Redox-Responsive Core-Cross-Linked Block Copolymer Micelles for

Jan 22, 2018 - Significant reduction in IC50 was observed by DOX-loaded CCMs toward drug-resistant 231R cancer cell lines, which was further improved ...
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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 5318−5330

Redox-Responsive Core-Cross-Linked Block Copolymer Micelles for Overcoming Multidrug Resistance in Cancer Cells Chiranjit Maiti,† Sheetal Parida,‡ Shibayan Kayal,† Saikat Maiti,† Mahitosh Mandal,‡ and Dibakar Dhara*,† †

Department of Chemistry and ‡School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721302, India

ACS Appl. Mater. Interfaces 2018.10:5318-5330. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/21/19. For personal use only.

S Supporting Information *

ABSTRACT: Success of chemotherapy as a treatment for cancer has been often inhibited by multidrug resistance (MDR) of the cancer cells. There is a clear need to generate strategies to overcome this resistance. In this work, we have developed redox-responsive and core-cross-linked micellar nanocarriers using poly(ethylene glycol)-block-poly(2(methacryloyloxy)ethyl 5-(1,2-dithiolan-3-yl)pentanoate) diblock copolymers (PEG-b-PLAHEMA) with tunable swelling properties for the delivery of drugs toward drug-sensitive MDA-MB-231 and drug-resistant MDA-MB-231 (231R) cancer cells. PEG-b-PLAHEMA containing varying number of 2-(methacryloyloxy)ethyl 5-(1,2-dithiolan-3-yl)pentanoate (LAHEMA) units were synthesized by employing the reversible addition-fragmentation chain transfer polymerization technique. The block copolymer self-assembly, cross-linking induced by reduction, and de-cross-linking triggered time-dependent controlled swelling of micelles were studied using dynamic light scattering, fluorescence spectroscopy, and transmission electron microscopy. In vitro cytotoxicity, cellular uptake efficiency, and glutathione-responsive anticancer activity of doxorubicin (DOX) encapsulated in core-cross-linked block copolymer micelles (CCMs) toward both drug-sensitive and drug-resistant cancer cell lines were evaluated. Significant reduction in IC50 was observed by DOX-loaded CCMs toward drug-resistant 231R cancer cell lines, which was further improved by coencapsulating DOX and verapamil (a P-glycoprotein inhibitor) in CCMs. Thus, these reduction-sensitive biocompatible CCMs with tunable swelling property are very promising in overcoming MDR in cancer cells. KEYWORDS: RAFT polymerization, nanoparticles, self-assembly, stimuli-responsive polymers, biocompatible polymers, drug delivery, apoptosis, glutathione, doxorubicin, verapamil

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INTRODUCTION Amphiphilic block copolymer nanoassemblies in aqueous media have been extensively explored for potential delivery of anticancer drugs because of their ability of noncovalently encapsulating water-insoluble hydrophobic drugs.1−3 These nanoassemblies are particularly useful because of their great stability and low critical aggregation concentrations (CACs) in comparison to those of their small molecular counterparts.4,5 The most commonly used polymeric nanoassemblies as drug delivery vehicles are micelles6−8 and vesicles (or polymerosomes).9,10 In particular, nanoassemblies containing a poly(ethylene glycol) (PEG) hydrophilic shell provide some distinctive advantages, such as excellent biocompatibility, decreased side effects, and increased drug availability that results from prolonged circulation time in the bloodstream, which helps encapsulated drug molecules to selectively accumulate in tumor tissues due to the so-called enhanced permeability and retention effect.11−13 However, it should be noted that the clinical success of above-described polymeric nanoassemblies is limited by slow and inefficient drug release at the pathological site.14,15 For this purpose, it is important that © 2018 American Chemical Society

these self-assembled polymeric nanoassemblies should be designed in such a manner that these are sufficiently stable under extracellular environments as well as possess the capability of releasing their contents on reaching their target site in response to intracellular stimuli, such as pH,16 temperature,17 redox agent,18 and enzyme.19 This will enable these polymeric nanoassemblies to release the drug inside the tumor cells in fast and efficient way.20−22 Among these stimuliresponsive nanoassemblies, reduction-sensitive micelles have aroused great interest among researchers in the past several years because of their fascinating potential to accomplish rapid and efficient intracellular drug delivery in cancer cells.23−25 Thiol−disulfide exchange reactions that are readily reversible are known to play a vital role in maintaining the proper biological functions, including enzymatic activity and redox cycles, of living cells.26,27 Glutathione (GSH), a tripeptide produced in mammalian cells by the reduction of nicotinamide Received: November 30, 2017 Accepted: January 22, 2018 Published: January 22, 2018 5318

DOI: 10.1021/acsami.7b18245 ACS Appl. Mater. Interfaces 2018, 10, 5318−5330

Research Article

ACS Applied Materials & Interfaces

diblock copolymers (PEG-b-PLAHEMA) to control the release rate of doxorubicin and thereby its accumulation in the cell nuclear region. The in vitro cytotoxicity, cellular uptake efficiency, and intracellular drug release behavior in drugsensitive and drug-resistant cancer cells after treatment of CCMs loaded with DOX (and verapamil, a chemosensitizer) were extensively evaluated, and the most promising composition of the block copolymers that was capable of overcoming multidrug resistance (MDR) in breast cancer cells was identified.

adenine dinucleotide phosphate and GSH reductase, is found to be the most abundant low-molecular-weight biological thiolsource. Disulfide bonds in a molecule can be reversibly cleaved to form the respective thiols in the presence of GSH whose concentration in the cytosol and cell nucleus of tumor tissues is around 10 mM, which is about 10 times higher compared to that in normal tissues and about 100 times higher than the concentration found in blood plasma (∼10 μM).28−30 The reduction-sensitive nanoassemblies are susceptible to cleavage under a reductive environment involving thiol− disulfide exchange reactions as compared with their reduction-insensitive counterparts.31,32 Additionally, non-cross-linked polymeric micellar nanostructures have a major disadvantage owing to their tendency to disassemble on dilution below a certain concentration, resulting in instability and uncontrolled drug release when injected into the body.33,34 Therefore, design and implementation of versatile strategies where the micellar cores can undergo cross-linking without the involvement of any external cross-linker should enable selective tailoring of the nanomaterials for a number of applications in various fields related to delivery of drugs and other biomolecules. Multidrug resistance (MDR) is a major obstacle to the success factor for the treatment of patients with malignancies.35,36 Although MDR in cancer cells may get generated through many molecular mechanisms, P-glycoprotein (P-gp) is an important transporter and the best known protein involved in MDR being overexpressed in the cytosol and cell surface of MDR tumor, which results in adenosine 5′-triphosphatedependent effluxing of different anticancer drugs like doxorubicin (DOX) and paclitaxel into the extracellular space.37,38 The MDR effect can be overcome by increasing the concentration of the drug or treating various classes of chemosensitizers like calcium channel blockers and inhibitors, which block the drug efflux facilitated by membrane transporter P-gp. However, this may result in strong side effects and unacceptable toxicity when used at the required concentrations.39,40 Drug carriers based on polymer nanoassemblies have shown the capability to enhancing the efficacy of anticancer drugs by targeting tumors, which is obtained by their prolonged and systematic circulation time that increases their accumulation in the diseased area.41,42 Loading a combination of a chemosensitizer and a chemotherapy drug in polymeric nanoassemblies may also provide good opportunities for overcoming MDR.43−45 Moreover, an effective control of the drug release rate has been reported to be extremely important for the treatment of different stages of leukemia because it reduces the required dose, thus reducing the side effects.46 An appropriate drug delivery system (DDS) with a specific release rate and release period of the drug may enhance the therapeutic factor and result in optimal clinical outcome in cancer therapy. In this work, we have employed micellar nanocarriers from poly(ethylene glycol)-block-poly(2-(methacryloyloxy)ethyl 5(1,2-dithiolan-3-yl)pentanoate) diblock copolymers (PEG-bPLAHEMA) containing varying number of units of 2(methacryloyloxy)ethyl 5-(1,2-dithiolan-3-yl)pentanoate (LAHEMA) as drug delivery systems (DDSs) for delivering doxorubicin (DOX), a typical chemotherapeutic agent, into drug-resistant cancer cells. The block copolymer micelles were cross-linked, and the swelling property of these core-crosslinked block copolymer micelles (CCMs) was tuned under an elevated reductive environment that is often associated with cancer cells by varying the number of units of LAHEMA in the

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EXPERIMENTAL SECTION

Materials. DL-α-Lipoic acid (LA), 2-hydroxyethyl methacrylate (HEMA), N,N′-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), L-glutathione (GSH) reduced, and doxorubicin hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Poly(ethylene glycol) monomethyl ether (PEG, molar mass 5000 g/mol; Sigma-Aldrich) was dried by azeotropic distillation from anhydrous toluene. 2,2′Azobisisobutyronitrile (AIBN, Sigma-Aldrich) was used after crystallizing twice from methanol. Synthesis of S-1-dodecyl-S′-(α,α′dimethyl-α″-acetic acid) trithiocarbonate (DDMAT) was done according to an earlier report.47,48 Nile red (from Exciton, Dayton, OH) stock solution was prepared in methanol. 4′,6-Diamidino-2phenylindole dihydrochloride (DAPI) and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Fetal bovine serum and Dulbecco’s minimum essential medium (DMEM) were procured from Gibco, and Sigma-Aldrich, respectively. ApopTag, in situ apoptosis detection kit, was purchased from Promega, Madison, WI. Milli-Q water was used for all experiments. Cell Lines. Human breast cancer cell line MDA-MB-231 was procured from the National Center for Cell Science (Pune, India), maintained in a 5% CO2 atmosphere, at 37 °C and 95% humidity in DMEM (Gibco-BRL, Rockville, MD) media supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL). DAPI staining was utilized to detect the mycoplasma status of all cell lines. The multidrug-resistant cell line (231R) was established by stepwise and continuous exposure of the parental MDA-MB-231 cells to an increasing concentration of doxorubicin hydrochloride and 5FU for 6−8 months, which were routinely cultured and maintained in their respective media. Synthesis of Block Copolymers. Synthesis of PEG Macro-Chain Transfer Agent (CTA) (Scheme 1). PEG macro-CTA was synthesized by a coupling reaction using poly(ethylene glycol) monomethyl ether (PEG, molecular weight (MW) 5000 g/mol). Subsequent to azeotropic distillation with toluene at 70 °C under high vacuum, PEG was reacted with DDMAT in dry DCM as a solvent and in the presence of DCC and catalytic amount of DMAP. PEG (3 g, 0.6 mmol), DDMAT (0.26 g, 0.7 mmol), and DMAP (0.015 g, 0.12 mmol) were taken in a 100 mL round-bottomed flask (100 mL), dry dichloromethane (DCM) (35 mL) was added as a solvent, and the reaction mixture was kept in an ice-water bath with stirring for 30 min under constant bubbling of N2 gas throughout the entire reaction. To this solution, DCC (0.15 g, 0.73 mmol) in 5 mL dry DCM was added dropwise over 1 h with vigorous stirring. The reaction was allowed to proceed in an ice-cold environment for an additional 1 h and then was stirred at room temperature for overnight. The resulting insoluble N,N′-dicyclohexylurea was filtered using a G4 silica crucible, and the filtrate was concentrated to yield a yellowish waxy solid. The product was further purified by dialyzing against methanol using cellulose membranes (cutoff value of MW ∼ 3.5 kDa). Dialysis was continued for 1 day with the outside solvent being changed thrice from the dialysis container, and finally the required solution was dried under high vacuum. The purified product was analyzed by NMR spectroscopy (see Figure S1, Supporting Information) and gel permeation chromatography (GPC) (Figure 2). Synthesis of 2-(Methacryloyloxy)ethyl 5-(1,2-dithiolan-3-yl)pentanoate (LAHEMA) (Scheme 1). Monomer LAHEMA was 5319

DOI: 10.1021/acsami.7b18245 ACS Appl. Mater. Interfaces 2018, 10, 5318−5330

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Figure 1. 1H NMR spectra (in CDCl3) of PEG-b-PLAHEMA52 containing 52 units of LAHEMA. solvent changed at every 4 h interval. The product was recovered by evaporating CHCl3 using a rotary evaporator and drying under high vacuum. The final purified copolymers were analyzed by 1H NMR spectroscopy and GPC (Figures 1 and 2).

synthesized by the carbodiimide coupling reaction of DL-α-lipoic acid (LA) with 2-hydroxyethyl methacrylate (HEMA) in the presence of DCC and catalytic amount of DMAP in the dry DCM solvent following the procedure reported earlier with some modification.49,50 HEMA (0.35 g, 2.6 mmol) and DMAP (0.029 g, 0.24 mmol) were dissolved in dry DCM (20 mL) in an oven-dried 100 mL doublenecked round-bottomed flask containing a magnetic stir bar. The solution was kept in an ice-water bath with constant bubbling of N2 gas through the entire reaction medium and stirred for 30 min. To this solution, DCC (0.49 g, 0.24 mmol) in 5 mL DCM followed by lipoic acid (0.5 g, 2.4 mmol) in 5 mL DCM with a gap of 30 min were added dropwise with constant stirring. The reaction mixture was allowed to proceed in an ice-cold environment for an additional 1 h and then was stirred at room temperature for overnight. The resulting insoluble N,N′-dicyclohexylurea was filtered using a G4 silica crucible, and the filtrate was concentrated to yield a yellowish liquid. Finally, the product was purified using column chromatography on silica using 10% ethyl acetate in hexane as the eluent to yield a bright yellow liquid and stored at low temperature (−20 °C) to maintain the stability of monomer LAHEMA for several months. The purified product was analyzed by NMR spectroscopy (see Figures S2 and S3, Supporting Information). Synthesis of PEG-b-PLAHEMA (Scheme 1). For the synthesis of the diblock copolymer, we have polymerized monomer LAHEMA in the presence of PEG macro-CTA (MW 5350 g/mol) and AIBN as initiator in dry 1,4-dioxane at 70 °C. The reaction content were added to a septa-sealed single-necked round-bottomed flask (10 mL) with a magnetic stir bar and deoxygenated by purging N2 gas for 20 min on an ice-water bath. The polymerization reactions were carried out using three different ratios of monomer LAHEMA to PEG macro-CTA to obtain diblock copolymers (PEG-b-PLAHEMA) containing varying number of LAHEMA units, whereas the molar ratio of [macro-CTA] to [initiator] was kept constant at 3:1. The reaction was carried out for 12 h and then quenched by cooling the reaction mixture in a liquid nitrogen bath. The mixture was then diluted by appropriate amounts of CHCl3 and further dialyzed against CHCl3 using cellulose membranes (cutoff value of MW ∼ 10 kDa) for 16 h with the outside

Figure 2. GPC chromatograms of the PEG macro-CTA and the diblock copolymers (PEG-b-PLAHEMA) containing different numbers of LAHEMA units. Instrumentation and Methods. NMR Spectroscopy. Recording of 1H NMR and 13C NMR spectra was acquired in CDCl3 or dimethyl sulfoxide (DMSO)-d6 using a Bruker DPX spectrometer operating at 400/600 and 100 MHz at 25 °C, respectively, with the residual solvent signal being used as an internal standard for mode locking. Gel Permeation Chromatography (GPC). GPC (Shimadzu) was utilized to determine the molecular weight and dispersity (Đ = Mw/ Mn) of the polymers using an refractive index detector and high5320

DOI: 10.1021/acsami.7b18245 ACS Appl. Mater. Interfaces 2018, 10, 5318−5330

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ACS Applied Materials & Interfaces Scheme 1. Synthesis of Diblock Copolymer PEG-b-PLAHEMA Using the RAFT Polymerization Technique

Drug Encapsulation. Prior to drug encapsulation, doxorubicin hydrochloride (1 mg/mL) was neutralized in water with a stoichiometric amount of 10 mM sodium hydroxide. The resulting solution was lyophilized, redissolved in ethanol, and filtered using a membrane filter (pore size ∼ 0.2 μm) for removing the precipitated salt. This DOX base in ethanol medium was used as a stock solution. To load DOX, stock polymer solutions (1 mg/mL) in PB (pH 7.4, 10 mM) prepared by the solvent exchange method (described earlier), DOX (0.3 mg/mL) in ethanol, and weighted amount of GSH (maintaining GSH concentration 10 mol % with respect to the LAHEMA unit in PB) were added and stirred for 12 h under dark conditions at room temperature. The solution was then placed in a dialysis tube possessing cellulose membrane (MW cutoff value of 10 kDa) and dialyzed against 10 mol % GSH containing PB (pH 7.4) for 24 h. The buffer was replaced in every 6 h interval to remove the nonencapsulated DOX and ethanol. The quantity of DOX encapsulated in the cross-linked micelles was determined by UV−vis spectroscopy. Before choosing a particular ratio of drug and micelles during loading, we have screened the loading content and loading efficiency of DOX with varying DOX concentration at a fixed micellar (BCP26) concentration. The data is presented in Figure S5, Supporting Information. On the basis of these data, the ratio of DOX to polymer micelles was fixed at 0.3:1.0 (by wt) for further drug loading experiments. To maintain parity, the same ratio was used for drug loading for the other two nanocarriers (BCP52 and BCP83) as well. For coencapsulation of DOX and verapamil, 30 μL of DOX solution (5 mg/mL in DMSO) and 20 μL of verapamil solution (5 mg/mL in DMSO) were added in polymer solutions (1 mg/mL in 10 mM PB at pH 7.4). Then, required amount of GSH was added for cross-linking to happen. The solutions were stirred for 12 h under dark conditions at room temperature. Dialysis was performed to remove the nonencapsulated drug and DMSO. Cell Viability Assay. Cell viability was determined by the MTT assay for investigating the effect of free polymer on the growth of breast cancer cell line MDA-MB-231 and also on HaCat cells (normal cells) in a time- and dose-dependent manner. Cells in the logarithmic phase (1 × 104 cells/well) were seeded in 96-well tissue culture plates and were allowed to grow for 16 h at 5% CO2 and 37 °C. Subsequently, the cells were treated with the free polymers for 72 h so that the final effective concentrations of the polymers were varied between 0 and 2.5 mg/mL. The MTT dye reduction assay was performed at 540 nm with few modifications in the protocol used by Younes et al.52 The time-dependent curves of free DOX and DOX encapsulated in cross-linked micelles were analyzed using Prism software (GraphPad Prism 5 software). Cellular Uptake Studies. Uptake of DOX encapsulated in crosslinked micelles was qualitatively analyzed by fluorescence imaging in a time-dependent manner over a period of 6 h.

performance liquid chromatography-grade dimethylformamide (DMF) as a mobile phase, at a flow rate of 1 mL/min. The molecular weights were determined relative to PEG standards. Structural Characterization. Transmission electron microscopy (TEM) measurements were performed for structural analysis of selfassembled polymeric aggregates using a JEOL model JEM 2100 transmission electron microscope at an operating voltage of 80 kV. Dynamic light scattering (DLS) measurements were performed to determine the size and size distribution of the self-assembled polymeric aggregates using a Malvern Nano ZS instrument equipped with a temperature-controlled sample chamber using a 4 mW He−Ne laser (λ = 632.8 nm). During data recording, scattering photons were collected at a fixed detector angle of 173° in this instrumental setup to avoid effects of high-concentration measurements. The scattering intensity obtained from each sample was processed by instrumental software, and it provided the hydrodynamic diameter (Dh) and size distribution in terms of polydispersity index (PDI). The Dh values of polymeric aggregates were estimated from the intensity autocorrelation function of time-dependent fluctuation in intensity, and Dh is defined as Dh = kBT/3πηD, where kB is the Boltzmann constant, η is the viscosity of the solvent at absolute temperature, and D is the translational diffusion coefficient. Absorbance and Fluorescence Measurements. The UV−vis absorbance was measured using a Shimadzu (model number, UV2450) spectrophotometer, and steady-state fluorescence spectra were recorded using Hitachi (model no. F-7000) and Jobin Yvon-Spex Fluorolog-3 spectrofluorimeters, respectively. All of the measurements were performed at 25 °C using a quartz cuvette of 1 cm path length. Determination of Critical Aggregation Concentration (CAC). CACs of the block copolymers were determined using Nile red as a hydrophobic fluorescent probe.51 First, stock polymer solutions of 0.1 mg/mL concentration were prepared by the solvent exchange method. A weighted amount of the respective polymer in DMF was added dropwise to an appropriate volume of phosphate buffer (PB) (10 mM, pH 7.4) with constant stirring for 30 min. Then, the resulting dispersion was dialyzed against phosphate buffer using cellulose membranes (MW cutoff value of 10 kDa) for 24 h with frequent change of the outside buffer solution in every 6 h interval. After that, a methanolic stock solution of Nile red (1.83 mM) was taken in several vials and the solvent was removed by evaporation. Different amounts of polymer solutions (0.1 mg/mL) in phosphate buffer (PB) were added to each of these vials, and the final volume (1 mL) was made up with the required volume of buffer to get a series of solutions with polymer concentrations varying from 0 to 0.01 mg/mL, in which the Nile red concentration remained constant. Each solution was sonicated for 10 min and allowed to settle for overnight. Fluorescence was recorded at an excitation wavelength of 550 nm, and fluorescence intensity at the emission maxima was plotted against the polymer concentration. The inflection point in the observed plot was considered as the CAC. 5321

DOI: 10.1021/acsami.7b18245 ACS Appl. Mater. Interfaces 2018, 10, 5318−5330

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Table 1. Results from the RAFT Polymerization of LAHEMA in the Presence of PEG Macro-CTA in 1,4-Dioxane at 70 °C

a

polymer composition

polymer abbreviation

[M]/[macro-CTA]

conversiona (%)

Mn,NMRb (g/mol)

Mn,GPCc (g/mol)

Đc

PEG macro-CTA PEG-b-PLAHEMA26 PEG-b-PLAHEMA52 PEG-b-PLAHEMA83

PEG macro-CTA BCP26 BCP52 BCP83

30 60 100

68 62 53

5400 13 600 21 900 31 700

5500 12 100 23 500 35 800

1.05 1.12 1.18 1.26

Determined gravimetrically. bCalculated from 1H NMR spectroscopy. cObtained from GPC.

Cytotoxicity and Induction of Apoptosis. To investigate the timedependent effect of free DOX and DOX encapsulated in cross-linked polymer micelles on the growth of MDA-MB-231 and multidrugresistant MDA-MB-231 (231R) cells, the MTT dye reduction assay was performed as described in the above procedure. The timedependent curves of free DOX and DOX encapsulated in cross-linked micelles were analyzed using Prism software (GraphPad Prism 5 software). Induction of apoptosis and changes in morphology associated with it were studied by dUTP nick-end labeling (TUNEL) staining. Cells were fixed in 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked in 2% bovine serum albumin, and TUNEL stained as per instructions given by the manufacturers. The cells were analyzed using confocal laser scanning microscopy (Olympus FluoView FV1000, version 1.7.1.0) using an appropriate wavelength. The images were taken and digitized using FLUOVIEW 1000 imaging software (version 1.2.4.0).

self-assemble in water and form stable nanostructures above a certain concentration, known as critical aggregation concentration (CAC). Self-association and solution properties of PEGb-PLAHEMA diblock copolymers, namely, BCP26, BCP52, and BCP83, were studied using DLS, TEM, and hydrophobic fluorescent probe encapsulation studies. First, block copolymer nanostructures were prepared using the solvent exchange method. Briefly, a polymer solubilized in minimum volume of DMF was added dropwise to the aqueous phosphate buffer (PB) solution of pH 7.4 at 25 °C under stirring, followed by dialysis for 24 h to remove unwanted organic solvent molecules. DLS measurements revealed that the block copolymers formed colloidal nanoassemblies with sizes ranging from 75 to 170 nm and polydispersities of 0.10−0.20 (Figure 3). TEM micro-

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RESULTS AND DISCUSSION Synthesis of the Diblock Copolymers. In the present work, well-defined block copolymers were synthesized by the controlled radical polymerization technique, namely, reversible addition-fragmentation chain transfer (RAFT), using methacrylate monomer LAHEMA and long-chain poly(ethylene glycol)-based macro-CTA, as shown in Scheme 1. First, PEG macro-CTA was prepared by the carbodiimide coupling reaction with DDMAT, a chain transfer agent (CTA). DDMAT is a versatile CTA for various acrylate and methacrylate monomers, which have been previously reported to prepare a series of well-defined diblock copolymers.53,54 End group analysis by 1H NMR spectroscopy of the prepared PEG macro-CTA confirms very high conversion (≥90%) and number-average molecular weight (Mn) of 5500 g/mol with dispersity (Đ) = 1.05. In the next step, as-synthesized PEG macro-CTA was utilized for the preparation of block copolymers with varying number of LAHEMA repeat units by carrying out the polymerization reaction at varying monomer to macro-CTA ratios. The number-average molecular weight and composition of the synthesized block copolymers were determined by 1H NMR spectroscopy from the relative intensities of the protons at 3.17 ppm in the poly(LAHEMA) block and methylene protons adjacent to the ester oxygen of the PEG block with chemical shift value at 3.65 ppm. 1H NMR spectra of one of the representative block copolymers PEG-bPLAHEMA52 is shown in Figure 1. The number in the subscript next to PLAHEMA represents the number of LAHEMA units in the block copolymers as quantified from 1 H NMR spectroscopy. Gel permeation chromatography (GPC) analysis revealed monomodal distributions for all of the block copolymers with narrow dispersity (Đ), ensuring good control over polymerization (Figure 2). Detailed polymerization conditions and characterization results are summarized in Table 1. Micelle Formation from PEG-b-PLAHEMA Diblock Copolymers. Diblock copolymers containing hydrophilic and hydrophobic blocks in the same chain are expected to

Figure 3. Size distribution profiles obtained from dynamic light scattering measurements of PEG-b-PLAHEMA copolymers containing different units of LAHEMA.

graphs revealed the formation of near-spherical aggregates of sizes that are in close agreement with those obtained from DLS measurements, suggesting the formation of micelle-like aggregates (Figure 4). In the case of BCP83, having highest hydrophobic content in the polymer backbone, some intermicellar association was also observed (Figure 4c). From Figures 3 and 4, it can be inferred that the size of the micelles increased with the number of hydrophobic units in the block copolymers that formed the core of the micelles. For example, the sizes obtained from DLS studies (Figure 3) were 76, 106, and 167 nm for BCP26, BCP52, and BCP83 respectively, whereas the sizes from TEM analysis (Figure 4) for the same set of copolymers were approximately 65, 100, and 155 nm, respectively. These data show that the sizes obtained from DLS and TEM match fairly well. Additionally, it was observed from TEM images (Figure 4) that on increasing the hydrophobic PLAHEMA content the core of the micelles became increasingly dense, as shown by darker images in TEM, indicating an increase in the compactness of the micellar core. 5322

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Figure 4. TEM images of diblock copolymers (PEG-b-PLAHEMA) containing different units of LAHEMA, namely, (a) BCP26, (b) BCP52, and (c) BCP83.

respectively, which are comparable to the CMC values of similar copolymers described previously.55,56 Cross-Linking and De-Cross-Linking of PEG-b-PLAHEMA Micelles. PEG-b-PLAHEMA block copolymer micelles were conveniently cross-linked by ring-opening of the 1,2dithiolane moiety based on thiol−disulfide exchange in PB (pH 7.4, 10 mM) by introducing 10 μM GSH, the concentration of GSH generally found in the blood plasma and extracellular environment.57,58 Under the catalysis of GSH, few 1,2dithiolane rings present in the LAHEMA unit in the polymer opened to yield dihydrolipoyl (the reduced form of lipoyl) groups. Subsequently, the exchange reaction between the generated dihydrolipoyl groups and the disulfide bonds of other 1,2-dithiolane rings present in the PLAHEMA block resulted in the formation of linear disulfide bonds between the different lipoyl groups in the hydrophobic core of the micelles. DLS measurements revealed that the size of the micelle decreased in this reaction compared to that of their non-cross-linked counterparts by 10−20 nm, indicating the existence of crosslinking. These core-cross-linked micelles (CCMs) displayed very low polydispersity index of 0.03−0.06 (Figure 6a, Table 2). TEM micrographs also demonstrated that the CCMs had a more compact spherical morphology compared to that of their non-cross-linked counterparts and the size distribution was close to that determined by DLS (Figure 6c, Table 2). Owing to cross-linking, the polymer micelles (after crosslinking) should retain their structural integrity even on manyfold dilution, unlike non-cross-linked micelles.59 We have studied the structural stability of CCMs by DLS against extensive dilution. Notably, CCMs upon dilution below the critical micellar concentrations of the corresponding block copolymers showed a slight increase in micelle size and maintained a low PDI. Figure 6a shows the data for core-crosslinked BCP52 micelles (BCP52CCM). In contrast, the parent non-cross-linked micelles under similar dilution dissociated to form unimers with the intensity-average hydrodynamic diameter (Dh) of 5.8 nm (Figure 6a). This observation further confirmed the cross-linking of the micellar core and formation of the CCMs. The reduction sensitivity of the CCMs was investigated by monitoring the time-dependent change in micelle sizes in response to 10 mM GSH in PB buffer (pH 7.4, 10 mM) (Figures 6b, S4a,b and Table 2). This concentration of GSH is found in the cytosol and cell nucleus of tumor tissues at 37 °C.26−28 The results from DLS showed that excess GSH caused a significant increase in size along with an increase in PDI values for all of the three CCMs, indicating the occurrence of de-cross-linking-induced micellar swelling and concomitant

This increased compactness could be due to increased hydrophobic interactions between the PLAHEMA blocks in the core. We have also determined the critical micelle concentration (CMC) of diblock copolymers using Nile red as a hydrophobic fluorescent probe.48,51 In Figure 5, the Nile red emission

Figure 5. Representative plot of the emission intensity of Nile red versus log of polymer concentration at the emission maxima (λex = 550 nm) for determining the critical micelle concentration (CMC) of diblock copolymer BCP52. The inset shows emission spectra of Nile red for two different polymer concentrations.

intensity (λex = 550 nm) was plotted with varying copolymer concentration from 0 to 0.01 g/L in which the Nile red concentration was fixed. In the absence of polymer, Nile red showed very weak emission because of very low aqueous solubility. On increasing the polymer concentration, the intensity of Nile red emission increased significantly in a nonlinear fashion with a simultaneous blue shift of the emission maxima (please see the inset of Figure 5) due to encapsulation of Nile red in the hydrophobic core generated by the selfassembly of the block copolymers. The intensity of Nile red emission was plotted against the polymer concentration, which resulted in an inflection point upon extrapolating the intensities to regions of low and high concentrations. The concentration corresponding to the inflection point was considered as the critical micellar concentration (CMC) of the block copolymer. In the present study, the CMCs for BCP26, BCP52, and BCP83 were determined to be 5.0, 2.0, and 0.65 mg/L, 5323

DOI: 10.1021/acsami.7b18245 ACS Appl. Mater. Interfaces 2018, 10, 5318−5330

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Figure 6. (a) Stability (hydrodynamic size and PDI) of the cross-linked (PEG-b-PLAHEMA52) micelles versus the non-cross-linked control measured by DLS. (b) Change of size distribution profiles of cross-linked BCP52 micelles with time in response to 10 mM GSH in PB (pH 7.4, 10 mM) at 37 °C. TEM image of cross-linked (PEG-b-PLAHEMA52) micelles (c) and (d) their swelling in response to 10 mM GSH up to 96 h in PB (pH 7.4, 10 mM) at 37 °C.

than that in the other two (BCP52CCM and BCP83CCM) and almost got saturated within 24 h. In the case of BCP52CCM, slow and prolonged swelling was observed up to 96 h but BCP83CCM shows very less swelling even up to 72 h of incubation. This de-cross-linking-induced swelling property of the three sets of CCMs can be attributed to the increased hydrophobicity with the increasing molecular weight of the PLAHEMA block as well as GSH permeability to the disulfide bonds present in the hydrophobic micellar core. Furthermore, the above results also suggest that PEG-b-PLAHEMA CCMs exhibit superior colloidal stability under extracellular conditions, whereas they undergo controlled swelling with a tunable rate under reductive conditions, mimicking the cytoplasm and cell nucleus of tumor tissues. Biological Studies of DOX Encapsulated in CoreCross-Linked Block Copolymer Micelles (CCMs) in Sensitive as Well as Drug-Resistant Breast Cancer Cells. To assess the safety of the three sets of CCMs for drug delivery applications, in vitro cytotoxicity of these micelles was evaluated by the MTT dye reduction assay on both normal cell lines HaCat and on breast cancer cells MDA-MB-231. All of the three CCMs showed no obvious cytotoxicity up to 2.5 mg/mL concentration even after culturing for 72 h (Figure 7), which established excellent biocompatibility of the present set

Table 2. Swelling Property of Cross-Linked Block Copolymers Micelles (CCMs) in Terms of Size Distribution Determined Using DLS in the Presence of 10 mM GSH Containing PB (pH 7.4) at 37 °Ca time of swelling (h) 0 (before cross-linking) 0 (after cross-linking) 12 24 48 72 96

BCP26CCM [Dh (PDI)]

BCP52CCM [Dh (PDI)]

BCP83CCM [Dh (PDI)]

76 60 405 499 535 711 775

106 96 142 420 686 886 955

167 151 175 283 411 599 861

(0.153) (0.036) (0.432) (0.471) (0.561) (0.536) (0.924)

(0.118) (0.041) (0.145) (0.255) (0.286) (0.462) (0.617)

(0.177) (0.055) (0.106) (0.114) (0.121) (0.177) (0.391)

a

The size data for the micelles before cross-linking are also included here for comparison.

increased hydrophilicity of the micellar core resulting from the conversion of each disulfide bond into two hydrophilic thiol groups. The TEM study of BCP52CCM and its de-crosslinking-induced swelling after 96 h also supports the observation from DLS study (Figure 6d). From the PDI value and size increase in the time-dependent DLS study (Table 2), it also appears that the rate of de-cross-linkinginduced swelling in the case of BCP26CCM was much faster 5324

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Figure 7. Cytotoxicity of the CCMs on (a) normal cell line HaCat and (b) breast cancer cell line MDA-MB-231 after 72 h of incubation.

Figure 8. Reduction-triggered release of (a) DOX and (b) verapamil from CCMs in the presence of 10 mM GSH in PB buffer (pH 7.4, 10 mM) at 37 °C. Release from BCP52CCM in the absence of 10 mM GSH is plotted as control.

Figure 9. Qualitative examination of the time-dependent cellular uptake of cross-linked micelle-encapsulated DOX at a fixed polymer concentration of 0.25 mg/mL. The BCP26, BCP52, and BCP83 series indicate data for BCP26CCM, BCP52CCM, and BCP83CCM, respectively.

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Figure 10. In vitro cytotoxicity of all of the three cross-linked block copolymer micelles encapsulating DOX: BCP26CCM (left), BCP52CCM (middle), and BCP83CCM (right) with different incubation times against MDA-MB-231 cells (top) and 231R cells (bottom).

Table 3. IC50 Value of DOX in the Formulation of All Three CCMs in Both the Sensitive and Resistant Cancer Cell Linesa IC50 (μM) polymer

incubation time (h)

free DOX

DOX-loaded BCP26CCM

DOX-loaded BCP52CCM

DOX-loaded BCP83CCM

DOX + verapamil-loaded BCP26CCM DOX + verapamil-loaded BCP52CCM DOX + verapamil-loaded BCP83CCM a

24 48 72 24 48 72 24 48 72 24 48 72 24 48 72

MDA-MB-231 3.147 1.462 0.7870 2.097 1.580 0.777 0.9585 0.3852 0.2495 2.969 2.478 1.154

± ± ± ± ± ± ± ± ± ± ± ±

0.97 0.96 0.98 0.99 0.96 0.98 0.95 0.91 0.89 0.98 0.99 0.97

multidrug-resistant 231R 14.79 ± 0.98 5.131 ± 0.96 2.3 ± 0.95 2.9 ± 0.90 1.705 ± 0.96 1.502 ± 0.94 1.088 ± 0.44 0.3690 ± 0.96 0.1722 ± 0.89 1.748 ± 0.95 1.581 ± 0.88 0.8711 ± 0.97 0.9592 0.07276 0.5871

IC50 is the concentration of DOX required to cause 50% cell death. The IC50 data for free DOX are also included here for comparison.

BCP26CCM, BCP52CCM, and BCP83CCM, respectively. We have also studied the release kinetics of encapsulated DOX and verapamil from the CCMs containing both DOX and verapamil in response to elevated reductive environment (10 mM GSH) in PB buffer (pH 7.4, 10 mM). Figure 8a,b clearly revealed de-cross-linking-induced controlled micellar swelling with a tunable drug release rate for the designed CCMs. Before finding out the efficacy of DOX-loaded CCMs, it was necessary to establish the MDR in experimental cell line 231R. For this, breast cancer cell lines MDA-MB-231 and multidrugresistant cell line 231R were treated with varying concentrations of free DOX for different incubation times. IC50 values were determined by the MTT assay, and data are found to be as follows: in sensitive breast cancer cells, 3.147 ± 0.972, 1.462 ± 0.96, and 0.7870 ± 0.975 μM for 24, 48, and 72 h, respectively and in resistant breast cancer cells, 14.79 ± 0.98, 5.131 ± 0.96, and 2.3 ± 0.95 μM for 24, 48, and 72 h, respectively. The IC50

of CCMs. We have loaded DOX, a model hydrophobic anticancer drug, into three CCMs. The loading contents (LCs) of DOX inside these CCMs were calculated using equation LC (%) =

weight of DOX into the micelle weight of micelle taken

× 100 and were found to be

16.6, 23.8, and 19.2% in the case of BCP26CCM, BCP52CCM, and BCP83CCM, respectively. It is generally accepted that the polymer micelles with a higher hydrophobic content should solubilize more hydrophobic drug.60 Lower solubility of DOX in BCP83CCM in comparison to that in BCP52CCM could be due to the low permeability of DOX inside the core of BCP83CCM. In the present study, we also aimed at overcoming multidrug resistance in breast cancer cells by coencapsulating verapamil in cross-linked polymeric micelles along with DOX. The coencapsulation study showed that the drug loading contents (LCs) of DOX were 7.5, 10.7, and 8.9%, whereas those of verapamil were 6.3, 7.8, and 8.3% in 5326

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Figure 11. Viability of 231R cells after incubation with CCMs encapsulating DOX and verapamil at different times of incubation: (a) BCP26CCM, (b) BCP52CCM, and (c) BCP83CCM.

swelling) from BCP52CCM was slow and sustained, which results in overcoming the resistance of the 231R cells. These results show the potential of these CCMs as drug delivery vehicles toward MDR cancer cells. From Table 3, we can also see that for drug-sensitive cells (MDA-MB-231) the IC50 value for 24 h has reduced significantly, at least by 30% in the case of BCP26CCM and BCP52CCM. However, the IC50 values for 48 and 72 h did not show significant decrease for BCP26CCM and BCP83CCM, whereas they were decreased by about 70% in BCP52CCM. Doxorubicin hydrochloride is widely used as a very potent chemotherapeutic drug that acts by inducing G2/M arrest in cells. The IC50 values were calculated using the MTT assay, which is based on the metabolic activity of the cells. A decrease in IC50 in 24 h demonstrates the improved efficiency of the drug formulation. Slightly higher IC50 values in 48 and 72 h do not necessarily mean that the residual cells are viable but could be due to increased metabolic activity as a result of stress induced by the treatment while in the phase of growth arrest. It could also be due to early release of the drug once DOX-loaded CCMs were taken up by the cells. However, in MDR cells, the cells do not retain the drug and hence respond poorly to the free drug itself. The use of core-cross-linked polymeric micelles improved the drug uptake and retention, thus reducing its effective dose. Verapamil, the well-known calcium channel blocker, has been proved to reverse multidrug resistance in cancer cells by directly binding P-glycoprotein. From the MTT assay, it was observed that verapamil alone had no significant toxicity to 231R cells up to 20 μM for 72 h of incubation (data not shown).64 On encapsulating verapamil, which has a similar loading efficiency to that of DOX, the required dose of DOX on 231R cells was significantly reduced (Figure 11). The effective IC50 values of CCMs encapsulating both DOX and verapamil was found to be significantly lower compared to the IC50 values for CCMs containing only DOX (Table 3). These results show that the potential of DOX-loaded CCMs as drug delivery vehicles toward MDR cancer cells could be further improved by coloading verapamil along with DOX in the CCMs. Nuclear fragmentation and induction of apoptosis on treatment of multidrug-resistant 231R cells were microscopically examined by TUNEL staining. TUNEL-positive green nuclei are indicating apoptotic cell death. On treatment of 231R cells with sublethal dose (about three-fourth of IC50) of free DOX, there was no significant apoptosis induction up to 24 h. TUNEL-positive green nuclei were observed only after 48 h of treatment, and the proportion was significant only after 72 h of treatment (Figure 12, upper row). However, on treatment with

values were found to be almost 5-fold higher for cell line 231R compared to those for the sensitive cell. This is likely due to the overexpression of P-glycoprotein, an efflux pump protein on the cell membrane of drug-resistant cell line 231R, which pumps the drug out.61 To confirm passive accumulation of these CCMs inside cancer cells, a time-dependent cellular uptake study was conducted using DOX encapsulated in CCMs in drug-resistant cell line 231R and inherent fluorescence of DOX was used as a tracer to monitor cellular accumulation qualitatively. Figure 9 shows increased accumulation of the drug in and around the cell over a period of 6 h for all of the micelles. We have further studied the potential of these CCMs encapsulating DOX and their de-cross-linking-induced controlled micellar swelling with tunable rates under reductive conditions that mimic the reductive conditions of the cytoplasm and cell nucleus in tumor tissues. Intracellular DOX release properties of all of the three CCMs were evaluated against both MDA-MB-231 and 231R cell lines in a dose- and time-dependent manner. Enhanced therapeutic efficiency, evident from reduced IC50, in both sensitive and resistant cell lines was observed in all of the three CCMs encapsulating DOX. The respective time-dependent IC50 values in each case were obtained from Figure 10 and have been shown in Table 3. A faster swelling in case of BCP26CCM may be responsible for the medium IC50 value after 24 h. The IC50 value in case of BCP83CCM seemed to be high even after incubation of 72 h for both the cell lines, whereas a slow and sustained drug release is evident from the IC50 values of BCP52CCM for both sensitive and resistant cell lines. Recent successes in the development of cancer therapy drugs have generated renewed focus on the development of new drug delivery systems for delivering drugs in a site-, dose-, and timedependent manner.62,63 As multidrug resistance (MDR) in case of cancer is quite a complex process, involving interaction between genes with their environment, appropriate selections of dose and exposure time are the two important factors toward the enhancement of efficacy and improvement of patient compliance. A careful analysis of the data presented in Table 3 provides us with some interesting inferences. The efficacy of BCP52CCM is significantly better than that of free DOX, whereas the efficacies of BCP26CCM and BCP83CCM are similar and inferior, respectively, compared to those of free DOX toward sensitive cells. However, the efficacies of all of the three DOX-loaded CCMs were significantly improved compared to those of free DOX toward resistant 231R cells. Of the three CCMs, the improvement in the IC50 value was highest for BCP52CCM. As discussed earlier, the GSH-induced release of DOX due to de-cross-linking (and subsequent 5327

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Figure 12. Representative confocal laser scanning microscopy images (20× magnification) of 231R cells treated with free DOX, DOX-loaded BCP52CCM, and DOX- and verapamil-loaded BCP52CCM for indicated time periods.

■

BCP52CCM (the most efficient of the three polymers)encapsulated DOX with a similar dose, apoptosis was evidently induced in 24 h and the proportion of apoptotic nuclei significantly increased after 48 h (Figure 12, lower row). In addition, the results were markedly improved on treating with a combination of DOX and verapamil encapsulated into BCP52CCM.

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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.7b18245. NMR spectra of macro-CTA and synthesized monomer LAHEMA and loading content and loading efficiency with varying DOX concentration and few DLS measurements (PDF)

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CONCLUSIONS

In the present work, we have successfully demonstrated the synthesis of reduction-responsive PEG containing block copolymers with varying number of LAHEMA repeat units by RAFT polymerization. These block copolymers exhibit selfassembling behavior in aqueous medium, forming micellar nanostructures, the core of which can undergo cross-linking in the presence of catalytic amount of GSH mimicking that of the GSH concentration in extracellular environments. The crosslinking imparted stability to the core-cross-linked micellar nanostructures (CCMs) against dilution, which is expected to make them stable during systemic circulation post-injection. The CCMs were found to be biocompatible, a feature that is expected due to the presence of PEG in the shells of the CCMs. Of the three CCMs, BCP52CCM was found to have improved efficacy of delivering drug into sensitive MDA-MB-231 cancer cells. Furthermore, all of these CCMs loaded with anticancer drug DOX showed improved efficacy in delivering DOX into drug-resistant 231R cancer cells due to controlled release of DOX from the CCMs induced by controlled swelling of the core that resulted from GSH-triggered de-cross-linking. The concentration of GSH used for de-cross-linking was relevant to the prevailing GSH concentration in the intracellular compartments such as the cytoplasm and the cell nucleus in leukemic tissues. The efficacy of the DOX-loaded CCMs as drug delivery vehicles toward MDR cancer cells was found to be further improved by coloading verapamil along with DOX in the CCMs. Thus, the PEG-b-PLAHEMA core-cross-linked micelles reported in this work provide an option for stable, biocompatible nanocarriers for delivering anticancer drugs to both sensitive and resistant cancer cells.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. Tel: +91-3222-282326. Fax: +91-3222-282252. ORCID

Dibakar Dhara: 0000-0003-2574-5378 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from Science and Engineering Research Board, Department of Science and Technology, Government of India (Project Ref No. EMR/2016/007040) is acknowledged. Authors also thank the Indian Institute of Technology, Kharagpur, for funding the purchase of a DLS-Zeta and a multi-detector GPC instrument through competitive research infrastructure seed grants (project codes ADA and NPA with institute approval numbers IIT/SRIC/CHY/ADA/2014-15/18 and IIT/SRIC/CHY/NPA/2014-15/81, respectively). The authors thank Aditya Parekh for his help in preparation of MDR cells. C.M. and S.K. acknowledge UGC, New Delhi, and IIT Kharagpur, respectively, for Research Fellowships.

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REFERENCES

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DOI: 10.1021/acsami.7b18245 ACS Appl. Mater. Interfaces 2018, 10, 5318−5330