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Thiol-responsive block copolymer nanocarriers exhibiting tunable release with morphology changes

Qian Zhang,a Samuel Aleksanian,a Seung Man Nohb and Jung Kwon Oh*a

Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX

DOI: 10.1039/b000000x

New thiol-responsive nanocarriers of amphiphilic block copolymers consisting of a pendent disulfide-labeled methacrylate polymer block (PHMssEt) and a hydrophilic poly(ethylene oxide) (PEO) block were reported. These well-controlled block copolymers were synthesized by atom transfer radical polymerization (ATRP) of a new pendent disulfide-functionalized methacrylate (HMssEt) in the presence of PEO-Br macroinitiator. Due to its amphiphilic nature, the PEO-b-PHMssEt with narrow molecular weight distribution self-assembled in aqueous solution to form monomodal micellar aggregates with PHMssEt cores surrounded with hydrophilic PEO coronas. In response to thiols, the disulfide linkages were cleaved, and thus self-assembled micelles were either converted to core-crosslinked micelles or destabilized to further disintegrate, depending on the amount of added thiols. Such change in morphology led to tunable release of encapsulated model drugs in aqueous solutions.

Introduction

In recent years aqueous micellar aggregates of well-defined amphiphilic block copolymers (ABPs) exhibiting stimuli-responsive (bio)degradation (SRD) have been extensively explored due to their tremendous potential as multifunctional drug delivery nanocarriers.[1-4] Nanoparticles exhibiting SRD show great promise as nanotherapeutics due to their controllable and tunable response to external triggers. This enables the enhanced release of encapsulated therapeutics into targeted cells while facilitating the removal of empty vehicles after the release. Furthermore, SRD has been utilized to tune the morphologies of self-assembled nanostructures.[5-6] Of external stimuli typically including oxidative,[7] enzymatic reaction,[8] low pH,[9-13] and light,[14-15] reductive reaction employing disulfide-thiol platform is particularly promising because disulfide linkages are cleaved to the corresponding thiols in response to thiols or reducing agents.[16] In biological systems, glutathione (GSH, a tripeptide containing cysteine) which plays an important role in cellular metabolism is found at millimolar concentrations in cells,[17-18] and at further elevated levels in cancer cells.[19]

a Department of Chemistry and Biochemistry and Center for Nanoscience Research (CENR), Concordia University, Montreal, Quebec, Canada H4B 1R6; E-mail: john.oh@concordia.ca

b PPG Industries Korea , Cheonan 330-912 & Department of Chemical and Biological Engineering, Korea University, Seoul 136-713, Republic of Korea

†Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/b000000x/

A variety of thiol-responsive degradable ABPs and their nanosized assemblies have been reported.[20-21] For example, mono-cleavable micelles having single disulfide bonds in the middle of symmetric triblock copolymers[22-24] and sheddable micelles having single disulfides at the junctions of diblock copolymers[25-36] have been explored. These micelles are characterized as block copolymers having single disulfides along the main polymer chains. Multi-cleavable micelles are also designed with block copolymers having disulfides positioned in the hydrophobic blocks as having multiple repeats or groups along the main chains. Previous reports from our research group[37-38] and others[39-41] have shown that these backbone multi-cleavable micelles exhibit rapid and controlled release of encapsulated biomolecules including fluorescent dyes and anticancer drugs through main chain degradation mechanisms. Furthermore, the degradation can be tuned with varying densities of disulfides[42-43] and degrees of crystallinity.[44]

Another promising class of multi-cleavable micelles employs block or copolymers having pendent disulfide linkages as side chains. The design of these pendent multi-cleavable micelles is advantageous in that they can be converted to core-crosslinked micelles or nanogels with disulfide crosslinks through intra and inter-chain thiol-disulfide polyexchange reactions in response to reductive reactions. Such core-crosslinking provides enhanced colloidal stability as well as prevents premature release of encapsulated drugs during the circulation in the body.[45-46] A further advantage of utilizing such reversible redox reaction is that the newly-formed disulfide crosslinks are cleaved in the presence of excess thiols. Such change in hydrophobic/hydrophilic balance causes crosslinked micelles to dissociate, thus enhancing the release of encapsulated drugs in targeted cells. Several crosslinked nanogels labeled with pendent disulfides have been developed. These nanogels are mostly based on random copolymers prepared through post-modification of dextrans with lipoic acids[47-48] and radical copolymerization of pendent disulfide-labeled methacrylates.[49-51] However, only few reports describe the synthesis of block copolymers and pendent multi-cleavable micelles of our interests. An example includes well-controlled ABPs consisting of poly(N-(2-hydroxypropyl) methacrylamide) and poly(2-(pyridyldisulfide)ethyl methacrylate) prepared by reversible addition fragmentation chain transfer (RAFT) polymerization.[52] Further, there are still needs to design new types of degradable micelles for better understanding of structure-property relationship between morphological variance and stimuli-responsive degradation.

In this work, we have developed new thiol-responsive nanocarriers based on well-controlled ABPs consisting of a pendent disulfide-labeled methacrylate polymer block and a hydrophilic poly(ethylene oxide) (PEO) block, exhibiting tunable release of encapsulated model drugs with change in morphology as a result of thiol-triggered degradation. The reductively-cleavable ABPs here were synthesized by atom transfer radical polymerization (ATRP), a successful living/controlled radical polymerization, of a new pendent disulfide-functionalized methacrylate (HMssEt) in the presence of PEO-Br macroinitiator, yielding PEO-b-PHMssEt (Scheme 1). PEO is a biocompatible polymer with low toxicity that is well known to prevent nonspecific protein adsorption, and has been FDA-approved for clinical use. [53-54] The PHMssEt block is designed to be responsive to thiols in hydrophobic micellar cores. Thus, in response to different amounts of thiols, self-assembled micelles with spherical morphology were either converted to core-crosslinked micelles or destabilized to further disintegrate. Such change in morphology (here, core-crosslinked micelles or destabilization-driven aggregates) led to tunable release of encapsulated model drugs in aqueous solution (Scheme 2).

Experimental

Materials and instrumentation

3,3'-Dithiodipropionic acid (ss-DCOOH), 2-hydroxyethyl methacrylate (HEMA), N,N(-dicyclohexyl carbodiimide (DCC), 4-dimethylaminopyridine (DMAP), copper(I) bromide (CuBr, >99.99%), N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA, >98.0%), methanol (MeOH), and ethanol (EtOH) from Aldrich and DL-dithiothreitol (DTT, 99%) from Acros Organics were purchased and used as received. A water-soluble macroinitiator, PEO-functionalized bromoisobutyrate (PEO5000-Br, EO units DP ( 113), was synthesized using a reported procedure.[55] Poly(ethylene glycol) monomethyl ether (PEO5000-OH, Aldrich, 20 g, 4 mmol) reacted with 2-bromo-2-methylpropionic acid (98%, Acros, 1 g, 6.0 mmol) in the presence of DCC (0.9 g, 4.4 mmol), and a catalytic amount of DMAP in CH2Cl2 (150 mL). The product was mixed with THF, isolated from dispersion by vacuum filtration, and then dried in a vacuum oven at 30 (C for 12 h. In this way, residues such as excess DCC, DMAP, and dicyclohexyl urea as a by-product could be removed from the final product.

1H-NMR spectra were recorded using a 500 MHz Varian spectrometer. The CDCl3 singlet at 7.26 ppm and DMSO-d6 quintet at 2.5 ppm were selected as the reference standard. Spectral features are tabulated in the following order: chemical shift (ppm); multiplicity (s - singlet, d - doublet, t – triplet, m - complex multiple); number of protons; position of protons. Molecular weight and molecular weight distribution were determined by gel permeation chromatography (GPC). The first, a Viscotek GPC, was equipped with a VE1122 pump and a refractive index (RI) detector, two PolyAnalytik columns (PAS-103L and 106L) were used with THF as an eluent at 30 (C at a flow rate of 1 mL/min. The second was an Agilent GPC with a 1260 Infinity Isocratic Pump and a RI detector, two Agilent columns (PLgel mixed-D and mixed-C) were used with DMF containing 0.1 mol% LiBr as an eluent at 50 (C at a flow rate of 1 mL/min. Linear polystyrene (PSt) and poly(methyl methacrylate) (PMMA) standards from Fluka were used for calibration. Aliquots of polymer samples were dissolved in either THF or DMF/0.1 mol% LiBr. The clear solutions were filtered using a 0.25 (m PTFE filter to remove any THF-insoluble species. A drop of anisole was added as a flow rate marker. Monomer conversion was determined using 1H-NMR in CDCl3.

Synthesis of HMssEt methacrylate bearing a pendent disulfide linkage

In the first step to synthesize HMss-COOH, a clear solution of DCC (18.0 g, 87.2 mmol) in THF (35 mL) was drop wise added into a mixture consisting of HEMA (10.5 g, 80.9 mmol), ss-DCOOH (70.0 g, 332.9 mmol), DMAP (0.88 g, 7.2 mmol), and THF (700 mL) in an ice bath for 40 min under vigorous stirring. The resulting mixture was kept at 0 (C for 30 min, and then stirred at room temperature for 12 hrs. The formed solids (dicyclohexyl urea) as by-products were removed by a vacuum filtration and the organic solvents were removed using a rotary evaporator at 40 (C. The resulting residues were mixed with chloroform (280 mL) and solids (presumably unreacted ss-DCOOH) were removed by vacuum filtration. Chloroform was removed by rotary evaporation to form residues containing HMss-COOH.

In the second step, DCC (20.0 g, 96.9 mmol) dissolved in chloroform (30 mL) was added drop wise to a solution consisting of the oily residues (synthesized in the first step), DMAP (0.12 g, 0.98 mmol), EtOH (19 g, 412.4 mmol), and chloroform (80 mL) in an ice bath. The resulting mixture was kept in the ice bath for 20 min and then stirred at room temperature for 12 hrs. The formed solids (dicyclohexyl urea) were removed by vacuum filtration. The organic solution was washed with 0.2 M aqueous HCl solution (75 mL) and aqueous saturated NaHCO3 solution (75 mL) twice, and then dried over MgSO4. Solvents were removed by rotary evaporation and the product was purified by silica column chromatography with a mixture of ethyl aceta te/hexane (1/9 v/v). The product was collected as the second of the total two bands off a silica gel column. The product was isolated by evaporation of solvents in reduced pressure to form an oily residue (HMssEt). Yield = 11.9 g (42%). Rf = 0.36 on silica (3/7 ethyl acetate/hexane). 1H-NMR (CDCl3, ppm) 6.12 (s, 1H, H2C=C-), 5.59 (s, 1H, H2C=C-), 4.35 (s, 4H, -C(O)OCH2CH2O(O)C-), 4.15 (q, 2H,-C(O)OCH2CH3), 2,91 (t, 4H, -CH2SSCH2-), 2.76 (t, 2H, -O(O)CCH2CH2SS-), 2.71 (t, 2H, -SSCH2CH2C(O)O-), 1.94 (s, 3H, H2C=C(CH3)-), 1.26 (t, 3H, -C(O)OCH2CH3). 13C-NMR (CDCl3, ppm): 171.5, 171.3, 166.9, 135.8, 126.0, 62.4, 62.2, 60.7, 34.1, 33.9, 33.2, 32.9, 18.2, 14.1. Mass calculated for (C14H22O6S2Na+): 373.07555. Found: 373.07554.

Synthesis and purification of PEO-b-PHMssEt using ATRP

A procedure for ATRP of HMssEt catalyzed with CuBr/PMDETA complex in the presence of PEO-Br macroinitiator was carried out in anisole at 50 (C. PEO-Br (0.30 g, 0.058 mmol), HMssEt (1.2 g, 3.42 mmol), PMDETA (4.5 mg, 0.026 mmol), and anisole (4.0 mL) were mixed in a 25 mL Schlenk flask. The resulting mixture was deoxygenated by three freeze-pump-thaw cycles. The reaction flask was filled with nitrogen and then CuBr (2.9 mg, 0.02 mmol) was added to the frozen solution quickly. The flask was closed, evacuated with vacuum and backfilled with nitrogen three times. The mixture was thawed and then the flask was immersed in an oil bath preheated at 50 (C to start polymerization. Aliquots were withdrawn at different time intervals to analyze molecular weight by GPC and conversion by 1H-NMR. Polymerization was stopped by cooling and exposing the reaction mixture to air. For purification, the as-synthesized polymer solution was passed through a basic alumina column to remove the copper complex, and then solvents were removed by rotary evaporation. The products were precipitated from hexane three times, and then dried in vacuum oven at room temperature for 18 hrs.

Determination of critical micellar concentration (CMC) by tensiometry

An aliquot of the purified, dried PEO-b-PHMssEt (15 mg) was dissolved in THF (2.5 mL). The clear polymer solution was drop-wise added into deionized water (15 mL). The resulting dispersion was kept under stirring overnight to remove THF, allowing for colloidally stable micellar dispersion at a concentration of 1.0 mg/mL. Then, aliquots of the aqueous stock solution were diluted with different amounts of deionized water, forming a series of aqueous PEO-b-PHMssEt solutions at various concentrations ranging from 10-6 to 1.0 mg/mL. A tensiometer was used to measure the pressure (mN/m) of the solutions as follows; an aliquot of each solution (600 (L) was carefully placed on each well and equilibrated before measurements. The tensiometer was calibrated using air and water.

Aqueous micellization of PEO-b-PHMssEt

For the preparation of micellar aggregates in aqueous solutions, deionized water was added drop-wise into clear polymer solutions consisting of aliquots of the purified, dried PEO-b-PHMssEt dissolved in THF (2.5 mL). The resulting dispersions were kept under stirring for 24 hrs to remove THF. For a micellar dispersion at 1 mg/mL, PEO-b-PHMssEt (15 mg) and water (15 mL) were used.

Transmission Electron Microscopy (TEM)

TEM images were taken using a Philips CM200 HR-TEM, operated at 200 kV electrons and equipped with thermionic LaB6 cathode filament, anti-contamination cold finger, Genesis EDAX system, and AMT V600 2k X2K CCD camera. The point-to-point resolution and the line resolution of the machine are 0.24 nm and 0.17 nm, respectively. To prepare specimens, the micellar dispersion at 1 mg/mL was dropped onto the TEM copper grids (400 mesh, carbon coated). The grids were dried in air.

Thiol-responsive degradation of PEO-b-PHMssEt in DMF

A stock solution of PEO-b-PHMssEt at 10 mg/mL was prepared by mixing PEO-b-PHMssEt (100 mg) with DMF (10 mL). Then, an aliquot of the stock solution (2 mL) was mixed with different amounts of DTT. The mixtures were stirred for 12 hrs to analyze molecular weight of PEO-b-PHMssEt by Agilent GPC/DMF and size and size distribution by DLS.

Thiol-responsive degradation of micellar aggregates in aqueous solution

Aliquots of micellar dispersion (1 mg/mL, 2 mL) were mixed with different amounts of DTT under stirring overnight. An aliquot was taken for DLS measurements. For DLS measurements in DMF, an aliquot of the mixture of aqueous micelles with and without DTT (100 (L) was mixed with DMF (900 (L). For GPC measurements, water was evaporated by rotary evaporation, and the residual polymers were dissolved in DMF/0.1 mole% LiBr.

Thiol-triggered release of NR from NR-loaded micelles

NR-loaded micelles were prepared as follows; a stock solution of NR in THF (5 mg/mL, 316 (L), PEO-b-PHMssEt (15.8 mg), and THF (2.5 mL) were mixed with water (15 mL). After the resulting mixture was stirred for 24 hrs to remove THF, free NR molecules were removed by filtration using a 0.45µm PES filter. The final concentration of PEO-b-PHMssEt was adjusted to be 1.0 mg/mL. The sample was then divided into three equivalent aliquots (3 mL each) in 20 mL vials. To two aliquots were added DTT (0.2 mg, 0.2 equivalents to disulfides, 0.2 mM) and DTT (4.95 mg, 5 equivalents to disulfides, 5 mM) and another aliquot was used as control without DTT under stirring. Their fluorescence spectra ((ex = 480 nm) were measured at different time intervals and fluorescence intensity at 610 nm was monitored.

Results and Discussion

Synthesis of PEO-b-PHMssEt and kinetic studies

First, a new methacrylate monomer bearing a pendent disulfide linkage (HMssEt) was synthesized in two steps. As described in Fig. 1 (upper), HEMA reacted with excess ss-DCOOH in THF through a carbodiimide coupling reaction, resulting in HMss-COOH intermediate. Without purification, the intermediate reacted with excess EtOH, and then column chromatography was used to purify HMssEt at 43% yield. As seen in Fig. 1 (lower), 1H-NMR shows typical peaks: two singlets at 5.6 and 6.2 ppm corresponding to vinyl protons (a) and triplets at 2.9 ppm corresponding to two methylene protons adjacent to disulfide (f, g). The 1H-NMR results, combined with those of 13C-NMR and high resolution mass spectroscopies, confirm the structure of HMssEt.

With successful synthesis and characterization of HMssEt, ATRP of HMssEt catalyzed with CuBr/PMDETA in the presence PEO-Br macroinitiator was carried out in anisole at 50 (C. ATRP conditions include [HMssEt]0/[PEO-Br]0/[CuBr]0/[PMDETA]0 = 60/1/0.35/0.45 and HMssEt/anisole = 0.4/1 wt/wt. For kinetic studies, aliquots were taken to determine monomer conversion using 1H-NMR and molecular weight and molecular weight distribution using GPC. Fig. S1a shows the first-order kinetic plot, reaching over 70% conversion in over 4 hrs. Such fast polymerization is presumably attributed to high concentration of active centers. However, molecular weight increased linearly with conversion and molecular weight distribution remained narrow with Mw/Mn < 1.1. Furthermore, the GPC traces evolved to high molecular weight region over the course of the polymerization (Fig. S1b). These results suggest that ATRP of HMssEt proceeded in a living fashion.

The as-synthesized polymer solutions were purified by passing through a basic aluminum oxide column to remove Cu species and precipitating from hexane to remove unreacted monomers, yielding PEO-b-PHMssEt with Mn = 21,700 g/mol and Mw/Mn =1.1 (Fig. 2a). 1H-NMR allows to determine the degree of polymerization (DP) of PHMssEt block to be 42 from integral ratios of peaks (d/EO, note the DP of PEO = 113), thus suggesting the synthesis of well-defined PEO113-b-PHMssEt42 block copolymer (Fig. 2b).

Aqueous micellization of well-defined PEO-b-PHMssEt

PEO-b-PHMssEt is amphiphilic and can form micellar aggregates consisting of a hydrophobic PHMssEt core containing pendent disulfide linkages surrounded with hydrophilic PEO coronas. To determine critical micellar concentration (CMC) of PEO-b-PHMssEt using tensiometry, a series of aqueous solutions of various concentrations of PEO-b-PHMssEt ranging from 10-6 to 0.1 mg/mL were prepared. As seen in Fig. S3, the pressure does not change at its lower concentration; however with an increasing concentration of PEO-b-PHMssEt the surface pressure began to increase, until a sharp rise around the CMC. From the two equations obtained by fitting each data set to a linear relationship, the CMC of PEO-b-PHMssEt was determined to be 49 (g/mL.

Aqueous micellization through self-assembly of PEO-b-PHMssEt using a solvent-evaporation method yielded colloidally stable micellar aggregates in aqueous solution. The size and morphology of degradable micelles were then examined using dynamic light scattering (DLS) and transmission electron microscopy (TEM) at a micellar concentration of 1.0 mg/mL; well above the CMC (95 (g/mL) (Fig. 3). DLS results indicate a hydrodynamic diameter = 50.9 ± 0.5 nm with a monomodal distribution. TEM images indicate spherical micelles with average diameter = 40.1 ± 6.7 nm, which is smaller than the size determined by DLS. The difference in micellar sizes between DLS and TEM can be attributed to the dehydrated state of the micelles.[56] These results suggest the formation of spherical monomodal micelles having PHMssEt cores surrounded with PEO coronas in water.

Thiol-responsive cleavage of pendent disulfide linkages of PEO-b-PHMssEt in DMF

The well-controlled PEO113-b-PHMssEt42 block copolymer contains pendent disulfide linkages (42 disulfides in each polymer chain). These disulfide linkages can be cleaved through thiol-disulfide exchange reactions in response to DTT (a thiol) in DMF as homogeneous media. Aliquots of PEO-b-PHMssEt were mixed with DTT whose amounts are defined as the mole equivalent ratios of DTT/disulfides. Fig. 4 shows GPC traces in DMF mixed with and without different amounts of DTT after 12 hrs. In the absence of DTT, PEO-b-HMssEt had Mn = 25,000 g/mol with monomodal molecular weight distribution, using GPC with DMF as an eluent. Note that this value is somewhat larger than that (Mn = 21,700 g/mol) determined by GPC with THF as eluent (Fig. 2). In the presence of DTT as the mole equivalent ratio of DTT/disulfide = 0.02/1, the GPC trace of the degraded PEO-b-PHMssEt became bimodal, having both low molecular weight species with Mn (17,000 g/mol and high molecular weight species with Mn ( 63,000 g/mol. When the DTT/disulfide ratio was increased to 0.5/1, the bimodal GPC trace had low molecular weight species with Mn (18,000 g/mol, which is similar to that with the DTT/disulfide = 0.02/1, and high molecular weight species with Mn ( 112,500 g/mol, which is significantly larger than that with the DTT/disulfide = 0.02/1. It should be noted that significant amounts of extremely branched polymers and crosslinked gels could be removed during filtration of polymer solutions prior to injection into GPC. When the DTT/disulfide ratio further increased to 2.5/1, the GPC trace of the degraded PEO-b-PHMssEt shows a monomodal molecular weight distribution with Mn = 21,700 g/mol, which is little bit smaller than that of PEO-b-PHMssEt in the absence of DTT (Mn = 25,000 g/mol). DLS measurements of the same mixtures in DMF were taken and are shown in Fig. 5. The diameter increased from 5.7 nm to 9-11 nm with DTT/disulfide 1/1), most disulfide linkages even including new disulfide crosslinks formed through intermolecular or intramolecular thiol-disulfide polyexchange reactions are cleaved, resulting in predominantly PEO-b-PHMSH containing pendent thiols, leading to decreasing sizes.

Thiol-responsive change in morphologies of micellar aggregates in aqueous solution

Self-assembled micellar aggregates of PEO-b-PHMssEt consist of hydrophobic PHMssEt cores surrounded with hydrophilic PEO coronas. Here, the response of PHMssEt blocks having pendent disulfide linkages in micellar cores to the different amounts of DTT in aqueous solutions was investigated. Fig. 6 show DLS and GPC results of micellar aggregates at different DTT concentrations. In the absence of DTT, the micellar aggregates had a monomodal distribution of both size and molecular weight. In the presence of 0.2 mole equivalent DTT, the DLS diagram shows a bimodal size distribution with a small population of large aggregates (d > 1 (m). The formation of such large aggregates could be attributed to destabilization of micelles caused by the cleavage of pendent disulfide linkages in micellar cores. Similar to the results in DMF, GPC traces of PEO-b-PHMssEt as micellar aggregates after the removal of water also show a bimodal distribution with an occurrence of highly branched or crosslinked polymers with Mn ( 887,000 g/mol. These high molecular weight species could be formed by thiol-disulfide polyexchange reactions in micellar cores. To further examine the formation of crosslinked micelles in the presence of 0.2 mole equivalent DTT, the aqueous micellar dispersion with and without 0.2 mole equivalent DTT were mixed with DMF, a good solvent to both PEO and PHMssEt blocks. As seen in Fig. S4, DLS results indicates the diameter ( 20 nm for the micelles with 0.2 mole equivalent DTT due to the occurrence of core-crosslinking, while < 3 nm for the micelles without DTT. These DLS and GPC results suggest the formation of mainly crosslinked micelles in the presence of ................
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