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A D-Peptide Ligand of Integrins for Simultaneously Targeting Angiogenic Blood Vasculature and Glioma Cells Yachao Ren, Changyou Zhan, Jie Gao, Mingfei Zhang, Xiaoli Wei, Man Ying, Zining Liu, and Weiyue Lu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00944 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Molecular Pharmaceutics

A D-Peptide Ligand of Integrins for Simultaneously Targeting Angiogenic Blood Vasculature and Glioma Cells Yachao Rena, b, Changyou Zhana, c, Jie Gaoa, Mingfei Zhanga, Xiaoli Weia, Man Yinga, Zining Liua, Weiyue Lu*,a, d a

Key Laboratory of Smart Drug Delivery of the Ministry of Education & Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, & State Key Laboratory of Medical Neurobiology, and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China. b

Harbin Medical University, Harbin, 1500813, China.

c

Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, & State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China. d

Minhang Branch, Zhongshan Hospital and Institute of Fudan-Minghang Acadimic Health System, Minghang Hospital, Fudan University, Shanghai 201199, & Institutes of Integrative Medicine of Fudan University, Shanghai 200040, China * Corresponding author: Weiyue Lu, 826 Zhangheng RD., Pudong District, Shanghai 201203, China. Email: [email protected], Tel: +86 21 51980006, Fax: +86 21 51980090

ABSTRACT: The current prognosis of glioma patients remains poor after intensive multimodal treatments, which is partially due to the existence of blood-brain tumor barrier (BBTB). In the present study, a novel “bifunctional ligand” (termed DVS) was developed by retro-inverso isomerization. DVS is a ligand of integrins highly expressed on glioma cells and tumor neovasculature. DVS exhibited exceptional stability in serum, and demonstrated significantly higher targeting efficiency for glioma and HUVEC cells compared with the parent L-peptide. As a result, DVS modified micelles (DVS-MS) exhibited high encapsulation efficiency of doxorubicin, ideal size distribution and sustained release behavior of the payload. In vivo studies showed DVS-MS could target and efficiently deliver fluorescence to tumor cells and tumor vasculature not only in the mice bearing subcutaneous tumors, but also in that bearing intracranial tumors. Moreover, doxorubicin loaded DVS modified micelles exerted potent tumor growth inhibitory activity against subcutaneous and intracranial human glioma in comparison to drug loaded plain micelles and LVS modified micelles. Therefore, DVS appears to be a suitable targeting ligand with potential applications for glioma targeted drug delivery. Keywords: glioma, tumor angiogenic vessels, DVS, micelle, active targeting 1. INTRODUCTION Cancer becomes a major threaten of public health and is one of the leading cause of death around the world. Glioblastoma multiforme (GBM) is the most common and lethal tumor of the central nervous system1. Irrespective of the intensive multimodal treatments including surgical resection and chemo/radiation therapy, the current prognosis of GBM patients remains dismal with a median survival of less than 15 months2. Only 3–5% of GBM patients can survive five years3. Because of extremely

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infiltrative nature of glioma, complete surgical resection of GBM is nearly impossible, leading to frequent tumor recurrence. Therefore, chemotherapy becomes essential in the treatment of GBM. Tumor microenvironment includes not only tumor cells, but also fibrotic tissue, various immune cells and tumor capillaries. GBM has been characterized as a highly angiogenic tumor4; the new blood vessels grow and infiltrate into the tumor, providing essential nutrients and oxygen and a route for tumor metastasis and recurrence5-8. It appears that targeting angiogenic vessels in glioma is a promising strategy. Appropriate targeting ligands that specifically bind to the tumor vessels and/or tumor cells are important for glioma targeted drug delivery. Integrins are a large family of cell adhesion receptors9-11, and have been exploited to achieve targeted drug delivery12-16. The integrin αvβ3 is over-expressed on cancer cells17and neovasulature9,12; integrin α6β1 is up-regulated in glioma cells18. LVS (VSWFSRHRYSPFAVS), a 15-residue peptide selected from a phage display library, interacts with integrin α6β1 with high affinity19. Integrin αvβ3 and integrin α6β1 are subtypes of integrins. The sequence identity between integrin αvβ3 and integrin α6β1 of α and β subunits are respective about 30% and 45%20,21. Thus, we hypothesize that L VS peptide may have a high binding affinity to not only integrin α6β1 but also integrin αvβ3. In addition, LVS consists of L-amino acids, thus is susceptible to proteolysis in plasma. We have previously verified that retro-inverso isomerization provides a useful method to develop stable peptide possessing comparable binding affinity to the parent L-peptide22-24. Thus, the retro-inverso isomer of LVS, termed D VS, would be able to bind intergins with high affinity and be fully resistant to proteolysis in blood circulation. In this study, we synthesized both LVS and DVS have a high binding affinity to integrin α6β1 and αvβ3 and assessed their binding affinities to integrins α6β1 and αvβ3. Glioma targeting efficiency of both peptides were evaluated. Furthermore, doxorubicin loaded VS peptides modified micelles (LVS-MS/DOX and D VS-MS/DOX) were prepared and their anti-glioma effects were assessed in vivo. 2. MATERIALS AND METHODS 2.1. Materials Integrin αvβ3 and Integrin α6β1 were purchased from R&D System, USA. Biacore series sensor chips CM5 and HBS-EP buffer were supplied by GE (Sweden). mPEG2000-PLA2000 and Mal-PEG3000-PLA2000 were acquired from advanced polymer materials Inc. (Montreal, Canada). Fluorescein-5-maleimide and 5-carboxyfluorescein (FAM) were supplied by Fanbo Biochemicals (Beijing, China). Near infrared dye DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide) were purchased from Invitrogen (Grand Island, NY). 4′,6-Diamidino-2-phenylindole (DAPI) was acquired from Roche (Switzerland). Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (United States). Rat anti-mouse CD31 was supplied by Abcam (United States). Goat anti-rat IgG (H&L), Dylight 594 conjugate was obtained from SantaCruz Biotechnology (United States). DOX hydrochloride was obtained from Zhejiang Haizheng Co. (Zhejiang, China). The U87 glioma cells, L929 fibroblast cells, HEK293 human embryonic kidney cell line and HUVEC human umbilical vein endothelial cells were acquired from the Shanghai Institute of Cell Biology. bEnd.3 brain capillary endothelial cells were supplied by ATCC (Manassas, VA). All cell lines were cultured in DMEM containing


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10% FBS, supplemented with 100 IU/mL penicillin and 100 µg/mL streptomycin at 37 °C under a humidified atmosphere containing 5% CO2. BALB/c nude mice (4-6 weeks age) were supplied by Shanghai SLAC laboratory animal center (Shanghai, China) or Charles River Laboratories (Beijing, China) and kept under SPF conditions. The protocol of all animal experiments were approved by the ethics committee of Fudan University. 2.2. Synthesis and Characterization of VS 2.2.1. Synthesis of VS L VS and DVS were synthesized by solid phase peptide synthesis using active ester chemistry to couple Fmoc-protected amino acid to the deprotected resin. LVS-FAM and DVS-FAM were synthesized by covalent conjugation of thiolated peptides with Fluorescein-5-maleimide. The products were purified by preparative C18 reversed-phase HPLC and molecular masses were determined by electrospray ionization mass spectrometry (ESI-MS). 2.2.2. Characterization of VS Peptides L VS and DVS were characterized at 0.1 mg/ml by circular dichroism spectrometry. The spectra were collected on a Chirascan spectropolarimeter using standard measurement parameters (e.g., temperature, 20 °C; wavelength, 190-260 nm; step resolution, 1 nm; response, 0.5 s; path length, 1 mm). 2.2.3. Stability of VS The stability of LVS and DVS was investigated in 25% mouse serum (diluted by PBS). 2 mg peptide was dissolved in 2 mL mouse serum solution. After different incubation times, 100 µL serum was sampled and mixed with 20 µL TCA to precipitate the serum proteins. Then, the mixture was stored at 4 °C for 20 min, and centrifuged at 12 000 rpm for 10 min. The residual peptides were quantified using reverse-phase HPLC. All experiments were carried out three times. 2.2.4. Surface Plasmon Resonance Assay The binding between VS peptides and two targeting protein (integrin α6β1 and integrin αvβ3 ) were assessed by surface plasmon resonance (SPR, Biacore 3000, Sweden). In brief, human integrin α6β1 and αvβ3 were covalently attached to the CM5 sensor chip. A mouse CD133 binding-peptide25, LQNAPRS (LS), was used as a control. LS, LVS and DVS were dissolved in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4). The samples were injected onto the integrin-immobilized CM5 sensor chip at a flow rate of 10 µL/min at 25 °C. Biacore 3000 evaluation software version 4.5 was used to calculate KD values. 2.3. In Vitro Cell Study of VS 2.3.1. Cellular Uptake HUVEC and U87 were seeded in confocal dishes or 12-well plates at 105 cells per well and were incubated for 24h at 37 °C. Then the medium was replaced with culture medium containing 10 µM LVS-FAM or DVS-FAM, which were incubated with the above cells at 37 °C for 4 h. Finally, the fluorescent intensity of cells was captured by confocal laser microscope and quantitated by flow cytometry. 2.3.2. Tumor Spheroid Penetration A U87 tumor spheroid was prepared to estimate the tumor-penetrating ability of FAM labeled peptide. Briefly, 2% low-melting temperature agarose was added into 48-well plates. After the agarose was set, U87 were seeded in the plates at a



concentration of 5 × 103 cells per well to form tumor spheroids. After a week, L VS-FAM or DVS-FAM molecules at a final concentration of 10 µM were added to the spheroids to incubate for 4 h. The spheroids were washed with PBS and fixed with 4% formaldehyde and then visualized with confocal laser microscope. 2.4. Preparation and Characterization of Micelles 2.4.1. Preparation of Micelles To prepare LVS and DVS modified micelles (LVS-MS and DVS-MS), L VS-PEG-PLA and DVS-PEG-PLA were synthesized through covalent conjugation according to the previous procedure26. For Coumarin-6 (C6) or DiR encapsulated micelles (including MS/C6, LVS-MS/C6, DVS-MS/C6, MS/DiR, LVS-MS/DiR and D VS-MS/ DiR), C6 or DiR was dissolved in CHCl3 together with mPEG2000-PLA or a mixture of mPEG2000-PLA/VS-PEG-PLA (95/5, by mol) to prepare thin film, which was hydrated with saline. The free fluorophores were removed by gel filtration column. For DOX loaded micelles (including MS/DOX, LVS-MS/DOX and D VS-MS/DOX), DOX·HCl was dissolved in purified water and NaHCO3 was added into to remove hydrochloride. Chloroform was used to extract free DOX from the reaction mixture and CHCl3 was removed by rotary evaporation. DOX was dissolved in CHCl3 together with mPEG2000-PLA or a mixture of mPEG2000-PLA/LVS-PEG-PLA or mPEG2000-PLA/DVS-PEG-PLA to prepare thin film, which was hydrated with saline and filtrate through membrane with 0.22µm pore to remove unencapsulated DOX. 2.4.2. Characterization of Micelles Dynamic light scattering (DLS) was used to determine the particle size and zeta potential of micelles, and each sample was tested in triplicate. The morphology of micelles were characterized by transmission electron microscope (TEM), after negative staining with 4% phosphotungstic acid. The encapsulation efficiency of micelles was investigated as previously reported24. The in vitro DOX release from micelles was measured at 37 °C using a dialysis method described previously24. 2.5. The Evalution of Effect of Micelles on the Glioma Spheroids 2.5.1. Tumor Spheroid Penetration U87 spheroids were incubated with various micelle formulations loading DOX at 10 µg/mL for 4 h. Then the spheroids were washed with PBS and fixed with 4% formaldehyde, and then visualized with a fluorescence microscopy. 2.5.2.The Study of Growth Inhibition The tumor spheroids were incubated with culture medium, DOX, MS/DOX, L VS-MS/DOX or DVS-MS/DOX. The major (dmax) and minor (dmin) diameter of each U87 glioma spheroids were measured using a microscope each day. The spheroid volume and volume ratio was calculated according to the previous report27 with the following equation: V=(π×dmax×dmin)/6 and R=(Vday i/Vday 0) × 100%. Where Vday i is the spheroids volume at the ith day after administration, and Vday 0 is the spheroids volume prior to treatment. 2.6.Pharmacokinetics Study The pharmacokinetic profiles of DOX-loaded micelles were measured using SD rats injected at 6 mg/kg (DOX to body weight) via tail vein. Blood was obtained from


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the retro-orbital sinus at different time points within 2 days. A 200 µL sample of serum was collected after centrifugation of the blood samples; then, 25 µL of internal standard solution (20 µg/mL daunorubicin hydrochloride) and 1 mL of extract (chloroform/methanol 4:1 of volume ratio) were added, vortexed, and centrifuged at 6000 g for 10 min. The lower layer (organic layer) was collected and dried by nitrogen gas. The residue was dissolved in 100 µL methanol to be analysed by HPLC. The pharmacokinetic profiles of micelles were analyzed using the software DAS2.0. 2.7. Animal Model U87 subcutaneous and intracranial tumor models were established to value the effect of the various micelle formulations on U87 animal model. To establish U87 tumor models, the trypsinized U87 cells were resuspended in cold PBS. For the subcutaneous tumor model, BALB/c nude mice were inoculated with 5.0 × 106 U87 cells on the right shoulder blade. For the orthotopic intracranial glioma model, a total of 5.0 × 105 U87 cells were implanted into the right hemisphere of the mouse brain (1.8 mm lateral, 0.6 mm anterior to the bregma with 3 mm depth). 2.8. In Vivo Tumor Targeting Study To assess tumor targeting specificity, the mice were administered with MS/DiR, VS-MS/DiR or DVS-MS/DiR for in vivo imaging observation when the subcutaneous tumor volume reached at least 200 mm3 or two weeks after inoculation of intracranial tumor cells. Then DiR was injected at a dose of 0.1 mg/kg of body weight. For the subcutaneous tumor model, at predetermined time points, the mice were sacrificed. The normal organs (heart, liver, spleen, lung, and kidney) and tumor tissues were collected to be observed using a live animal imaging system. For the intracranial tumor model, at 24 h postinjection, the mice were sacrificed. The normal organs and tumor tissues were collected to be detected. To investigate the targeting mechanism, the subcutaneous tumors 8 h after treatments with DVS-MS/C6 or the intracranial tumor 24 h after treatments with D VS-MS/C6 were snap-frozen. The tumor tissues were cut into 10-µm-thickness slices using Thermo Cryostat. Sections were incubated with rat anti-mouse CD31 following by goat anti-rat IgG conjugated to Dylight 594 to label the blood endothelial cells. Then slides were treated with DAPI for nuclear counterstaining, and imaged using a laser scanning confocal microscope. L

2.9. Study of In Vivo Antitumor Effect 2.9.1. Antitumor Study in Subcutaneous Tumor Models After the tumor volume reached 50-100 mm3, the model animal were randomly divided into five groups (n = 6) and received injections of saline, free DOX, MS/DOX, L VS-MS/DOX and DVS-MS/DOX at 2 mg/kg (DOX to body weight) every 3 days for five times. Tumor size and body weight of each mouse were determined every other day. The tumors were harvested and weighed, after the mice were sacrificed at 34 days postinoculation. Hearts were collected to evaluate heart damage after the treatment of various DOX formulations. Then formalin-fixed tumors and hearts were embedded in paraffin blocks to prepare hematoxylin and eosin (HE) stained tumor and heart sections, and then were visualized by optical microscope.



2.9.2. Antitumor Study in Intracranial Tumor Models The intracranial U87 tumor mice were randomly divided into 5 groups (n = 8) and were intravenously injected with saline, free DOX, MS/DOX, LVS-MS/DOX and D VS-MS/DOX (3 mg/kg DOX/mice/injection) via tail vein at 9, 12, 15, and 18 days after implantation. Survival time was monitored and Kaplan-Meier survival curves were plotted for each group. 3. RESULTS 3.1. Characterization of VS L

VS and DVS were synthesized by solid phase peptide synthesis. The purity and molecular weight of LVS and DVS were ascertained by HPLC and ESI-MS spectrometry. The determined molecular weights were consistent with their theoretical ones. The results of circular dichroism spectrometry indicated that both L VS and DVS harbored randomly coiled structure (Figure 1A). To evaluate peptide stability, the proteolysis of peptides was investigated in 25% rat serum. As shown in Figure 1B, DVS remains at almost 100% even after 24 h incubation; in contrast, LVS displayed very fast degradation. More than 50% was degraded in 4h and almost completely degraded in 10 h. To access whether or not DVS is capable of interacting with both integrins α6β1 and αvβ3, SPR-based binding assays were conducted. As shown in table S1, KD value of L VS and DVS to integrin αvβ3 was respective 11.0 nM and 78.3 nM, and that to integrin α6β1 was 39.8 nM and 21.9 nM. KD value of control peptide (LS) to both integrins was only 17.0 µM and 22.3 µM. These data demonstrated that LVS and DVS had a high binding affinity to both integrins α6β1 and αvβ3. KD value of LVS and DVS to both integrins is comparable. 3.2. In Vitro Cell Study of VS U87 cells were used as target cell line, while L929 and HEK293 cells were used as control cell lines. c(RGDyK) was used as a control peptide. Intracellular fluorescence intensity of LVS-FAM in glioma cells was 2.6 and 1.8 times higher than that in L929 and HEK293 cells (Supplementary Figure S1). These findings showed that LVS-FAM was taken up by glioma cells and is significantly higher than by control cell lines, suggesting LVS could specifically recognize glioma cells. Moreover, targeting of LVS was better than that of RGD (Supplementary Figure S1). The targeting of VS peptide was evaluated by U87 cells and HUVEC cells. As shown in Figure 2A, VS could be taken up by U87 cells and HUVEC cells, suggesting that VS peptides held the potential to target brain cancer cells and neovasculature. Notably, DVS exhibited significant higher endocytosis efficiency than did LVS. The confocal images were further confirmed by the data of flow cytometry (Figure 2B). The percentage of fluorescein in positive cells after treatments with L VS-FAM and DVS-FAM was 64.09% and 87.26% for U87 cells, and 40.50% and 87.31% for HUVEC cells, and the targeting of DVS-FAM for U87 and HUVEC cells were obviously significant than LVS-FAM. These results demonstrated that LVS were easily degraded by plasma proteases, therefore partially losing its targeting capacities; while DVS was much more stable. U87 tumor spheroids were applied to imitate in vivo status of glioma and to observe the penetrating ability of DVS by confocal laser microscopy. As indicated in Figure


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2C, by distribution and fluorescence intensity properties, the penetration of DVS-FAM was obviously deeper than that of LVS-FAM in glioma spheroids. Collectively, these results confirmed that the DVS not only increases uptake by U87 cells and HUVEC cells, but also effectively improved the efficacy of the tumor penetration. 3.3. Characterization of Micelles L

VS-PEG-PLA and DVS-PEG-PLA were obtained through covalent conjugation between maleimide group of mal-PEG-PLA and thiol group of VS peptide and its structure was validated by the NMR spectra in Figure S2. The particle size, polydispersity index (PDI) and zeta potential of various micelles were listed in Table S2. Peptide modified micelles showed slightly lower zeta potential. The encapsulation efficiency of MS/DOX, LVS-MS/DOX and DVS-MS/DOX was respective 74.07 ± 2.87%, 79.32 ± 5.72% and 78.69 ± 3.22%, and drug loading of MS/DOX, L VS-MS/DOX and DVS-MS/DOX was respective 10.3 ± 1.23%, 10.8 ± 1.58% and 11.2 ± 1.62%. DOX loaded micelles with or without modification were of similar encapsulation efficiency and drug loading. The TEM images displayed uniform spherical shapes of MS/DOX, LVS-MS/DOX and DVS-MS/DOX with a good dispersity (Figure S3A). These results demonstrated that the presence of LVS and DVS on the surface of micelles executed no significant effect on the physical properties of micelles. The average size of all DOX loaded micelles were about 22 nm (Figure S3B and Table S2), which was beneficial for delivering drugs to the tumorous tissues28,29. The surface potential of various DOX loaded micelle formulations was ranging from 10 to 14 mV and this positive surface charge would enhance the interaction with the electronegative cell membrane, leading to the endocytosis by cells30. The release profiles of DOX from different micelles were investigated in two different buffer solutions (pH 5.0 and 7.4). As shown in Figure S3C, the release of DOX from different formulations was similar. DOX release from micelles at pH 5.0 was much faster than that at pH 7.4. 3.4. Distribution of Micelles In Vitro and In Vivo In this study, the U87 glioma spheroids were incubated with various micelle formulations when it became compact and homogeneous after 7 days culture. In Figure S4, the images of glioma spheroids showed that fluorescent intensity of tumor spheroid treated with DVS-MS/DOX was obviously greater than that of free DOX, MS/DOX and LVS-MS/DOX, which were consistent with the results of tumor spheroid penetration of VS peptides, suggesting the importance of the stability of the targeting ligand. The pharmacokinetic profiles of DOX in all micelles-treated mice were compared with free DOX-treated mice (Figure S5). Pharmacokinetic parameters of all DOX formulations were evaluated by using the software DAS2.0. The half-lives (t1/2), area under concentration-time curve (AUC), and mean residence time (MRT) were increased notably, compared with those of the free DOX solution. Moreover, the modification of LVS or DVS did not influence the pharmacokinetic profiles of micelles significantly. It is imperative to investigate the targeting of micelles modified with VS in vivo after systemic administration, although the in vitro study described above proved the specificity of VS peptides. The mice bearing U87 subcutaneous tumor were injected with MS/DiR, LVS-MS/DiR or DVS-MS/DiR. At predetermined time points, the level



of fluorescence in the tumor tissues or normal organs (heart, liver, spleen, lung, and kidney) were observed by in vivo imaging system. In tumor tissues from mice injected with DVS-MS/DiR, the strong fluorescence was detected from 8 h to 24 h (Figure S6). However, only weak fluorescence was observed in tumor tissues from mice treated with MS/DiR or LVS-MS/DiR (Figure S6). In addition, the semi-quantitative data indicated the highest fluorescence intensity in tumor in DVS-MS group (Figure 3A), which was about 3.06-fold of that of MS and 1.86-fold of that of LVS-MS at 8 h. The targeting ability of VS-MS delivery system was also confirmed in the intracranial U87 tumor model. The model mice were administered with MS/DiR, L VS-MS/DiR or DVS-MS/DiR and then were imaged with the in vivo imaging system. The ex vivo fluorescent image of the excised brain tumor demonstrated that preferential accumulation of fluorescence was evident in the brain tumor of the mice treated with DVS-MS compared with that treated with MS or LVS-MS (Figure 4A), which was consistent with the results obtained from the subcutaneous tumor model. The quantitative measurements of the fluorescence intensity in these tumors were displayed in Figure 4B. In order to clarify the targeting mechanism, we sought to determine whether the tumor-targeting described above including specificity to tumor angiogenic vessels in in vivo model. The immunofluorescence analysis of the subcutaneous and intracranial tumors was performed using tumor angiogenesis maker CD31. As shown in Fig. (3B and 4C), abundant green fluorescence signal was observed in tumor cells. More importantly, DVS-MS/C6 delivery was not limited to tumor cells, but was also obvious in tumor angiogenic vessels. Taken together, these results in mice bearing subcutaneous and intracranial U87 tumor confirmed not only the tumor specificity of DVS-MS, but also its ability to target and efficiently deliver the payload into tumor cells and tumor angiogenic vessels after systemic administration. 3.5. Anti-tumor Effect In Vitro and In Vivo The inhibitory effects of various treatments on the U87 glioma spheroids were investigated in this study. Figure S7 represented the tumor spheroid volume change ratios after treatment of DOX, MS/DOX, LVS-MS/DOX and DVS-MS/DOX, respectively. Compared with the control group, all treatment groups exhibited significant inhibitory effect on glioma spheroids. However, there were no significant differences of the tumor spheroid volume change ratios among free DOX, MS/DOX and LVS-MS/DOX. In contrast, DVS-MS/DOX significantly inhibited growth of the 3D glioma spheroids, suggesting that DVS-MS/DOX may improve therapeutic effect in vivo, due to the tumor spheroid mimics the microenvironment of in vivo solid tumor. The data presented above shows that DVS-MS/DOX significantly inhibited growth of the 3D glioma spheroids. To evaluate the potential clinical application of this formulation, we investigated the anti-tumor efficacy of DOX-loaded VS-MS in vivo using BALB/C nude mice bearing subcutaneous U87 tumor. The tumor growth inhibition of mice in different groups were compared and the results were shown in Figure 5A. All groups with DOX treatment exhibited significant inhibitory effect on tumor growth compared with the saline control group. However, the growth of tumor was more significantly suppressed by the treatment of LVS-MS/DOX and D VS-MS/DOX, suggesting that VS-MS could deliver more drug into glioma tissue via the receptor mediated endocytosis. More interestingly, DVS-MS/DOX displayed even


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higher tumor growth inhibitory effects than LVS-MS/DOX. At the end of the test, the average weight of isolated tumors of all groups further confirmed the tumor volume results (Figure 5B). The images of the tumors H&E staining also validated DVS-MS groups could result in the greatest necrosis areas among all groups (Figure 5C). The anti-tumor effects of DVS-MS were in good accordance with the tendency of cellular uptake in vitro, the tumor drug accumulation in vivo and the in vitro 3D glioma spheroids experiment. As the above in vivo study indicated obvious response with DVS-MS/DOX in subcutaneous U87 model, we intend to evaluate the effect of DVS-MS/DOX on tumor growth in an intracranial model. The survival of the tumor bearing mice treated with various micelle formulations was presented as the Kaplan-Meier survival curve (Figure 5D) and the median survival time of mice in the group of saline control, free DOX, MS/DOX, LVS-MS/DOX and DVS-MS/DOX was 22.5, 23.5, 24, 25.5 and 29 days, respectively. Compared to the untreated mice, there were an only minimal increase in survival after treatment with free DOX (p = 0.4684) or MS/DOX (p = 0.0604). In contrast, VS-modified micelles were significantly more effective in prolonging mice survival (p < 0.05) than the saline group, however, the median survival of the mice administered with DVS-MS/DOX (p = 0.001) was much longer than that with LVS-MS/DOX (p = 0.0384). Moreover, the median survival time of mice in DVS-MS/DOX group prolongs 1.17 times than that in LVS-MS/DOX group by equation, that is, ratio = (TD - TC)/(TL - TC) = (29-22.5)/(25.5-22.5) = 2.17. TC, TL and TD is the median survival time of mice in the group of saline control, L VS-MS/DOX and DVS-MS/DOX, respectively. Such in vivo anti-glioma effects confirmed that DVS-MS/DOX could better improve the chemotherapeutic efficacy of the intracranial U87 glioma treatment, which was consistent with the above tumor growth inhibition results. 3.6. The Toxicity of DOX Micelles In addition, the toxicity of all DOX formulations was also evaluated. The body weight change, as an indicator of systemic toxicity, was investigated in all experimental mice. The body weight increased in all the groups, but the saline group increased quickly, partly owing to the rapid growth of the implanted tumor. However, the body weight of animals administered with DOX showed relative significantly slow growth compared with that with DVS-MS/DOX, clearly suggesting that the free DOX was more severely toxic than the DOX-loaded DVS modified micelles (Figure 6A). Cardiotoxicity is one of the major side effects of DOX, thus the heart tissues of the mice treated with various DOX formulations were further evaluated by H&E staining. After staining with H&E, the free DOX caused inflammatory response and slight damage to cardiac muscle, and no obvious cardiac toxicity was observed in any micelle groups (Figure 6B). Overall, DVS-modified micelles could improve antitumor efficacy in vivo with low cardiac toxicity by increasing accumulation in tumors. 4. DISCUSSION Despite current therapies for glioma are effective in the initial phase of treatment, recurrence occurs frequently. So far, a great deal of efforts have been made on glioma-targeted therapy. A number of studies have focused on using a single targeting ligand to modify a drug delivery system31. As tumor tissue contains not only cancer cells but also neovesculature, multitype receptors are uniquely expressed or



upregulated. Simultaneous targeting two receptors at disease site could lead to better affinity and specificity for drug delivery carriers in target cells32. Therefore, for glioma therapies, a more practical approach would be able to simultaneously target cancer cells and neovasulature. Ligands are an imperative part of the targeted drug delivery system; however, cell surface receptors are dissimilar for different cells33. Integrins are cell adhesion molecules that are heterodimeric molecules by an α and an β subunit. Integrins directly or indirectly influence cell migration, cell proliferation, cell growth, angiogenesis, cell survival, invasion and tumorigenesis34. They have been exploited to the targeted drug delivery in several forms. The integrin αvβ3 and integrin α6β1 are indicated in the studies described above to be overexpressed on cancer cells, tumor angiogenic vessels. Interestingly, targeting studies in molecular and cell levels demonstrated that LVS had a high binding affinity to both integrins α6β1 and αvβ3 and can efficiently target glioma cells and neovasculature. However, the stability of targeting moiety in circulation in vivo is critical to achieve better targeting effect. It was found that peptides consisting of L-amino acids are susceptible to proteolysis in plasma and thus readily lose their targeting capability24. Cyclization, partial D-amino acid substitution and retro-inverso isomerization methods are often adopted to increase peptide stability35. DVS was designed by retro-inverso isomerization. We hypothesize that DVS would possess higher targeting efficiency by overcoming the protease degradation in plasma. DVS demonstrated high binding affinity to both receptors (integrin αvβ3 and integrin α6β1) and could target not only glioma cells but also tumor angiogenic vessels. Consequently, DVS could be a potential a “bifunctional ligand” for glioma targeted therapy. A variety of nano-sized drug delivery systems are currently explored for anticancer therapy. Polymeric micelles are among the most extensively used delivery systems owing to the ability to prolong circulation time, enhanced drug solubility, decreased side effects, improved drug bioavailability and passive targeting to the tumor region by the enhanced permeability and retention (EPR) effect36-38. PEG-PLA is an amphiphilic copolymer that was apt to form micelles and has been approved for medical application in Korea39-41. Thus, DVS peptide was conjugated to PEG-PLA to prepare DVS-MS to investigate the feasibility of DVS peptide as target molecular of nanocarrier. The results of characterization of micelles indicated that the conjugation of DVS into the surface of micelles did not influence on size, morphology and release behavior of micelles. It was reported that nanocarrier with the smaller size (~20 nm) could rapidly distributed throughout the tumor tissue28,29. In our study, all the micelles with an average diameter of about 20 nm is also more suitable for passive targeting to tumor tissue by EPR effect42. Such carrier size is also favorable for efficiently transporting the BBB43. Although nano delivery system could target cancer tissue by passive targeting, how to realize active targeting to achive better targeting efficiency. Targeting ligands play a key role in the active targeted drug delivery systems to the cancer sites. We found that DVS could be a potential a “bifunctional ligand”. Then we hypothesize that micelles modified with DVS would possess the similar targeting property. After administering various micelle formulations, we found that DVS-MS could target and efficiently deliver payload to tumor cells and tumor vasculature in the mice bearing subcutaneous tumors. More importantly, the targeting was not limited to the subcutaneous tumor model, but was also observed in the intracranial tumor model. However, the brain tumors comprise solid tumors and the avascular regions that prevent drugs from penetrating into the center of tumor44,45. In vitro 3D glioma spheroids were prepared to mimic the avascular regions and to evaluate the


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penetration of nano drug delivery systems. Results showed that the DVS-MS could effectively penetrate into the U87 tumor spheroids and reduce their volumes in comparison to the control group, free drug, or other micelle formulations. The chemotherapy is a vital means for cancer patients in clinic by suppressing tumor growth46. DOX is a widely used chemotherapeutic agent due to its wide anticancer spectrum47,48. However, the severe side effects of DOX such as dose-related cardiotoxicity and drug-resistance impede its clinical application49,50. In our work, DOX was loaded into micelles. Our present results indicated that the DOX loaded micelles with DVS modification could induce not only better anti-glioma effects in vivo, but also less side effects in comparison to free drug or other DOX loaded micelle formulations. 5. CONCLUSION In summary, we designed stable DVS by retro-inverso isomerization with potent multi-targeting to glioma and tumor angiogenic vessels to overcome enzymatic barriers. The function of DVS peptides as ligands of Integrin αVβ3 and α6β1 was experimentally validated, and tumor targeting efficacy was evaluated. DVS was modified to the surface of micelles to construct a stabilized multifunctional targeted drug delivery systems. DVS-MS could target and efficiently deliver fluorescence to tumor cells and tumor vasculature not only in the mice bearing subcutaneous tumors, but also in that bearing intracranial tumors. Moreover, doxorubicin loaded DVS modified micelles exerted potent tumor growth inhibitory activity against subcutaneous and intracranial human glioblastoma in comparison to drug loaded plain micelles and LVS modified micelles. DVS-MS delivery system has potential value in improving the anti-glioma efficacy of doxorubicin. SUPPORTING INFORMATION Table S1-S2 Figures S1-S7

ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (973 Program, No.2013CB932500), National Natural Science Foundation of China (No.81773657, No.81690263 and No.81473149), Shanghai Education Commission Major Project (2017-01-07-00-07-E00052) and Shanghai international science and technology cooperation project (No.16430723800).

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Figure 1. (A) Circular dichroism spectra of peptides. LVS and DVS respectively dissolved in water was diluted by phosphate buffer (pH 7.4) and the circular dichroism spectrum was obtained on a Chirascan spectropolarimeter. (B) Stability of peptides. LVS and DVS was respectively incubated with 25% rat plasma at a concentration of 0.1 mg/mL at 37°C for different time periods, and the remaining peptide amounts were quantified by HPLC.



Figure 2. In vitro cellular uptake and tumor spheroid penetration of VS peptides. U87 and HUVEC was respectively incubated with 10 µM fluorescein labeled VS peptides at 37°C for 4 h, followed by DAPI staining and rinse with phosphate buffered saline. Intracellular fluorescence was detected by (A) confocal laser microscopy and (B) flow cytometry, Scale bar = 20 µm. (C) Tumor spheroid penetration of fluorescein labeled VS peptides was examined by confocal microscopy, with a 10 µm interval between consecutive slides; the bar indicates 100 µm.


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Figure 3. The fluorescent intensity and immunofluorescence analysis of micelles in the subcutaneous glioma-bearing mice. (A) The fluorescent intensity of the tumor per unit area at different time point. MS, VS-MS labeled with DiR were injected into the tail vein of mice. At 1, 4, 8 and 24 h postinjection, mice were sacrificed (n = 3) and the tumor tissues were dissected for ex vivo fluorescence imaging. (B) The immunofluorescence images of tumor tissues. Tumor tissues were dissected 8 h after injection with DVS-MS/C6. The tumor tissues were cut into 10-µm-thick slices, stained with an anti-CD31 antibody, then treated with DAPI and analyzed using confocal microscopy, Scale bar = 20 µm.



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Figure 4. In vivo near infrared fluorescence imaging and immunofluorescence images of micelles in the U87 intracranial mice. (A) Ex vivo fluorescent image of tumors of mice sacrificed at the end of test 24 h post-injection with MS/DiR, LVS-MS/DiR or

D

VS-MS/DiR via tail vein. (B)

Quantitative analysis of the intensity of DiR fluorescence signal in tumor (* p < 0.05, ** p < 0.01). (C) Mice were injected with DVS-MS/C6, 24 h after injection, the brain tumor tissues were cut into 10-µm-thick slices, stained with anti-CD31 antibody, then treated with DAPI and analyzed using confocal microscopy, Scale bar = 20 µm.


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Figure 5. Anti-tumor effect of micelles in the subcutaneous glioma model and in the

intracranial model. (A) Tumor growth curve of mice treated with different formulations. Relative tumor volume = tumor volume/primary tumor volume. P = 0.048: DVS-MS/DOX group vs LVS-MS/DOX group, *P = 0.037 or 0.035 and 0.001: vs free DOX or MS/DOX and saline groups. Data shown as mean ± SD (n = 6). (B) Tumor weight, and (C) H&E staining images of tumor (×200) after different DOX formulations treatment. (D) Kaplan-Meier survival curves of the bearing intracranial U87 glioma nude mice (n = 8) that were treated with saline control, free DOX, MS/DOX, LVS-MS/DOX and DVS-MS/DOX.



Figure 6. The toxicity of DOX micelles. (A) Body weight curves of nude mice bearing U87 subcutaneous tumor after treated with different DOX formulations. Data shown as mean ± SD (n = 6, * p < 0.05). (B) H&E staining images of heart (×200) after different DOX formulations treatment.


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Graphic for manuscript 338x190mm (300 x 300 DPI)


A d-Peptide Ligand of Integrins for Simultaneously ... - ACS Publications

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