Modular Design and Facile Synthesis of Enzyme-Responsive Peptide

Aug 26, 2016 - Construction of efficient doxorubicin (DOX) delivery systems addressing a cascade of physiological barriers remains a great challenge f...
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Modular Design and Facile Synthesis of Enzyme-Responsive PeptideLinked Block Copolymers for Efficient Delivery of Doxorubicin Wendong Ke,†,‡ Junjie Li,†,‡ Kaijie Zhao,‡ Zengshi Zha,‡ Yu Han,‡ Yuheng Wang,‡ Wei Yin,‡,§ Ping Zhang,‡,∥ and Zhishen Ge*,‡ ‡

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, Anhui China § Department of Pharmacology, Xin Hua University of Anhui, Hefei, China ∥ Department of Chemistry, Anhui Science and Technology University, Anhui Fengyang 233100, China S Supporting Information *

ABSTRACT: Construction of efficient doxorubicin (DOX) delivery systems addressing a cascade of physiological barriers remains a great challenge for better therapeutic efficacy of tumors. Herein, we design welldefined enzyme-responsive peptide-linked block copolymer, PEG-GPLGVRGDG-P(BLA-co-Asp) [PEG and P(BLA-co-Asp) are poly(ethylene glycol) and partially hydrolyzed poly(β-benzyl L-aspartate) (PBLA), respectively] (P3), with modular functionality for efficient delivery of DOX. The block copolymers were successfully obtained via click reaction to introduce peptide (alkynyl-GPLGVRGDG) into the end of PEG for initiating ring-opening polymerization of β-benzyl L-aspartate N-carboxyanhydride (BLA-NCA) by terminal amino groups followed by partial hydrolysis of PBLA segments. P3 micelle was demonstrated to encapsulate DOX efficiently through synergistic effect of benzyl group-based hydrophobic and carboxyl moiety-based electrostatic interactions. Effective matrix metalloproteinase-2 (MMP-2)-triggered cleavage of peptide for dePEGylation of P3 micelles was confirmed and residual RGD ligands were retained on the surfaces. Against HT1080 cells overexpressing MMP-2, DOX-loaded P3 micelles showed approximately 4-fold increase of the cellular internalization amount as compared with free DOX and half maximal inhibitory concentration (IC50) value of DOX-loaded P3 micelles was determined to be 0.38 μg/mL compared with 0.66 μg/mL of free DOX due to MMP-triggered dePEGylation, RGD-mediated cellular uptake, and rapid drug release inside cells. Binding and penetration evaluation toward HT1080 multicellular tumor spheroids (MCTs) confirmed high affinity and deep penetration of P3 micelles in tumor tissues. This modular design of enzyme-responsive block copolymers represents an effective strategy to construct intelligent drug delivery vehicles for addressing a cascade of delivery barriers.

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INTRODUCTION

many malignant tumors. As compared with free DOX, these DOX formulations dramatically prolonged the blood circulation and improved tumor accumulation, which finally enhanced the therapeutic efficacy and reduced side effects.6 However, the delivery systems have to face a cascade of physiological barriers in the delivery journey to the target sites primarily including long circulation, accumulation, tissue penetration, cellular internalization, and rapid intracellular drug release.7−9 The above systems just addressed partial barriers, finally resulting in relatively modest therapeutic efficacy.4,5,10 Higher efficiency of DOX delivery depends on more elaborate delivery system design to overcome all the primary barriers efficiently.

Doxorubicin (DOX) as one of most frequently used anticancer drugs showed potent treatment ability toward many cancers through intercalating DNA of cells. But the clinical application of administration dosage was limited by the extremely serious adverse effect of life-threatening heart damage.1,2 Therefore, a suitable delivery system that could efficiently deliver the drug into the cancer cells is critically essential for clinical utility of DOX. Several carriers have been proceeded into clinical applications or trials. For example, PEGylated DOX-loaded liposomes (Doxil) was the first nanomedicine approved by the US food and drug administration (FDA).3 DOX-conjugated poly(N-(2-hydroxypropyl)methacrylamine (PHPMA) linked by enzymatically cleavable peptide (PK1) 4 and DOXconjugated poly(ethylene glycol) (PEG)-poly(amino acid) via amide bonds (NK911)5 are also in the process of clinical trials. All of them displayed potent treatment performance against © XXXX American Chemical Society

Received: July 4, 2016 Revised: August 11, 2016

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DOI: 10.1021/acs.biomac.6b00997 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

Scheme 1. Schematic Illustration of Formation of DOX-Loaded Enzyme (MMP)-Responsive Block Copolymer Micelles, as Well as Sequential Triggered dePEGylation and RGD-Mediated Cellular Internalization for Intracellular Drug Release and Cell Killing

Scheme 2. Synthetic Routes Employed for the Preparation of the MMP-Responsive Block Copolymer, PEG-GPLGVRGDGP(BLA-co-Asp)

widely used as the effective trigger for tumor imaging, drug release, or dePEGylation for enhanced cellular internalization,25−33 because MMP enzymes play an essential role in the process of tumor growth and metastasis.34 MMP stimuli could provide the trigger in the level of tumor tissue, which showed evident advantages over stimuli of intracellular environment. Typically, cell-penetrating peptides (TAT) are buried by the MMP-cleavable peptide-linked long PEG on the surface of the drug-loaded liposomes.27,35 In tumor tissues, TAT is exposed to the cell membranes through cleavage of peptide linkage for dePEGylation by highly overexpressed MMP. However, for this system, it is difficult to ensure that all the TAT molecules are protected efficiently before dePEGylation to eliminate the nonspecific interaction with blood component. Herein, we propose a modular design strategy of MMPresponsive peptide-linked block copolymers by incorporating several functional modules into one system to satisfy sequential demands in the process of DOX delivery (Scheme 1). We

Stimuli-responsive nanocarriers attracted great attention in recent years since the nanoparticle structures or surface properties can be modulated in response to the specific environment.11−15 Exogenous stimuli (e.g., temperature, light, ultrasounds) and endogenous stimuli (e.g., upregulated enzymes in diseased tissues, tumoral pH, endo/lysosomal pH, redox potential) have been explored as the triggers to achieve promoted cellular internalization, endosomal escape, or drug release of the nanoparticles after long blood circulation and high tumor accumulation. For DOX delivery, various elaborately engineered stimuli-responsive systems have been developed addressing more barriers in the process of delivery.16−23 However, facile preparation of a structurally well-defined system with multifunction simultaneously addressing a cascade of physiological barriers remains a great challenge. Among various stimuli, the endogenous ones on the basis of diseased microenvironments show more site-specificity and targeting.24 Notably, upregulated enzymes in tumor tissues, particularly, matrix metalloproteinases (MMPs), have been B

DOI: 10.1021/acs.biomac.6b00997 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

size distributions, and zeta potentials of the nanoparticles at 25 °C and the measured scattering angle was fixed at 173°. A F-4600 (Hitachi) spectrofluorometer was used to obtain fluorescence spectra. Transmission electron microscopy (TEM) samples were prepared through adding a drop of micelles solution to copper TEM grids and dried overnight. Hitachi H-7650 electron microscope was used to obtain transmission electron microscopy (TEM) images at an acceleration voltage of 100 kV. Leica TCS SP5 microscope was used to acquire confocal laser scanning microscopy (CLSM) images. Synthesis of PEG-GPLGVRG-NH2 and PEG-GPLGVRGDG-NH2. PEG113-N3 (0.3 g, 0.06 mmol), peptide alkynyl-GPLGVRGDG (62 mg, 0.072 mmo1), DMF (3 mL), and PMDETA (31 mg, 0.18 mmol) were added into a Schlenk flask. The mixture was frozen to solid in liquid nitrogen and it was degassed to vacuum and backfilled with N2. With the protection of N2, CuBr (26 mg, 0.18 mmol) was added. The system was degassed for two more times by freeze−pump−thaw cycles followed by seal under vacuum. The reaction was performed at 40 °C for 24 h. After that the mixture was precipitated into cold diethyl ether. The crude product was collected by centrifugation and then drying under vacuum overnight. The crude product was redissolved in water, added into a dialysis bag (MWCO: 3500 Da), and dialyzed for 3 days against distilled water. After lyophilization, PEG-GPLGVRGDG-NH2 was obtained as a solid powder (0.22 g, yield: 62.7%; Mn,GPC = 5.3 kDa, Mw/Mn = 1.07). PEG-GPLGVRG-NH2 was also synthesized according to a similar procedure (0.24 g, yield: 68.2%; Mn,GPC = 5.3 kDa, Mw/Mn = 1.05). Synthesis of Block Copolymers, PEG−PBLA, PEG-GPLGVRGPBLA, and PEG-GPLGVRGDG-PBLA. Ring-opening polymerization of BLA-NCA was initiated by the terminal amino group of PEG-NH2. First, PEG-NH2 initiator was dissolved in about 0.5 mL of CH2Cl2, and then an excess amount of benzene was added. After lyophilization the anhydrous white powder was obtained. Then, lyophilized PEG-NH2 (0.250 g, 0.05 mmol) was dissolved in 4 mL of anhydrous CH2Cl2 followed by addition of BLA-NCA (0.623 g, 2.5 mmol) predissolved in 2 mL of anhydrous DMF, and the reaction was stirred for 3 days at room temperature in a glovebox. The resulting polymer was then precipitated into 50 mL of cold diethyl ether. The product was collected by centrifugation. The above-mentioned dissolution− precipitation cycle was repeated twice. The final products were dried under vacuum, yielding a white solid powder (0.69 g, yield: 90.1%). Block copolymers, PEG-GPLGVRG-PBLA and PEG-GPLGVRGDGPBLA, were also obtained according to similar procedures by using PEG-GPLGVRG-NH2 and PEG-GPLGVRGDG-NH2 as the initiator, respectively. The degrees of polymerization of PBLA segments were calculated to be 46, 45, and 42 for PEG−PBLA, PEG-GPLGVRGPBLA and PEG-GPLGVRGDG-PBLA, respectively, according to 1H NMR analysis using DMSO-d6 as the solvent. Thus, the block copolymers were denoted as PEG113-PBLA46 (P4), PEG113-GPLGVRG-PBLA45, and PEG113-GPLGVRGDG-PBLA42, respectively. Synthesis of PEG-P(BLA-co-PAsp), PEG-GPLGVRG-P(BLA-coPAsp), and PEG-GPLGVRGDG-P(BLA-co-PAsp). The block copolymers, PEG-P(BLA-co-PAsp), PEG-GPLGVRG-P(BLA-co-PAsp), and PEG-GPLGVRGDG-P(BLA-co-PAsp), were synthesized via partial hydrolysis of PEG−PBLA, PEG-GPLGVRG-PBLA, and PEG-GPLGVRGDG-PBLA, respectively. The hydrolysis degrees of BLA block were controlled according to the reported procedure.39 Typically, PEG−PBLA (144.3 mg, 0.01 mmol) block copolymer was dissolved in chloroform (2 mL). The polymer solution was then treated with 1 mL of NaOH (6 mg 0.15 mmol) solution in a mixture of water, methanol, and 2-propanol (1:2:2, v/v/v), vigorously stirring at 0 °C for 15 min. Glacial acetic acid (100 μL) was added to stop the reaction. Then the mixture was poured into 20 mL of diethyl ether to get a white precipitate. The precipitate was dispersed in 5 mL of DMSO and added into a dialysis bag (MWCO: 3500 Da) to dialyze against distilled water for 24 h. A white powder was obtained after lyophilization. According to 1H NMR analysis, the degree of PAsp block was calculated to be 16 by using DMSO-d6 as the solvent. Thus, the block copolymer was denoted as PEG113-P(BLA0.65-co-Asp0.35)46 (P1). According to the similar procedure, block copolymers, PEG113GPLGVRG-P(BLA0.73-co-PAsp0.27)45 (P2) and PEG-GPLGVRGDG-

facilely synthesized MMP-responsive peptide-linked block copolymers, PEG-GPLGVRGDG-P(BLA-co-Asp) (PEG and P(BLA-co-Asp) are poly(ethylene glycol) and partially hydrolyzed poly(β-benzyl L-aspartate) (PBLA), respectively) (P3)) via click chemistry to modify the end group of PEG into peptide bearing terminal amino groups, which was used to initiate the polymerization of β-benzyl L-aspartate N-carboxyanhydride (BLA-NCA), followed by partial hydrolysis of PBLA segments (Scheme 2). For DOX encapsulation, the synergistic effect of hydrophobic interactions and electrostatic interactions not only improved the drug loading efficiency but also increased the stability of the nanoparticles. In the presence of MMP-2, with the gradual dePEGylation by cleavage of the amido bond between G and V, the residual RGD on the surface will act as a ligand to interact with cell membrane to facilitate the cellular internalization. In the endo/lysosomes with lower pH and abundant esterase, the drug release can be achieved quickly and efficiently, and finally induced high cytotoxicity. Each module in this design plays an important role for encapsulation and delivery of DOX, which achieved highefficiency DOX encapsulation, high stability in aqueous solution with large dilution or in the presence of salt and serum, MMP-triggered dePEGylation, and cellular internalization, as well as intracellular triggered drug release.

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MATERIALS AND METHODS

Materials. Anhydrous tetrahydrofuran (THF) was obtained by distillation over sodium/benzophenone. Anhydrous dimethyl sulfoxide (DMSO) and hexane were obtained by distilling after drying over calcium hydride (CaH2). N,N,N′,N″,N″-Pentamethyldiethylenetriamine (PMDETA, 98%), N,N-dimethylformamide (DMF), copper(I) bromide (CuBr, 99%), esterase from porcine liver, and DOX·HCl were purchased from Sigma-Aldrich. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), trypsin, 4,6diamidino-2-phenylindole (DAPI), and 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Recombinant human MMP-2 (purity > 95%, the specific activity > 1000 pmol/min/ μg) was purchased from Sino Biological Inc. (Beijing, China). pAminophenyl mercuric acid (APMA) was purchased from Genmed Scientifics Inc., U.S.A. G(propargylglycine)-PLGVRG (alkynyl-GPLGVRG, purity 95.36% from HPLC) and G(propargylglycine)-PLGVRGDG (alkynyl-GPLGVRGDG, purity 97.86% from HPLC) peptides were purchased from China Peptides Co., Ltd. (Shanghai, China). αMethoxy-ω-amine-poly(ethylene glycol) (PEG113-NH2) was purchased from Jenkem Technology). α-Methoxy-ω-azido-poly(ethylene glycol) (PEG113-N3) and β-benzyl-L-aspartate N-carboxyanhydride (BLANCA) were synthesized on the basis of previously reported procedures.36,37 Human fibrosarcoma cell line (HT1080) and mouse 4T1 breast tumor cell line were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). TCNB buffer (50 mM Tris, pH 7.4, 10 mM calcium chloride, 150 mM sodium chloride, 0.05% BRIJ 35) was freshly prepared before use. Characterization. 1H NMR spectra were recorded on a Bruker AV300 spectrometer. CDCl3 or DMSO-d6 were used as the solvent. Gel permeation chromatography (GPC) was used to analyze the molecular weights and molecular weight distributions (Mw/Mn) of the polymers. The GPC was equipped with G1316A PL gel columns, an Agilent G1362A differential refractive index detector (set at 30 °C) and an Agilent 1260 pump. Before measurements, all the BLA units were hydrolyzed to carboxyl groups followed by treatment with chlorotrimethylsilane to eliminate the interaction with column according to reported procedure.38 A series of low-polydispersity PEG standards were employed for calibration. DMF (HPLC grade) at a flow rate of 1.0 mL/min with 1 g/L LiBr was used as eluent. A zetasizer (Nano ZS) instrument which was equipped with a He−Ne ion laser (λ = 633 nm) was used to measure the particle sizes, particle C

DOI: 10.1021/acs.biomac.6b00997 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

Table 1. Characterization of the Block Copolymers and the Corresponding DOX-Loaded Micelles That Were Formed at the Weight Ratio of 10:1 (Polymer/DOX·HCl) IC50 (μg/mL) polymer P1 P2 P3 P4 P5 DOX·HCl

a

PEG113-P(BLA0.65-co-Asp0.35)46 PEG113-GPLGVRG-P(BLA0.73-co-Asp0.27)45 PEG113-GPLGVRGDG-P(BLA0.70-co-sp0.30)42 PEG113-PBLA46 PEG113-PAsp46

b

Mn (kDa)

Mw/Mnb

c

c

size (nm)

PSD

13.0 13.8 13.3 14.4 10.3

1.23 1.28 1.27 1.23 1.23

42.4 37.8 43.5 48.9 66.8

0.25 0.21 0.22 0.18 0.17

d

e

DLE (%)

DLC (wt %)

HT1080

4T1

79.5 82.4 83.6 1.8 89.7

7.2 7.5 7.6 0.5 8.9

0.87 0.56 0.38

2.30 1.94 2.51

0.66

1.49

a

The degrees of polymerization (DPs) of the polymers were determined from the 1H NMR analysis. bDetermined by GPC analysis using DMF as the eluent. To eliminate the interaction between GPC column and carboxyl groups, all the BLA units were hydrolyzed to carboxyl groups, followed by treatment with chlorotrimethylsilane before measurements. cThe particle size and size distribution (PSD) were determined by DLS. dDrug loading efficacy. eDrug loading content. P(BLA0.70-co-PAsp0.30)42 (P3) were also synthesized. As a control, the complete hydrolysis of PBLA block was also performed for PEG113PBLA46 and the polymer, PEG113-PAsp46 (P5), was also obtained. GPC measurements using DMF as the eluent were performed. To eliminate the interaction between GPC column and carboxyl groups, all the BLA unites were hydrolyzed to carboxyl groups followed by treatment with chlorotrimethylsilane before measurements. The results were summarized in Table 1. MMP-2-Triggered dePEGylation. The dePEGylation of P1, P2 and P3 was conducted on the basis of the manufacturer’s Activity Assay protocol in the presence of MMP-2. Briefly, the polymeric micelles of P1 (5 mg), P2 (5.52 mg), and P3 (5.52 mg) were separately prepared in 2 mL of TCNB buffer, then MMP-2 (2 mL, 2 μg/mL in TCNB buffer) activated by APMA before use was added. The time-dependent changes in particle size, zeta potential, and particle size distribution were monitored by DLS at predetermined intervals. Simultaneously, the samples were took out from the mixtures and lyophilized for investigation by GPC measurements. The dePEGylation profiles of the micelles were analyzed from the GPC traces measured at varying time points. DOX Encapsulation. DOX encapsulation was performed via nanoprecipitation method. To determine the optimal drug loading and encapsulation efficiency, nanoparticles were prepared at various DOX· HCl/polymer weight ratios. Briefly, polymers (5 mg) and DOX·HCl (0, 0.25, 0.33, 0.5, 1.0, or 1.7 mg) were dissolved in 0.5 mL of DMSO and injected quickly into 4.5 mL of PBS (10 mmol, pH 7.4). The resulting micelles solutions were added into a dialysis membrane (MWCO 3500 Da) and dialyzed against PBS (pH 7.4) for 4 h to remove DMSO, while unloaded DOX·HCl was removed by the method of combined centrifugation with ultrafiltration (MWCO 50 kDa). The obtained DOX-loaded nanoparticles were stored in 4 °C refrigerator. After the samples were lyophilized and redissolved in DMSO, the drug loading content (DLC) and drug loading efficiency (DLE) of DOX·HCl were calculated by fluorescence intensity at the emission wavelength at 593 nm (excitation at 480 nm). Drug loading content (DLC, wt %) = [(wt of loaded drug)/(wt of total micelles)] × 100; Drug loading efficiency (DLE, wt %) = [(wt of loaded drug)/(wt of total added drug)] × 100. In Vitro Drug Release. The dialysis bag (MWCO 3500 Da) was used to study the release profiles of DOX from DOX-encapsulated P1, P2, and P3 micelles at 37 °C in corresponding media. Typically, 2 mL of DOX-loaded micelle PBS solutions (containing 0.2 mg of DOX) were loaded in a dialysis bag preswelled with hot distilled water which has a molecular cutoff of 3500 Da and then dialyzed against 18 mL PBS at pH 7.4 or pH 5.0 (10 mM). A total of 1 mL of release media was taken out from the released tube and then 1 mL of fresh medium was added at certain time intervals. DOX·HCl released profile was analyzed according to fluorescence measurement date with the emission at 593 nm and excitation at 480 nm. The above-mentioned experiments were carried out in triplicate, and the final results were the average data with standard deviations.

The release profiles in the presence of esterase (0.5 mg/mL) or MMP-2 (1 μg/mL) were also investigated according to a similar procedure. For the release in the presence of the esterase (from porcine liver), the esterase was added into the DOX-loaded micelles solutions at the beginning of release. For that in the presence of MMP2, the release experiments were performed in TCNB buffer at pH 7.4 and MMP-2 was also added at the beginning of release. Cellular Uptake Measurements by Flow Cytometry. 4T1 cells or HT1080 cells (1 × 105) were cultured separately in each well of a 24-well plate supplemented with 1 mL of DMEM containing 10% FBS under humidified 5% CO2 atmosphere for 1 day at 37 °C. Then, various samples which have a DOX concentration of 10 μg/mL were added then incubated for another 24 h, respectively. Subsequently the each well was washed with cold PBS to remove the extracellular residual DOX formulation and medium completely. Then small amounts of trypsin was added to digest and detach them. Then cells were suspended in 1 mL of cold PBS and measured by flow cytometry (EasyCyte2, 484 nm/591 nm). Flowjo software was used to analyze the date. Cytotoxicity Assays. The cytotoxicity of various micelles (DOXloaded P1, P2, and P3 micelles) and free DOX·HCl were evaluated against 4T1 cells or HT1080 cells by MTT assays. First, 1 × 104 4T1 cells and HT1080 cells were cultured separately in each well of a 96well plate supplied with 100 μL of DMEM containing 10% FBS under humidified 5% CO2 atmosphere for 24 h at 37 °C. Then 90 μL of fresh medium containing 10% FBS was added to replace the medium taken out before. After that, a total of 10 μL of DOX-loaded micelles or free DOX·HCl in PBS buffer (10 mM, pH 7.4) were added to yield the final DOX-equivalent concentrations in the range of 1 × 10−3 to 10 μg/mL, followed by another 24 h incubation. Each sample was replicated in four wells. In MTT assay, 100 μL of fresh medium was added to replace the aspirated medium completely. Then 20 μL freshly prepared 5 mg/mL of MTT PBS solution was added into each well, then incubated for another 4 h at 37 °C. After that, the medium in each well was removed completely and 200 μL of DMSO was added and incubated for another 30 min. A microplate reader was used to measure the absorbance of each well with the wavelength fixed at 490 nm. GraphPad Prism software was used to calculate the half maximal inhibitory concentrations (IC50) values according to MTT results. Intracellular Distribution of DOX by CLSM. 4T1 cells or HT1080 cells (5 × 104) in 500 μL of DMEM with FBS (10%) were cultured in each well of a 4-well glass-bottom Petri dish and incubated overnight at 37 °C under humidified 5% CO2 atmosphere. Then, DMEM was aspirated and an equal volume of fresh medium containing free DOX·HCl, P1, P2, or P3 at a DOX concentration equal to 10 μg/mL were added, respectively. Followed by 24 h incubation, ice-cold PBS was added to wash the cells for three times and then 4′,6-diamidino-2-phenylindole (DAPI) was added to each well to stain the cell nucleus before imaging. Then, the redundant DAPI was removed completely by washing three times by ice-cold PBS and the images were obtained under CLSM. D

DOI: 10.1021/acs.biomac.6b00997 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules Penetration and Binding Ability in Multicellular Tumor Spheroids (MCTs). 4T1 or HT1080 MCTs were established according to the previous reported method.40 The MCTs with the diameter of around 100 μm were picked out and incubated with free DOX·HCl, P1, P2, and P3 micelles at a DOX concentration equal to 20 μg/mL for 48 h under humidified 5% CO2 atmosphere at 37 °C, respectively. Then MCTs were washed carefully with cold PBS and observed under CLSM. A total of 10 MCTs of each formulation were chosen to quantify the fluorescence intensity of DOX by image J software and the final results were performed as mean and standard deviation. Statistical Analysis. All the quantitative data were presented as mean ± standard deviation (SD). P-value calculated by Student’s t-test lower than 0.05 was considered statistically significant.

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RESULTS AND DISCUSSION Polymer Synthesis. MMP-2-responsive peptide-linked block copolymer P3 was synthesized via a multistep reaction (Scheme 2). Peptide, alkynyl-GPLGVRGDG, containing alkynyl groups, was designed containing MMP-responsive PLGVR moiety, which has been demonstrated to be cleaved at the site of the amido bond between G and V.41 Click reaction between terminal azide group of PEG-N3 and alkynylGPLGVRGDG was performed. The complete disappearance of azide group was confirmed by FT-IR spectra indicating the efficient attachment of peptide onto PEG polymers (Figure S1). The 1H NMR analysis also confirmed the quantitative transformation of the terminal groups by comparing the signals of methyl groups on peptide (b) and methylene peaks of PEG (a) (Figure S2B). The final peptide-modified polymer, PEGGPLGVRGDG-NH2, with amino groups as the terminals, was further used as the initiator to initiate the polymerization of BLA-NCA. After partial hydrolysis of PBLA segments, the final peptide-linked block copolymer P3 was obtained. Hydrolysis degree of the PBLA segments can be controlled by the added amount of NaOH solution and confirmed through 1H NMR analysis (Figure 1A).39 Approximately 30% hydrolysis fraction of PBLA segments of PEG-GPLGVRGDG-PBLA was selected as the typical block copolymer for the followed investigation. After complete hydrolysis and protection of carboxyl groups by chlorotrimethylsilane, GPC trace showed a unimodal curve without obvious shoulder in the position of PEG, indicating high-efficiency initiation and chain propagation of polymerization in a controlled manner (Figure 1B). To highlight the function of each segment of P3 for DOX delivery, we also synthesized the block copolymers containing comparable PAsp ratios without peptide linkage (P1) or with shorter GPLGVRG linkage (P2) as the controls. Moreover, no hydrolysis (P4) and complete hydrolysis (P5) of the block copolymer, PEG−PBLA, were also used as the controls. All the polymers were summarized in Table 1. Self-Assembly and MMP-Triggered DePEGylation of P3. Next, P3 block copolymer was self-assembled into micelles and put into MMP-2 cleavage of PEG shells. The critical micelle concentration (CMC) of P3 in aqueous solution was first determined to be 9.3 mg/L by using pyrene as the probe (Figure S3). Moreover, the MMP-2-responsive property of the block copolymer was evaluated. As shown in Figure 1B, after a 4 h reaction, approximately 50% PEG was detached in the presence of 1 μg/mL MMP-2. Then, the cleavage rate decreased significantly with 70% PEG detachment after 16 h cleavage. This result suggested that PEG surface on the micelles can be cleaved efficiently by MMP-2. Notably, the peptide sequence of PLGVR has been verified to be cleaved by many

Figure 1. (A) 1H NMR spectrum of the block copolymer, PEGGPLGVRGDG-P(BLA-co-Asp) (P3) in DMSO-d6. (B) DMF GPC traces of P3 (left) and quantified PEG release (right) after treatment with 1 μg/mL MMP-2 at pH 7.4.

kinds of MMP enzymes at the site between G and V.28,41 The residual VRGDG was speculated to be retained on the surface of the micelles. Drug Encapsulation and Release. DOX·HCl molecules were encapsulated into P3 micelles through two interactions which were expected to synergistically promote the encapsulation. At pH 7.4, negatively charged PAsp segments could interact with positively charged amino groups on DOX·HCl via electrostatic interactions. Simultaneously, PBLA segments could provide a hydrophobic environment and π−π interaction to increase the encapsulation of DOX·HCl molecules. Initially, we tried to use P4 micelles to encapsulate DOX·HCl, and the final results showed significantly low DLE (1.8%). Presumably, the hydrophobic cores of the micelles were unfavorable for the hydrophilic DOX·HCl encapsulation. Next, the weight ratios of DOX·HCl/P3 in the range from 1/20 to 1/3 were used for DOX encapsulation via facile nanoprecipitation from DMSO to phosphate buffer saline (PBS) buffer (pH 7.4). When DOX· HCl/P3 was lower than 1:10, DLEs were higher than 80%, revealing the synergistic interactions significantly promoted the DOX encapsulation (Tables 1 and S1). The drug loading efficiency decreased significantly with DOX·HCl/P3 higher than 1:10. We finally selected the DOX·HCl/P3 weight ratio of 1:10 with DLE of 83.6% as the typical system to perform the following investigation. Notably, for P1 and P2 micelles, the DLEs were also similar to that of P3 micelles (Table 1). Size characterization of DOX-loaded P3 micelles by DLS indicated number-average size of 43.5 nm with relatively narrow size distribution. TEM images showed relatively uniform spherical morphology (Figure 2). The stability of the DOX-loaded micelles in various conditions was further investigated. After incubation for 3 days in aqueous solution with 150 mM NaCl, DMEM containing 10% FBS, or a 100-fold dilution, the size and size distribution of the DOX-loaded P3 micelles almost maintained constant without significant variations, which E

DOI: 10.1021/acs.biomac.6b00997 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

due to the fact that protonation of carboxyl groups of PAsp component reduced the interaction between the micellar cores and DOX molecules. Moreover, we further used MMP-2 and esterase enzymes as well as pH values to simulate the MMPoverexpressed tumor extracellular environment and the endo/ lysosomal intracellular conditions, respectively (Figure 3B). In the presence of esterase (0.5 mg/mL) or MMP-2 (1 μg/mL) at pH 7.4, the drug release rates were relatively slow exhibiting approximately 30% release within 24 h. Surprisingly, in the presence of esterase (0.5 mg/mL) at pH 5.0, almost 80% DOX was released from the micelles within 8 h presumably due to hydrolysis of benzyl groups by esterase and reduced electrostatic interactions at pH 5.0. Given that abundant hydrolases exist in the endo/lysosomes with low pH values, it is rationalized to speculate that the encapsulated drugs can be released rapidly and specifically inside cells. Cellular Uptake and Cytotoxicity. Cellular uptake of DOX-loaded micelles was further investigated against HT1080 cells which have been demonstrated to express high concentration of several MMP enzymes including MMP-2 (Figure S5) and MMP-7 which can all cleave PLGVR efficiently.28,42 Various DOX formulations were coincubated with HT1080 cells at the DOX-equivalent concentration of 10 μg/mL. Flow cytometry was used to analyze the cellular uptake by the fluorescence intensity of DOX (Figure 4A). DOX·HCl molecules showed high cellular internalization. The P1 delivery system lacking peptide linkage showed much lower cellular uptake efficiency compared with free DOX. For GPLGVRGlinked P2 micelles, dePEGylation occurred in the presence of MMP-2, which reduced the PEG hindrance and increased the cellular internalization as compared with P1 micelles. Notably, cellular uptake efficiency of P2 micelles was still lower than free DOX. To our surprise, for GPLGVRGDG-linked P3 micelles, approximately 4-fold increase of the cellular internalization amount was observed as compared with free DOX determined by fluorescence intensity. The results confirmed that residual RGD on the surface of the nanoparticles in the presence of MMP-2 mediated the cellular internalization significantly. 4T1 cell lines were considered to express much lower concentration of MMP-2 enzyme which was used as the negative control (Figure S5). In 4T1 cells, P1, P2, and P3 showed no significant differences of cellular uptake amount, which was significantly lower than free DOX (Figure S7). After cellular internalization, intracellular distribution of the micelles was further investigated by CLSM with nucleus stained by DAPI (Figure 4B). After incubating HT1080 cells with various DOX formulations for 24 h, P3 micelles showed remarkably strong red fluorescence intensity in the cell nucleus as compared with the other polymeric micelles (P1 and P2) and free DOX, which was consistent with the results obtained from cytometry flow analysis. Notably, the internalized DOX molecules were predominantly distributed in the cell nucleus indicating that the encapsulated drugs could be released inside cells effectively and entered the nucleus efficiently. However, for 4T1 cells, free DOX showed highest fluorescence intensity, while P1, P2, and P3 block copolymer micelles showed similar fluorescence intensities, which were much lower than free DOX (Figure S7). We further assessed the cytotoxicity of the DOX-loaded micelles against HT1080 cells (Figure 4C). The three blank micelles without DOX were first investigated, which showed negligible toxicity toward cells (Figure S6). With the DOXequivalent concentrations increasing, the cytotoxicity of all DOX formulations increased dramatically. Notably, at DOX-

Figure 2. (A) Size distribution and (B) morphology of DOX-loaded P3 nanoparticles analyzed by DLS and TEM, respectively. The DOX encapsulation was performed at the P3/DOX·HCl weight ratio of 10:1.

suggested high stability of the DOX-loaded micelles in the physiological condition (Figure S4). Notably, for polymer P5, although the DLE was very high for encapsulation of DOX·HCl (Table 1), the formed nanoparticles were unstable for long incubation time in salty solution. Zeta-potential measurements showed the DOX-loaded micelles were negatively charged (Table S1). We next evaluated the drug release profiles of the DOXloaded micelles. As shown in Figure 3A, at pH 7.4 in PBS buffer, less than 10% DOX was released within 24 h, revealing high stability of the micelles, which is favorable to minimize the adverse side effects of DOX in the delivery journey. When acidity of pH 5.0 was applied, approximately 40% DOX was released after 8 h indicating higher drug release rate presumably

Figure 3. Drug release profiles of DOX-loaded P3 micelles in various conditions, (A) pH 5.0 or pH 7.4 and (B) in the presence of varying enzymes (MMP-2 and esterase) at pH 7.4 or 5.0 at the initial DOX· HCl concentration of 0.1 mg/mL. F

DOI: 10.1021/acs.biomac.6b00997 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

cellular uptake, and drug release.43−45 For DOX-loaded micelles, we can determine the binding and diffusion clearly through measuring the relative fluorescence intensity and distribution inside MCTs. As shown in Figure 5, the equatorial

Figure 5. (A) Typical images of equatorial cross-section slices of HT1080 MCTs after incubation with various DOX formulations at DOX-equivalent concentration of 20 μg/mL (a: free DOX, b: P2 micelles, c: P1 micelles, d: P3 micelles). The inset images are HT1080 MCTs recorded by using optical microscopy. The scale bars represent 50 μm. (B) Quantified average red fluorescence intensity of the images after subtracting the background intensity (a: free DOX, b: P2 micelles, c: P1 micelles, d: P3 micelles). The data are expressed as mean ± sd (n = 10). **P < 0.01, ***P < 0.005 (t-test).

Figure 4. (A) Flow cytometry analysis of HT1080 cells after incubation with various DOX formulations at various DOX-equivalent concentrations (a: free DOX, b: P2 micelles, c: P1 micelles, d: P3 micelles, e: blank control). (B) CLSM observation of HT1080 cells incubated with various DOX formulations at various DOX-equivalent concentrations (a: free DOX, b: P1 micelles, c: P2 micelles, d: P3 micelles). The cell nuclei are stained by DAPI (blue) and DOX is red. The scale bars represent 10 μm. (C) Cytotoxicity of various DOX formulations against HT1080 cells at the DOX-equivalent concentration of 10 μg/mL (a: free DOX, b: P1 micelles, c: P2 micelles, d: P3 micelles). The data are expressed as mean ± sd (n = 3). *P < 0.05 (ttest).

cross-section slices of HT1080 MCTs showed P3 micelles could bind efficiently onto the MCTs and penetrate deeply exhibiting strong fluorescence intensity in the center. In comparison, free DOX molecules mainly distributed in the peripheral edge of MCTs. P1 and P2 micelles showed significantly weak MCTs binding. On the other hand, we also tested the performance of the DOX-loaded micelles incubated in 4T1MCTs, the micelles (P1, P2, and P3) showed similar MCTs binding and penetration efficiency. No significant differences of fluorescence intensity inside MCTs could be observed (Figure S8). The results verified that DOX-loaded P3 micelles showed efficient binding and penetration efficiency of MCTs overexpressing MMP enzymes after MMP-triggered dePEGylation and exposure of RGD ligands.

equivalent concentration of 5 and 10 μg/mL, P3 micelles showed significantly higher cytotoxicity compared with free DOX. The cytotoxicity results can be rationalized by highefficiency of cellular uptake and triggered drug release of P3 micelles. Half maximal inhibitory concentration (IC50) values of DOX-loaded P3 micelles were determined to be 0.38 μg/mL compared with 0.66 μg/mL of free DOX (Table 1). In contrast, toward 4T1 cells, P1, P2, and P3 micelles showed similar cytotoxicity at various DOX-equivalent concentrations without significant differences of IC50 values (Table 1 and Figure S7). Penetration and Distribution in MCTs. To investigate the tissue binding and penetration capability of the DOXloaded micelles, we established HT1080 and 4T1MCTs. Threedimension in vitro tumor model of MCTs was recognized as an important tool to investigate intratumoral drug delivery by nanocarriers, including binding to tumor, extracellular diffusion,

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CONCLUSIONS In summary, we presented a modular design strategy of block copolymer micelles for DOX delivery. Block copolymer P3 consisting of PEG, GPLGVRGDG linkage, and P(BLA-co-Asp), was finely designed and facilely synthesized to address a cascade physiological barriers for efficient DOX delivery. MMP-2triggered dePEGylation to detach PEG-GPLG could overcome PEG dilemma in the extracellular environment and simultaneously residual RGD moieties that retained on the nanoparticle surfaces mediated cellular uptake. P(BLA-co-Asp) block G

DOI: 10.1021/acs.biomac.6b00997 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

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containing benzyl and carboxyl groups not only enhanced DOX encapsulation efficiency and stability of the micelles but also favored acid and esterase-triggered rapid drug release. DOXloaded P3 micelles showed higher cellular uptake and cytotoxicity than free DOX toward MMP-2-overexpressed HT1080 cells. Taking into account better performance of P3 micelles in MCTs binding and penetration, P3 micelles are expected to be an efficient in vivo DOX delivery system and corresponding evaluation is underway. Moreover, this modular anticancer drug delivery system design strategy with welldefined structures represents an efficient construction method which can be easily extended to the design of other anticancer drug delivery systems.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b00997. FT-IR and 1NMR characterization; CMC and micelles stability measurements; MMP-2 level; cytotoxicity of the polymers; cellular uptake against 4T1 cells; penetration in 4T1MCTs (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

These authors contributed equally (W.K. and J. L.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support from National Natural Science Foundation of China (NNSFC) Project (51273188), A Foundation for the Author of National Excellent Doctoral Dissertation of P.R. China (FANEDD; 201224), and the Fundamental Research Funds for the Central Universities (WK3450000002, WK2060200012) is gratefully acknowledged.

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REFERENCES

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DOI: 10.1021/acs.biomac.6b00997 Biomacromolecules XXXX, XXX, XXX−XXX