RGD and Interleukin-13 Peptide Functionalized Nanoparticles for

Feb 12, 2014 - IRNPs also displayed the strongest penetration ability according to ... Mao , Changyou Zhan , Cao Xie , Huitong Ruan , Man Ying , Jianf...
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RGD and Interleukin-13 Peptide Functionalized Nanoparticles for Enhanced Glioblastoma Cells and Neovasculature Dual Targeting Delivery and Elevated Tumor Penetration Huile Gao, Yang Xiong, Shuang Zhang, Zhi Yang, Shijie Cao, and Xinguo Jiang* Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education, Department of Pharmaceutics Sciences, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China ABSTRACT: As the most common malignant brain tumors, glioblastoma multiforme (GBM) was characterized by angiogenesis and tumor cells proliferation. Dual targeting to neovasculature and GBM cells could deliver cargoes to these two kinds of cells, leading to a combination treatment. In this study, polymeric nanoparticles were functionalized with RGD and interleukin-13 peptide (IRNPs) to construct a neovasculature and tumor cell dual targeting delivery system in which RGD could target αvβ3 on neovasculature and interleukin-13 peptide could target IL13Rα2 on GBM cells. In vitro, interleukin-13 peptide and RGD could enhance the uptake by corresponding cells (C6 and human umbilical vein endothelial cells). Due to the expression of both receptors on C6 cells, RGD also could enhance the uptake by C6 cells. Through receptor labeling, it clearly showed that αvβ3 could mediate the internalization of RGD modified nanoparticles and IL13Rα2 could mediate the internalization of interleukin-13 peptide modified nanoparticles. The ligand functionalization also resulted in a modification on endocytosis pathways, which changed the main endocytosis pathways from macropinocytosis for unmodified nanoparticles to clathrin-mediated endocytosis for IRNPs. IRNPs also displayed the strongest penetration ability according to tumor spheroid analysis. In vivo, IRNPs could effectively deliver cargoes to GBM with higher intensity than monomodified nanoparticles. After CD31-staining, it demonstrated IRNPs could target both neovasculature and GBM cells. In conclusion, IRNPs showed promising ability in dual targeting both neovasculature and GBM cells. KEYWORDS: glioma, interleukin-13 peptide, RGD, neovasculature, dual targeting delivery

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INTRODUCTION Chemotherapy is one of the most common and essential strategies in the treatment of malignant tumors; however, the clinical outcome is usually poor with serious side effects owing to the short blood circulation half-life, poor physicochemical properties, low concentration in diseased tissues, and high toxicity to normal tissues. To conquer these problems, degradable polymeric nanoparticulated systems gain much attention owing to their ability to carry drugs with high loading capacity, mask initial properties of cargoes, prolong circulation half-life after PEGylation, and target specifically diseased tissues and cells through surface modification with ligands.1−6 Although many studies have focused on enabling nanoparticulated systems with excellent properties, it is far from ideal as to how to effectively treat malignant tumors. Glioma remains a serious threat and is widely incurable. Glioblastoma multiforme (GBM), accounting 70% of all malignant gliomas, are highly aggressive with a 5-year survival rate lower than 5%.7,8 In spite of the fact that angiogenesis is one of the main features of tumor tissues and GBM is among the most highly vascularized tumors,9,10 the application of antiangiogenesis treatment is shadowed by the reactive © 2014 American Chemical Society

resistance for the proapoptotic effect of chemotherapy as well as enhanced metastatic and invasive potential of tumor cells because of the activation of the hypoxic response.11,12 This concern could be addressed by the combination of antiangiogenesis and antitumor therapy, and several strategies were developed for this purpose, such as codelivery of anti-VEGF gene (antiangiogenesis agent) and doxorubicin (antitumor agent).9,13,14 However, most strategies were focused on delivery of two agents with different functions. In this study, a novel strategy was presented that using dual targeting delivery system to deliver cargoes to both neovasculature and GBM cells for combinational therapy. RGD, a tripeptide of arginine-glycine-aspartic acid, could specifically bind with αvβ3 which were overexpressed on neovascular endothelial cells.15,16 Thus this study utilized RGD as a neovasculature-targeting ligand. As a derivation from interleukin-13, only the binding domain with IL13Rα2 (a Received: Revised: Accepted: Published: 1042

December 15, 2013 February 8, 2014 February 12, 2014 February 12, 2014 dx.doi.org/10.1021/mp400751g | Mol. Pharmaceutics 2014, 11, 1042−1052

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purchased from Beyotime (Haimen, China). The C6 and HUVEC was obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). RFP-C6 was obtained from Shanghai SBO Medical Biotechnology Co., Ltd. (Shanghai, China). Dylight647 conjugated donkey antirabbit IgG and Cy3 conjugated donkey antigoat IgG were purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA, U. S. A.). Rabbit anti-CD31 polyclonal antibody and rabbit anti-integrin-β3 polyclonal antibody were purchased from Abcam Ltd. (Hong Kong, China). Goat antiIL-13Rα2 (N-20) polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U. S. A.). All of the other chemicals were analytical reagent grades and purchased from Sinopharm Chemical Reagent (Shanghai, China). BALB/c nude mice (male, 4−5 weeks, 18−22 g) were obtained from the Shanghai Laboratory Animal Research Center (Shanghai, China) and maintained under standard housing conditions. All animal experiments were carried out in accordance with protocols evaluated and approved by the ethics committee of Fudan University. Preparation Nanoparticles. PEG-PCL nanoparticles (NPs) were prepared by emulsion/solvent evaporation method described previously.21 Briefly, 28 mg of MPEG-PCL, 1 mg of HOOC-PEG-PCL, and 1 mg of MAL-PEG-PCL were dissolved in 1 mL of dichloromethane and then added into 5 mL of 0.6% sodium cholate hydrate solution. The mixture was pulse sonicated for 75 s at 200 W on ice using a probe sonicator (Scientz Biotechnology Co. Ltd., China). Then, the emulsion was applied to rotary evaporator to remove the dichloromethane and the NPs were condensed to a fixed concentration by ultrafiltration at 4000g. For the IL-13p conjugation (ILNPs), the carboxyl unit of NPs was activated by EDC and NHS in pH 6.0 MES buffer for 0.5 h. The MES buffer was then replaced by pH 7.4 PBS using a Hitrap desalting column and 50 μg of IL-13p in 1 mL of pH 7.4 PBS was added into the NPs suspension and stirred for 4 h in the dark. For the RGD conjugation (RNPs and IRNPs), 50 μg of RGD was added to NPs or ILNPs suspension and stirred for 6 h in the dark. The product was then applied to ultrafiltration to remove the unconjugated IL-13p and RGD, and the nanoparticles were collected. DiR- and coumarin-6-loaded IRNPs were prepared with the same procedure except the materials were dissolved in 1 mL of dichloromethane containing 30 μg of coumarin-6 or 600 μg of DiR, respectively. Characterization of NPs. Particle size and ζ potential were determined by dynamic light scattering (DLS) using a Malvern Zeta Sizer (Malvern, NanoZS, UK). Particle morphology was detected by transmission electron microscope (TEM) (H-600, Hitachi, Japan) after negative staining with 2% sodium phosphotungstate solution. Receptor Expression. HUVEC and C6 cells were lysed using RPMI buffer containing 1 mM PMSF. The protein concentration was determined using the BCA method. Equivalent amounts of protein were boiled for 5 min in loading buffer and then separated by 10% SDS polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes. The membranes were blocked for 1 h in TBS containing 4% low-fat milk. Membranes were then probed with specific antibodies recognizing the target proteins, and the proteins were visualized using an ECL reagent.

glioma restricted receptor) was reserved in the sequence of interleukin-12 peptide (IL-13p),17,18 making IL-13p an ideal GBM cell-targeting ligand. In combination, RGD and IL-13p were dual-functionalized onto polymeric nanoparticles (IRNPs) for the dual targeting of neovasculature and GBM cells (Figure 1). Although we had utilized these strategies to deliver

Figure 1. Elucidation of the preparation and targeting effect of IRNPs. Particles were prepared from functional PEG-PCL by the emulsion/ solvent evaporation method. RGD was conjugated onto NPs through maleimide and IL-13p was conjugated onto NPs through carboxylation. Particles could target the tumor through both the EPR effect and αvβ3-mediated endocytosis by neovasculature. The internalization of IRNPs into tumor cells was mediated by both IL13Rα2 and αvβ3.

chemotherapeutic for GBM treatment, which showed well anti-GBM effect;19 the cell response mechanism, targeting efficiency, and dual targeting mechanism were still not clear. To elucidate the targeting ability, in vitro cellular uptake was performed on human umbilical vein endothelial cells (HUVEC) and C6 cells, and the receptor expression on these cells were determined by Western blot. The internalization procedure and mechanism were further elucidated using several markers and inhibitors. Then in vivo imaging was performed to evaluate the GBM targeting ability and neovasuclature and GBM cells were labeled to determine whether the IRNPs could target to both cells or not.

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EXPERIMENTAL SECTION Materials. RGD and IL-13p were synthesized by Chinapeptide Biotech Co. Ltd. (Shanghai, China). DTX was purchased from Knowshine (Shanghai, China). Methoxy poly(ethyleneglycol)-poly(ε-caprolactone) (MPEG-PCL) (Mw: 3k−15k) and carboxyl poly(ethylene glycol)-poly(ε-caprolactone) (HOOC-PEG-PCL) (Mw: 3.4k−15k) were synthesized as previously described.20 N-(3-Dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC), N-hydroxy-succinimide (NHS), and coumarin-6 were purchased from Sigma (Saint Louis, MO, U. S. A.). 1,1′-Dioctadecyl-3,3,3′,3′tetramethylindotricarbocyanine iodide (DiR) was obtained from Biotium (Hayward, CA). Plastic cell culture dishes and plates were purchased from Wuxi NEST Biotechnology Co. Ltd. (Wuxi, China). Dulbecco’s Modified Eagle Medium (high glucose) cell culture medium (DMEM) and fetal bovine serum (FBS) was purchased from Life technologies (Grand Island, NY, U. S. A.). 4,6-Diamidino-2-phenylindole (DAPI) was 1043

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In Vivo Imaging. GBM bearing mice were established as described previously.23 Mice were anesthetized and fixed on a stereotaxic apparatus. A 5 μL suspension containing 5 × 105 RFP-C6 cells was slowly injected into the right corpus striatum of the nude mice. Ten days later, 2 mg/kg of DiR-loaded NPs or ILNPs were injected into the GBM-bearing mice. The distribution of fluorescence was observed by an IVIS spectrum in vivo imaging system (Caliper, MA, U. S. A.) 24 h postinjection. Mice were sacrificed at 24 h and the ex vivo image of the brain was also captured at that time. Brain distribution. The coumarin-6-loaded particles were injected into the GBM-bearing mice through the tail vein at a concentration of 100 μg/kg according to coumarin-6. After 2 h, the mice were anesthetized and the hearts were perfused with saline followed by 4% paraformaldehyde. The brain was removed for the preparation of consecutive frozen sections of 5 μm thicknesses. Microvessels were stained with goat antiCD31 antibody followed with dylight647-conjugated goat antirabbit IgG according to previous established procedure.21 The distribution of fluorescence was observed by a confocal microscope (TCS SP5, Leica, Germany). Statistical Analysis. Data were presented as mean ± SD. Statistical differences in cell uptake and in vivo imaging were determined by Student’s t-test.

Cellular uptake. C6 cells and HUVEC in the logarithmic growth phase were seeded into 6-cm glass-bottom dishes at a density of 1 × 104 cells/mL. Twenty four hours later, 100 μg/ mL of coumarin-6-loaded NPs, ILNPs, RNPs, or IRNPs were added into the wells and incubated for 1 h. The adsorptive and free particles were removed by washing with ice-cold PBS at 4 °C for 5 min. After nuclei stained by 0.5 μg/mL of DAPI for 5 min, the fluorescent images of cells was obtained by a confocal microscope (TCS SP5, Leica, Germany). Colocalization of Particles with Receptors. After being incubated with different kinds of particles for 1 h, C6 cells were fixed by 4% paraformaldehyde and then rinsed with 1% Triton X-100 (used for permeation of the membrane of cells). After being washed by Tris-buffered saline (TBS, pH 7.2), cells were stained by goat anti-IL13Rα2 (1:200) and rabbit anti-αvβ3 (1: 200) at room temperature for 1 h. After being washed with TBS three times, cells were labeled with Dylight647-conjugated donkey antirabbit IgG (1:500) and Cy3-conjugated donkey antigoat IgG (1:500) for 0.5 h at room temperature. After the cells were washed three times by TBS, the nuclei of the cells were stained by 0.5 μg/mL DAPI for 5 min. The fluorescence of cells was observed through confocal microscope (TCS SP5, Leica, Germany). Subcellular Location. C6 cells and HUVEC were seeded in the glass-bottom dishes at a density of 1 × 104 per dish. After a 24 h incubation period, cells were treated for 30 min or 2 h with 100 μg/mL coumarin-6-loaded NPs, ILNPs, IRNPs, and RNPs in DMEM with makers of endolysosomal compartments (Lysotracker Red DND-99, 50 nmol/L). After nuclei were stained by DAPI (0.5 μg/mL) for 5 min and washed three times, cells were fixed and mounted in a fluorescent mounting medium. Images were observed with confocal microscope (TCS SP5, Leica, Germany). Uptake Mechanism. C6 cells (2 × 105 cells per 1 mL/ well) were seeded in 12-well plates. Forty-eight hours later, cells were treated with 100 μg/mL of coumarin-6-loaded NPs or IRNPs and various inhibitors for 60 min: PBS (control), 20 μg/ mL chlorpromazine, 2 μmol/L phenylarsine oxide, 10 μg/mL filipin, 40 μmol/L Cytochalasin D, 450 mmol/L sucrose, 0.1% w/v sodium azide, 200 nmol/L monensin, 50 μmol/L nacodazole, and 20 μg/mL brefeldin A (BFA). After being washed with ice-cold PBS three times, cells were harvested and resuspended in 0.5 mL PBS. The mean fluorescence intensity was observed by flow cytometry (BD, FACS Aria Cell Sorter, U. S. A.). To more precisely determine the involvement of clathrin-mediated endocytosis and macropinocytosis, C6 cells in 12-well plates were treated with coumarin-6-loaded NPs or IRNPs in the presence of inhibitors with different concentrations: 1.25, 5, or 20 μg/mL chlorpromazine, 450 mmol/L sucrose, 2.5, 10, or 40 μmol/L cytochalasin D, or 3.125, 12.5, or 50 μmol/L nocodazole. After 1 h incubation, cells were handled as described above. Tumor Spheroid Penetration. C6 three-dimensional spheroids were established as previously described.22 C6 cells (1 × 105/mL) were seeded in 96-well plate precoated with 2% low melting point agarose. Seven days later, coumarin-6-loaded NPs, ILNPs, IRNPs, and RNPs were added to the wells with C6 spheroids at a concentration of 200 μg/mL. After 12 h incubation, C6 spheroids were washed with PBS three times and fixed with 4% paraformaldehyde overnight. The fluorescent intensity of different depths within C6 spheroids was observed through confocal microscope (TCS SP5, Leica, Germany).

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RESULTS AND DISCUSSION Characterization of NPs. In this study, we utilized MPEGPCL to form nanoparticles. Its biocompatibility and low toxicity made it attractive for nanoformulations.20,24 The median lethal dose (LD50) of MPEG-PCL is 1.47 g/kg,25 which is much higher than the dose used in our experiments (approximately 100 mg/kg of MPEG-PCL). The particle sizes of NPs, ILNPs, IRNPs, and RNPs were around 120 nm and generally spherical according to TEM (Table 1 and Figure 2), which was suitable Table 1. Characterization of Different Kinds of NPs DiR-NPs DiR-ILNPs DiR-IRTNPs DiR-RNPs

particle size (nm)

PDI

ζ potential (mV)

± ± ± ±

0.187 0.195 0.204 0.187

−10.23 −9.98 −10.29 −9.67

103.1 110.5 121.3 120.1

40.8 46.8 59.8 53.1

for drug delivery because particles with a size from 5 to 200 nm generally exhibited better transporting efficiency.26 ζ potentials of particles were approximately −10 mV, which could contribute to decreased rate of reticuloendothelial system uptake and longer blood circulation time compared to charged (about ±40 mV) particles.27,28 Receptor Expression. The expression of IL13Rα2 and αvβ3 was evaluated on both HUVEC and C6 cells (Figure 3). It showed high αvβ3 expression on HUVEC cells, which was consistent with previous results,29,30 making HUVEC a widely used model for angiogenesis targeting delivery and treatment.13 Additionally, the expression of αvβ3 on C6 was relatively high. However, there was high IL13Rα2 expression on C6 cells but low expression on HUVEC cells, which was consistent with previous studies that showed IL13Rα2 was a glioma-restricted receptor.18,31 Thus, this study utilized C6 cells and HUVEC for the evaluation of tumor cell and neovascular targeting efficiency of IRNPs. Cellular Uptake. According to our experiments, the release of coumarin-6 from NPs was lower than 1% after 24 h 1044

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Figure 2. (A) Morphology of IRNPs obtained by TEM after negative staining with 2% sodium phosphotungstate solution; the bar is 100 nm. (B) Particle size of IRNPs determined by DLS.

neovasculature targeting effect (Figure 4B). IL-13p, targeting for GBM restricted receptor IL-13Rα2, could not significantly increase the uptake by HUVEC, which was consistent with the low IL-13Rα2 expression in HUVEC as determined by Western blot. RGD could significantly enhance the HUVEC uptake owing to the high expression of αvβ3 in HUVEC. This experiment demonstrated IRNPs could target both GBM cells (C6) and neovasculature (HUVEC). However, because of the expression of both receptors on C6 cells, only C6 cells were used in the following studies on colocalization with receptors and endocytosis mechanism. Colocalization of Particles with Receptors. To further determine the targeting efficiency of different kinds of nanoparticles, C6 cells were stained with anti-IL13Rα2 antibody and anti-αvβ3 antibody (Figure 5). Conjugation with IL-13p facilitated the uptake of ILNPs by C6 cells, which was much colocalized with IL-13 Rα2, suggesting the IL-13p modification could enhance the uptake through the IL13Rα2mediated pathway. RNPs displayed an obvious colocalization with αvβ3, demonstrating the integrin receptor αvβ3 could mediate the internalization of RGD modified nanoparticles. Combining the effect of RGD and IL-13p, IRNPs displayed the highest cellular uptake, which was well colocalized with both receptors, indicating the dual modification could improve the

Figure 3. IL13Rα2 and αvβ3 expression of HUVEC and C6 cells. GADPH was used as control.

incubation; thus, the fluorescent distribution could represent the behavior of particles.32 To identify the targeting effect of the particles, C6 GBM cells and HUVEC were used. IL-13p and RGD could significantly increase the C6 uptake (Figure 4A) owing to the relatively high expression levels of IL13Rα2 and αvβ3 on C6 cells, which was consistent with other studies and our previous study.31,33−35 HUVEC was used to identify the

Figure 4. (A)C6 cell and (B) HUVEC uptake of 100 μg/mL coumarin-6-loaded NPs, ILNPs, IRNPs, or RNPs for 1 h. The bar in (A) represent 25 μm, and the bar in (B) represents 50 μm. 1045

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Figure 5. Colocalization of different kinds of particles with receptors of IL13Rα2 and αvβ3 after a 1 h incubation period. Blue represents nuclei stained by DAPI, green represents particles labeled by coumarin-6, red presents IL13Rα2 stained by anti-IL13Rα2 antibody, violet represents αvβ3 stained by anti-αvβ3 antibody, and the bar represents 10 μm.

Figure 6. Subcellular location of different formulations with endosomes. (A) Incubation of different particles with C6 cells for 0.5 h. (B) Incubation of different particles with C6 cells for 2 h. Green represents particles that tracked by coumarin-6, red represents endosomes labeled by Lysotracker Red, and the bar represents 5 μm.

associated with the endosomes. However, after a 2 h incubation period, most of the ILNPs, RNPs, and IRNPs distributed in cytoplasm rather than endosomes, suggesting the particles could escape from endosomes after a 2 h incubation period. In the contrast, NPs were still mostly colocalized with endosomes, suggesting the modification IL-13p or RGD could increase not only the cell uptake but also the endosome escape, which was consistent with many other studies.37−40 Consistent results were obtained in HUVEC (Figure 7). The cellular uptakes of IRNPs and RNPs were higher than those of NPs and ILNPs. There were obviously colocalization of particles with endosomes after 0.5 h of incubation, suggesting the uptake was involved in endosomes. When the incubation time was extended to 2 h, much of the fluorescence was

cell uptake through respective receptors and lead to an improvement on the total amount of uptake. Several other dual-modification drug delivery systems also showed similar phenomenon,29,32,36 which demonstrated the dual targeting delivery systems were superior in transporting cargoes into cells. Subcellular Location. Endocytosis was often involved in endosomes. After a 30 min incubation period, there was obviously higher uptake of ILNPs and RNPs than NPs in bEnd.3 cells (Figure 6A), whereas the uptake of IRNPs was even higher than that of ILNPs and RNPs. These results were consistent with cellular uptake results. All kinds of nanoparticles were mostly colocalized with the endosomes, which were marked by LysoTracker Red, indicating the cell uptake were 1046

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Figure 7. Subcellular localization of different formulations with endosomes. (A) Incubation of different particles with HUVEC for 0.5 h. (B) Incubation of different particles with HUVEC for 2 h. Green represents particles that tracked by coumarin-6, red represents endosomes labeled by Lysotracker Red, and the bar represents 10 μm.

Figure 8. (A) Uptake mechanism of NPs and IRNPs by C6 cells. (B) Cellular uptake of NPs and IRNPs at the presentation of different concentrations of inhibitors for clathrin-mediated endocytosis. (C) C6 cells uptake of NPs and IRNPs at the presentation of different concentrations of inhibitors for macropinocytosis. The results were showed in relative uptake compared to control (n = 3). *p < 0.05 vs control; #p < 0.01 between NPs and IRNPs.

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Figure 9. (A) Penetration through C6 spheroids after 12 h incubation. The pictures showed the fluorescent images of tumor spheroids from the bottom every 15 μm for 105 μm; the bar represents 200 μm. (B) Average fluorescent intensity of center of spheroids at 105 μm (n = 3). *p < 0.05, **p < 0.01.

Figure 10. In vivo imaging of brain tumor bearing mice 24 h after injection with DiR-loaded IRNPs. (A) Whole body imaging of mice 24 h after injection with DiR loaded nanoparticles. (B) Ex vivo imaging of brains from mice treated with DiR loaded nanoparticles. The upper was observed from the front side, and the lower was observed from the reverse side. (C) Semiquantitative fluorescent intensity of brain tumors. (D) Ex vivo imaging of various tissues. (E) Semiquantitative fluorescent intensity of vary tissues. The unit for fluorescent intensity in (C) and (E) is ×109 p/sec/ cm2/sr/(μW/cm2).

ent pathway.31,42 Additionally, this pathway was also affected by the property of ligands, such as density, length, and rigidity of the ligands.43 The Golgi apparatus and lysosomes have important roles in both intracellular cargo transport and disposition.39,44 In this study, both BFA, which disrupts the Golgi apparatus and intracellular trafficking, and monensin, a lysosome inhibitor, significantly decreased the uptake of IRNPs, indicating that both the Golgi apparatus and lysosomes are significantly involved in intracellular transport of IRNPs.

distributed in cytoplasm, suggesting the particles could escape from endosomes, which was consistent with other studies.31,41 Uptake Mechanism. The cellular uptake of NPs and IRNPs was through energy-dependent endocytosis because it was reduced to 93.4% and 76.7% of the control after energy depletion by sodium azide (Figure 8A). The effect on uptake of IRNPs was significantly greater than that of NPs because IRNPs were mostly internalized by receptors mediated active uptake, as demonstrated above, which was an energy-depend1048

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Figure 11. Distribution of various particles in brain tumors and colocalization with microvessels (indicated by white arrows) 2 h after injection. Nuclei were stained by DAPI, particles were marked with coumarin-6, brain tumor cells were RFP-C6, and microvessels were stained by CD31 antibody. The bar represents 100 μm.

D and nocodazole, the results were opposite (Figure 8C). These results clearly indicated the modification with IL13-p and RGD changed the main uptake pathway from macropinocytosis to receptor mediated endocytosis, which can explain the enhanced uptake of IRNPs by C6 cells, displaying good consistency with previous publications.31 Tumor Spheroid Penetration. Three-dimensional tumor spheroids have been proposed to targeted delivery and therapy evaluation.22,47−49 These tumor spheroids are increasingly utilized as models of intermediate complexity between in vitro cultured cells and in vivo tumors because of several advantages, including poor drug penetration, altered protein expression and enzyme activity, and viable rim with gradients of oxygen tension, nutrients, catabolites and cell proliferation.50−53 Thus, this study utilized tumor spheroid to evaluate the penetration ability of IRNPs. Although there was high fluorescent intensity in the bottom of C6 spheroids of all groups (Figure 9A), the penetration effect was not the same. At 105 μm to the bottom, fluorescence of NPs was only distributed in the periphery of C6 spheroids, whereas there was obvious distribution in the core of C6 spheroids of ILNPs and RNPs, demonstrating the conjugation with IL-13p and RGD could enhance tumor penetration effect as well as C6 cell uptake, which was consistent with a previous study.54 Comodification of IL-13p and RGD onto nanoparticles led to further improvement on the penetration ability. The average fluorescent intensity in the core of spheroids (with an area of 8 × 104 μm2) was further quantitatively analyzed to more clearly elucidate the difference in penetration ability (Figure 9B). The fluorescent intensity of ILNPs and RNPs was 1.97- and 2.51fold higher than that of NPs respectively. IRNPs displayed

However, the Golgi apparatus and lysosomes may not be obviously involved in the uptake of NPs. These results were consistent with the subcellular localization study. The endocytosis was obviously involved in the uptake of both NPs and IRNPs because phenylarsine oxide, an inhibitor of endocytosis, greatly reduced the cell uptake to 50.4% and 37.7% respectively. There were at least four basic mechanisms involved in endocytosis: caveolae-mediated endocytosis, clathrin-mediated endocytosis, macropinocytosis, and clathrin and caveolae-independent endocytosis.24,45 In this study, sucrose, chlorpromazine (a specific inhibitor of clathrin-coated pits formation24), and filipin (the block agents of caveolaeassociated endocytosis37) could all significantly decrease the IRNPs uptake with a higher degree than the NPs uptake, indicating that these two pathways were obviously involved in the IRNPs uptake. However, cytochalasin D (microtubuledisrupting agents38) and nocodazole (an inhibitor of macropinocytosis) could significantly inhibit the uptake of NPs rather than IRNPs, suggesting macropinocytosis and clathrin- and caveolae-independent endocytosis were more important in the uptake procedure of NPs rather than that of IRNPs, which was mainly due to the IL-13p and RGD modification on IRNPs. To further elucidate the difference in internalization between NPs and IRNPs, Several inhibitors were selected to determine the concentration-related cellular uptake inhibition. Clathrinmediated endocytosis was the most important pathway for receptor-mediated endocytosis.46 Results showed that even at low concentration (5 μg/mL), chlorpromazine could effectively decrease the uptake of IRNPs. However, the chlorpromazine presented no effect on the uptake of NPs (Figure 8B). Another inhibitor, glucose, displayed the same results. For cytochalasin 1049

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IRNPs could target to GBM with high efficiency, which colocalized with both GBM cells and neovessels.

highest penetration ability, which was 3.94-fold higher than that of NPs and was significantly higher than that of ILNPs and NPs. The result was consistent with other studies, which showed targeting ligands modification can facilitate the penetration of nanoparticles into tumor spheroids.47,48,55−57 In Vivo Imaging. Twenty-four hours after the injection of DiR-loaded NPs, obviously there was localization of NPs in the brain (Figure 10A). Modification with IL-13p or RGD could increase the fluorescent intensity in brain, which was owing to the GBM targeting and neovasculature targeting effect. Comodification with IL-13p and RGD further improve the GBM targeting efficiency. Through ex vivo imaging of brain (Figure 10B), it showed that most particles were distributed in GBM site, which was because of the EPR effect of GBM and the integrate BBB in normal brain. The fluorescent intensity in GBM was consistent with in vivo imaging result. Through semiquantitative assay (Figure 10C), it showed the intensity of ILNPs and RNPs in GBM was 2.40- and 2.91-fold, respectively, of that of NPs, which was consistent with previous results.31,58,59 The intensity of IRNPs in GBM was 3.82-fold of that of NPs, further demonstrating the comodification of IL13p and RGD could increase the GBM targeting effect, which proved the superiority of dual targeting delivery systems.60,61 However, there were no significant differences among NPs, ILNPs, RNPs, and IRNPs in the distribution in normal organs (Figure 10D and E). Most particles distributed in liver, spleen, and kidney because these organs were the main organs to eliminate foreign materials from bodies.62 The modification with IL-13p and RGD did not obviously affect the in vivo circulation of particles. The in vivo study further demonstrated that tumor cell and neovasculature dual targeting could further improve the localization of delivery systems in GBM site, which could deliver more cargoes or therapeutics to GBM and lead to a better anti-GBM effect. Distribution in GBM. To further elucidate the distribution of particles in GBM bearing brains, frozen sections were prepared and neovessels were stained by anti-CD31 antibody (Figure 11). There was a low intensity of NPs in the GBM site, which was due to the poor targeting efficiency. ILNPs could target to GBM cells, leading to considerably higher localization in GBM cells, which was consistent with in vivo imaging study. However, rare ILNPs colocalized with neovessels owing to the low expression of IL13R2 on neovessels.63 There was also a relatively high intensity of RNPs in the GBM site and considerable colocalization with neovessels, which was because of the high αvβ3 expression on neovessels. Meanwhile, some of RNPs were distributed in GBM cells, which was consistent with cellular uptake study and was due to the expression of αvβ3 on GBM cells. Combining the effect of IL-13p and RGD, IRNPs showed the highest localization in GBM site. IRNPs could be observed both in GBM cells and neovessels, indicating IRNPs successfully presented their dual targeting effect.

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AUTHOR INFORMATION

Corresponding Author

*X. Jiang. E-mail: [email protected]. Phone: +86-2151980067. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, 2013CB932502), National Science and Technology Major Project (2012ZX09304004), and Department of Health of Zhejiang Province (2013KYA134).

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CONCLUSION In this study, we established a tumor cell and neovasculature dual targeting delivery system: IRNPs. A cellular uptake study demonstrated modification with IL-13p and RGD could enhance the uptake amount through corresponding receptors. The internalization of IRNPs was obviously involved in endosomes, which was mainly mediated by clathrin-mediated pathway that was different from unmodified NPs. Meanwhile, the modification with IL-13p and RGD significantly enhanced the tumor spheroid penetration. In vivo imaging demonstrated 1050

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