d-Retroenantiomer of Quorum-Sensing Peptide-Modified Polymeric

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D‑Retroenantiomer

of Quorum-Sensing Peptide-Modified Polymeric Micelles for Brain Tumor-Targeted Drug Delivery

Danni Ran,†,⊥ Jiani Mao,†,⊥ Changyou Zhan,†,§,∥ Cao Xie,† Huitong Ruan,† Man Ying,† Jianfen Zhou,† Wan-Liang Lu,‡ 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 Kay Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Science, Peking University, Beijing 100191, China § Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China ∥ State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China S Supporting Information *

ABSTRACT: Compared to that of other tumors, various barriers, such as the blood−brain barrier (BBB), enzymatic barriers, and the blood−brain tumor barrier, severely impede the successful treatment of gliomas. Peptide ligands were frequently used as targeting moieties to mediate brain tumortargeted drug delivery. LWSW (SYPGWSW) is a recently reported quorum-sensing (QS) peptide that is able to efficiently cross the BBB. Even though linear LWSW traverses the BBB in vitro, its in vivo targeting ability has been greatly impaired due to proteolysis. Here, we developed a stable peptide, D WSW (DWDSDWDGDPDYDS), using the retro-inverso isomerization technique to achieve an enhanced antiglioma effect. In vitro studies have demonstrated that both the LWSW and DWSW peptides possessed excellent tumor-homing properties and barrierpenetration abilities, whereas DWSW exhibited exceptional stability in serum and maintained its targeting ability after serum preincubation. In vivo, DWSW-modified probes and micelles accumulated more efficiently in the glioma region in comparison with LWSW-modified probes and micelles because of full resistance to proteolysis in blood circulation. As expected, DWSWmodified paclitaxel (PTX)-loaded micelles (DWSW Micelle/PTX) exhibited the longest median survival time among gliomabearing nude mice. Our results suggested that the QS peptide appears to be a promising targeting moiety, with potential applications in glioma-targeted drug delivery. KEYWORDS: quorum-sensing peptides, stability, drug-delivery system, glioblastoma, blood−brain barrier

1. INTRODUCTION Bacteria communicate with each other by secreting chemical signal molecules into the surrounding environment during their growth processes.1,2 When the number of signal molecules reach a certain threshold level, biological effects, such as regulation of gene expression, biofilm formation, or bioluminescence, will be exerted.3,43,4 This phenomenon is called bacterial quorum sensing (QS). QS peptides are a class of biologically attractive molecules. They have a wide variety of structures and display many functions.2,4−6 Recently, QS has received widespread attention because it can influence cancer and different central nervous system-related disorders, such as anxiety, depression, and autism.7−13 For example, Salmonella produces anticancer proteins with the potential to kill tumors through QS systems.14 Some QS peptides have also been reported to cross the blood−brain barrier (BBB).15 LWSW (also named PhrCACET1) is a recently reported peptide originating from Clostridum acetobutylicum. It could effectively cross the BBB and showed a measured brain clearance.16 Currently, many researchers focus on peptides acting in brain and their therapeutic potential.5,17 WSW peptides could be © 2017 American Chemical Society

ideal targeting moieties to facilitate brain-targeted drug delivery. In addition, WSW peptides are able to target glioma cells and tumor neovasculature, suggesting that WSW peptides may be useful ligands for intracranial glioma targeting. Glioblastoma multiforme (GBM) is one of the most aggressive brain cancers and severely threatens human health.18 The standard treatments of GBM include surgical resection, radiotherapy, and chemotherapy. However, the prognosis of GBM patients remains unsatisfactory, and the median survival of GBM patients is around 15 months.19 Considering the infiltrative growth of glioma, conservative resection is generally conducted in a clinic due to the complexity of the brain functions.20 In recent years, active targeting drug-delivery systems have been widely studied to achieve effective delivery of chemotherapeutics to the tumor region for enhanced antitumor efficacy and reduced toxicity.21 Gliomas are different from peripheral tumors due to their location. Numerous Received: March 11, 2017 Accepted: May 26, 2017 Published: May 26, 2017 25672

DOI: 10.1021/acsami.7b03518 ACS Appl. Mater. Interfaces 2017, 9, 25672−25682

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ACS Applied Materials & Interfaces

Figure 1. Schematic illustration of QS peptide-mediated glioma-targeted drug delivery. Micelles are designed to penetrate the BBB and BBTB and then target the vasculogenic mimicry (VM) and tumor cells. DWSW micelles are superior to LWSW micelles because DWSW exhibits exceptional stability in blood circulation. Ltd. (Beijing, China). Paclitaxel (PTX) was supplied by Dalian Meilun Biology Technology Co. Ltd. Rat serum was supplied by Guangzhou Jianlun Biotechnology Co. (Guangzhou, China). Rat brain capillary endothelial cells (BCECs) were primarily cultured by our laboratory. U87 cells were obtained from ATCC (Manassas, VA). Human umbilical vascular endothelial cells (HUVECs) were supplied from Shanghai Institute of Cell Biology (Shanghai, China). Dulbecco’s modified Eagle medium (DMEM), penicillin−streptomycin, and fetal bovine serum were purchased from Gibco (Carlsbad, CA). All animal experimental protocols were evaluated and approved by the ethics committee of Fudan University. Sprague-Dawley (SD) rats and male BALB/c nude mice (4−6 weeks of age) were provided by BK Lab Animal Ltd. (Shanghai, China) and kept under specific pathogen free conditions. 2.2. Synthesis and Characterization of Materials. The DWSW (DWDSDWDGDPDYDS) and LWSW (SYPGWSW) peptides were synthesized according to a previous report.23 Cysteine was inserted at the C terminal to provide a thiol group for functionalization. The obtained crude products were purified by preparative reverse phase high-performance liquid chromatography (RP-HPLC). After purification, both the peptides were labeled with fluorescein-5-maleimide and cyanine 7-maleimide via covalent conjugation with sulfhydrylmaleimide. Analytical HPLC and electrospray ionization mass spectrometry (ESI-MS) were used to monitor the reaction process and ascertain the molecular masses of the reaction products. DWSWPEG3000-PLA2000 and LWSW-PEG3000-PLA2000 were synthesized through covalent conjugation according to the previous procedure.24 2.3. Preparation and Characterization of Polymeric Micelles. Blank micelles and WSW peptide-modified micelles were prepared by thin-film hydration. For drug and fluorescence loading, fluorescein and PTX were dissolved in the organic solvent together with the membrane materials. The unloaded drug and fluorescein were removed using a 0.1 μm filter membrane. The particle sizes and polydispersity indexes (PDIs) of different micelles were measured by the dynamic light scattering method (n =

biological barriers, including BBB/blood−brain tumor barrier (BBTB) and enzymatic barriers, severely hamper the accumulation of drugs or drug-delivery systems in the glioma region.22 In the present study, we studied the brain-targeting efficiency of WSW peptides and WSW-modified drug-delivery systems. To overcome the poor proteolytic stability of L-peptides, we developed a retro-inverso isomer of the LWSW peptide (DWSW) and hypothesized that DWSW and LWSW would possess comparable binding affinities to QS receptors, thus facilitating the BBB penetration of micelles and achieving effective glioma-targeted drug delivery (Figure 1). The in vitro and in vivo BBTB penetration efficiencies of both peptides and peptide-modified drug-delivery systems were also assessed.

2. EXPERIMENTAL SECTION 2.1. Materials. MBHA resin was supplied by Sigma-Aldrich (St. Louis, MO). Boc-protected α-amino acids were supplied by Kanglong Biotech Ltd. (Changzhou, China). mPEG2000-PLA2000 and MALPEG3000-PLA2000 were obtained from Yare Biotech (Shanghai, China). Phenol red-free Matrigel Matrix was supplied by BD Biosciences (San Diego, CA). Methanol, acetonitrile, and dimethyl sulfoxide (DMSO) were obtained from Merck (Darmstadt, Germany). 4′,6-Diamidino-2phenylindole, dihydrochloride (DAPI) was supplied by Beyotime (Shanghai, China). Cyanine 7 maleimide (Cy7) was purchased from Shanghai Seebio Biotech (Shanghai, China). Sulfo-Cyanine 7 was purchased from LiTTLE-PA Sciences Inc. (Wuhan, China). LysoTracker Red DND-99 and 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide (DiR) were supplied by Invitrogen (Grand Island, NY). Anti-M6PR antibody (ab2733), anti-EEA1 antibody (ab2900), and goat anti-rabbit IgG H&L (Alexa Fluoy 594) were obtained from Abcam (Cambridge, MA). Collagen I Rat Tail Natural was obtained from BD BioCoat (San Diego, CA). DNase I, collagenase, and EBM-2 were from Beijing Lablead Biotech Co., 25673


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collected at predetermined time points and detected by a fluorescence microplate reader. 2.8. In Vitro BBTB Penetration Assays. The in vitro BBTB model was established according to the literature.23 An HUVEC monolayer was used to mimic the BBTB in vitro. Briefly, HUVECs were seeded in the upper chamber and U87 cells were seeded in the basal chamber. After 3 days, the medium in the apical chamber was replaced by various 5 mg/L Coumarin-6-loaded micelles (diluted with DMEM). Solutions collected from the lower compartment after different incubation periods were detected by a fluorescence microplate reader. 2.9. In Vitro Evaluation of Brain Tumor-Targeting Ability. Tumor spheroids were cultured according to the literature.32 After 7 days, tumor spheroids were moved to the basal chamber of Transwell, containing established BBB or BBTB models. The medium in the apical chamber was replaced by various 5 mg/L Coumarin-6-loaded micelles or micelles preincubated with 50% rat plasma. Then, the tumor spheroids were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and visualized with a confocal microscope using tomoscan. 2.10. Evaluation of the Targeting Ability in the Intracranial Glioma-Bearing Mice Model. The orthotopic intracranial U87 tumor model was established as previously reported.17,33 Twelve nude mice were randomly divided into four groups and injected with 200 μL of LWSW-Cy7, DWSW-Cy7, and free Cy7 in normal saline solutions at a concentration of 0.01 μM through the tail vein (n = 3). Mice administered normal saline were used as controls. At predetermined time points, fluorescence imaging of the brain was conducted using an in vivo imaging system (IVIS Spectrum, Caliper). The brain and other organs were dissected for imaging after 2 h. The fluorescence intensity was analyzed using Living Image Software. To observe the targeting ability of peptide-mediated drug-delivery systems, the near-infrared fluorescence probe DiR was loaded into the micelles. Mice were allocated as aforementioned and respectively administered LWSW Micelle/DiR, DWSW Micelle/DiR, and Micelle/ DiR; they were then imaged at 2, 4, 8, and 24 h. In addition, the brain and other organs were dissected for imaging after 24 h. The fluorescence intensity was analyzed using Living Image Software. 2.11. Immunofluorescence Assay. Nude mice bearing intracranial U87 tumors were intravenously administered with Coumarin-6loaded micelles, LWSW micelles, and DWSW micelles. After 4 h, their brains were harvested and frozen in TissueTek O.C.T. compound after dehydration. Then, the brains were cut into 10 μm thick sections and stained with 300 nM DAPI and anti-CD31 antibody, as previously reported. The dissected organs were also stained with DAPI for observation of in vivo distribution of different micelle formulations. The sections were examined under a confocal laser scanning microscope. 2.12. In Vitro Evaluation of Antiglioma Efficacy. The in vitro antiglioma efficacy was evaluated through the MTT assay and destruction of tube formation. For the MTT assay, U87 cells and HUVECs were seeded into 96-well plates (3 × 103 cells per well). After 24 h, the culture medium was replaced by different concentrations of PTX formulations and cultured for another 72 h. At last, 20 μL of MTT solutions were added to each well and incubated for another 4 h. The percentage of cell viability was determined on the basis of absorbance at 490 nm by a plate reader (Power Wave XS; Bio-TEK). Untreated cells were used as the control. The toxicity of WSW peptides and their modified functional materials were also evaluated by the MTT assay. Endothelial cells are able of autogenously forming vessel-like tube networks on Matrigel in vitro, which is an important feature in the process of angiogenesis.34 Meanwhile, some capillary vessels originating from U87 cells were also developed during tumorigenesis and defined as vasculogenic mimicry (VM).35 For the destruction of tube formation, HUVECs and U87 cells were cultured in the Matrigel as previously reported and then treated with different PTX-loaded micelles. After incubation for 12 h, the tube structures were observed under an inverted phase contrast microscope (DMI4000 B; Leica, Germany). The length of the tubes

3). The encapsulation efficiency (EE%) and load efficiency (LC%) of PTX were calculated as previously reported.25 The in vitro release profile of different PTX-loaded micelle formulations was studied in 0.1 mM, pH 7.4 phosphate-buffered saline (PBS) with 0.5% of the surfactant Tween 80. The release media were removed at scheduled time points. The concentration of PTX was quantified by RP-HPLC. The concentrations of Coumarin-6 and DiR were determined by a fluorescence microplate reader (TECAN Infinite 200 PRO NanoQuant) at Ex/Em 494/522 and 741/776 nm, respectively. 2.4. In Vitro Study of Peptide Stability. It is reported that the L WSW peptide had good in vitro metabolic stability in plasma during the experimental time frame. Therefore, we performed the in vitro peptide stability study to compare the stability of two WSW peptides.16 DWSW and LWSW were dissolved in distilled water (0.5 mg/mL). Each solution (120 μL) was mixed with 1.08 mL of 50% sterile rat serum (diluted with PBS). After incubation at 37 °C, at each predetermined point 20 μL of 15% (w/v) trichloroacetic acid was mixed with 100 μL of reaction fluids and stored at 4 °C for 30 min, followed by 15 min of centrifugation at 12 000 rpm. The supernatant (20 μL) was sampled and determined by RP-HPLC. 2.5. Cellular Internalization Study of Peptides. Three different kinds of cells were used to investigate the peptides’ targeting ability, including primary BCECs, which were the main component of the BBB; U87 cells, which were the most representative glioma cell line; and HUVECs, which were used as a model of tumor neovascular endothelial cells. The cells were seeded into four-chambered confocal discs for observation or 12-well plates for flow cytometry. After 12 h, the culture medium was discarded and replaced with 5 μM fluoresceinlabeled peptides in DMEM and incubated for another 4 h (for BCECss, the incubation time should be 12 h). The fluorescent intensity was captured by flow cytometry or imaged by confocal microscopy after rinsing three times with PBS. Because the QS ligands interact with QS receptors in consistent with traditional concepts of receptor binding and different pathways have already been proposed to describe the receptor-mediated transcytosis.26 Meanwhile, the endosome was always involved in receptor-mediated endocytosis processes. Therefore, a colocation assay was further performed to determine the subcellular fate of the WSW peptide. Lysosomes and endosomes were tracked using LysoTracker Red DND-99 and early/late endosome tracker and then imaged by a laser scanning confocal laser microscope.27−29 The Manders’ overlap coefficient is used to quantify the colocalization rate (0.4 implies that 40% of both selected channels colocalize). 2.6. Endocytic Pathway Assays. The internalization mechanism of WSW micelles on HUVECs, U87 cells, and BCECs was investigated by endocytic pathway inhibition assays. The cells were preincubated for 30 min with different inhibitors at the following concentrations: BFA, 5 mg/L; chlorpromazine hydrochloride, 10 mg/L; colchicine, 4 mg/L; cytochalasin A, 5 mg/L; filipin, 5 mg/L; nocodazole, 20 μM; genistein, 200 μM; NaN3, 10 mM; methyl-β-cyclodextrin, 2.5 mM; and monensin, 200 mM, and with 200 mg/L of the LWSW and DWSW peptide (all from Aladdin except the peptides). They were then incubated with Coumarin-6-loaded LWSW micelles (LWSW Micelle/ C6) and DWSW micelles (DWSW Micelle/C6) at a concentration of 5 mg/L for 1 h at 37 °C. Quantitative analysis of cellular uptake of the micelles was carried out as previously reported.25 2.7. In Vitro BBB Penetration Assays. The in vitro BBB model was established according to the literature.30,31 The BCEC monolayer was used to mimic the BBB in vitro. Briefly, the upper chamber of Transwell was precoated with rat tail collagen overnight (0.3 mg/mL). The cortices of 6 week old SD rats were dissected and minced. Subsequently, the tissues were digested using collagenase/DNase I for 1.5 h at 37 °C. The capillaries were obtained by BSA density-gradient centrifugation, and the capillary fragments were seeded on collagencoated wells. The successful establishment was confirmed by measuring the transendothelial electrical resistance (TEER). Monolayers with a TEER over 300 Ω·cm2 were used for further experiments. To evaluate BBB penetration, 50 μM fluorescein-labeled peptides or 5 mg/L Coumarin-6-loaded micelles in DMEM were added to the upper chamber of Transwell. The solutions in the lower compartment were 25674


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ACS Applied Materials & Interfaces Table 1. Characterization of Different Nanoparticles (Data Represent Mean ± SE, n = 3) micelle

size (nm)

PDI

EE%

LC%

Micelle/PTX L WSW Micelle/PTX D WSW Micelle/PTX

23.17 ± 0.24 22.54 ± 0.21 22.89 ± 0.31

0.110 ± 0.015 0.078 ± 0.012 0.135 ± 0.011

88.65 ± 2.98 87.82 ± 4.86 86.74 ± 3.35

21.01 ± 0.56 20.85 ± 0.91 20.65 ± 0.63

Figure 2. Confocal observation and quantitative analysis of WSW peptides endocytosed by BCECs (A, B), HUVECs (C, D), and U87 cells (E, F). Fluorescein-labeled WSW peptides (5 μM) were incubated with different cells at 37 °C for 1 h, followed by lysosome staining. After fixation and nuclear staining, the cells were observed under a confocal microscope. was counted using Image J software within three randomly selected areas. 2.13. In Vivo Evaluation of Antiglioma Efficacy. The BALB/c nude mice model bearing intracranial glioma was established as previously reported.33 The mice were randomly allocated into five groups (n = 8) and treated with saline, Taxol, Micelle/PTX, LWSW Micelle/PTX, and DWSW Micelle/PTX, respectively, via the caudal vein with a PTX dose of 24 mg/kg at 6, 9, 12, and 15 days post implantation, respectively. The survival time was monitored and Graphpad was applied to plot the Kaplan−Meier survival curve for each group. After drug administration, the mice were sacrificed and immunohistochemical studies were performed on paraffin-embedded tissue sections as previously reported.23 The terminal deoxynucleoti-

dyltransferase-mediated dUTP nick end-labeling (TUNEL) assay was performed to evaluate apoptosis according to the manufacturer’s instructions. For evaluation of angiogenesis, CD31/PAS dual staining was performed.36 Three equal-sized fields were randomly chosen, and the positive cells were counted using image pro plus 5 software. 2.14. Statistical Analysis. The data are presented as mean ± SE (n = 3). Graphpad prism 6.0 software was used to perform comparisons among different groups using two-way analysis of variance analysis. p < 0.05 was considered significant.

3. RESULTS AND DISCUSSION Uncontrollable proliferation and profound neurological damage make glioma one of the most deadly brain diseases.37 The 25675


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Figure 3. Transcytosis of Coumarin-6-loaded plain micelles, DWSW Micelle/C6, and LWSW Micelle/C6 in the model of BBB (A, B) and BBTB (D, E). Uptake of U87 tumor spheroids with or without serum preincubation in the coculture models of BBB/U87 tumor spheroids (C) and BBTB/U87 tumor spheroids (F). Confocal microscopy was performed to examine tumor spheroid penetration. Mean ± SD, n = 3, **p < 0.01, ***p < 0.001.

micelles are classic nanocarriers for the delivery of various therapeutic agents. It has already been approved by the Food and Drug Administration for its safety and biocompatibility. In this study, the particle size of the micelles was around 22 nm. Meanwhile, the PDI, EE%, and LC% of the micelles were also investigated (Table 1). The release profiles of PTX from different micelle formulations were similar (Figure S4), suggesting that the drug-loading capacity of the PEG−PLA micelles was not affected by modification of the WSW peptide. There was negligible change in those physiological properties of micelles after peptide modification, indicating that modification of the peptide exerted inconspicuous effects on the micelles. 3.2. Cellular Uptake and Intracellular Distribution of WSW Peptides. BCEC, HUVECs, and U87 cells were treated with 5 μM fluorescein-labeled LWSW and DWSW (FAM was used as the negative control), and the results of cellular uptake are shown in Figure 2. These results indicated that WSW peptides were taken up by primary BCECs, tumor cells, and tumor-related blood vessel cells, suggesting that WSW peptides could specifically recognize BCECs and glioma-related cells. The results of flow cytometry indicated that the DWSW peptide exhibited a higher endocytosis efficiency than that of the LWSW peptide, which may be explained by the higher targeting efficiency of DWSW in comparison to that of the parent Lpeptide (Figure 2B,D,F). The two peptides were mostly colocalized with lysosome in the three different types of cells (Figure 2A,C,E), suggesting that intracellular WSW peptides followed the lysosome process. Anti-EEA1 and anti-M6PR antibodies were used to label early and late endosomes, respectively. Colocalization of the WSW peptides with the late endosome was evident and increased from 2 to 12 h (Figure S5). These results indicated that intracellular WSW peptides

failure of antiglioma therapy is mainly ascribed to its physiological and pathological barriers, including the BBB, BBTB, complex tumor microenvironment, and malignant tumor cells, among many others. In the previous reports, a variety of glioma-targeted drug-delivery systems have been developed to improve the therapeutic outcomes by overcoming one or more barrier.17,29,38 However, a few of them can overcome both physiological and pathological barriers. Recent studies have shown that QS peptides have great potential in cancer therapy for the treatment of many tumors and some of the QS peptides can selectively transverse the BBB.10,16 Our groups have already applied the retro-inverso strategy to convert linear peptide ligands comprising L-amino acids into the D configuration, which significantly improved the targeting efficiency.17,23,39 On the basis of previous researches and inspired by the systemic glioma-targeted drug-delivery strategy, we successfully developed QS peptide-modified micelles, achieving effectively glioma-targeted drug delivery.38 3.1. Characterization of Peptides and PeptideModified Micelles. Two WSW peptides were synthesized and ascertained by RP-HPLC and ESI-MS (Figure S1). The serum stability of the peptides are shown in Figure S2, indicating that DWSW possessed better stability in blood serum. In the 1H NMR spectrum (Figure S3) of MAL-PEG3000PLA2000, the peaks at 3.7−3.8 ppm were from repeat units of PEG and the signal at 7.0 ppm was the characteristic peak of the maleimide group. The chemical shift at δH 2.48 was the residual solvent signal of DMSO (DMSO-d6), and δH 3.33 was the proton signal of water. Protons at δH 2.66 and δH 3.76 were attributed to i and h in the structure, respectively. The disappearance of the maleimide peak (d) in the spectra suggested that the maleimide group completely reacted with the thiol group of LWSW-Cys or DWSW-Cys. PEG−PLA 25676


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Figure 4. In vivo-targeting ability of WSW peptides and their corresponding micelles in glioma-bearing mice. (A) In vivo fluorescence imaging of intracranial U87 glioma-bearing mice at predetermined time points after intravenous injection with DiR-loaded micelles. (B) Ex vivo imaging of the dissected brain and tumor at 24 h after intravenous injection with DiR-loaded micelles. (C) Ex vivo near-infrared imaging of the dissected brain and tumor 2 h after injection with peptide-Cy7 probes. (D) Semiquantitative region of interest (ROI) analysis of the mean fluorescence intensity from the DiR-labeled micelles in the brain at 24 h after intravenous injection with DiR-loaded micelles. (E) Semiquantitative ROI analysis of in vivo fluorescence images at different time points in the tumor from peptide-Cy7 probes (mean ± SD, n = 3). *p < 0.05, ***p < 0.001.

followed the early endosome (2 h), late endosome (12 h), and lysosome process. 3.3. Cellular Uptake Mechanism of WSW PeptideModified Micelles. The association mechanism underlying the internalization of nanoparticles on targeted cells was crucial for the actively targeted drug-delivery system.40,41 The endocytosis of WSW-modified micelles in several types of cells may depend on different endocytic mechanisms. To clarify the endocytosis mechanisms of WSW peptide-modified micelles, experiments were performed in the presence of various endocytosis inhibitors. As shown in Table S1, the associations of LWSW Micelle/C6 and DWSW Micelle/C6 in HUVECs, U87 cells, and BCECs were all endosome-involving and energy-dependent. In addition, we tested the internalization of LWSW Micelle/C6 and DWSW Micelle/C6 in the presence of an unlabeled WSW peptide, demonstrating that the QS receptor could be blocked by the WSW peptide. The presence of M-β-CD, an inhibitor of lipid raft-mediated endocytosis, significantly reduced the cellular uptake of L WSW Micelle/C6 and DWSW Micelle/C6, indicating that the lipid raft mediated the endocytosis process of WSW peptide-modified micelles. In addition, caveolae and golgi apparatus were also partially involved in the endocytosis of two WSW peptide-modified micelles in U87 cells and HUVECs. The internalization of LWSW Micelle/C6 and DWSW Micelle/ C6 on BCECs could be inhibited by Cyto-D, suggesting that the transportation of WSW Micelle/C6 in the BBB was also mediated by macropinocytosis. These results indicated that the endocytic pathway of WSW-modified micelles may refer to the caveolae/lipid raft-mediated endocytosis and the involvement of the energy-dependent macropinocytosis pathway. All of these results indicated that the change in the configuration the

of WSW peptide would not influence the cellular endocytosis of micelles. 3.4. Brain Tumor-Targeting Ability of WSW Micelles in Vitro. In vitro BBB and BBTB models were established to evaluate the barrier-penetrating ability of the WSW micelles in vitro. As shown in Figure S6, two WSW peptides could efficiently cross the BBB, and the process was energy dependent. In addition, both the WSW peptide-modified micelles could effectively penetrate the in vitro BBB and BBTB models. As shown in Figure 3, WSW modification significantly increased transcytosis of the micelles that traversed the BBB and BBTB. After 4 h, 5.213 ± 0.018% of the LWSWmodified micelles and 5.097 ± 0.054% of the DWSW modified micelles crossed the BBB, which was significantly higher than that of plain micelles (3.853 ± 0.052%). In the BBTB model, 4.453 ± 0.072% of the LWSW-modified micelles and 4.513 ± 0.096% of the DWSW-modified micelles traversed the BBTB after 4 h compared with unmodified micelles (3.767 ± 0.023%). The brain tumor-targeting efficiency in vitro could also be observed in the coculture model of BBB/U87 tumor spheroids and BBTB/U87 tumor spheroids. After preincubation with 50% mouse serum, the LWSW peptide was degraded by serum and the LWSW-modified micelles lost their BBB and BBTB crossing and tumor spheroid-targeting capabilities, whereas the DWSWmodified micelles still retained their targeting and barrierpenetration abilities (Figure 3C,F). These results further indicated the stability and superiority of the D-peptide. 3.5. Brain- and Tumor-Targeting Abilities of WSW Peptides and Their Corresponding Micelles in Vivo. To confirm whether WSW peptides could selectively penetrate the BBB and its efficiency, ICR mice were used. The results showed that both the WSW peptides could cross the BBB. The DWSW peptide exhibited a higher accumulation efficiency in the brain 25677


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Figure 5. Distribution of Coumarin-6-loaded plain micelles and WSW peptide-modified micelles in the tumors of glioma-bearing mice (A) and other organs (B) 4 h post injection. Red: blood vessels (anti-CD31 stained); blue: cell nuclei (DAPI stained); green: Coumarin-6-labeled micelles; white arrows indicate where colocalization occurs.

compared to that of the LWSW peptide (Figure S7), which may be correlated to the high stability of DWSW in blood circulation. In the glioma-bearing mice, a dramatic difference could be observed 15 min after administration of the Cy7 probe (Figure 4E). As for peptide-modified micelles, a difference could be observed at each predetermined point (Figure 4A). The results of ex vivo imaging also confirmed that the DWSWmodified micelles in the glioma region have the highest fluorescence intensity (Figure 4B,D). Quantification assays of the fluorescent intensities in different organs are shown in Figure S8. The peptides mainly distributed in kidney, whereas the peptide-modified formulations mainly distributed in the liver. The results of immunofluorescence showed that WSW peptide-modified micelles could colocalize with CD31, suggesting that it could not only target tumor cells but also target neovasculature (Figure 5A). Quantification of the colocalization of micelles with CD31 was analyzed by ImagePro Plus from three randomly selected microscope-captured images. The statistical analysis of colocalization is shown in Figure S9. Furthermore, as shown in Figure 5B, the distribution of WSW-modified micelles in other organs had no selectivity, for its random distribution in the heart, liver, spleen, lung, and kidney. 3.6. Destruction of VM Channels and the Neovasculature Network. The Matrigel-based tube formation assay was used to evaluate the destruction effect of PTX formulations (Figure 6A). HUVECs and U87 cells can autogenously form vessel-like tube networks after 24 h of incubation on a Matrigel matrix. However, treatment with

various PTX formulations markedly inhibited tube formation of the HUVEC and U87 cells, resulting in less elongated and broken tubes. In addition, LWSW Micelle/PTX and DWSW Micelle/PTX displayed a higher activity in inhibiting tube formation and U87 VM on Matrigel than that of Micelle/PTX. 3.7. Cytotoxicity of WSW Micelles in Vitro. The MTT assay was used to evaluate the in vitro cytotoxicity of various PTX formulations (Figure 6D,E). DWSW Micelle/PTX exhibited the strongest antiproliferative effect, registering an IC50 values of 0.054 μM (U87 cells) and 0.343 μM (HUVECs). The IC50 values for unmodified micelles and LWSW-modified micelles against U87 cells were 0.478 and 0.273 μM, respectively; whereas for HUVECs, the values were 0.811 and 0.323 μM. The higher cytotoxicity of DWSW Micelle/PTX may be related to an increase in cellular uptake. In addition, WSW peptides and their functional materials showed no significant toxicity against U87 cells (average cell viability of over 90%) (Figure S10). 3.8. In Vivo Antitumor Efficacy of WSW Micelles. The median survival of nude mice bearing intracranial gliomas was used as an index to evaluate the antiglioma effect of PTX micelles. Figure 7 shows that treatment with Micelle/PTX or free taxol at a dose of 24 mg/kg body weight (at 6, 9, 12, and 15 days post implantation) could only slightly prolong the survival. The median survival for the Micelle/PTX- and free taxol-treated groups was 21 and 20 days, respectively, compared to that for the saline-treated group (19 days). However, the median survival of the groups treated with DWSW Micelle/PTX (27.5 days, a 3.25 times prolonged median survival compared to 25678


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Figure 6. Therapeutic efficacy of PTX-loaded micelles in vitro. Effect of various PTX-loaded micelles on U87 VM formation and HUVEC tube formation (A). Quantitative data of U87 VM formation (B) and HUVEC tube formation (C) on treatment with different PTX-loaded formulations. Data are presented as percentages of the control group, which was set at 100%, (mean ± SD, n = 3) *p < 0.05, **p < 0.01. Cytotoxicity of various PTX-loaded micelles against U87 cells (D) and HUVECs (E) was assessed using the MTT assay after 72 h of incubation (mean ± SD, n = 3). The IC50 values were calculated using GraphPad Prism 6. X axis: logarithm of PTX concentration; Y axis: percentage of viable cells.

that of the Micelle/PTX-treated group) and LWSW Micelle/ PTX (25 days, a 2 times prolonged median survival compared to that of the Micelle/PTX-treated group) was much longer than that of the Micelle/PTX-treated group. The brain slice in Figure S11 shows that two WSW peptide-mediated drugdelivery systems could significantly reduce the tumor size. These results indicated that micelles modified with the WSW peptide exhibited an improved antiglioma effect compared to that of plain micelles and DWSW Micelle/PTX achieved the highest antiglioma efficacy. It appeared that DWSW Micelle/ PTX treatment induced more apoptosis in tumors (2.5%) in comparison to that induced by other groups (saline: 0.1%, Taxol: 0.15%, Micelle/PTX: 0.2%, DWSW Micelle/PTX: 1.2%). Additionally, DWSW Micelle/PTX achieved the highest inhibitive effect against angiogenesis (Taxol: 51%, Micelle/

PTX: 50%, LWSW Micelle/PTX: 63%, DWSW Micelle/PTX: 82%) and VM channels. These results are consistent with the survival curves and validated that the enhanced antitumor effects were attributed to DWSW modification (Figure 8). Our results showed that WSW peptide-modified functional materials endowed micelles with the abilities of binding to and penetrating the BBB and BBTB. The retro-inverso isomerization strategy was applied to improve the stability of the WSW peptides. The cocultured BBB (or BBTB)/U87 tumor spheroid model was also constructed to confirm the superiority of the DWSW peptide. The DWSW peptide could transverse the BBB more efficient than the LWSW peptide both in healthy mice and in glioma-bearing mice. DWSW could remain intact after traversing the BBB and enzymatic barrier, exerting targeting capability to the glioma site. WSW-modified micelles 25679


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Figure 7. Kaplan−Meier survival curves of nude mice bearing intracranial U87 gliomas (n = 8) that received four doses of different PTX formulations or saline (at 6, 9, 12, and 15 days post implantation). *p < 0.05, ***p < 0.001, log-rank analysis.

Figure 8. Immunohistochemical examination of tumor sections from animals for CD31/PAS (A, C) and TUNEL assays (B, D). Red arrows in the image represent positive staining for CD31- and TUNEL-positive cells. The number of CD31-positive cells and TUNEL-positive cells in tumors was counted at 40× magnification using image pro plus software, and Graphpad Prism 6 was used for statistical treatment. Mean ± SD, n = 3, **p < 0.01 ***p < 0.001.

prepared with DWSW peptide modification on the surface. The prepared DWSW micelles showed multifunctional targeting of the brain, tumor cells, and angiogenic blood vessels and enhanced penetration capability. When loaded with PTX, D WSW micelles demonstrated the strongest antiangiogenesis and antitumor effects in vitro and in vivo, which may be attributed to its better serum stability and higher tumor accumulation. The micelle-based drug-delivery system developed in the current study could effectively overcome multiple barriers and precisely target the tumor site, validating its promising value in improving the antiglioma efficacy of PTX, and might therefore provide an efficient method for tumor therapy.

loading PTX demonstrated good inhibitory effects against U87 cells, HUVECs, VM, and HUVEC three-dimensional tubes in vitro. By specifically targeting tumor cells, tumor angiogenesis, and VM, DWSW Micelle/PTX significantly prolonged the survival time compared with other groups and achieved the best antitumor effect.

4. CONCLUSIONS In conclusion, we utilized a QS peptide that could efficiently and selectively penetrate the BBB as a targeting ligand for the first time and designed a stable DWSW peptide by the retroinverso strategy. To construct a stabilized multifunctional targeted drug-delivery system, PEGylated micelles were 25680


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b03518. Chromatogram and ESI-MS graph of LWSW-Cys and D WSW-Cys; peptide stability; NMR spectra of MALPEG-PLA, LWSW-PEG-PLA, and DWSW-PEG-PLA; effect of various inhibitors on the endocytosis of WSWmodified micelles in BCECs, HUVECs, and U87 cells; release profile of PTX from different PTX-loaded micelle formulations; colocalization of endocytosed LWSW and D WSW with early (EEA1)/late (M6PR) endosome in BCECs after 2 or 12 h of incubation; transcytosis efficiency of DWSW and LWSW in the in vitro BBB model at 4 or 37 °C; ex vivo imaging of dissected tissues of the mice treated with DWSW fluorescein and LWSW fluorescein and of the negative control (FAM) at 15, 30, and 60 min post injection; distribution of WSW peptides and their corresponding micelles in glioma-bearing mice; statistical analysis of different micelle formulations colocalized with the blood vessel marker CD31; safety evaluation of WSW peptides and their functional materials; brain slices of mice after treatment with various micelle formulations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 21 5198 0006. Fax: +86 21 5198 0090. ORCID

Weiyue Lu: 0000-0001-8003-2675 Author Contributions ⊥

D.R. and J.M. contributed equally to this work.

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, No. 2013CB932500), the National Natural Science Foundation of China (No. 81690263 and No. 81473149), and the Shanghai International Science and Technology Cooperation Project (No. 16430723800).

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REFERENCES

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d-Retroenantiomer of Quorum-Sensing Peptide-Modified Polymeric

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