (B) DOX-AuNPs-GNPs incubated with MMP-2 (300 ng/mL) for 24 h, and (C) DOX-AuNPs. DLS data of (D) DOX-AuNPs-GNPs, (E) DOX-AuNPs-GNPs incubated with MMP-2 (300 ng/mL) for 24 h, and (F) DOX-AuNPs.
Figure 3. Fluorescence spectra of DOX-AuNPs at (A) pH 7.4 and (B) pH 5.0. Fluorescence spectra of DOX-AuNPs-GNPs at (D) pH 7.4 and (E) pH 5.0. Cumulative release of (C) DOX-AuNPs and (F) DOX-AuNPs-GNPs at pH 7.4 and 5.0.
3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Modified DOX. 1 H NMR spectra of products in each step was presented in Figure 1. The representative functional groups could be found in the spectra, suggesting the products were synthesized successfully. 3.2. Preparation and Characterization of Different Nanoparticles. The particle size of GNPs and AuNPs was 91.6 and 20.9 nm at the beginning. After being modified by DOX, the particle size of DOX-AuNPs and DOX-AuNPsGNPs was 32.4 nm (Figure 2C,F) and 131.1 nm (Figure 2A,D). The increase of DOX-AuNPs-GNPs particle size was mainly because of the connection of DOX-AuNPs to GNPs. The typical TEM image of DOX-AuNPs-GNPs further indicated that the DOX-AuNPs-GNPs were successfully fabricated (Figure 2A). The particle size of DOX-AuNPsGNPs shrank to 46.6 nm after incubation with MMP-2, which was demonstrated by TEM and DLS (Figure 2B,E). The obvious degradation of gelatin of DOX-AuNPs-GNPs was showed in TEM images; this size-shrinkable ability could lead
to tumor deep penetration, which has been indicated in an early study.6 The detailed particle size and zeta potential were listed in Table S1. 3.3. In Vitro Release. Fluorescence scanning was carried out to measure the release of DOX from these formulations. The fluorescence of DOX was quenched when it was connected on the surface of AuNPs because of the nanosurface energy transfer.35,36 Therefore, the release of DOX from different formulations in vitro could be determined via measuring the fluorescence intensity of DOX. As shown in Figure 3, all the groups presented a very low fluorescence intensity of DOX at 0 h, suggesting that DOX did not break down from the AuNPs. With the extension of time, the fluorescence intensity of DOX on DOX-AuNPs and DOX-AuNPs-GNPs increased accordingly at both pH 7.4 and 5.0, suggesting that release of DOX was time-dependent. In addition, as the incubation time was prolonged, the fluorescence intensity of both DOX-AuNPs and DOX-AuNPs-GNPs at pH 5.0 compared to those at pH 7.4 was significantly elevated, indicating that hydrolysis of hydrazine occurred in a pH-dependent manner.37 Furthermore, D
DOI: 10.1021/acsami.5b09391 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 4. Cellular uptake and apoptosis. (A) Confocal images of cellular uptake on 4T1 cells after being treated with DOX-AuNPs, DOX-AuNPsGNPs, and iRGD + DOX-AuNPs-GNPs for 1 h. (B) Cellular uptake of different groups on 4T1 cells for 1 h, measured by flow cytometer. (C) Percentage of apoptotic and necrotic cells of treated 4T1 cells induced by DOX-AuNPs, DOX-AuNPs-GNPs, and iRGD + DOX-AuNPs-GNPs tested by Annexin V-FITC and PI staining assay. Bar represents 50 μm. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, among the marked groups and the iRGD + DOX-AuNPs-GNPs group.
Figure 5. Distribution of different nanoparticles in tumor and normal tissues. (A) 4T1 tumors, 24 h postinjection with DOX, DOX-AuNPs, DOXAuNPs-GNPs, and iRGD + DOX-AuNPs-GNPs. (B) Semiquantitative distribution of tumors. (C) Ex vivo images of heart, liver, spleen, lung, and kidney. (D) Quantitative distribution of DOX in tumor and normal tissues. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, among the marked groups and iRGD + DOX-AuNPs-GNPs group.
the 48 h cumulative release ratio from DOX-AuNPs and DOXAuNPs-GNPs was 73.2 and 85.2%, respectively, under pH 5.0, whereas that under pH 7.4 was only 13.6 and 19.4%, respectively (Figure 3C,F). Because the pH of endosome/ lysosome is nearly 5.0, the pH-responsive property enabled DOX be specifically and almost completely released in tumor cells after the internalization of the small-size DOX-AuNPs,
which is favorable for the efficient delivery of DOX into tumor cells.38 These results were similar to our early researches.7,21,29 3.4. Cellular Uptake and Apoptosis. To study the homing and cell penetration ability of iRGD peptide that was cotreated with particles, cellular uptake study was carried out. Both confocal images (Figure 4A) and flow cytometer data (Figure 4B) suggested that DOX-AuNPs presented higher uptake efficiency than DOX-AuNPs-GNPs; this could be E
DOI: 10.1021/acsami.5b09391 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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determine the formulation’s target effect and particle’s distribution in tumor. As showed in Figure 6, the accumulation of DOX-AuNPs group was much greater than that of free DOX because DOX-AuNPs benefited from the EPR effect. However, compared with DOX-AuNPs, DOX-AuNPs-GNPs displayed
attributed to the fact that cellular uptake of cells is in an sizedependent manner and that small-sized DOX-AuNPs contributed to a higher intracellular uptake efficiency than the larger DOX-AuNPs-GNPs.5 When coincubating with iRGD, the fluorescence intensity of DOX was significantly higher than that of DOX-AuNPs and DOX-AuNPs-GNPs, indicating that cellular uptake of DOX-AuNPs-GNPs was extremely enhanced. In addition, similar results were obtained with regard to apoptosis (Figure 4C). After incubation for 24 h, the percentage of apoptotic and necrotic cells of the DOXAuNPs, DOX-AuNPs-GNPs, and iRGD + DOX-AuNPs-GNPs groups was 94.6, 91.0, and 97.5%, respectively. DOX-AuNPs group showed higher percentage of apoptosis and necrotic cells than that of DOX-AuNPs-GNPs, but when coadministering iRGD with DOX-AuNPs-GNPs, the iRGD + DOX-AuNPsGNPs group displayed the highest percentage of apoptosis and necrotic cells. The apoptosis result was consistent with the MTT result (Figure S1). According to the MTT assay, all of the formulations were cytotoxic to 4T1 cells, and the cell apoptosis rate was proportional to the concentration of DOX. Among these formulations, the iRGD + DOX-AuNPs-GNPs group showed lower cell viability than that of other groups in a series of concentrations. This was owing to αvβ3 and NRP-1 receptors being overexpressed in 4T1 cells: iRGD could home to tumor cells through the αvβ3 receptor; then, a motif with C-terminal was shown to NRP-1 receptor after protease hydrolysis on cell membrane surface.39 Next, the endocytic bulk transport pathway was triggered by NRP-1-mediated “bystander effect”; thus, the cellular uptake of nanocarriers that was simply administered together with iRGD was enhanced.10,40 The enhanced cellular cytotoxicity may be due to the increase of DOX-AuNPs-GNPs’ uptake efficiency when coadministering with iRGD.41 3.5. Ex Vivo Imaging. To validate the tumor-targeting and -penetrating ability of different formulations, ex vivo and in vivo imaging was used. The iRGD + DOX-AuNPs-GNPs group showed the highest accumulation efficiency and displayed the strongest fluorescent intensity in tumor compared to those of other groups (Figures 5A and S2), which was supplemented by semiquantitative data obtained from tumors (Figure 5B), indicating that DOX-AuNPs-GNPs could be efficiently delivered to 4T1 tumor tissue. In addition, the distribution of normal tissue showed that liver was the major metabolic organ of DOX-AuNPs-GNPs when coadministering with iRGD (Figure 5C). These results were consistent with the quantitative distribution of 4T1 tumor (Figure 5D). The DOX concentration in tumor of iRGD + DOX-AuNPs-GNPs group (0.53 μg/g) was 43 and 71% higher than that of the DOX-AuNPs-GNPs group (0.37 μg/g) and DOX-AuNPs group (0.24 μg/g), respectively. When coadministering with free iRGD peptide, the penetration of DOX-AuNPs-GNPs from tumor vasculature and tumor cells was increased, owing to overexpression of αvβ3 and NRP-1.3,11,12,14 The DOX-AuNPsGNPs were degraded in the tumor interstitial space by overexpressed MMP-2 and released small-sized DOX-AuNPs when the nanoparticles spilled from blood vessels. Then, the small sized DOX-AuNPs penetrated to the deep region of tumor, leading to the highest accumulation in tumor tissue.7 All of these results confirmed that iRGD could home to 4T1 tumor tissue and significantly enhance DOX-AuNPs-GNPs’ accumulation in 4T1 tumor. 3.6. In Vivo Slice Distribution. Tumor slices were stained with anti-NRP-1, anti-αvβ3 and anti-CD34 antibody to
Figure 6. Fluorescence distribution of DOX, DOX-AuNPs, DOXAuNPs-GNPs, and iRGD + DOX-AuNPs-GNPs in 4T1 tumors at 24 h after tail injection for in vivo imaging. Tumor tissues were stained with (A) anti-NRP-1, (B) anti-αvβ3, and (C) anti-CD34 antibodies; bar represents 40 μm. F
DOI: 10.1021/acsami.5b09391 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 7. Antitumor effect. (A) Tumor weight after treatment. (B) 4T1 tumor images at the end of treatment. (C) Average tumor volumes during the treatment; *, P < 0.05; **, P < 0.01; and ***, P < 0.001, among the marked groups and iRGD + DOX-AuNPs-GNPs group. (D) HE staining and TUNEL staining of tumor slices from different groups; the red arrows point to the dyed typical apoptotic bodies.
higher fluorescence, indicating that the size-shrinkable DOXAuNPs-GNPs may benefit from its large size, being retained more in tumors than are small-sized DOX-AuNPs.7 Furthermore, iRGD + DOX-AuNPs-GNPs group presented the highest fluorescence and best accumulation in deep tumor region (Figure 6). Because of overexpression of αvβ3 and NRP1 in 4T1 tumor (Figure 6A,B), iRGD peptide could increase the permeability of tumor vasculature and tumor cell, leading more DOX-AuNPs-GNPs being spilled from the vessels and shrunk to obtain deep tumor penetration. Tumor neovessels were stained by anti-CD34 antibody to evaluate the tumor penetration ability. Because of enhanced access to the extravascular of DOX-AuNPs-GNPs, more nanoparticles in iRGD + DOX-AuNPs-GNPs group could shrink from large to small size to reach the deep location of tumor where was far away from the anti-CD34-stained neovessels. Furthermore, because the penetration of tumor cell was enhanced by iRGD peptide, obviously more internalization of nanoparticles and DOX release was found in the iRGD + DOX-AuNPs-GNPs group than in the DOX-AuNPs-GNPs group. The distribution of all the formulations was also evaluated in normal tissues (Figure S3). The DOX-AuNPs, DOX-AuNPs-GNPs, and iRGD + DOX-AuNPs-GNPs groups showed lower distribution in hearts than did the free DOX treated group, indicating that the heart toxicity of DOX could be decreased by functionalizing onto AuNPs and AuNPs-GNPs (Figure S4). Furthermore, the DOX-AuNPs showed higher distribution in lung than did other groups, maybe owing to the smaller size.42 TEM was used to determine the in vivo distribution of DOXAuNPs-GNPs and iRGD + DOX-AuNPs-GNPs (Figure S5). Consistent with the fluorescent distribution, there were few
AuNPs-GNPs accumulated in tumor tissue, whereas after coinjecting with iRGD, the accumulation AuNPs-GNPs was much higher. 3.7. Antitumor Efficiency. As showed in Figure 7A−C, 4T1-bearing mice in the free DOX group showed an undesirable antitumor efficiency compared with that of the saline group, although the free DOX group presented an obvious apoptosis of 4T1 cells in vitro (Figure S1), which was owing to the poor accumulation in tumor. Additionally, the DOX-AuNPs and DOX-AuNPs-GNPs groups showed better antitumor efficiency than the free DOX group because they can target tumor by EPR effect, but their antitumor efficiency was limited by their poor uptake and penetration efficiency. Furthermore, the iRGD + DOX-AuNPs-GNPs group presented the best antitumor effect compared with those of other groups, owing to its deeper tumor penetration though a shrinkable size and coadministration with iRGD, which could target αvβ3 integrin and NRP-1 receptors in the tumor vasculature and tumor cells to increase the penetration of tumor vascular and tumor tissue, thus enhancing the therapeutic effect of DOXAuNPs-GNPs.10,41 HE staining and TUNEL staining were used to investigate further the cell apoptosis in tumor (Figure 7D). Because of poor targeting and accumulation efficiency, the DOX group presented more complete tumor cell shape and fewer apoptotic bodies than other groups. However, compared with the DOXAuNPs-GNPs group, the DOX-AuNPs group presented fewer apoptosis cells, which indicated that size-shrinkable DOXAuNPs-GNPs had a better antitumor effect than that of smallsized DOX-AuNPs. Also, the iRGD + DOX-AuNPs-GNPs group showed lower density of live tumor cells and more G
DOI: 10.1021/acsami.5b09391 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
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apoptotic and necrotic cells than did the DOX-AuNPs-GNPs group because of the homing and penetrating effect of iRGD.41 Consistent with the treatment results, the iRGD + DOXAuNPs-GNPs group presented a favorable antitumor effect.
4. CONCLUSIONS In this study, we put forward a strategy in which we coinjected tumor penetration peptide iRGD with multistage tumormicroenvironment-responsive DOX-AuNPs-GNPs to enhance tumor penetration and tumor treatment efficiency. After injection, DOX-AuNPs-GNPs could shrink from 131.1 to 46.6 nm triggered by MMP-2, and DOX can release from AuNPs-GNPs in a pH-dependent manner. In vitro, DOXAuNPs-GNPs combined with iRGD showed higher cellular uptake and cytotoxicity on 4T1 cells than did giving DOXAuNPs-GNPs alone. In vivo, the iRGD + DOX-AuNPs-GNPs group also presented a higher accumulation of drugs in 4T1 tumor. Therefore, iRGD coinjected with DOX-AuNPs-GNPs displayed the best antitumor effect, and it may lead to an excellent treatment in the future.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b09391. Particle size and polydipersity index (PDI) of different nanoparticles; cell toxicity study of DOX, DOX-AuNPs, DOX-AuNPs-GNPs and iRGD + DOX-AuNPs-GNPs on 4T1 cells; in vivo image of 4T1 bearing mice 24 h after being treated with different formulations; fluorescence distribution in heart, liver, spleen, lung, and kidney from DOX, DOX-AuNPs, DOX-AuNPs-GNPs and iRGD + DOX-AuNPs-GNPs groups; heart HE staining of different groups after the treatment; and TEM images of tumor obtained from DOX-AuNPs-GNPs and iRGD + DOX-AuNPs-GNPs treated mice. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81402866 and 31571016). REFERENCES
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DOI: 10.1021/acsami.5b09391 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
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DOI: 10.1021/acsami.5b09391 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
