Tumor Microenvironment and Angiogenic Blood Vessels Dual

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Tumor Microenvironment and Angiogenic Blood Vessels DualTargeting for Enhanced Anti-Glioma Therapy Quanyin Hu, Ting Kang, Jingxian Feng, Qianqian Zhu, Tianze Jiang, Jianhui Yao, Xinguo Jiang, and Jun Chen* Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Lane 826, Zhangheng Road, Shanghai 201203, People’s Republic of China ABSTRACT: Advances in active targeting drug delivery system (DDS) have revolutionized glioma diagnosis and therapy. However, the lack of the sufficient targets on glioma cells and limited penetration capability of DDS have significantly compromised the treatment efficacy. In this study, by taking advantages of the abundant extracellular matrix-derived heparan sulfate proteoglycan (HSPG) and enhanced tumor penetration ability mediated by neuropilin-1 (NRP-1) protein, we reported the ATWLPPR and CGKRK peptide dual-decorated nanoparticulate DDS (designated AC-NP) to achieve angiogenic blood vessels and tumor microenvironment dual-targeting effect. The resulted AC-NP displayed the particle size of 123 ± 19.47 nm. Enhanced cellular association of AC-NP was achieved on HUVEC cells and U87MG cells. AC-NP was internalized via caveolin- and lipid raft-mediated mechanism with the involvement of energy and lysosome in HUVEC cells and via caveolin- and lipid raft-mediated pathway with the participation of energy, microtubulin, and lysosome in U87MG cells. After loading with anticancer drug, paclitaxel (PTX), the enhanced apoptosis induction and antiproliferative activity were achieved by AC-NP. Furthermore, in vitro U87MG tumor spheroids assays showed a deeper penetration and an enhanced inhibitory effect against the U87MG tumor spheroids achieved by AC-NP. In vivo animal experiment showed that decoration of AC peptide on the nanoparticulate DDS resulted in extensive accumulation at glioma site and improved anti-glioma efficacy. Collectively, the results suggested that AC-NP holds great promise to serve as an effective tumor blood vessel and tumor microenvironment dual-targeting DDS with enhanced penetration capability, holding great potential in improving anti-glioma efficacy. KEYWORDS: glioma, tumor microenvironment, neuropilin-1, heparan sulfate, dual-targeting, penetration

1. INTRODUCTION The treatment for glioma, which is the most frequent primary malignant brain tumor with a life expectancy of less than 2 years after diagnosis, remains a big challenge.1,2 Despite the great advances in surgery and radiotherapy, the glioma patients still have an extremely poor prognosis and a high risk of recurrence due to the highly proliferative, infiltrative, and invasive nature of glioma cells.3,4 Chemotherapy is indispensable for subsequent treatment after surgery resection of glioma. However, the nonspecific distribution nature of chemotherapeutic dramatically impedes the application, compromising the treatment efficacy and leading to severe side effects.5,6 Building on the success of the emergence of nanotechnology, ligand-based active targeting nanoparticulate DDS for glioma therapy has attracted considerable attention and been developed to bridge the requirements of glioma treatments, such as improving the transportation of drugs across the blood brain barrier or selectively accumulating at glioma site.7−9 However, it is now increasingly acknowledged that glioma is © XXXX American Chemical Society

genetically heterogeneous and complex, and shows variations in the expression of the biomarkers postulated to be important for active targeting strategy.10,11 For example, the dysregulated transferrin receptor expression in glioma cells is often only 3− 5-fold higher than that in normal cells.12,13 This inadequate expression of the receptors on glioma tissue usually compromised the efficacy of monotargeting glioma drug delivery. Furthermore, the high interstitial pressure in the glioma tissue and physical distance away from blood vessels for glioma cell significantly impede the entrance of drug to the entire tumor region even after the extravasation of nanoparticle from blood vessels, which serves as a formidable barrier to glioma therapy and a driving force leading to glioma recurrence and multidrug resistance.14,15 To augment the anti-glioma therapy efficacy, it is essential to develop a multiple-targeting Received: July 6, 2016 Accepted: August 31, 2016

A

DOI: 10.1021/acsami.6b08239 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. (A) Preparation of AC peptide conjugated PEG−PLA nanoparticles. The ATWLLPPR peptide and CGKRK peptide were coupled to overcome the tumor microenvironment barrier and increase the tumor parenchyma penetration. (B) After administration, AC-decorated nanoparticles are capable of targeting tumor blood vessel endothelial cells through utilizing the specific affinity between ATWLPPL peptide and NRP-1, and targeting tumor cells by taking advantage of the high selectivity of CGKRK and HSPG with enhanced penetration.

compared to normal tissue.25 Therefore, the high abundance of HSPG expression can provide the ideal targeted substrate for active targeting DDS. CGKRK peptide, which was discovered by phage display, was found to specifically bind HSPG overexpressed in the tumor microenvironment with high affinity and specificity.26 Additionally, the high cellular internalization contributed by the transmembrane effect in an energy and HSPG receptor-mediated manner of CGKRK peptide provided high binding efficiency and made it more favorable for tumor microenvironment targeted drug delivery.27 To further enhance the vascular extravasation from the blood vessels and penetration capability of DDS, we applied ATWLPPR peptide, which is identified as the substrate of NRP-1 with high affinity and specificity, as the dual targeting ligand.28,29 NRP-1 is overexpressed on the endothelial cells of glioma blood vessels and glioma cells, with a relatively low expression in normal tissues. Furthermore, as a transmembrane protein, NRP-1 is responsible for mediating the transportation of molecules across the blood vessels.30,31 Additionally, the Cterminal carboxyl group in the ATWLPPR peptide is responsible for the binding with NRP-1 receptor and inducing vascular and tissue permeability.32 By taking advantage of the highly selective and specific recognition of ATWLPPR peptide and NRP-1, functionalizing drug delivery system with ATWLPPR peptide could initiate a specific trafficking pathway through the blood vessels, enhancing tumor blood vessels extravasation and tumor parenchyma penetration through the NRP-1-dependent internalization pathway. In the present study, we decorated the drug delivery system with the dual-targeting ligand by coupling ATWLLPPR peptide and CGKRK peptide with a GYG linker to form a new sequence CGKRKGYGATWLLPPR (AC peptide), which is

drug delivery system with improved tumor penetration capability. Tumor microenvironment, which is composed of surrounding extracellular matrix, glycosaminoglycans, proteins, immune cells, and fibroblast, etc., has been gradually recognized as a key contributor for cancer progression and invasion, protection of the tumor from host immunity, and considered as the essential “soil” for the “seed” (the tumor cells) to grow.16−18 The most important feature of the tumor microenvironment is the abundant content and organization of the extracellular matrix (ECM), which plays a vital role in inhibiting the entrance of drug delivery system as well as a main pathway for drug clearance and metabolism.19 Therefore, ECM-based active targeting DDS can make a significant contribution to destruct the supportive environment in maintaining glioma morphology and growth and is expected to enhance the retention time of the accumulated drug. To date, the tumor microenvironment with overexpression of the various biomarkers has been recognized as the promising target, and tumor microenvironment-targeting DDS has been actively pursued to enhance antiglioma therapy, including fibronectins-targeting DDS,12 tumor microenvironment-responsive DDS,20,21 and tumor microenvironment-remodeling strategy.22 Heparan sulfate proteoglycan (HSPG), which is an important component of the extracellular matrix, is composed of core protein, glycosidic linkages, and several linear heparan sulfates (HS).23 Through interacting with adjacent proteins, growth factors, and cytokines, HSPG can regulate the signal transduction between ECM and cancer cells, and thereafter further affects the adhesion, migration, proliferation, and differentiation of cancer cells.24 Additionally, it has been found that in tumor microenvironment, HSPG was highly upregulated when B


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adding the same volume of medium. The PTX concentration was determined by HPLC as described previously.35 2.6. Cellular Association. To access the protein expression on the HUVEC cells and U87MG cells, a Western blotting assay was performed.36 Briefly, RIPA buffer was added to cells and incubated for 30 min. After centrifugation, the protein was dissolved in PBS and subjected to BCA assay for concentration determination. The protein then was mixed with loading buffer, boiled, separated by 10% SDSpage, and transferred to nitrocellulose membrane. Afterward, the membrane was blocked, incubated with specific primary antibodies, and mixed with HRP-conjugated secondary antibodies after washing with TBS-T. Finally, the membrane was applied to enhanced chemiluminescence procedures for visualization. The cellular association was first investigated by the qualitative fluorescence uptake. Briefly, 1 × 105 cells were seeded in a well of the plate and incubated for 24 h. Afterward, the culture medium was replaced by the coumarin-6-labeled NP, ATWLPPR-NP, CGKRK-NP, and AC-NP. After 1 h of incubation, the cells were fixed, stained, and visualized by fluorescent microscopy (Leica DMI4000 B, Germany). For quantitative study, cells were incubated with coumarin-6-loaded NP, ATWLPPR-NP, CGKRK-NP, and AC-NP for 1 h. The cells then were treated as above-mentioned and evaluated by a KineticScan HCS Reader (version 3.1, Cellomics Inc., Pittsburgh, PA). For timedependent experiment, the cells were incubated with 200 ng/mL coumarin-6-loaded NP, ATWLPPR-NP, CGKRK-NP, and AC-NP with different time points. 2.7. Internalization Mechanism of AC-NP in HUVEC Cells and U87MG Cells. HUVEC cells and U87MG cells were seeded in 96-well plates and premixed with different endocytosis inhibitor37 for 1 h, followed by the addition of coumarin-6-loaded AC-NP (coumarin-6 200 ng/mL). The fluorescence intensity then was quantitatively analyzed. The cells without treatment of endocytosis inhibitor served as the control. 2.8. Apoptosis Assay. The apoptosis induction capability of ACNP was qualitatively and quantitatively studied on U87MG cells as previously reported.38 The morphology of the nuclei after being treated with Taxol, NP-PTX, ATWLPPR-NP-PTX, CGKRK-NP-PTX, and AC-NP-PTX (PTX concentration 100 ng/mL) for 24 h was evaluated by fluorescent microscope. The quantitative apoptotic cells after incubation with Taxol, NP-PTX, ATWLPPR-NP-PTX, CGKRKNP-PTX, and AC-NP-PTX (PTX concentration 100 ng/mL) for 24 h were applied to Annexin V-FITC and PI protocols and determined by a flow cytometer (FACS Calibur, BD, U.S.). 2.9. Cytotoxicity Assay. 5 × 103 cells/well of U87MG cells were seeded into a 96-well plate. Afterward, Taxol, NP-PTX ATWLPPRNP-PTX, CGKRK-NP-PTX, and AC-NP-PTX were mixed with the cells for 72 h. The cells then were applied to a Cell Counting Kit-8 assay, and fluorescence absorbance was determined. 2.10. Tumor Spheroids Assay. Tumor spheroids were prepared as described previously39,40 and incubated with coumarin-6-loaded NP, ATWLPPR-NP, CGKRK-NP, and AC-NP (coumarin-6 concentration 400 ng/mL) for 4 h. The tumor spheroids then were washed with PBS, fixed, and visualized by laser scanning confocal microscopy. Fifteen tumor spheroids were randomly divided into five groups and treated with DMEM, Taxol, NP-PTX, ATWLPPR-NP-PTX, CGKRKNP-PTX, and AC-NP-PTX (PTX concentration 200 ng/mL) every 2 days for a week. Afterward, the treated tumor spheroids were subjected to an invert microscope (Chongqing Optical & Electrical Instrument, Co., Ltd., Chongqing, China) for observation. 2.11. In Vivo Biodistribution. The intracranial glioma-bearing mice model was established as described previously.41 Twelve mice were randomly divided into four groups and injected with DiR-loaded NP, ATWLPPR-NP, CGKRK-NP, and AC-NP (200 μL, DiR concentration 1 mg/kg). The biodistributions of NPs were imaged 2, 6, 12, and 24 h via an In Vivo IVIS spectrum imaging system (PerkinElmer, U.S.). Furthermore, the brains and other organs were also taken out for imaging after 24 h. The fluorescence intensities were analyzed via Living Image Software. 2.12. Glioma Biodistribution. Nude mice bearing intracranial U87MG tumors were intravenously administered with coumarin-6-

expected to be able to bind NRP-1 receptor and heparan sulfate proteoglycan simultaneously, leading to improved targeting efficiency and tumor penetration capability (Figure 1). To justify our hypothesis, PEG−PLA nanoparticles were prepared and modified with AC peptide through chemical interaction between cysteine on the peptide and maleimide group on the materials. In vitro cellular uptake and in vivo biodistribution of AC-NP was systematically evaluated using fluorescence tracking probe coumarin-6 and DiR. Using PTX as the model drug, the anti-glioma efficacy of AC-NP-PTX was assessed on glioma bearing nude mice.

2. MATERIALS AND METHODS 2.1. Materials. Methoxy-poly(ethylene glycol) 3000-poly(lactic acid) 34000 (MePEG−PLA) and maleimide-poly(ethylene glycol) 3400poly(lactic acid) 34000 (Male-PEG−PLA) were synthesized as described previously.30 ATWLPPR peptide, CGKRK peptide, and AC peptide (CGKRKGYGATWLPPR) were purchased from Shanghai Dechi Bioscience Co., Ltd. (Shanghai, China). PTX was provided by Shanghai Jinhe Biotechnology Co., Ltd. (Shanghai, China). Taxol (marketed product of PTX) was obtained from Bristol-Myers Squibb (China) Investment Co., Ltd. (Shanghai, China). Fluorescence probes (Coumarin-6, DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine Iodide) and DAPI (4,6-diamidino-2-phenylindole)) were obtained from Sigma−Aldrich (St. Louis, MO). Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Alexa Fluor 594 conjugated antimouse CD31 antibody was purchased from abcam (Boston, MA). Annexin V-FITC Apoptosis Detection Kit I was obtained from BD Bioscience (San Diego, CA). Anti CD31 antibody, anti-NRP-1 primary antibody, antiheparan sulfate proteoglycan 2 antibody, antibeta actin antibody, and rabbit antihuman IgG were purchased from Abcam Co. (Boston, MA). Other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used without further purification. 2.2. Cell Lines and Animals. Human umbilical vein endothelial cells (HUVEC cells) were purchased from Cascade Biologics (Portland, OR). U87MG cells were obtained from the Cell Institute of Chinese Academy of Sciences (Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), trypsin, and penicillin−streptomycin were purchased from Gibco BRL (Carlsbad, CA). Balb/c nude mice (male, 4 weeks) were provided by BK Lab Anima Ltd. (Shanghai, China). All animals were treated in accordance with guidelines approved by the ethics committee of Fudan University (Shanghai, China). 2.3. Preparation of AC Peptide-Modified Nanoparticles. The nanoparticles were prepared as described previously.33,34 In brief, 1 mL of dichloromethane was added to the blend of 2.5 mg of Male-PEG− PLA, 22.5 mg of MePEG−PLA, followed by the addition of 2 mL of 1% sodium cholate aqueous solution. After sonication with a probe sonicator (Ningbo Scientz Biotechnology Co. Ltd., China), the emulsion was stirred with 0.5% sodium cholate solution for 8 min. Subsequently, the emulsion was evaporated and concentrated. The nanoparticles were obtained after discarding supernatant. The ATWLPPR peptide, CGKRK peptide, or AC peptide was then incubated with nanoparticle for functionalization. 2.4. Characterization of AC Peptide-Modified Nanoparticles. The particle sizes and zeta potentials of unmodified NP, ATWLPPRNP, CGKRK-NP, and AC-NP were evaluated by a Malvern Zeta Sizer Nano series (Malvern Instruments, Worcestershire, UK). The morphologies were visualized by transmission electron microscopy (TEM) (H-600, Hitachi, Japan). 2.5. In Vitro Release of PTX from NP Formulations. For the PTX release study, Taxol, NP-PTX, ATWLPPR-NP-PTX, CGKRKNP-PTX, and AC-NP-PTX were added into 1 mL of PBS and embedded in 39 mL of release medium, and shaken at 120 rpm for 72 h. At every time point, 0.2 mL of solution was withdrawn, followed by C


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ACS Applied Materials & Interfaces loaded NP, ATWLPPR-NP, CGKRK-NP, and AC-NP. Three hours later, the brains were harvested, frozen, sectioned, stained with DAPI and anti-CD31 antibody, and visualized by the confocal microscope. 2.13. Anti-Glioma Efficacy. The glioma-bearing mice were treated with saline, Taxol, NP-PTX, ATWLPPR-NP-PTX, CGKRKNP-PTX, and AC-NP-PTX (PTX concentration 5 mg/kg), every 3 days for 2 weeks. Afterward, the survival time of each group was recorded. 2.14. Statistical Analysis. Statistical analysis was applied to GraphPad Prism 5.0. Two-group comparison was conducted with unpaired student’s t-test, and multiple-group analysis was calculated with one-way ANOVA with Bonferroni tests. P < 0.05 was considered as the statistical difference.

formulation with over 90% of cumulative PTX release within 6 h. 3.3. Cellular Association. The expressions of NRP-1 and heparan sulfate were evaluated on both HUVEC and U87MG cells (Figure 2C). It showed high NRP-1 expression on HUVEC cells. However, the expression of NRP-1 was relatively lower on U87MG cells as compared to that on HUVEC cells. Additionally, the expression of heparan sulfate on U87MG cells was higher than that on HUVEC cells. To evaluate the tumor blood vessels and tumor homing capabilities of AC-NP, HUVEC and U87MG cells were selected as the cellular models. As shown in Figure 3A, the stronger fluorescence intensity was observed by ATWLPPR-NP and AC-NP as compared to that of NP and CGKRK-NP on HUVEC cells. Additionally, CGKRK-NP and AC-NP displayed the enhanced association on U87MG cells as compared to NP and ATWLPPR-NP (Figure 3B). The quantitative results were in good accordance with qualitative fluorescence intensities. The cellular association of AC-NP was 2.0-fold and 1.5-fold higher than that of NP and CGKRK-NP on HUVEC cells, respectively (Figure 3C,D). The cellular association of AC-NP was 2.1-fold and 1.7-fold higher than that of NP and ATWLPPR-NP on U87MG cells, respectively. Additionally, the association of AC-NP was displayed in a time-dependent manner, which was higher than NP and CGKRK-NP on HUVEC cells and higher than NP and ATWLPPR-NP on U87MG cells at every study time interval (Figure 3E,F). 3.4. Internalization Mechanism of AC-NP on HUVEC Cells and U87MG Cells. The association of AC-NP on HUVEC cells was inhibited by genistein (p < 0.01), NaN3 (p < 0.01), M-β-CD (p < 0.001), and monensin (p < 0.01). Additionally, the pretreated free ATWLPPR and CGKRK peptide significantly restrained the association of AC-NP on HUVEC cells (Table 2, Figure 4A). For U87MG cells, Cyto-D (p < 0.001), BFA (p < 0.001), genistein (p < 0.01), NaN3 (p < 0.001), M-β-CD (p < 0.001), and monensin (p < 0.01) significantly decreased the association of AC-NP. Moreover, the pretreated free CGKRK peptide significantly inhibited the association of AC-NP on U87MG cells (Table 2, Figure 4B). 3.5. Apoptosis and Cytotoxicity Assay. The apoptosisnducing capabilities of different PTX formulations were evaluated using fluorescence microscopy and flow cytometry. The fluorescence images displayed the nuclei morphology changes after treatment with different PTX formulations. ACNP treated U87MG cells showed the most fractured nuclei with apoptotic bodies appearing (Figure 5A). Additionally, the quantitative results demonstrated the early apoptotic rate of 20.76 ± 2.79% for AC-NP-PTX, which was significantly higher than that of ATWLPPR-NP-PTX (13.85 ± 2.93%) (p < 0.01), NP-PTX (12.09 ± 2.87%) (p < 0.01), and Taxol (12.92 ± 3.13%) (p < 0.01) on U87MG cells, respectively. Additionally, the early apoptotic rate of CGKRK-NP-PTX (21.48 ± 3.67%) was similar to that of AC-NP-PTX on U87MG cells (Figure 5B). As shown in Figure 5C, AC-NP-PTX displayed the strongest cytotoxicity with IC50 of 20.1 nM as compared to ATWLPPRNP-PTX (72.6 nM), NP-PTX (102.6 nM), and Taxol (130.6 nM). Besides, the IC50 value of AC-NP-PTX was similar to that of CGKRK-NP-PTX (20.5 nM). 3.6. Tumor Spheroids Penetration Assay. 3D tumor spheroids have been increasingly selected as the in vitro tumor

3. RESULTS 3.1. Characterization of AC Peptide-Modified Nanoparticles. The particle sizes of blank NP, ATWLPPR-NP, CGKRK-NP, and AC-NP were 102 ± 9.3, 113 ± 15.3, 119 ± 16.2, and 123 ± 19.5 nm, respectively, with the unique spheral structure observed by TEM (Figure 2A). The zeta potentials of blank NP, ATWLPPR-NP, CGKRK-NP, and AC-NP were −37.5 ± 3.75, −31.6 ± 4.35, −14.6 ± 5.47, and −11.4 ± 4.29 mV, respectively (Table 1).

Figure 2. (A) The morphologies of unmodified NP, ATWLPPR-NP, CGKRK-NP, and AC-NP observed by TEM. Scale bar: 200 nm. (B) In vitro cumulative release of PTX from Taxol, NP-PTX, ATWLPPRNP-PTX, CGKRK-NP-PTX, and AC-NP-PTX. (C) The expression of NRP-1 and HSPG on HUVEC and U87MG cell lines.

Table 1. Characterization of NP, ATWLPPR-NP, CGKRKNP, and AC-NPa nanoparticles NP ATWLPPR-NP CGKRK-NP AC-NP a

particle size (nm) 102 113 119 123

± ± ± ±

9.35 15.32 16.24 19.47

polydispersity index (PI) 0.13 0.14 0.17 0.15

± ± ± ±

0.08 0.08 0.09 0.07

zeta potential (mV) −37.5 −31.6 −14.6 −11.4

± ± ± ±

3.75 4.35 5.47 4.29

Data represented mean ± SD (n = 3).

3.2. In Vitro PTX Release Profile. As shown in Figure 2B, similar sustained release profiles were observed in NP-PTX, ATWLPPR-NP-PTX, CGKRK-NP-PTX, and AC-NP-PTX. The cumulative release percentages of PTX were 78.3%, 77.1%, 79.4%, and 75.6% in 72 h, respectively. By contrast, a burst release behavior of PTX was achieved in Taxol D


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Figure 3. Fluorescence images of the HUVEC cells (A) and U87MG cells (B) after being treated with coumarin-6 loaded NP, ATWLPPR-NP, CGKRK-NP, and AC-NP (coumarin-6 concentration 200 μg/mL) for 1 h. Scale bar: 100 μm. The quantitative HUVEC cells (C) and U87MG cells (D) association after incubation with coumarin-6-labeled NP, ATWLPPR-NP, CGKRK-NP, and AC-NP (coumarin-6 concentration ranging from 25 to 600 μg/mL) for 1 h. Time-dependent association analysis after being treated for different time intervals on HUVEC cells (E) and U87MG cells (F). *p < 0.05, **p < 0.01, ***p < 0.001 indicated that the cellular association of AC-NP was significantly higher than that of CGKRK-NP on HUVEC cells and ATWLPPR-NP on U87MG cells and NP on both cells, and #p < 0.05, ##p < 0.01, ###p < 0.001 indicated that the cellular association of CGKRK-NP was significantly higher than that of NP on HUVEC cells.

models to study the penetration of the drug delivery system.42 In this study, we employed U87MG tumor spheroids to evaluate the penetration capability of AC-NP. As displayed in Figure 5D, the high expression of HSPG on the U87MG tumor spheroids was demonstrated, which validated that U87MG tumor spheroids could serve as the solid tumor model with mimicry of tumor microenvironment. As shown in Figure 6, the tumor spheroids treated with ACNP displayed stronger fluorescence intensity as compared to those treated with ATWLPPR-NP and NP, but similar to CGKRK-NP. Additionally, the penetration depth of AC-NP was about 105 μm and significantly deeper than that of ATWLPPR-NP and NP. 3.7. Tumor Spheroids Inhibition Assay. The growth inhibition of U87MG tumor spheroids was evaluated after

being treated with drug-free DMEM, NP-PTX, ATWLPPRNP-PTX, CGKRK-NP-PTX, and AC-NP-PTX. The tumor spheroids treated with DMEM displayed the increased volume with the compact cellular conjunction. However, the tumor spheroids treated with PTX formulations exhibited the shrinkage of size. Moreover, the tumor spheroid with exposure of AC-NP-PTX and CGKRK-NP-PTX showed the smallest size with loss of three-dimensional structure and resulted in cellular fragments at day 6 as compared to other PTX formulations treatment (Figure 7). 3.8. In Vivo Biodistribution. To assess the in vivo targeting capability of AC-NP, the intracranial U87MG tumorsbearing mice model was established and the targetability of ACNP was evaluated via a noninvasive optical imaging system. The mice treated with AC-NP exhibited a much stronger E


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fluorescence signal at the glioma site at every imaging time point when compared to NP, ATWLPPR-NP, and CGKRK-NP (Figure 8A−D). Ex vivo imaging confirmed the highest fluorescence intensity of AC-NP treatment at the glioma site (Figure 8E). Furthermore, the quantitative fluorescence intensity analysis confirmed the higher glioma accumulation of AC-NP than NP, ATWLPPR-NP, and CGKRK-NP (Figure 8F). 3.9. Glioma Biodistribution. As shown in Figure 9A, the fluorescence signal was barely observed for NP at the site of glioma. In contrast, the higher distribution was achieved by the NP formulations decorated with active targeting ligands. Additionally, AC-NP exhibited the strongest fluorescence intensity at the glioma site as compared to ATWLPPR-NP and CGKRK-NP. Of note, the colocalization of the NP formulations and CD31-stained blood vessels substantiated the extravasation of the nanoparticle from the blood vessels and accumulated at the glioma site. 3.10. Anti-Glioma Efficacy. As shown in Figure 10, the medium survival was 22 days for those mice treated with saline,

Table 2. Function of Various Inhibitors on the Endocytosis of AC-NP on HUVEC Cells and U87MG Cells inhibitors chlorpromazine colchicines cyto-D BFA filipin genistein NaN3 M-β-CD moneisin nacodazole ATWLPPR CGKRK

function clathrin-mediated internalization microtubulin depolymerization macropinocytosis inhibitor golgi apparatus inhibitor caveolae-mediated endocytosis caveolae-mediated endocytosis energy inhibitor lipid draft inhibitor endosome inhibitor macropinocytosis inhibitor NRP-1-mediated internalization HSPG-mediated internalization

HUVEC cells

U87MG cells

−

−

−

−

− − −

+ + −

+

+

+ + + − +

+ + + − −

+

+

Figure 4. Quantitative analysis (mean ± SD (n = 3)) of the HUVEC cells (A) and U87MG cells (B) uptake efficiency of AC-NP after being preincubated with various endocytosis inhibitors. The cells without inhibitors treatment served as the control. *p < 0.05, **p < 0.01, ***p < 0.001 indicated the significant difference between treated groups and control. F


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Figure 5. (A) The nuclei morphologies of U87MG cells after treatment with different PTX formulations. Scale bar: 100 μm. (B) Quantitative analysis of apoptotic cells after staining with Annexin V-FITC and PI. (C) Cell viability of U87MG cells after being incubated with Taxol, NP-PTX, ATWLPPR-NP-PTX, CGKRK-NP-PTX, and AC-NP-PTX for 72 h. (D) Immunostaining of HSPG in U87MG tumor spheroids. Red arrows represented the HSPG. Scale bar: 50 μm.

4. DISCUSSION To date, an active targeting nanoparticulate drug delivery system has attracted considerable attention in anti-glioma drug delivery.43−45 Dual-targeting DDS has been enormously developed to specifically deliver the drug to the glioma tissue and overcome various physiological barriers, including blood brain barrier (BBB) and blood brain tumor barrier (BBTB).11,46−48 By taking advantage of BBB/glioma and neovasculature/glioma dual-targeting capabilities, the current dual-targeting strategies significantly enhance the drug availability at the glioma site and improve the anti-glioma treatment efficacy.49,50 However, the limited density of the receptors on the targeting tissue/cells limits the efficiency of these ligand-based dual-targeting systems.21 Tumor microenvironment plays a crucial role in facilitating tumor cell proliferation and metastasis, compromising the chemotherapy efficacy and inducing acquired drug resistance.51 Furthermore, the expression of chemokines and proteins in the tumor microenvironment is significantly higher than that of tumor cells, offering a more efficient way for tumor targeting drug delivery.52,53 In this study, AC peptide composed of CGKRK and ATWLPPR peptides was functionalized on the nanoparticulate DDS for enhanced anti-glioma therapy. Heparan sulfate proteoglycan, an important component of the extracellular matrix, served as the target of CGKRK-decorated DDS. CGKRK peptide could selectively bind to HSPG with

Figure 6. Z-stack fluorescence images of U87MG spheroids after incubation with coumarin-6-loaded different nanoparticles for 4 h. Scale bar: 100 μm.

29 days for Taxol, 31 days for NP, 41.5 days for ATWLPPRNP, 42 days for CGKRK-NP, and 51 days for AC-NP. Additionally, the survival time of mice with AC-NP treatment was significantly longer than that of CGKRK-NP (p < 0.01), ATWLPPR-NP (p < 0.01), NP (p < 0.001), Taxol (p < 0.001), and saline (p < 0.001). G


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that on CGKRK-NP, which is reasoned as the moderate expression of HSPG on HUVEC cells (Figure 2C). Similarly, the relative lower uptake of ATWLPPR-NP on U87MG cells as compared to AC-NP and CGKRK-NP could be attributed to the moderate expression of NRP-1 on the U87MG cells. The different expression levels of NRP-1 and HSPG on HUVEC and U87MG cells resulted in the diverse association of the various NP formulations, while the AC peptide composed of ATWLPPR and CGKRK peptide could combine the advantages and exhibited superior uptake on both HUVEC and U87MG cells. Collectively, the formation of AC peptide did not impair the individual bioactivity of each peptide, but synergistically combined the advantages of individual peptide. The interaction mechanism underlying the internalization of nanoparticles on targeted cells was crucial for the active targeting drug delivery system, which established the foundation for enhanced intracellular drug accumulation.56,57 In this study, the transport pathway of AC-NP was investigated in the presence of various endocytic inhibitors. As shown in Figure 4, the internalization of AC-NP on HUVEC cells was restrained by genistein, NaN3, M-β-CD, and monensin, indicating that the endocytosis mechanism was mediated by caveolin and lipid raft with the involvement of energy and lysosome. Additionally, the internalization of AC-NP on U87MG cells was inhibited by Cyto-D, BFA, genistein, NaN3, M-β-CD, and monensin, suggesting that the transportation of AC-NP was mediated by caveolin and lipid raft with the involvement of energy, microtubulin, and lysosome. Additionally, the pretreated ATWLPPR peptide and CGKRK peptide significantly decreased the uptake of AC-NP on HUVEC cells and U87MG cells, respectively, revealing that the endocytosis process was also mediated by the overexpressed receptors, NRP-1 and HSPG. After loading with anticancer drug, PTX, the apoptosisinduced capability and cytotoxicity of AC-NP-PTX were evaluated on U87MG cells. As shown in Figure 5, the flow cytometry analysis demonstrated that AC-NP-PTX and CGKRK-NP-PTX induced more apoptotic U87MG cells as compared to ATWLPPR-NP-PTX and NP-PTX, which could be due to the higher cellular accumulation of PTX via the CGKRK peptide-mediated endocytosis. Additionally, the IC50 value of AC-NP-PTX was 20.1 nM, which was significantly lower than that of ATWLPPR-NP-PTX, NP-PTX, and Taxol, suggesting cytotoxicity of PTX was substantially increased following the encapsulation in AC-NP, which can be ascribed to the enhanced cellular internalization mediated by the binding activity between AC peptide and overexpressed HSPG on U87MG cells. Tumor spheroids, microscaled and spherical cell clusters, are the most versatile and tumor-physiology mimicking platform for evaluation of drug delivery and penetration in solid tumor.58 Herein, a U87MG 3D tumor spheroid was prepared and employed as the solid tumor model to study the penetration capability and antitumor efficacy of AC-NP. First, the overexpression of HSPG in the tumor spheroids was demonstrated using immunostaining, which substantiated the mimicry of tumor microenvironment using tumor spheroids. As shown in Figure 7, the U87MG tumor spheroids treated with AC-NP and CGKRK-NP exhibited much stronger fluorescence intensity and deeper penetration depth when compared to that of ATWLPPR-NP and NP, indicating the conjugation of AC peptide and CGKRK-NP could facilitate the penetration of ACNP and CGKRK-NP in U87MG tumor spheroids. However,

Figure 7. Morphologies of U87MG glioma spheroids treated with Taxol, NP-PTX, ATWLPPR-NP-PTX, CGKRK-NP-PTX, and ACNP-PTX (PTX concentration 200 ng/mL) on different days.

high affinity and specificity. To further enhance the vascular extravasation of the drug delivery system, ATWLPPR peptide that can bind to the “shuttle protein” NRP-1 overexpressed on the endothelial cells was also conjugated on the surface of nanoparticulate DDS. It is expected that ATWLPPR and CGKRK peptides coconjugated nanoparticles (AC-NP) hold vast promise in mediating extensive extravasation from tumor blood vessels, specific and efficient tumor targeting for antiglioma therapy. In this study, we used the GYG linker to conjugate the ATWLPPR peptide and CGKRK peptide together to form AC peptide. The obtained AC-NP-PTX displayed an average size of 123 nm, which was below the cutoff size threshold (∼200 nm) of leaky tumor blood vessels and suitable for glioma targeting drug delivery.54,55 Additionally, the increased zeta potentials of CGKRK-NP and AC-NP were attributed to the positively charged amino acids in CGKRK, suggesting that the peptide was successfully conjugated. Furthermore, the decoration of AC peptide did not change the sustained release profile of the nanoparticles. To evaluate the tumor blood vessel and tumor cells dualtargeting effects, HUVEC cells with the overexpression of NRP1 receptors and U87MG cells with the overexpression of HSPG were selected as the cell models. A concentration- and timedependent internalization manner was observed for all NP formulations on both HUVEC cells and U87MG cells. Interestingly, the internalization efficiencies of AC-NP and ATWLPPR-NP on HUVEC cells were similar, which was attributed to the enhanced uptake mediated by the binding capability of ATWLPPR and NRP-1 protein. The uptake of AC-NP and ATWLPPR-NP on HUVEC cells is higher than H


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Figure 8. Whole body fluorescence imaging of U87MG glioma-bearing mice after being administrated DiR-loaded NP (A), ATWLPPR-NP (B), CGKRK-NP (C), and AC-NP (D) at different time intervals and the fluorescence images of the organs (from left to right: heart, liver, spleen, lung, kidney). The brain images after 24 h administration (E). The quantified assay of the fluorescent intensities in different organs (F). White arrow: glioma site. Data were presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 demonstrated the significant difference between ACNP groups and other treated groups.

Figure 10. Survival curve of U87MG bearing-mice following the treatment of saline, Taxol, NP-PTX, ATWLPPR-NP-PTX, CGKRKNP-PTX, and AC-NP-PTX (PTX dose 5 mg/kg). Figure 9. In vivo glioma distribution of (A) NP, (B) ATWLLPPR-NP, (C) CGKRK-NP, and (D) AC-NP after treatment for 3 h. All of the white arrows represented nanoparticles. Green: nanoparticles. Red: CD31-stained blood vessels. Blue: cancer cell nuclei. Scale bar: 50 μm.

accumulation and penetration of ATWLPPR-NP. The in vitro antitumor spheroids growth experiment also confirmed the targeting capability of AC-NP-PTX and CGKRK-NP-PTX with stronger inhibitory efficiency when compared to ATWLPPRNP-PTX, NP-PTX, and Taxol. Furthermore, the disappearance of a compact 3D structure and emergence of the cellular

the target of ATWLPPR peptide is NRP-1, which is moderately expressed on the U87MG cells, leading to the limited I



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fragments validated the superiority of anti-glioma effects of ACNP-PTX and CGKRK-NP-PTX. Collectively, the enhanced penetration capability and improved antitumor efficacy mediated by decoration of AC peptide substantiated the good preservation of targeting activity of CGKRK peptide, promoting a promising approach for anti-glioma chemotherapy. In vivo tumor homing capability of AC-NP was evaluated on the intracranial glioma-bearing mice model. As shown in Figure 8, a much stronger fluorescence intensity was achieved at the tumor site for mice administered with AC-NP at every experimental time point and ex vivo glioma tissues when compared to that of mice injected with ATWLPPR-NP, CGKRK-NP, and unmodified NP. Furthermore, the distribution of nanoparticles at glioma site confirmed the enhanced accumulation of AC-NP as compared to CGKRK-NP, ATWLPPR-NP, and unmodified NP (Figure 9). These results suggested that AC-NP could synergistically combine the individual advantage of CGKRK peptide and ATWLPPR peptide by taking advantages of HSPG-mediated tumor microenvironment targeting and NRP-1-mediated tumor blood vessel targeting and enhanced penetration, leading to superior accumulation of nanoparticulate DDS at the glioma site. The anti-glioma efficacy of AC-NP-PTX was studied by investigating the survival time of U87MG bearing-mice after different treatment. The survival results demonstrated a longest survival time achieved by the treatment with AC-NP-PTX (51 days), significantly longer than those mice treated with CGKRK-NP-PTX (42 days, **p < 0.001), ATWLPPR-NPPTX (41.5 days, **p < 0.01), NP-PTX (31 days, ***p < 0.001), Taxol (29 days, ***p < 0.001), and saline (22 days, ***p < 0.001). Such improved anti-glioma efficiency of ACNP-PTX was contributed by the superior dual-targeting efficiency, leading to the enhanced drug availability at the glioma site due to the AC peptide modification.

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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 Key Basic Research Program (Grant 2013CB932500), and the National Natural Science Foundation of China (Grant 81373353).

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5. CONCLUSION In summary, we constructed an AC peptide (composed of ATWLPPR peptide and CGKRK peptide) functionalized PEG−PLA nanoparticulate DDS to achieve angiogenic blood vessels and tumor microenvironment dual-targeting effect and enhanced penetration capability. The resulting AC-NP with the particle size of 123 ± 19.5 nm exhibited enhanced cellular association on HUVEC cells and U87MG cells, and improved the apoptosis-induction activity and cytotoxicity after loading with PTX. The internalization of AC-NP was internalized via caveolin- and lipid raft-mediated mechanism with the involvement of energy and lysosome on HUVEC cells and caveolin- and lipid raft-mediated pathway with the participation of energy, microtubulin, and lysosome on U87MG cells. Furthermore, in vitro U87MG tumor spheroids assays showed a deep penetration and an enhanced inhibitory effect on the U87MG tumor spheroids achieved by AC-NP. In vivo nearinfrared (NIR) imaging and glioma distribution revealed that AC-NP displayed a superior tumor targeting and higher accumulation within the tumor. Survival experiments demonstrated the superior anti-glioma efficacy of AC-NP. The results demonstrated that AC-NP holds great promise in dual-targeting tumor blood vessels and tumor microenvironment with enhanced penetration capability. J


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Tumor Microenvironment and Angiogenic Blood Vessels Dual

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