Stabilized Heptapeptide A7R for Enhanced Multifunctional Liposome

May 19, 2016 - To improve the in vivo stability and targeting efficacy, the retro inverso isomer of LA7R (DA7R) was developed for glioma-targeted drug...
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Stabilized Heptapeptide A7R for Enhanced Multifunctional Liposome-Based Tumor-Targeted Drug Delivery Man Ying,† Qing Shen,†,‡ Yu Liu,† Zhiqiang Yan,§ Xiaoli Wei,†,∥ Changyou Zhan,†,⊥ Jie Gao,† Cao Xie,† Bingxin Yao,† and Weiyue Lu*,†,‡,∥ †

Department of Pharmaceutics, School of Pharmacy, Fudan University & Key Laboratory of Smart Drug Delivery, (Fudan University), Ministry of Education, Shanghai 201203, China ‡ State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China § Institute of Biomedical Engineering and Technology, Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of chemistry and molecular engineering, East China Normal University, Shanghai 200062, China ∥ State Key Laboratory of Medical Neurobiology, The Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China ⊥ Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China S Supporting Information *

ABSTRACT: LA7R (ATWLPPR) is a heptapeptide with high binding affinity in vitro to vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1) overexpressed on glioma, glioma vasculogenic mimicry and neovasculature. However, its tumor targeting efficacy is significantly reduced in vivo due to proteolysis in blood circulation. To improve the in vivo stability and targeting efficacy, the retro inverso isomer of LA7R (DA7R) was developed for gliomatargeted drug delivery. DA7R was expected to have a similar binding affinity to its receptors in vitro (VEGFR2 and NRP-1), which was experimentally confirmed. In vivo, DA7R-modified liposomes achieved improved glioma-targeted efficiency than did LA7R-modified liposomes. After loading a chemotherapeutic agent (doxorubicin), D A7R-modified liposomes significantly inhibited subcutaneous model tumor in comparison to free doxorubicin, plain liposomes and LA7R-modified liposomes. In summary, the present study presented the potential of a proteolytically stable D-peptide ligand for in vivo tumor-targeted drug delivery. KEYWORDS: DA7R, liposomes, stability, tumor, targeted drug delivery

1. INTRODUCTION

targeting, enhanced antitumor efficacy and reduced systemic toxicity. Tumor targeting moieties, such as small molecules, peptides, antibodies and their fragments, play a key role in tumor targeting.7,8 Among those targeting moieties, tumorhoming peptides are highly attractive due to their high binding affinities and specficities to receptors/antigens and low risks of immunogenicity.9 Heptapeptide LA7R (ATWLPPR), which was identified by phage display, could bind to vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1) with high affinities and specificities.10,11 VEGFR2 and NRP-1 are overexpressed in various tumors and are implicated in tumor angiogenesis, growth and metastasis.12,13 Moreover, recent studies revealed that VEGFR2 participates in the formation of VM of glioma.14 Therefore, VEGFR2 and NRP-1 become ideal targets for glioma-targeted drug delivery. LA7R would be able to target

Cancer is emerging to be a leading cause of death and severely impairs human health. Conventional chemotherapy has been widely used in cancer therapy, but in most cases results in unsatisfactory clinical outcomes due to severe side effects and poor accumulation in tumor and/or tumor-related tissues. In addition, angiogenesis is a complex biological process in tumor tissues, playing a vital role in tumor growth, evasion, and metastasis.1 Vasculogenic mimicry (VM), which is a special form of tumor vasculature, comprises aggressive tumor cells instead of endothelial cells2 and exists in various types of tumors (such as glioma).3 Since VM is tightly associated with poor prognosis of chemotherapy, anti-VM treatment holds much potential to improve the clinical outcomes of cancer therapy.4 Blood tumor barrier (BTB) presents an additional obstacle to effective accumulation of chemotherapeutic agents in tumor and/or tumor-related tissues, exacerbating the failure of conventional chemotherapy.5,6 Targeted drug delivery systems (TDDS) have received increasing scrutiny owing to their advantages of precise tumor © XXXX American Chemical Society

Received: January 31, 2016 Accepted: May 12, 2016

A

DOI: 10.1021/acsami.6b01300 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Schematic illustration of multifunctional liposome-based tumor-targeted drug delivery systems. Liposomes are designed to specifically target to neovasculature, tumor cells and VM. DA7R-liposomes are superior to LA7R-liposomes to achieve these because DA7R exhibits exceptional stability and remains targeting ability in blood circulation. Grove, PA, USA). Mouse serum was supplied by Guangzhou Jianlun Biotechnology Co. (Guangzhou, China). Anticancer drug doxorubicin hydrochloride (DOX) was provided by Zhejiang Haizheng Co. (Zhejiang, China). U87 (human glioblastoma cells) and HUVECs (human umbilical vein endothelial cells) were provided by Shanghai Institute of Cell Biology. Both cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco) containing 10% FBS (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2. Male BALB/c nude mice of 4−6 weeks age were supplied by Shanghai SLAC laboratory animal Co. LTD (Shanghai, China) and housed under SPF conditions. 2.2. Synthesis of Peptide, Fluorescein-Labeled Peptides, and Functional Materials. DA7R and LA7R peptides were synthesized through the standard solid phase synthesis method utilizing active ester chemistry to couple Boc-protected amino acid to the deprotected resin. Dimethylformamide (DMF) was used as both the coupling and flow wash solvent throughout the cycles. The Boc protecting groups were removed with 100% trifluoroactic acid (TFA). Meanwhile, synthetic process was monitored by the ninhydrin test. The crude peptides cleaved from resin with HF were purified to homogeneity and confirmed by HPLC and ESI-MS. Fluorescein-labeled DA7R and LA7R were synthesized through sulfhydryl-maleimide covalently conjugation. Briefly, 5 mg A7R-Cys was dissolved in phosphate buffer (0.1 M, pH = 7.2), 2 mg fluorescein5-maleimide in DMF was added and gently stirred for 2 h at room temperature in the dark. The target product was purified via preparative C18 reverse-phase HPLC. Pure product was ascertained by HPLC and ESI-MS. The functional material, DA7R-PEG3400-DSPE was synthesized via sulfhydryl-maleimide coupling method. In brief, 15 mg of maleimidePEG3400-DSPE (mal-PEG3400-DSPE) was dissolved in DMF and 4 mg of DA7R-Cys was dissolved in phosphate buffer (0.1 M, pH = 7.2). The solutions were mixed and stirred at room temperature. Excessive D A7R-Cys was removed by dialysis against distilled water. The solution was lyophilized to obtain pure DA7R-PEG3400-DSPE, which was characterized by 1H NMR. LA7R-PEG3400-DSPE were synthesized and characterized using the same protocol. 2.3. Preparation and Characterization of Liposomes. Fluorescence-labeled liposomes without targeting moiety (LS), with D A7R-PEG3400-DSPE modification (DA7R-LS) or LA7R-PEG3400DSPE modification (LA7R-LS) were prepared using the thin-film hydration and extrusion method as previously reported procedure.23 A mixture of HSPC/cholesterol/mPEG2000-DSPE (molar ratio = 50/45/ 5) or HSPC/cholesterol/mPEG 2000 -DSPE/A7R-PEG 3400 -DSPE (molar ratio = 50/45/3/2) in CHCl3 solution was evaporated to

tumor cells, tumor angiogenesis, VM and cross the BTB by binding to VEGFR2 and NRP-1, thus achieving glioma-targeted delivery of the nanocarriers through multiple pathways. Even though LA7R has been exploited for tumor angiogenesis imaging and in vitro antitumor therapy,15,16 its in vivo applications would be limited because L-peptides are subject to proteolysis in blood circulation, leading to a short harf life in vivo. At present, a variety of methods have been explored to enhance the proteolyic stability of peptides, including N-to-C cyclization and designing peptidometics.17−22 A retro-inverso peptide is made up of D-amino acids in a reversed sequence and, when extended, assumes a side chain topology similar to that of its parent molecule but with inverted amide peptide bonds. Retro-inverso peptides are fully resistant to proteolysis because D-amino acids can not be recognized by enzymes. Here, we hypothesized that the retro-inverso isomer of LA7R (termed DA7R) would possess the comparable binding affinities to receptors (VEGFR2 and NRP-1) as LA7R, thus achieve effective glioma-targeting drug delivery (Figure 1). To verify this hypothesis, targeting efficiency of both A7R peptides and peptide-modified liposomes were investigated in vitro and in vivo. The antitumor efficacy of doxorubicin (DOX) encapsulated liposomes was assessed in a subcutaneous glioma-bearing nude mouse model.

2. EXPERIMENTAL SECTION 2.1. Materials. Near infrared dye DiR was purchased from Invitrogen (Grand Island, NY). Fluorescein-5-maleimide was supplied by Fanbo Biochemicals (Beijing, China). DAPI was from Roche (Basel, Switzerland). mPEG2000-DSPE and HSPC (Hydrogenated soy phosphatidylcholine) were supplied by Lipoid GmbH (Ludwigshafen, Germany). Cholesterol was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). FAM (5-carboxyfluorescein) as well as Sephadex G50 were obtained from Sigma (St. Louis, MO). Mal-PEG3400-DSPE was provided by Laysan Bio Co. (Arab, AL). Growth factor-reduced Matrigel matrix was obtained from BD Biosciences (San Diego, CA, USA). Purified recombinant human VEGFR2 and NRP-1 were obtained from R&D Systems (Minneapolis, MN). Rabbit anti-neuropilin 1 antibody, rabbit anti-VEGF receptor 2 antibody and rabbit anti-CD31 antibody were purchased from Abcam (USA). Dylight 647-conjugated donkey antirabbit antibody was obtained from Jackson Immunoresearch Laboratories, Inc. (West B

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ACS Applied Materials & Interfaces form a thin film and dried under vacuum for complete removal of CHCl3. The dried film was gently hydrated with FAM solution at 60 °C and extruded through a series of polycarbonate membranes with the pore size ranging from 200 to 50 nm by an Avanti Mini Extruder (Avanti Polar Lipids). Unloaded FAM was removed by gel filtration with normal saline. DiR-loaded liposomes were prepared by the same method except that DiR was included in the thin film. DOX-loaded liposomes were prepared using a traditional ammonium sulfate gradient loading method in accordance with the previous report.24 A transmission electron microscopy (TEM) was utilized to characterize the morphology of liposomes. Meanwhile, the particle size distribution of various liposomes was measured by dynamic light scattering (DLS) method (Malvern Zetasizer 3000, n = 3). The leakage of FAM-loaded or DiR-loaded liposomes was measured using the dialysis method with phosphate buffer (pH 7.4) according to the previous report.25 The concentrations of FAM and DiR were determined by a fluorescence spectrophotometer (Cary Eclipse, Agilent, Australia) at Ex/Em 494/522 nm and 741/776 nm, respectively. 2.4. Molecular Docking Study. The crystal structure of VEGFR2 (pdb code 1y6a) and b1b2 domain of NRP-1(pdb cod 2orz) was used for the molecular docking study. The missing residues in the crystal structures were added by the software Discovery Studio3.5. The threedimensional structure model of LA7R for the docking study was get from NMR data (pdb code 2jp5). The three-dimensional structure model of DA7R was derived from LA7R. The residues of LA7R were replaced with D-amino acids in a reversed sequence, and energy minimization was also performed with Discovery Studio3.5 to get the final model of DA7R. The software Hex v6.12 was applied to initially explore the peptides binding modes, and the program RosettaDock was applied for further exploring the best binding conformation of our designed peptide in pocket of receptors.26,27 The program RosettaDock contains the parameters of D-amino acids for flexible peptide docking. All the resulting binding conformations of the peptides were submitted to the program X-score 1.2 to predict the binding affinity of the peptides.28 2.5. Surface Plasmon Resonance (SPR) Analysis. To assess the binding affinities of A7R peptides with VEGFR2 and NRP-1, binding analysis was conducted using Biacore T200 system (GE Healthcare). Recombinant human NRP-1 or recombinant human VEGFR2 was coupled to the CM5 sensor chip according to the standard amine coupling procedure. DA7R and LA7R were dissolved in HBS-EP buffer at defined concentrations and injected for recording resonance changes to assess the binding affinity. Data were analyzed using the Biacore software (GE Healthcare). 2.6. Ex Vivo Stability of Peptides. One hundred microliters of D A7R or LA7R solution in pure water was mixed with 0.9 mL 25% sterile mouse serum (diluted with phosphate buffered saline). After incubation at 37 °C for 0, 0.25, 0.5, 1, 2, and 4 h, one hundred microliters mixture was sampled and mixed with 20 μL of 15% trichloroacetate aqueous solution. After stored at 4 °C for 20 min, the mixture was centrifuged at 13000 rpm for 10 min and the peptide content in the supernatant was analyzed by HPLC. 2.7. Establishment of the in Vitro VM Model and Tumor Spheroids. Twenty-four-well plates were coated with 100 μL growth factor-reduced Matrigel. HUVECs and U87 cells were gently suspended and seeded onto the three-dimensional Matrigel overnight for tube formation. Tubes formed by U87 cells in Matrigel were utilized as an in vitro VM model.14,29 To culture tumor spheroids, U87 cells were suspended in cell culture medium and seeded on agarosecoated 48-well plates at a density of 2000 per well and kept steady for 10 days. 2.8. Cellular Uptake of Fluorescein-Labeled Peptides or FAM-Loaded Liposomes. HUVECs and U87 cells were treated with fluorescein-labeled peptides or FAM-loaded liposomes (dye concentrations were 5 μM in all cases) in DMEM with 10% FBS for 4 h at 37 °C. Cellular uptake was visualized by a laser scanning confocal microscope (TCS SP5, Leica, Germany) after staining with DAPI and rinsing with phosphate buffered saline (PBS) for three times. For quantitative analysis, cells were trypsinized and suspended in PBS and

fluorescence-positive cells were counted by flow cytometry (FACSAria, BD, USA). In vitro VM model was incubated with fluorescein-labeled peptides or FAM-loaded liposomes (dye concentrations were 5 μM in all cases) for 4 h. After staining the nuclei with DAPI, fluorescence was visualized by a fluorescence microscope (DMI4000 B, Leica, Germany). U87 tumor spheroids were treated with 5 μM FAMloaded liposomes in DMEM containing 10% FBS for 4 h. After PBS rinsing and 4% paraformaldehyde fixation, the tumor spheroids were imaged by a confocal laser microscope. 2.9. Targeting Ability on BTB/U87 Tumor Spheroids Coculture Model. HUVECs/U87 coculture model (BTB model) was set up in accordance with the previous report.30 Briefly, U87 cells were plated into the chamber plates of Transwell at a density of 5 × 104 per well and HUVECs were seeded on the upper inserts of Transwell plates at a density of 2 × 104. The insert of Transwell was transferred to the chamber containing U87 tumor spheroids after 3 days. Twenty micromolar FAM-loaded liposomes dispersed in DMEM containing 10% FBS were added into each apical chamber. After 4 h incubation, U87 tumor spheroids were rinsed with PBS for three times and fixed with 4% paraformaldehyde for 30 min. Cellular uptake of fluorescence was visualized by a confocal laser microscope. 2.10. In Vitro Cytotoxicity Assay. MTT assay was used to measure the in vitro cytotoxicity of DOX-loaded liposomes against HUVECs and U87 cells. Cells were seeded into 96-well plates at a density of 3000 cells per well and cultured overnight. Then cells were treated with culture mediums containing DOX-loaded liposomes at a series of DOX concentrations ranging from 7.81 × 10−10 to 12.8 × 10−6 M for 4 h, and then the culture medium was changed to DOXfree DMEM with 10% FBS. After 68 h culture, 20 μL MTT solution was added to each well. After 4 h incubation, the percentage of cell viability was determined on the basis of absorbance at 490 nm by a plate reader (PowerWave XS, Bio-TEK, USA). Untreated cells were used as a control. 2.11. Inhibition of in Vitro VM Tube Formation. HUVECs and U87 cells cultured in the Matrigel (as described in 2.7) were treated with 1 μM DOX-loaded liposomes. Cells treated with drug-free DMEM were served as the control group. After incubation for 12 h, tube structures were observed under an inverted phase contrast microscope (DMI4000 B, Leica, Germany). The amount of tubes was counted within three randomly selected areas.31 2.12. Tumor Targeting Ability of DiR-Loaded Liposomes or Peptides. Male BALB/c nude mice bearing subcutaneous U87 xenograft tumors were used for tumor targeting evaluation. DiRloaded liposomes were injected into these model mice via tail vein. At defined time points, whole body fluorescence imaging was conducted using an in vivo imaging system (IVIS Spectrum, Caliper, USA). Mice were sacrificed and the organs and tumors were dissected for ex vivo fluorescence imaging. Fluorescein-labeled peptides were injected into subcutaneous U87 xenograft-bearing mice for peptides tumor-homing study. After 1 h, mice were put to death for further ex vivo fluorescence imaging of tumors. 2.13. Immunofluorescence Analysis. To understand the targeting mechanism of A7R modified liposomes, immunofluorescence assay was performed. FAM-loaded liposomes were intravenously administered to subcutaneous U87 xenograft bearing mice. After 4 h, mice were sacrificed and tumors were dissected for further study. Tumors were fixed with 4% paraformaldehyde overnight and dehydrated with 30% sucrose. Tumors were frozen with O.C.T. and cut into 10 μm slices. Sections were fixed with ice-cold acetone for 10 min at 4 °C, rinsed with PBS and blocked with bovine serum albumin (BSA) for 1 h. Microvessels were labeled with anti-CD31 antibody. VEGFR2 and NRP-1 receptors were stained with anti-VEGF receptor 2 antibody and anti-Neuropilin 1 antibody, respectively. The slices were stained with Dylight 647-conjugated secondary antibody and DAPI for nuclear counterstain. The distribution of fluorescence was visualized by a confocal microscope. 2.14. In Vivo Antitumor Growth Effect. Subcutaneous U87 xenograft bearing mice were established for antitumor activity. When C

DOI: 10.1021/acsami.6b01300 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. Binding modes between peptide and receptor. VEGFR2 (A) and NRP-1 (D) were shown in cartoon. The residues interacting with A7R peptides were denoted in pink. LA7R and DA7R were displayed in green and yellow, respectively. Interaction of recombinant human VEGFR2(C) or NRP-1 (F) with A7R peptides was assessed by SPR. Experimentally determined and predicted free energies of peptides binding with VEGFR2 (B) and NRP-1(E) (ΔG = 1.3636 log Ki). the average tumor volume reached about 100 mm3, mice were randomly divided into 5 groups (6 mice per group). DA7R-LS/DOX, L A7R-LS/DOX, LS/DOX, free DOX, and saline were injected into mice via tail vein at day 0, 3, 6, 9, and 12 with a doxorubicin dose of 2 mg/kg (10 mg/kg in total). Body weights and tumor volumes were monitored every 2 days. Tumor volume was measured using the formula: tumor volume = 0.5 (a × b2), where a and b refer to the largest and smallest diameter, respectively. Relative tumor volume = tumor volume at each time point/tumor volume at day 0. Relative body weight = body weight at each time point/body weight at day 0. After 18 days of treatment, mice were put to death and tumors were dissected for weighing and immunohistochemical analysis. 2.15. Immunohistochemical Analysis. Tumors were fixed with 4.0% paraformaldehyde, paraffin-embedded, and sectioned. TUNEL assay was used to detect apoptotic cells in tumor tissues according to the manufacture’s protocol.32 CD31/PAS dual staining was performed to observe the microvessels.33 All the sections were counterstained by hematoxylin and eosin. TUNEL-positive cells or CD31-positive cells in three slices were counted. 2.16. Statistical Analysis. Data were presented as mean ± SD unless otherwise indicated. Comparison among different groups was conducted by one-way ANOVA with Bonferroni correction. p < 0.05 was considered statistically significant.

Molecular docking study was conducted to understand the binding modes. The computational model of DA7R was constructed by mutating the L-amino acids of LA7R to Damino acids with a reversed sequence (Figure S1). Both peptides were docked to the crystal structure of VEGFR2 (Figure S2A). A7R peptides deeply penetrated into the clef between two domains of VEGFR2. LA7R and DA7R adopted similar binding conformations. Arg in LA7R and DArg in DA7R located at the outer edge of the binding pocket and formed ionic interactions with Asp1054 of VEGFR2. The indole fragment of Trp in LA7R formed hydrophobic interaction with Ala864, Phe916, Leu1033 and Phe1045 of VEGFR2 (Figure 2A). The side chain of DTrp in DA7R slightly shifted and docked into the hydrophobic pocket formed by Ala864, Phe916 and Leu1033. In addition, the binding energy of both A7R peptides was also predicted by X-score, which was consistent with the experimental results (Figure 2B). The binding mode of both A7R peptides for NRP-1 was also constructed. Both peptides located at the tip of the b1 domain of NRP-1 (Figure S2B). Arg in LA7R and DArg in DA7R formed ionic interactions with Asp320 of NRP-1 (Figure 2D), which was mostly conserved in NPR-1 binding modes.34,35 DArg in D A7R also formed ionic interaction with Glu319 and ionic-π interaction with Tyr297. The binding energy predicted by Xscore was consistent with the experimental data (Figure 2E). As a whole, stabilized heptapeptide DA7R demonstrated similar binding affinity to VEGFR2 and NRP-1 when compared with its parent peptide LA7R. 3.2. In Vitro and in Vivo Tumor Targeting Ability and Stability of A7R Peptides. U87 cells and HUVECs overexpressing VEGFR2 and NRP-1 were used as the models of tumor cells and tumor neovascular endothelial cells, respectively.36 To investigate the targeting efficacy of A7R

3. RESULTS 3.1. Interaction Properties of A7R Peptides with Receptors. A7R peptides, including DA7R and LA7R, were synthesized through solid-phase peptide synthesis. SPR assay was conducted to assess the binding affinities of A7R peptides with VEGFR2 and NRP-1. LA7R and DA7R displayed similar binding affinity to VEGFR2. Their equilibrium dissociation constant (KD) was respective 9.29 and 8.41 nM (Figure 2B, C). L A7R and DA7R also demonstrated high binding affinity to NRP-1, with KD values of 6.62 nM and 2.31 nM, respectively (Figure 2E, F). D

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ACS Applied Materials & Interfaces peptides, U87 cells, and HUVECs were treated with 5 μM fluorescein-labeled DA7R and LA7R at 37 °C. DA7R and LA7R could be effectively internalized into U87 cells and HUVECs (Figures 3A and 3B). The flow cytometry results indicated that

in vivo targeting evaluation. Characterization data including transmission electron microscope (TEM) images of DOXloaded liposomes are shown in Table S2 and Figure S6. The size of all liposomes was around 100 nm, suggesting that LA7R or DA7R modification did not influence the physical properties of liposomes. 3.4. Cellular Uptake of A7R-liposomes. LA7R-LS and D A7R-LS could be efficiently internalized by U87 cells, HUVECs and in vitro U87 VM model (Figures 4 and S4B).

Figure 3. In vitro and in vivo tumor targeting ability and stability of A7R peptides. Cellular uptake of A7R peptides by U87 cells (A) and HUVECs (B). (C) Stability of A7R peptides in 25% mouse serum at 37 °C determined by HPLC. (D) Mean fluorescence intensity of tumor in each group. Tumors were dissected 1 h after injection for ex vivo imaging and fluorescence measurement. Data are mean ± SD, n = 3. *p < 0.05, **p < 0.01.

both A7R peptides exhibited similar targeting ability in vitro (Figure S3). In the U87 cells vasculogenic mimicry model, both D A7R and LA7R could be taken up at a similar level (Figure S4A), indicating that both DA7R and LA7R possessed equal targeting efficacy to tumor cells, tumor angiogenesis and VM in vitro. However, DA7R displayed much higher stability in serum than LA7R (Figure 3C). LA7R was degraded in serum quickly and almost disappeared after 2 h, while DA7R demonstrated nearly no degradation after 4 h incubation. In the study of in vivo tumor targeting efficacy evaluation, DA7R displayed higher intratumoral fluorescence intensity than LA7R (Figure 3D). 3.3. Characterization of A7R-PEG3400-DSPE and A7RLiposomes. Functional materials A7R-PEG3400-DSPE, including DA7R-PEG3400-DSPE and LA7R-PEG3400-DSPE, were synthesized via sulfhydryl-maleimide coupling. In the 1H NMR spectrum (Figure S5) of Mal-PEG3400-DSPE, the solvent peak at 7.26 ppm was from CDCl3, and the multiple signals at 1.26 ppm were from the methylene protons of DSPE. The characteristic peaks at 3.7−3.8 ppm were from the repeat units of PEG and the signal at 6.7 ppm was the characteristic peak of the maleimide group. The maleimide peak disappeared in the spectra of DA7R-PEG3400-DSPE and LA7R-PEG3400-DSPE, suggesting that the maleimide group had completely reacted with the thiol group of DA7R-Cys and LA7R-Cys. Liposomes were fluorescently labeled with FAM or DiR for tumor targeting assay. The results of characterization of FAM or DiR-loaded liposomes were provided in Table S1. FAMloaded liposomes displayed only 4% leakage in PBS (pH 7.4) in 12 h, excluding any interference from released fluorescent probes in the following experiments. DiR-loaded liposomes showed no leakage of DiR in 50% mice serum within 24 h, indicating that the fluorescent signal was from liposomes in the

Figure 4. Targeting ability of A7R modified liposomes (A7R-LS) in U87 cells and HUVECs. U87 cells (A,C) and HUVECs (B,D) uptake of A7R-LS with and without preincubation with mouse serum. Quantitative cellular uptake by using flow cytometer. Scale bar represents 10 μm; ***p < 0.001.

Preincubation with mouse serum significantly undermined the cellular uptake of LA7R-LS, as the ratio of FAM-positive cells decreased from 90% to 12% for U87 cells and from 74% to 2% for HUVECs, respectively. However, for DA7R-LS, preincubation in mouse serum did not affect the intracellular uptake at all, suggesting that D-peptide ligand modified liposomes could keep their targeting ability in plasma. 3.5. Targeting Ability of A7R-Liposomes on BTB/U87 Tumor Spheroids in Vitro. After 4 h incubation, LA7R-LS and DA7R-LS distributed into tumor spheroids more evidently than LS (Figure 5A), indicating that the modification of A7R not only increased the U87 cell uptake but also enhanced the tumor spheroids penetration efficiency. To better imitate the blood tumor barriers, a BTB/U87 tumor spheroids coculture model was set up to explore the targeting ability and transcytosis efficiency of various liposomes. As shown in Figure 5B, both LA7R-LS and DA7R-LS crossed the BTB and targeted tumor spheroids. However, after preincubation with 50% mouse serum, LA7R-LS lost almost all BTB crossing and tumor spheroids-targeting capabilities, whereas DA7R-LS exhibited no significant decrease of cellular uptake in tumor spheroids (Figure 5C). These results again emphasized the advantage of the stabilized targeting ligand. 3.6. Targeting Ability of A7R-Liposomes in Vivo. It was evident that DA7R-LS displayed significantly higher accumuE

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than did LA7R-LS in the tumor region 4 h after intravenous injection (Figures 7 and S7), while unmodified liposomes were

Figure 7. Distribution of the FAM-loaded liposomes in tumor tissue of subcutaneous xenografts bearing mice 4 h after i.v. administration. Frozen sections were examined under a confocal microscope. Blood vessels were labeled with anti-CD31 (red) and the nuclei was stained with DAPI (blue). Green represented the liposomes. Bars represent 50 μm.

Figure 5. Uptake of FAM-loaded LS, LA7R-LS, and DA7R-LS by U87 tumor spheroids. (A) U87 tumor spheroids uptake of different liposomal formulations after 4 h. (B−C) Uptake of tumor spheroids without or with preincubation with mouse serum in the BTB/U87 tumor spheroid coculture model. Tumor spheroid penetration was examined by a confocal microscope, with a 5 μm interval between consecutive slides.

unable to achieve obvious accumulation in tumor. This result was consistent with those from whole animal fluorescence imaging (Figure 6). A7R-LS could partially colocalize with tumor blood vessel marker CD31 (Figure 7), indicating that A7R-LS could bind with tumor neovasculature, extravasate and accumulate in the tumor parenchyma. As expected, A7R-LS completely colocalized with VEGFR2 and NRP-1. It was clear that A7R peptides (especially DA7R) could facilitate tumor localization of liposomes in VEGFR2- and/or NRP-1-positive cells. 3.7. Cytotoxicity of A7R-Liposomes in Vitro. The in vitro cytotoxicity of various DOX-loaded liposomes against U87 cells and HUVECs was measured using MTT assay. It was evident that all DOX formulations could inhibit cell growth (Figure 8A, B). DA7R-LS/DOX displayed an IC50 values of 0.62

lation in subcutaneous U87 tumor than did LA7R-LS and plain liposomes (LS) (Figure 6A). Semiquantitative ROI analysis revealed that DA7R modified liposomes led to the highest intratumoral fluorescence intensity (Figure 6B). Figure 6C illustrates the characteristic ex vivo images of main organs and tumors 24 h after intravenous injection. Semiquantitative analysis of ex vivo images also showed strong fluorescence accumulation in livers and tumors, in contrast to limited accumulation in heart and other organs (Figure 6D). To investigate the intratumoral distribution of liposomes, immunofluorescence analysis of the frozen U87 tumor sections was conducted. DA7R-LS demonstrated higher accumulation

Figure 6. Targeting ability of A7R modified liposomes in subcutaneous U87 tumors. (A) In vivo fluorescence imaging of subcutaneous U87 tumor 24 h after intravenous injection with DiR-labeled liposomes. (B) Semiquantitative ROI analysis of in vivo fluorescent images at different time points in the tumor (mean ± SD, n = 3). (C) Ex vivo near-infrared imaging of harvested main organs and tumors 24 h postinjection. (D) Semiquantitative ROI analysis of the mean fluorescence intensity from the DiR-labeled liposomes in tumor and organs (Mean ± SD, n = 3). ***p < 0.001. F

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Figure 8. Antitumor efficacy of DOX-loaded liposomes in vitro. Cytotoxicity of different DOX-loaded liposomes against U87 cells (A) and HUVEC cells (B) was assessed after 72 h incubation (mean ± SD, n = 3). (C−D) Effect of various DOX-loaded liposomes on HUVEC tube formation and U87 VM formation. The quantitative data of the HUVEC tube formation (C) and U87 VM formation (D) treated by various DOX-loaded liposomes. Cells treated with drug-free DMEM served as the control. Data are presented as the percentages of the control group, which was set at 100%. *p < 0.05, ***p < 0.001.

Figure 9. Antitumor effects of DOX-loaded liposomes in vivo. (A) Growth curves of subcutaneous U87 tumor in nude mice receiving various DOX formulations. A dosage of 2 mg/kg (with respect to DOX) was administered intravenously for all mice on the day 0, 3, 6, 9, and 12, and the tumor volume was measured at the predeterminated time points. (B) Changes in mice body weights at different time points. Relative body weight = body weight at each time point/body weight at day 0. (C) The weight of excised tumor masses. (D) Photographs of tumors from each group at the end of treatments (day 18). Data represent mean ± SD, n = 6 (*p < 0.05, ***p < 0.001).

μM against U87 cells, significantly lower than that of LA7R-LS/ DOX and LS/DOX. The cytotoxicity of DA7R-LS/DOX against HUVECs was also significantly enhanced in comparison to LA7R-LS/DOX and LS/DOX. These results indicated that the cytotoxic effect of DOX-loaded liposomes was effectively enhanced by modification of DA7R. 3.8. Destroying Ability of A7R-Liposomes on U87 VM Channels and HUVEC Tubes in Vitro. The effects of different DOX-loaded liposomes on the formation of HUVECs tubes and U87 VM were evaluated (Figure 8C−D, Figure S8).

HUVECs subjected to DMEM treatment (blank control) formed extensive and enclosed tube networks. DA7R-LS/DOX displayed higher activity in inhibiting tube formation than did LS/DOX and LA7R-LS/DOX. In addition, DA7R-LS/DOX also demonstrated efficient inhibition on U87 VM on Matrigel. 3.9. Antitumor Efficacy of A7R-Liposomes in Vivo. The in vivo antitumor activity of various liposomes was evaluated in nude mice bearing subcutaneous U87 tumors. Nude mice were randomly grouped (6 mice per group) when the average tumor volume reached about 100 mm3. DA7R-LS/DOX, LA7R-LS/ G

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ACS Applied Materials & Interfaces

Figure 10. Immunohistochemical analysis of the tumor tissues of the mice bearing subcutaneous U87 tumor after various treatments. (A) Analysis of tumor slices treated with various DOX-loaded liposomes. TUNEL immunohistochemical staining, the upper panel; CD31/PAS dual staining, the lower panel. Scale bar represents 100 μm. (B) Quantification of the percentage of TUNEL positive apoptotic cells in tumor treated with different DOX formulations. Cells from three random fields were counted for each tumor slice under a light microscope. (C) Quantification of angiogenesis inhibition in tumors with different DOX formulations in comparison to saline group. Three random fields were selcted under a light microscope. Data are mean ± SD *p < 0.05, **p < 0.01.

therapeutic effects. However, the stability problem of the targeting ligands is usually overlooked. To circumvent the elimination by the reticuloendothelial system, actively targeted drug delivery systems have to be modified to prolong blood circulation. PEGylation has been proven to be effective for prolonged circulation. However, during the prolonged circulation process in vivo, targeting ligands are completely exposed to countless enzymes in blood, which may induce the deactivation of the targeting ligands. It is well-known that linear peptide ligands comprising L-amino acids are susceptible to proteolytic degradation in blood circulation,17−19,37 necessitating stabilization of peptide targeting ligands in TDDS. LA7R, which is a linear sequence-peptide, binds effectively to NRP-1 and VEGFR2 and holds promise in tumor imaging and tumor angiogenesis molecular imaging.15,38,39 Those short-time activetargeted imaging could be feasible, regardless of enzymatic degradation of L-amino peptides. However, only stable ligands could keep the targeting ability in long blood circulation for therapeutic purposes. Current methods for improving peptide stability include retro-inverso isomerization, cyclization as well as partial nonnatural amino acid modification.40 In the present study, we synthesized a retro-inverso isomer of LA7R to improve in vivo stability and tumor-homing capability. The binding affinity of A7R peptides with targeting receptors was evaluated by SPR and molecular docking study, which validated similar binding properties of LA7R and DA7R with NRP-1 and VEGFR2. Moreover, LA7R and DA7R demonstrated comparable cellular uptake efficiency in U87 cells, HUVECs and U87 VM model. Most importantly, DA7R exhibited excellent stability in mouse serum, while LA7R was quickly degraded. Furthermore, DA7R displayed better intratumoral accumulation than did LA7R. All results indicated that stabilized A7R peptide with high receptor binding capacity was successfully achieved.

DOX, LS/DOX, free DOX, and saline were intravenously administered every 3 days for five times (total doxorubicin dose: 10 mg/kg), respectively. Tumors in the LA7R-LS/DOX group grew slower than that of the group receiving LS/DOX. In the group of mice receiving DA7R-LS/DOX, tumor ceased to grow and shrank gradually after treatments, indicating the superior tumor-suppression effect. Free DOX suppressed tumor growth to some extent but was less effective than all other experimental groups (Figure 9A). In addition, the body weight of mice in each group showed no significant difference (Figure 9B). The final weight of excised tumors at the ending point of treatment indicated that DA7R-LS/DOX displayed better antitumor effects than did LA7R-LS/DOX (Figure 9C−D). Tumor slices were stained with TUNEL kits to detect apoptosis and CD31/PAS for the assessment of angiogenesis and VM channels (Figure 10A). As shown in Figure 10B, LS/ DOX induced a certain degree of apoptosis and LA7R-LS/DOX treated tumors displayed more apoptosis. Remarkably, the percentage of TUNEL-positive cells in DA7R-LS/DOX group was 83%, which was higher than that in LA7R-LS/DOX group (50%). CD31-positive channels were counted for detection of angiogenesis structure (Figure 10C). Compared to the saline group, angiogenesis inhibition rate in DA7R-LS/DOX treated tumors was 82%, which was higher than that in other groups (DOX: 44%, LS/DOX: 55%, LA7R-LS/DOX 66%). CD31negative and PAS-positive channels were defined as VM structures. Less VM channels were observed in DA7R-LS/DOX group. These results validated that stabilized peptide ligand (DA7R) could induce remarkable antitumor activity of DOXloaded liposomes in comparison to the parental peptide LA7R modified DOX-loaded liposomes.

4. DISCUSSION Actively targeted drug delivery systems have been developed using a variety of targeting ligands to achieve satisfactory H

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ACS Applied Materials & Interfaces Next, LA7R-PEG3400-DSPE or DA7R-PEG3400-DSPE was incorporated in liposomes to construct multifunctional targeted drug delivery systems. The targeting capacity of DA7R-LS in serum was similar to that of DA7R-LS, indicating liposomes modified with DA7R could possess targeting abilities in blood plasma. Tumor spheroids were exploited to mimic the in vivo status of solid tumors and evaluate the penetrating ability of TDDS.41 Our results showed that A7R-lipid derivative conjugates endowed liposomes with binding and penetrating abilities to a certain extent. BTB limits the delivery of chemotherapeutic agent to brain tumor tissues.5 The BTB/ U87 tumor spheroids coculture model was also constructed to confirm the superiority of DA7R-LS. DA7R-LS distributed into model tumors more effectively than did LA7R-LS after preincubation in serum. A7R modified liposomes containing DOX demonstrated good inhibitory effects on U87 cells, HUVECs, VM, and HUVECs 3D tube in vitro. By specifically targeting to tumor cells, tumor angiogenesis and VM, DA7R-LS accomplished the best antitumor effect among the tested groups. Recent researches have revealed that cancer stem cells (CSC) play pivotal roles in tumor growth and relapse, leading to the failure of chemotherapy.42 Blood−brain barrier (BBB) is another therapeutic obstacle in the therapy of glioma, which remains intact, surrounding the infiltrative tumor edge.6 It is urgent to consider those barriers into targeting strategies to enhance antiglioma outcome in the near future. L A7R itself has been reported to possess antiangiogenic effects at a relative high dosage.43 Our preliminary study revealed that the minimal effective dosage for DA7R to display antiangiogenic effects was 10 mg/kg (data not shown here), higher than the dosage used in our study (3.5 mg/kg). Therefore, the inhibition of tumor growth reported here for A7R-modified liposomes are not likely from the possible contribution of peptides themselves.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86 21 5198 0094. Fax: +86 21 5198 0090. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (973 Program, 2013CB932500), National Natural Science Foundation of China (81273458 and 81473149), and N a t i o n a l S c i e n c e & T e c h n o lo g y M a j o r P r o j e c t (2012ZX09304004).

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REFERENCES

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5. CONCLUSION In summary, a D-peptide ligand DA7R and DA7R-modified liposomes were developed to overcome enzymatic barriers in vivo. The function of A7R peptides as ligands of VEGFR2 and NRP-1 was experimentally and computationally validated, and tumor targeting efficacy was evaluated in vitro and in vivo. D A7R significantly enhanced the tumor targeting efficiency of liposomes. DA7R-modified liposomes containing DOX suppressed tumor growth more effectively than LA7R-modified ones, which may be attributed to their better serum stability and higher tumor accumulation. On the basis of the promising performance of D-peptide as the targeting moiety, the present study paved a way for the design of proteolytically stable targeting materials for tumor targeted drug delivery.

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the FAM-loaded liposomes in tumor tissue; effect of various DOX-loaded liposomes on HUVEC tube formation and U87 VM formation; cellular uptake of fluorescein labeled A7R peptides by L929 cells (PDF)

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b01300. 3D structure model of LA7R and DA7R; binding modes of A7R peptides in VEGFR2 or NRP-1; flow cytometry results of cellular uptake of fluorescein-labeled A7R peptides; the NMR spectra of Mal-PEG3400-DSPE, LA7RPEG3400-DSPE and DA7R-PEG3400-DSPE; characterization of different fluorescently labeled liposomes; characterization of different doxorubicin liposomes; TEM images of DOX-loaded liposomes; distribution of I

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