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Jan 22, 2018 - School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur,. West Bengal 721302, India. •S Support...
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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18245 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 22, 2018

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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

School of Medical Science and Technology Indian Institute of Technology Kharagpur West Bengal 721302 India

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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 crosslinked micellar nano-carriers 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 towards drug-sensitive MDA-MB-231 and drugresistant 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 reversible addition-fragmentation chain transfer (RAFT) polymerization technique. The block copolymer self-assembly, cross-linking induced by reduction, and de-crosslinking 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 (GSH) responsive anticancer activity of doxorubicin (DOX) encapsulated core cross-linked block copolymer micelles (CCMs) towards both drug-sensitive and drug resistant cancer cell lines were evaluated. Significant reduction in IC50 was observed by DOX-loaded CCMs towards drug-resistant 231R cancer cell lines that was further improved by co-encapsulating DOX and Verapamil (a P-gp inhibitor) in CCMs. Thus, these reduction-sensitive biocompatible CCMs with tunable swelling property are very promising in overcoming MDR of 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 nano-assemblies in aqueous media have been extensively explored for potential delivery of anticancer drugs because of their ability to non-covalently encapsulate water-insoluble hydrophobic drugs.1-3 These nano-assemblies are particularly useful because of their great stability and low critical aggregation concentrations (CACs) in comparison to their small molecular counterparts.4-5 The most commonly used polymeric nano-assemblies as drug delivery vehicles are micelles6-8 and vesicles (or polymerosomes)9-10. In particular, nanoassemblies containing poly(ethylene glycol) (PEG) hydrophilic shell provide some distinctive advantages, such as excellent biocompatibility, decreased side effects, imcreased drug availability that results from prolonged circulation time in the bloodstream which helps encapsulated drug molecules to selectively accumulate in tumour tissues due to the so-called enhanced permeability and retention (EPR) effect.11-13 However, it should be noted that the clinical success of above described polymeric nano-assemblies are limited by slow and inefficient drug release at the pathological site.14-15 For this purpose, it is important that these self-assembled polymeric nano-assemblies 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 pH16, temperature17, redox agent18, and enzyme19 etc. This will enable these polymeric nanoassemblies to release drug inside the tumor cells in fast and efficient way.20-22 Among these stimuli responsive nano-assemblies, 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

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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 NADPH and GSH reductase, is found to be the most abundant low-molecular-weight biological thiol-source. Disulfide bonds in a molecule can be reversibly cleaved to form the respective thiols in 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 normal tissues and about 100 times higher than the concentration found in blood plasma (~10 µM).28-30 The reduction-sensitive nano-assemblies are susceptible to cleavage under a reductive environment involving thiol-disulfide exchange reactions as compared to their reductioninsensitive counterparts.31-32 Additionally, uncross-linked polymeric micellar nanostructures have 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 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 ATP dependent effluxing of different anticancer drugs like doxorubicin and paclitaxel into the extracellular space.37-38 MDR effect can be overcome by increasing concentration of the drug or treating various classes of chemosensitizer like calcium channel

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blockers and inhibitors which block the drug efflux facilitated by membrane transporter P-gp. However, this may results in strong side effects and unacceptable toxicity when used at the required concentrations.

39-40

Drug carriers based on polymer nano-assembles have shown the

capability to enhance the efficacy of anticancer drugs by targeting tumors which is obtained by their prolong 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 nano-assemblies may also provide good opportunities for overcoming MDR.43-45 Moreover, an effective control of drug release rate has been reported to be extremely important for 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 nano-carriers from poly(ethylene glycol)-blockpoly(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-3yl)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 cross-linked block copolymer micelles (CCMs) was tuned under elevated reductive environment that is often associated with cancer cells by varying the number of units of LAHEMA in the diblock copolymers (PEG-bPLAHEMA) to control the release rate of doxorubicin and thereby it’s accumulation in the cell nucleus region. The in vitro cytotoxicity, cellular uptake efficiency and intracellular drug release behavior in drug-sensitive and drug-resistant cancer cells after treatment of CCMs loaded with

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DOX (and Verapamil, a chemosensitizer) were extensively evaluated and the most promising composition of the block copolymers was identified that was capable of overcoming multidrug resistance (MDR) in breast cancer cells.

EXPERIMENTAL SECTION: Materials. DL-α-Lipoic acid (LA), 2-hydroxyethyl methacrylate (HEMA), N,N′dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), L-glutathione reduced (GSH), doxorubicin hydrochloride were purchased from Sigma-Aldrich (St. Loius, MO, USA) and used without further purification. Poly(ethylene glycol) monomethyl ether (PEG, molar mass 5,000 g mol-1, 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, USA) stock solution was prepared in methanol. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) as well as 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from SigmaAldrich. Fetal bovine serum and Dulbecco's Minimum Essential Medium (DMEM) were procured from Gibco, USA and Sigma-Aldrich respectively. ApopTag, in situ apoptosis detection kit was purchased from Promega, Madison, WI, USA. Mlili-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 5% CO2 atmosphere, at 37 °C and 95% humidity in DMEM (Gibco-BRL, Rockville, MD) media supplemented with 10% heat inactivated FBS (Gibco-BRL). DAPI staining procedures was utilized to detect the mycoplasma

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status of all cell lines. 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 and were routinely cultured and maintained in their respective media. Synthesis of the Block Copolymers Synthesis of PEG macro-CTA (Scheme 1): PEG macro-CTA was synthesized by a coupling reaction using poly(ethylene glycol) monomethyl ether (PEG, MW 5,000 gmol-1). Subsequent to azeotropic distillation with toluene at 70 °C under high vacuum, PEG was reacted with DDMAT in dry DCM as solvent and in 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 r.b. flask (100 mL) and added dry DCM (35 mL) as solvent and the reaction mixture was kept in 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.

Scheme 1. Synthesis of diblock copolymer (PEG-b-PLAHEMA) using RAFT polymerization technique.

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for an additional 1 h and then was stirred at room temperature for overnight. The resulting insoluble N,N′-dicyclohexylurea was filtered using G4 silica crucible and the filtrate was concentrated to yield a yellowish waxy solid. The product was further purified by dialysing against methanol using cellulose membranes (cutoff value of MW~3.5 kDa). Dialysis was continued for one 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 (please see Supporting Information - Figure S1) 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 synthesized by 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 dry DCM solvent following to 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 double necked 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 drop wise with constant stirring. The reaction mixture allowed to proceed in an ice-cold environment for an additional 1 h and then was stirred for at room temperature overnight. The resulting insoluble N,N′-dicyclohexylurea was filtered using 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 in low temperature (-20 °C) to maintain the monomer LAHEMA

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stability for several months. The purified product was analyzed by NMR spectroscopy (please see Supporting Information - Figure S2 and S3). Synthesis of PEG-b-PLAHEMA (Scheme 1): For the synthesis of diblock copolymer we have polymerized the monomer LAHEMA in presence of PEG macro-CTA (MW 5,350 gmol-1) 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 in three different ratios of monomer LAHEMA to PEG macro-CTA to obtain diblock copolymers (PEG-b-PLAHEMA) containing varying number of LAHEMA units while the molar ratios of [macro CTA]/[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 (cut-off value of MW~10 kDa) for 16 h with the outside solvent changed at every 4 h interval. The product was recovered by evaporating the CHCl3 by rotary evaporator and drying under high vacuum. The final purified copolymers were analyzed by 1H NMR spectroscopy and GPC (Figure 1 and 2). Instrumentation and Methods. NMR Spectroscopy. Recording of 1H NMR and 13C NMR spectra were acquired in CDCl3 or DMSO-d6 using Bruker DPX spectrometer operating at 400/600 and 100 MHz at 25 ⁰C, 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 (Ð = Mw/Mn) of the polymers using RI detector and HPLC

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grade dimethylformamide (DMF) as 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 done for structural analysis of self-assembled polymeric aggregates using JEOL model JEM 2100 transmission electron microscope at an operating voltage 80 kV. Dynamic light scattering (DLS) measurements were performed to determine size and size distribution of the selfassembled polymeric aggregates using Malvern Nano ZS instrument equipped with a temperature-controlled sample chamber by 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 in order 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 ( ) and size distribution in terms of polydispersity index (PDI). The  values of polymeric aggregates were estimated from the intensity autocorrelation function of time-dependent fluctuation in intensity where  is defined as  =  ⁄3  where  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, UV-2450) spectrophotometer and steady-state fluorescence spectra were collected using Hitachi (model no. F-7000) and Jobin Yvon-Spex Fluorolog-3 spectrofluorimeter, respectively. All the measurements were performed at 25 oC using a quartz cuvette of 1 cm path length. Determination of Critical Aggregation Concentration (CAC): CAC of the block copolymers were determined by using Nile Red as hydrophobic fluorescent probe.51 At first, stock polymer

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solutions of concentration 0.1 mg/mL were prepared by solvent exchange method. Weighted amount of respective polymer in DMF was added dropwise to an appropriate volume of phosphate buffer (10 mM, pH 7.4) with constant stirring for 30 min. Then the resulting dispersion was dialyzed against phosphate buffer using cellulose membranes (MW cut-off value of 10 kDa) for 24 h with frequent change of 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 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 required volume of buffer to get a series of solutions with polymer concentrations varying from 0 to 0.01 mg/ml, in which 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 FL intensity at the emission maxima was plotted against polymer concentration. The inflection point in the observed plot, was considered as the CAC. 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, re-dissolved 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. In order to load DOX, stock polymer solutions (1 mg/mL) in PB (pH 7.4, 10 mM) prepared by 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 LAHEMA unit in PB) was added and stirred for 12 h in a dark condition at room temperature. The solution was then placed in a dialysis tube possessing cellulose membrane (MW cut-off value of 10 kDa) and dialyzed against 10 mol% GSH containing PB (pH 7.4) for 24 h. The buffer was replaced in

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every 6 h interval to remove the non-encapsulated 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 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. Based on these data, the ratio of DOX/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 co-encapsulation 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 in dark condition at room temperature. Dialysis was performed to remove non-encapsulated drug and DMSO. Cell Viability Assay: Cell viability by MTT assay was performed 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, 37 °C. Subsequently, the cells were treated for 72 hours with the free polymers so that the final effective concentrations of the polymers were varied between 0 to 2.5 mg/mL. MTT dye reduction assay was measured 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 cross-linked micelles were analyzed using Prism software (Graph Pad Prism 5 software).

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Cellular Uptake Studies: Uptake of DOX-encapsulated cross-linked micelles was qualitatively

analyzed by fluorescence imaging in time-dependent manner over a period of 6 hours. Cytotoxicity and Induction of Apoptosis: In order to investigate the time-dependent effect of free

DOX and DOX encapsulated cross-linked polymer micelles on the growth of MDA-MB-231 and multidrug resistant MDA-MB-231 (231R) cells, MTT dye reduction assay was performed as described in the above procedure. The time dependent curves of free DOX and DOX encapsulated cross-linked micelles were analyzed using Prism software (GraphPad Prism 5 software). Induction of apoptosis and the changes in morphology associated with it were studied by TUNEL staining. Cells were fixed in 3.7 % paraformaldehyde, permeabilized with 0.1 % Triton X-100, blocked in 2% BSA 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 appropriate wavelength. The images were taken and digitized using FLUOVIEW 1000 imaging software (Version 1.2.4.0).

RESULTS AND DISCUSSION: Synthesis of the Diblock Copolymers: In the present work, well-defined block copolymers were synthesized by controlled radical polymerization technique namely RAFT, using methacrylate monomer LAHEMA and long chain poly(ethylene glycol) based macro-CTA, as shown in Scheme 1. At first, the PEG macro-CTA was prepared by 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

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molecular weight (Mn) of 5,500 g mol-1 with dispersity (Ð) = 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 polymerization reaction at varying monomer to macro-CTA ratio. 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 ester oxygen of the PEG block with chemical shift value at 3.65 ppm. 1H NMR spectra of one of the representative block PEG-b-PLAHEMA52 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 1H NMR spectroscopy. Gel permeation chromatography (GPC) analysis revealed monomodal distributions for all the block copolymers with narrow dispersity (Ð), ensuring good control over the polymerization (Figure 2). Detailed polymerization conditions and characterization results are summarized in Table 1.

Figure 1: 1H NMR spectra (in CDCl3) of PEG-b-PLAHEMA52 containing 52 unit of LAHEMA.

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Figure 2: GPC chromatograms of the PEG macro-CTA and the diblock copolymers (PEG-b PLAHEMA) containing different number of LAHEMA units. Table 1: Results from the RAFT polymerization of LAHEMA in the presence of PEG macroCTA in 1,4-dioxane at 70 °C.

a

Mn,NMRb (gmol-1)

Mn,GPCc (gmol-1)

Ðc

-

5,400

5,500

1.05

30

68

13,600

12,100

1.12

BCP52

60

62

21,900

23,500

1.18

BCP83

100

53

31,700

35,800

1.26

Polymer composition

Polymer abbreviation

PEG macro CTA PEG-bPLAHEMA26 PEG-bPLAHEMA52 PEG-bPLAHEMA83

PEG macroCTA BCP26

-

[M] / [macro- Conversion CTA] (%)a

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

Micelle Formation from PEG-b-PLAHEMA Diblock Copolymers: Diblock copolymers containing hydrophilic and hydrophobic blocks in the same chain are expected to self-assemble in water and form stable nanostructures above a certain concentration, known as critical aggregation concentration (CAC). Self-association and solution properties of PEG-b-PLAHEMA diblock copolymers, namely BCP26, BCP52, and BCP83, were studied using DLS, TEM, and hydrophobic fluorescent probe encapsulation studies. At first, block copolymer nanostructures

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were prepared using 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 micrograph revealed the formation of near-spherical aggregates of size which are in close agreement with those obtained from DLS measurements suggesting formation of micelle-like aggregates (Figure 4). In case of BCP83, having highest hydrophobic content in the polymer backbone, some inter-micellar 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 size obtained from DLS studies (Figure 3) was 76 nm, 106 nm and 167 nm for BCP26, BCP52, BCP83 respectively, whereas the size from TEM analysis (Figure 4) for same set of copolymers was approximately 65 nm, 100 nm and 155 nm respectively. These data show that the size 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. This increased compactness could be due to increased hydrophobic interactions between the PLAHEMA blocks in the core. We have also determined critical micelle concentration (CMC) of diblock copolymers using Nile Red as a hydrophobic fluorescent probe.48, 51 In Figure 5, Nile Red emission intensity ( = 550 nm) was plotted with varying copolymer concentration from 0 to 0.01 g/L in which Nile Red concentration was fixed. In absence of polymer Nile Red showed a very weak emission due to

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Figure 3: Size distribution profiles obtained from dynamic light scattering measurement of PEG-bPLAHEMA copolymers containing different units of LAHEMA.

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

very low aqueous solubility. On increasing polymer concentration, Intensity of Nile Red emission increased significantly in a nonlinear fashion with 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 self-assembly of the block copolymers. Intensity of Nile Red emission was plotted against polymer concentration that 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)

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of the block copolymer. In the present study, the value of the CMC for BCP26, BCP52, and BCP83 were determined to be 5.0, 2.0 and 0.65 mg/L respectively which are comparable to the CMC values of similar copolymers described previously.55-56

Figure 5. Representative plot of emission intensity of Nile Red versus log of polymer concentration at the emission maxima ( = 550 nm) for determining the critical micelle concentration (CMC) of diblock copolymer BCP52. Inset shows emission spectra of Nile Red for two different polymer concentrations. 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 1,2-dithiolane moiety based on thiol–disulfide exchange in PB (pH 7.4, 10 mM) by introducing 10 µM of GSH, the concentration of GSH generally found in the blood plasma and extracellular environment.57-58 Under the catalysis of GSH, few 1,2-dithiolane ring present in the LAHEMA unit in the polymer opened to yield dihydrolipoyl (the reduced form of lipoyl) groups. Subsequently, exchange reaction between the generated dihydrolipoyl groups with the disulfide bonds of other 1,2dithiolane 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 measurement revealed that the size of the micelle decreased in this reaction compared to their

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non-cross-linked counterparts by 10-20 nm, indicating existence of cross-linking. These corecross-linked micelles (CCMs) displayed very low polydispersity index of 0.03-0.06 (Figure 6a, Table 2). TEM micrograph also demonstrated that the CCMs had more compact spherical morphology compared to their non-cross-linked counterparts and the size distribution was close to those determined by DLS (Figure 6c, Table 2).

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 with time of cross-linked BCP52 micelles 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 upto 96 h in PB (pH 7.4, 10 mM) at 37 °C.

Owing to cross-linking, the polymer micelles (after cross-linking) should retain their structural integrity even on many fold dilution, unlike non-cross-linked micelles.59 We have

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studied structural stability of CCMs by DLS against extensive dilution. Notably, CCMs upon dilution below the critical micellar concentrations of the corresponding block copolymers showed slight increase in micelle size and maintained a low PDI. Figure 6a shows the data for core cross-linked BCP52 micelles (BCP52CCM). In contrast, the parent non-cross-linked micelles under similar dilution dissociated to form unimers with intensity average hydrodynamic diameter ( ) 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 time dependent change in micelle sizes in response to 10 mM GSH in PB buffer (pH 7.4, 10 mM) (Figure 6b, Figure 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 significant size increase along with increase in PDI values for all the three CCMs indicating occurrence of de-cross-linking induced micellar swelling and concomitant increased hydrophilicity of the micellar core resulting from conversion of each disulfide bond into two hydrophilic thiol groups. TEM study of BCP52CCM and its de-cross-linking induced swelling after 96 h also supports the observation from DLS study (Figure 6d). From the PDI value and size increase in time dependent DLS study (Table 2), it also appears that the rate of de-crosslinking induced swelling in case of BCP26CCM was much faster than the other two (BCP52CCM and BCP83CCM ) and almost got saturated within 24 h. In case of BCP52CCM, slow and prolonged swelling was observed upto 96 h but BCP83CCM shows very less swelling even upto 72 h of incubation. This de-crosslinking induced swelling property of the three sets CCMs can be attributed from the increased hydrophobicity with increasing molecular weight of the PLAHEMA block as well as GSH permeability to the disulfide bonds present in the hydrophobic micellar core. Furthermore,

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above results also suggest that PEG-b-PLAHEMA CCMs exhibit superior colloidal stability under extracellular conditions whereas they undergo controlled swelling with tunable rate under reductive conditions, mimicking the cytoplasm and cell nucleus of tumor tissues. Table 2: Swelling property of cross-linked block copolymers micelles (CCMs) in terms of size distribution determined using DLS in presence of 10 mM GSH containing PB (pH 7.4) at 37 °C. The size data for the micelles before cross-linking are also included here for comparison. Time of swelling (h) 0 (before cross-linking) 0 (after cross-linking)

BCP26CCM [ (PDI)] 76 (0.153) 60 (0.036)

BCP52CCM [ (PDI)] 106 (0.118) 96 (0.041)

BCP83CCM [ (PDI)] 167 (0.177) 151 (0.055)

12

405 (0.432)

142 (0.145)

175 (0.106)

24 48 72

499 (0.471) 535 (0.561) 711 (0.536)

420 (0.255) 686 (0.286) 886 (0.462)

283 (0.114) 411 (0.121) 599 (0.177)

96

775 (0.924)

955 (0.617)

861 (0.391)

Biological studies of DOX encapsulated core cross-linked block copolymer micelles (CCMs) in sensitive as well as drug-resistant breast cancer cells: In order to assess the safety of the three sets of CCMs for drug delivery applications, in vitro cytotoxicity of these micelles were evaluated by MTT dye reduction assay in both normal cell lines HaCat and on breast cancer cells MDA-MB-231. All the three CCMs showed no obvious

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.

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cytotoxicity up to 2.5 mg/mL concentration even after culturing for 72 h (Figure 7) which established excellent biocompatibility of the present set of CCMs. We have loaded DOX, a model hydrophobic anticancer drug, into three CCMs. The loading capacities (LC) of DOX inside these CCMs were calculated using the following equations  % =

           !" 

× 100

and was found to be 16.6 %, 23.8 % and

19.2 % in case of BCP26CCM, BCP52CCM and BCP83CCM respectively. It is generally accepted that the polymer micelles with higher hydrophobic content should solubilize more hydrophobic drug.60 Lower solubility of DOX in BCP83CCM in comparison to BCP52CCM could be due to low permeability of DOX inside the core of BCP83CCM . In the present study, we also aimed to overcome multidrug resistance in breast cancer cells by co-encapsulating Verapamil in cross-linked polymeric micelles along with DOX. Co-encapsulation study showed that the drug loading content (LC) of DOX was 7.5%, 10.7% and 8.9% while that of Verapamil was 6.3%, 7.8% and 8.3% in BCP26CCM, BCP52CCM and BCP83CCM respectively. We have also studied 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 and 8b clearly revealed de-cross-linking induced controlled micellar swelling with tunable drug release rate for the designed CCMs.

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

Before finding out the efficacy of the DOX-loaded CCMs, it was necessary to establish the MDR of the experimental cell line 231R for this, breast cancer cell lines MDA-MB-231 and multidrug-resistant cell line 231R were treated with varying concentrations of free DOX for different incubation time. IC50 values were determined by MTT assay and data are found to be as follows - in sensitive breast cancer cells 3.147 ± 0.972 µM, 1.462 ± 0.96 µM and 0.7870 ± 0.975 µM for 24 h, 48 h and 72 h respectively; and in resistant breast cancer cells 14.79 ± 0.98 µM, 5.131 ± 0.96 µM and 2.3 ± 0.95 µM for 24 h, 48 h and 72 h respectively. The IC50 values were found to be almost five folds higher for cell line 231R compared to 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

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Figure 9: Qualitative examination of time dependent cellular uptake of cross-linked micelles 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.

To confirm passive accumulation of these CCMs inside cancer cells, a time dependent cellular uptake study was conducted using DOX encapsulated 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 hours for all the micelles. We have further studied the potential of these DOX encapsulated CCMs and their de-crosslinking induced controlled micellar swelling with tunable rates under a reductive condition that mimic the reductive condition of the cytoplasm and cell nucleus in tumor tissues. Intracellular DOX release properties of all the three CCMs were evaluated against both MDA-MB-231 and 231R cell line 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 the three DOX encapsulated CCMs. The respective time dependent IC50 values in each case were obtained from

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Figures 10 and have been shown in Table 3. A faster swelling BCP26CCM may be responsible for medium IC50 value after 24 h. 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 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, an appropriate selection of dose and exposure time are the two important factors towards enhancement of efficacy and improvement of patient compliance. A careful analysis of the data presented in the Table 3 provides us with some interesting inferences. Efficacy of BCP52CCM is significantly better than free DOX whereas the efficacy of BCP26CCM and BCP83CCM are similar and inferior respectively compared to free DOX towards sensitive cells. However, the efficacy of all the three 24h

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

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Table 3: IC50 value of DOX in the formulation of all three CCMs in both the sensitive and resistant cancer cell lines. IC50 is the concentration of DOX required to cause 50 % cell death. The IC50 data for free DOX are also included here for comparison.

Incubation time (h)

Polymer

Free DOX

DOX-loaded BCP26CCM

DOX-loaded BCP52CCM

DOX-loaded BCP83CCM DOX+Verapamil-loaded BCP26CCM DOX+Verapamil-loaded BCP52CCM DOX+Verapamil-loaded BCP83CCM

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

IC50 (µM) MDA-MB-231 Multidrug resistant 231R 3.147 ± 0.97 14.79 ± 0.98 1.462 ± 0.96 5.131 ± 0.96 0.7870 ± 0.98 2.3 ± 0.95 2.097 ± 0.99 2.9 ± 0.90 1.580 ± 0.96 1.705 ± 0.96 0.777 ± 0.98 1.502 ± 0.94 0.9585 ± 0.95 1.088 ± 0.44 0.3852 ± 0.91 0.3690 ± 0.96 0.2495 ± 0.89 0.1722 ± 0.89 2.969 ± 0.98 1.748 ± 0.95 2.478 ± 0.99 1.581 ± 0.88 1.154 ± 0.97 0.8711 ± 0.97 0.9592 0.07276 0.5871

DOX-loaded CCMs were significantly improved compared to free DOX in comparison to free DOX towards resistant 231R cells. Of the three CCMs, the improvement in the IC50 value was highest for BCP52CCM. As discussed earlier that the GSH induced release of DOX due to decross-linking (and subsequent swelling) was a slow and sustained from BCP52CCM which results in overcoming the resistance of the 231R cells. These results show the potential these CCMs as drug delivery vehicles towards MDR cancer cells. From the Table 3 we can also see that for drug sensitive cells (MDA-MB-231), the IC50 for 24 hours has reduced significantly, at least by 30% in case of BCP26CCM and BCP52CCM. However, the IC50 values for 48 and 72 hours did not show significant decrease for BCP26CCM and BCP83CCM, while it was decreased by about 70% in BCP52CCM. Doxorubicin

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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 MTT assay which is based on metabolic activity of the cells. A decrease in IC50 in 24 hours demonstrates the improved efficiency of the drug formulation. Slightly higher IC50 values in 48 and 72 hours 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 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 drug, hence respond poorly to the free drug itself. Use of core cross linked polymeric micelles improved the drug uptake and retention, hence, reducing its effective dose. Verapamil, the well-known calcium channel blocker has been proved to reverse multidrug resistance in cancer by directly binding p-glycoprotein. From MTT assay it was found 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 similar loading efficiency comparable to DOX, the required dose of DOX on 231R cells was significantly reduced (Figure 11). The effective IC50 values of CCMs encapsulated both DOX and verapamil was found to be significantly lower compared to the IC50 values for CCMs containing only DOX (Table 3). These

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

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results show the potential of the DOX-loaded CCMs as drug delivery vehicles towards MDR cancer cells could be further improved by co-loading verapamil along with DOX in the CCMs. Nuclear fragmentation and induction of apoptosis on treatment of multidrug resistant 231R cells was microscopically examined by TUNEL staining. TUNEL positive green nuclei are indicating of apoptotic cell death. On treatment of 231R cells with sublethal dose (about 3/4th 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 treatment(Figure 12, upper row). Whereas, on treatment with BCP52CCM (the most efficient of the three polymers) encapsulated DOX with 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.

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

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CONCLUSION 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 self-assembling behavior in aqueous medium, forming micellar nanostructures, the core of which can undergo cross-linking in presence of catalytic amount of GSH mimicking that of the GSH concentration in extracellular environments. The cross-linking imparted stability to the core cross-linked micellar nanostructures (CCMs) against dilution which are expected to make them stable during systemic circulation post-injection. The CCMs were found to be biocompatible, a feature that is expected due to 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 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-crosslinking. 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 towards MDR cancer cells was found to be further improved by co-loading Verapamil along with DOX in the CCMs. Thus the PEG-b-PLAHEMA core cross-linked micelles reported in this work provides an option for stable, biocompatible nano-carriers for delivering anticancer drugs to both sensitive and resistant cancer cells.

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ACKNOWLEDGEMENT: 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 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, NPA with institute approval numbers - IIT/SRIC/CHY/ADA/201415/18 and IIT/SRIC/CHY/NPA/2014-15/81 respectively). CM and SK acknowledge UGC, New Delhi and IIT Kharagpur respectively for Research Fellowships.

SUPPORTING INFORMATION. NMR Spectra of macro-CTA, synthesized monomer LAHEMA, loading content and loading efficiency with varying DOX conc. and few DLS are provided in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION *Corresponding Author: Email: [email protected], [email protected] Ph no: +91-3222-282326; Fax: +91-3222-282252

REFERENCES (1) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers As an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751-760. (2) Zhao, X.; Qi, M.; Liang, S.; Tian, K.; Zhou, T.; Jia, X.; Li, J.; Liu, P. Synthesis of Photo- and pH Dual-Sensitive Amphiphilic Copolymer PEG43-b-P(AA76-co-NBA35-co-tBA9) and Its

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Micellization as Leakage- Free Drug Delivery System for UV-Triggered Intracellular Delivery of Doxorubicin. ACS Appl. Mater. Interfaces 2016, 8, 22127-22134. (3) Chan, D.; Yu, A. C.; Appel, E. A. Single-Chain Polymeric Nanocarriers: A Platform for Determining

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Biomacromolecules 2017, 18, 1434-1439. (4) Davis, M. E.; Chen, Z.; Shin, D. M. Nanoparticle Therapeutics: An Emerging Treatment Modality for Cancer. Nat. Rev. Drug Discovery 2008, 7, 771-782. (5) van Dongen, S. F. M.; de Hoog, H.-P. M.; Peters, R. J. R. W.; Nallani, M.; Nolte, R. J. M.; vanHest, J. C. M. Biohybrid Polymer Capsules. Chem. Rev. 2009, 109, 6212-6274. (6) Moughton, A. O.; O’Reilly, R. K. Thermally Induced Micelle to Vesicle Morphology Transition for a Charged Chain End Diblock Copolymer. Chem. Commun. 2010, 46, 1091-1093. (7) Zhang, K.; Jia, Y-G.; Tsai, I-H.; Strandman, S.; Ren, L.; Hong, L.; Zhang, G.; Guan, Y.; Zhang, Y.; Zhu, X. X. “Bitter-Sweet” Polymeric Micelles Formed by Block Copolymers from Glucosamine and Cholic Acid. Biomacromolecules 2017, 18, 778-786. (8) Liu, X.; Tan, X.; Rao, R.; Ren, Y.; Li, Y.; Yang, X.; Liu, W. Self-Assembled PAEEP-PLLA Micelles with Varied Hydrophilic Block Lengths for Tumor Cell Targeting. ACS Appl. Mater. Interfaces 2016, 8, 23450-23462. (9) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Polymersomes: Tough Vesicles Made from Diblock Copolymers. Science 1999, 284, 1143-1146. (10) Luo, Z.; Li, Y.; Wang, B.; Jiang, J. pH-Sensitive Vesicles Formed by Amphiphilic Grafted Copolymers with Tunable Membrane Permeability for Drug Loading/Release: A Multiscale Simulation Study. Macromolecules 2016, 49, 6084-6094.

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(11) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor Vascular Permeability and the EPR Effect in Macromolecular Therapeutics: A Review. J. Control. Release 2000, 65, 271-284. (12) Nishiyama, N.; Okazaki, S.; Cabral, H.; Miyamoto, M.; Kato, Y.; Sugiyama, Y. Novel Cisplatin-Incorporated Polymeric Micelles Can Eradicate Solid Tumors in Mice. Cancer Res. 2003, 63, 8977-8983. (13) Stolnik, S.; Illum, L.; Davis, S. S. Long Circulating Micro particulate Drug Carriers. Adv. Drug Delivery Rev. 1995, 16, 195-214. (14) Gaucher, G.; Marchessault, R. H.; Leroux J. -C. Polyester Based Micelles and Nanoparticles for the Parenteral Delivery of Taxanes. J. Control. Release 2010, 143, 2-12. (15) Liu, G. –Y.; Chen, C. –J.; Ji, J. Biocompatible and Biodegradable Polymersomes as Delivery Vehicles in Biomedical Applications. Soft Matter 2012, 8, 8811-8821. (16) Du, J. Z.; Tang, Y. Q.; Lewis, A. L.; Armes, S. P. pH-Sensitive Vesicles Based on a Biocompatible Zwitterionic Diblock Copolymer. J. Am. Chem. Soc. 2005, 127, 17982-17983. (17) Banerjee, R.; Dhara, D. Functional Group-Dependent Self- Assembled Nanostructures from Thermo-Responsive Triblock Copolymers. Langmuir 2014, 30, 4137-4146. (18) Sun, J.; Chen, X. S.; Lu, T. C.; Liu, S.; Tian, H. Y.; Guo, Z. P.; Jing, X. B. Formation of Reversible Shell Cross-Linked Micelles from the Biodegradable Amphiphilic Diblock Copolymer Poly(l-cysteine)-block-Poly(l-lactide). Langmuir 2008, 24, 10099-10106. (19) Hu, J.; Zhang, G.; Liu, S. Enzyme-Responsive Polymeric Assemblies, Nanoparticles and Hydrogels. Chem. Soc. Rev. 2012, 41, 5933-5949. (20) Cheng, R.; Meng, F.; Deng, C.; Klok, H. –A.; Zhong, Z. Dual and Multi-Stimuli Responsive Polymeric Nanoparticles for Programmed Site-Specific Drug Delivery. Biomaterials 2013, 34, 3647-3657.

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