Biodegradable Glycopolymeric Micelles Obtained by RAFT-controlled

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Biodegradable Glycopolymeric Micelles Obtained by RAFTcontrolled Radical Ring-Opening Polymerization Sylvia Ganda,† Yanyan Jiang,† Donald S. Thomas,‡ Jeaniffer Eliezar,† and Martina H. Stenzel*,† †

Centre for Advanced Macromolecular Design, School of Chemistry, ‡NMR Facility, Mark Wainwright Analytical Centre, The University of New South Wales, UNSW, Sydney, NSW 2052, Australia S Supporting Information *

ABSTRACT: The design and synthesis of an entirely degradable glycopolymer micelle was presented. This design relies on the utilization of RAFT-controlled radical ring-opening polymerization (rROP) technique to afford multiple insertions of cleavable ester linkages onto the backbone of the corona. RAFT polymerization using a macroRAFT agent based on poly(ε-caprolactone) PCL was employed to control the polymerization of well-defined statistical glycopolymers of 1-O-acryloyl-2,3:4,5-di-Oisopropylidene-β-D-fructopyranose (1-O-AiPrFru) and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) monomer. Three block copolymers were synthesized to generate poly(ε-caprolactone)-b-poly[(1-O-acryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose)-co-(5,6-benzo-2-methylene-1,3-dioxepane)] (PCL-b-P[(1-O-AiPrFru)-co-(BMDO)]) with varying block lengths. Selfassembly of the deprotected block copolymers generated nonspherical egg shaped micelles, where the shorter chains PCL106-bP[(1-O-AFru)69-co-(BMDO)9] underwent self-assembly forming micelles with the hydrodynamic diameter (DH) of 106 nm. The biodegradation of these micelles were investigated via enzymatic degradation by Lipase Pseudomonas sp., indicating entirely degradable architectures, which are no longer visible via dynamic light scattering (DLS). SEC further confirmed the appearance of fragmented glycopolymeric units. In vitro cell proliferation assay of the micelles and their degradation products revealed no toxicity against healthy human fibroblast HS27 and breast cancer MDA-MB-231 cell lines. The polymer concentration range tested was up to 0.20 mg·mL−1 with the cell viabilities of ≥95%.



INTRODUCTION Glycopolymers as synthetic materials bearing natural carbohydrate-based derivatives have been long known and explored to deliver therapeutics in a targeted fashion. The structure of glycopolymers that mimic natural carbohydrates in biological systems offer myriad advantages. Carbohydrates are involved in many of the essential biological processes related to reproduction, inflammation, signal transmission, and biomolecular recognition events such as cell differentiation and infection. Building upon this foundation, carbohydrate-directed targeting has been used as a classic approach in promoting active targeting by utilizing the carbohydrate ligands that are recognized by the carbohydrate-binding proteins (receptors) on cell surfaces known as lectins.1 This activity induces cell− cell interactions, mediating an increase in cellular uptake via receptor-endocytosis.2 The prominent feature of cell recognition mediated by carbohydrate-based therapeutics draws major attention as a potential agent for targeted drug delivery. Although a single interaction is considerably weak, the advantages of using glycopolymers as drug carriers can be © XXXX American Chemical Society

extended through a collective binding effect, also known as cluster glycoside ef fect caused by multivalent ligands binding with lectins.3,4 Carbohydrate-coated nanoparticles accommodate extended advantages by not only promoting cell recognition as naturally occurring polysaccharide derivatives, but also in the ability to stimulate active targeting with carbohydrate binding proteins and transporters. A plethora of carbohydrate decorated nanoparticles have been demonstrated to promote selective and active targeting.5−8 Despite significant advances from synthetic and conceptual viewpoints followed by the promising outcomes of carbohydrate-based nanocarriers, the persisting issue of nondegradability of the glycopolymers may cause unwanted immune responses and toxicity, eventually preventing their translation toward in vivo application. The biomaterials intended for biomedical applications require definite biocompatibility and biodegradability.9 Previous studies reported Received: February 4, 2016 Revised: April 25, 2016

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DOI: 10.1021/acs.macromol.6b00266 Macromolecules XXXX, XXX, XXX−XXX

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the synthesis of partially biodegradable amphiphilic glycopolymer micelles comprising degradable poly(ε-caprolactone) block and various sugar moieties focusing on the delivery and release of doxorubicin in vitro,10 and the interaction between Concanavalin A lectin receptor that binds with different carbohydrate ligands.11 Ever since the pioneering work of Bailey and co-workers,12 cyclic monomers, specifically cyclic ketene acetals (CKA)s, have attracted extensive research interests in the use of radical ringopening polymerization (rROP) technique to incorporate degradable ester groups onto the polymer backbone enabling complete degradation. This approach was suggested to be the only pathway accessible to allow for the integration of ester units along the aliphatic backbone of vinyl polymers in a random order.13,14 However, alike the inherent characteristic of free radical polymerization (FRP), rROP requires an extra layer of control mechanism to enable the design and synthesis of well-defined complex macromolecular architectures with narrow molecular weight distribution. For this reason, various methods of reversible deactivation radical polymerization (RDRP) have been explored and implemented onto rROP to promote controlled-living polymerization characteristics to a library of cyclic ketene acetal monomers, such as 5,6-benzo-2methylene-1,3-dioxepane (BMDO). Among them include nitroxide-mediated polymerization (NMP),15−17 atom-transfer radical polymerization (ATRP)18−24 and reversible addition− fragmentation chain transfer (RAFT) polymerization.20,25−30 The investigation into the homopolymerization and copolymerization of CDRP mediated rROP have established an avenue in the design of new hydrolytically and enzymatically degradable materials. RAFT technique is widely established for its robustness and versatility in generating a high degree of structurally functional polymers with well-defined architecture and has been demonstrated excessively in the literature.31 The feasibility of RAFT to control rROP was demonstrated for the first time by Pan et al., reporting the quantitative conversion of BMDO resulting in aliphatic polyester with good control.26 The copolymerization of BMDO monomer via RAFT technique with common vinyl monomers such as methyl methacrylate,25 N,N-isopropylacrylamide,20 vinyl acetate,28 and poly(ethylene glycol methyl ether methacrylate) (PEGMA)32 has also been reported to successfully insert multiple hydrolytically degradable ester bonds on the polymeric backbone. Xiao et al. reported the synthesis of potentially degradable glycopolymers of 1,2:3,4-di-O-isopropylidene-6-O-(2′-formyl-4′-vinylphenyl)33 D-galactopyranose and BMDO via RAFT polymerization. In this work, we present the design of glycopolymer micelles that are fully degradable opening opportunities to the design of new nanoparticles for drug delivery. To our knowledge, this is the first report of a fully degradable glycopolymer micelle prepared with radical polymerization. The approach is based on the use of RAFT-mediated radical ring-opening polymerization of 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) and isopropylidene protected 1-O-acryloyl-β-D-fructopyranose using poly(ε-caprolactone) (PCL) macroRAFT agent. The incorporation of BMDO units into the main chain forms degradable links that can be degraded via enzymatic degradation with enzyme lipase Pseudomonas sp. In addition, the cytotoxicity against healthy human fibroblast HS27 and breast cancer MDA-MB-231 cell lines was investigated.

Article

EXPERIMENTAL SECTION

Chemicals. All chemicals were reagent grade purchased from Sigma-Aldrich and used as received unless stated otherwise. Deuterated NMR solvents (CDCl3, DMSO-d6, D2O and toluene-d8) were purchased from Cambridge Isotope Laboratories. 1-O-acryloyl2,3:4,5-di-O-isopropylidene-β-D-fructopyranose, 5,6-benzo-2-methylene-1,3-dioxepane (BMDO)34 and poly(ε-caprolactone)35 were synthesized as described previously with slight modifications. BMDO and ε-caprolactone monomers were distilled under reduced pressure. Anhydrous toluene was obtained from PureSolv MD 7 solvent purification system (Innovative Technology, Inc., Galway, Ireland) packed with activated alumina columns to remove water and trace impurities. General Procedures. Size Exclusion Chromatography (SEC). SEC was carried out using a Shimandzu modular system containing a DGU-12A degasser, a LC-10AT pump, a SIL-10AD automatic injector, a CTO-10A column oven, and a RID-10A differential refractive index detector. A PL 5.0 μm bead-size guard column (50 × 7.5 mm2) followed by four 300 × 7.8 mm linear PL (Styragel) columns (105, 104, 103 and 500 Å pore size) were used for the analyses. N,N-dimethylacetamide [DMAC, HPLC grade; 0.03% w/v LiBr, 0.05% 2,6-dibutyl-4-methylphenol (BHT)] with a flow rate of 1 mL·min−1 was used as the mobile phase with an injection volume of 50 μL at 50 °C. The unit calibration was conducted over commercially available narrow molecular weight distribution polystyrene standards (0.5−1000 kDa, Polymer Laboratories). Chromatograms were processed using Cirrus 2.0 software (Polymer Laboratories). Nuclear Magnetic Resonance (NMR) Spectroscopy. NMR was performed using either a Bruker Avance III 300, 5 mm BBFO probe (1H: 300.17 MHz, 13C: 75.48 MHz) or an Avance III 400, 5 mm BBFO probe (1H: 400.13 MHz, 13C: 100.62 MHz). NMR spectra were processed using either the Bruker TOPSPIN 3.2 software or MesRetNova NMR software. Samples were analyzed in either CDCl3, DMSO-d6 or D2O, except for the NMR in situ kinetics experiments where toluene-d8 was used as the polymerization solvent and lock material. All chemical shifts are stated in parts per million ppm (δ) relative to tetramethylsilane (δ = 0 ppm), referenced to the chemical shifts of residual solvent resonances (1H and 13C). Data is reported as follows: chemical shift [multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiplet), integration is reported as multiples of protons, proton identity, J (denotes the coupling constants and is measured in Hertz)]. Dynamic Light Scattering (DLS). These measurements employed a Malvern Zetasier Nano ZS instrument equipped with a 4 mV He−Ne laser operating at λ = 632 nm and noninvasive backscatter detection at 173°. Measurements were carried out in a disposable cuvette at 25 °C, provided 15 equilibration period prior to each set of measurements. For a given sample, a total of five measurements were conducted with the number of runs, attenuator, and path length being automatically adjusted by the instrument, depending on the sample quality. Transmission Electron Microscopy (TEM). This was carried out on a JEOL 1400 TEM with the beam voltage of 100 kV and a Gatan CCD for acquisition of digital image. Samples were prepared by depositing 1 drop of the solution mixture onto a copper grid. The grids were airdried and negatively stained with uranyl acetate (UA) solution for 5 min. Cell Culture. MDA-MB-231 (breast cancer) and HS27 (human fibroblast) cell lines were grown as monolayer cultures in cell culture flasks by using RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate and 1% of L-Glutamine-PenicillinStreptomycin solution (with 200 mM L-glutamine, 10,000 units penicillin and 10 mg·mL−1 streptomycin in 0.9% NaCl, sterile filtered). Penicillin was added as an antibiotic. Cell cultures were grown in a humidified atmosphere at 5% CO2 at 37 °C. The medium was routinely changed every 3 days. For cell subculture, cells grown in monolayer were released by washing with phosphate buffered saline (PBS) and detached by trypsin/EDTA treatment after they have reached confluence. The cells were then collected, centrifuged and resuspended in the new culture medium. B

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11.8 Hz), 4.38 (dd, 1H, 5, 3J5,6 = 2.6 Hz), 4.27 (m, 1H, 7), 4.16 (dd, 1H, 8′, 2J8′,8 = 11.8 Hz), 3.87 (m, 1H, 4), 1.57, 1.51, 1.41, 1.37 (4s, 12H, 4 × 9) (Figure S1, Supporting Information). Synthesis of 5,6-Benzo-2-(bromomethyl)-1,3-dioxepane.34 A 10.0 g sample of o-benzene dimethanol (72.4 mmol, 1.0 equiv) and 12.3 g bromodimethyl acetaldehyde (72.4 mmol, 1.0 equiv) were mixed in a predried nitrogen flask equipped with a Claisen bridge under argon atmosphere. Then, 0.16 g of p-toluenesulfonic acid (0.93 mmol) was added, and the mixture was heated to 120 °C for 18 h. Following, the product obtained was of dry solid and was left to cool down. The crude solid was dissolved in 60.0 mL of CHCl3 and washed with 30.0 mL of saturated NaHCO3− solution and water. The excess water was removed by drying over MgSO4. The solution was then filtered and solvent removed by evaporation under reduced pressure. The product obtained was recrystallized from 0.1 L cyclohexane to yield 14.5 g (82%). 1H NMR (300.17 MHz, CDCl3), δ (ppm): 7.16−7.24 (m, 4H), 5.12 (t, 1H, 3J = 5.12 Hz), 4.93 (d, 4H, 3J = 1.8 Hz), 3.46−3.44 (d, 2H, 3J = 5.2 Hz). Synthesis of 5,6-Benzo-2-methylene-1,3-dioxepane (BMDO).34 First, 14.47 g of 5,6-benzo-2-(bromomethyl)-1,3-dioxepane (59.54 mmol, 1 equiv) was dissolved in 100.0 mL of tert-butanol in a predried round-bottom Schlenk flask under nitrogen. Then, 6.83 g of potassium tert-butylate (60.86 mmol, 1.0 equiv) was added and the solution was heated to reflux for 18 h. The reaction mixture was left for cooling before the addition of 100.0 mL of Et2O to establish separation and filtered through vacuum filtration. The solvents were then evaporated with the resulting oil distilled at reduced pressure (2.6 mbar) at 107 °C to yield colorless liquid which solidified to form white crystal solids on standing. 1H NMR (400.13 MHz, toluene-d8), δ (ppm): 3.92 (s, 2H, B1), 4.76 (s, 4H, B2) 6.57−6.59 (m, 2H, Ar), 6.91−6.93 (m, 2H, Ar) (Figure S2, Supporting Information). Synthesis of Poly(ε-caprolactone) Macro-RAFT Agent.35 Poly(εcaprolactone) macro-RAFT agent was synthesized by adopting a procedure previously conducted by Bourissou et al. with slight modifications. The monomer ε-caprolactone was predistilled prior to reaction and anhydrous toluene was obtained from a MBraun solvent purification system packed with aluminum oxide reactor filter column. 0.933 g of εcaprolactone (8.18 mmol, 80.0 equiv) was added into a Schlenk flask equipped with a magnetic stirrer bar that was previously flame-dried under vacuum. Benzyl 2-hydroxyethyl carbonotrithioate (BHCT)37 RAFT agent (0.102 mmol, 1 equiv) as the initiator and anhydrous toluene were then added to the flask under inert atmosphere and mixed. The solvent was then removed under reduced pressure via rotary evaporator and was repeated for three times until all the solvent was removed. Subsequently, methanesulfonic acid (0.307 mmol, 3 equiv) was added to the flask under N2 flow to catalyze the reaction, followed by the addition of anhydrous toluene (0.5 M). The reaction flask was then immersed into a preheated oil bath at 30 °C and left to react under N2 atmosphere for 4 h. Finally, the reaction was quenched by the addition of N,N-diisopropylethylamine in excess. Synthesis of Poly[(1-O-AiPrFru)-co-(BMDO)] via RAFT Polymerization. Below is provided a typical procedure for the statistical radical ring-opening RAFT polymerization of 1-O-acryloyl-2,3:4,5-di-Oisopropylidene-β-D-fructopyranose (1-O-AiPrFru) monomer with 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) monomer. This general procedure was employed for a series of statistical glycopolymer syntheses by varying the monomer feed ratios. 1-O-Acryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (1-OAiPrFru) (0.307 g, 0.976 mmol), benzyl 2-hydroxyethyl carbonotrithioate RAFT agent (BHCT, 0.057 g, 0.00610 mmol), and 1,1′azobis(cyclohexanocarbonitrile) (VAZO88, 0.3 × 10−3 g, 1.22 × 10−3 mmol) were added into a Schlenk flask that was previously flamedried, equipped with a magnetic stirrer bar under N2 flow. The flask was transferred into the glovebox and 5,6-benzo-2-methylene-1,3dioxepane (BMDO, 0.0396 g, 0.244 mmol) was added, followed by the addition of 0.60 mL of anhydrous anisole to give [1-O-AiPrFru]0: [BMDO]0:[BHCT]:[VAZO88] = 80:20:1.0:0.1 with the total concentration of 2.0 M. The flask was then sealed with a glass stopper and deoxygenated by 3 cycles of freeze−pump−thaw until all

Cytotoxicity Test and SRB Assay. The cytotoxicity of glycopolymer micelles and their degradation products were determined by a standard sulforhodamine B colorimetric proliferation assay (SRB assay). After incubation with the enzyme, the degradation products were heated to 85 °C for 15 min in order to deactivate the enzyme. MDA-MB-231 (human breast carcinoma) and HS27 (human fibroblast noncancerous) cell lines were seeded at the corresponding density of 4,000 cells per well in 96-well microtiter plates followed by the addition 100 μL of RPMI-1640 culture medium per well and incubated at 37 °C in a 5% CO2 for 24 h. The specimens were sterilized by UV irradiation for 20 min before serially diluting (sequential 2 × dilution) with sterile water. For the cytotoxicity assay, the medium in the cell culture plate was discarded, and 100 μL of fresh 2 × concentrated RPMI-1640 medium was added to each well of the plate. The micelles and degradation products were added correspondingly into the plates at 100 μL per well. Sterile water was added to the nontreated cells as a control. The cells were incubated with the micelles for 48 h, and the cell viability was determined using SRB assay. The incubation with micelles was terminated by the addition of cold trichloroacetic acid (TCA) (10% w/v) for 30 min at 4 °C. After a complete washing with distilled water (5 times), the TCA-fixed cells were stained with 100 μL of 0.4% w/v sulforhodamine B (SRB) solution in 1% acetic acid (w/v) for 15 min. After staining, unbound dye was removed by washing with 1% acetic acid for five times, and plates were air-dried. Finally, the SRB was solubilized with 200 μL of 10 mM unbuffered Tris base to dissolve bounded dye, and the optical density was determined by using a multiwell scanning spectrophotometer at the wavelength of 490 nm. Dose−response curves were plotted accordingly where the values were expressed as percentage of control (nontreated cells were used as controls). The optical density was used to calculate cell viability.

cell viability (%) =

OD490,sample − OD490,blank OD490,control − OD490,blank

× 100

Synthesis. Protection of D-Fructose. Synthesis of 2,3:4,5-Di-Oisopropylidene-β-D-fructopyranose.36 Dry and finely powdered Dfructose (5.0 g, 27.75 mmol) was added to a cooled mixture of concentrated sulfuric acid (5.0 mL) and acetone (100.0 mL) in an Erlenmeyer flask. The suspended mixture was left stirring on a magnetic stirring plate at room temperature until all the sugar had dissolved. The solution was left sitting at room temperature for an additional 80 min before cooling with ice. Following, an ice-cold solution of 2.75 M NaOH (15.3 g, 1.1 equiv) was added gradually upon stirring. The crude product was a crystalline solid, isolated via recrystallization by dissolving in boiling ether, cooling and adding hexane to give the final product of rosette of needles. 1H NMR (300.17 MHz, DMSO-d6) δ (ppm): 5.06 (t, 1H, J = 5.8 Hz), 4.56 (dd, 1H, H-4, 3J4,3 = 2.5 Hz), 4.27 (d, 1H, H-3, 3J3,4 = 2.5 Hz), 4.21 (dd, 1H, H-5), 3.73 (dd, 1H, H-2, 2J2,2′ = 4.9 Hz), 3.53 (dd, 1H, H-2′, 2J2′,2 = 4.5 Hz), 3.39 (m, 2H, H-6), 1.44, 1.34, 1.27 (4s, 12H, 4 × CH3). Glycosylation of Protected D-Fructose Derivative. Synthesis of 1O-Acryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (1-OAiPrFru). First, 3.0 g (11.53 mmol) of 2,3:4,5-di-O-isopropylidene-βD-fructopyranose and 3.23 mL of triethylamine (Et3N, 23.05 mmol) were added to a three-neck round-bottom flask equipped with a magnetic stirrer bar and dissolved in anhydrous dichloromethane (270.0 mL) under argon atmosphere and cooled in an ice bath. Acryloyl chloride (1.44 mL, 5.85 mmol) dissolved in 90.0 mL of dry DCM was added dropwise with stirring under argon flow. The solution was allowed to warm to room temperature overnight. The reaction mixture was poured into ice-cold water and extracted three times with DCM (270.0 mL) and dried over Na2SO4. The solvent was removed by rotovapping to give a sticky orange residue, which was purified by silica gel column chromatography by using a mixture of ethyl acetate:n-hexane = 1:2 v/v % as the eluent to obtain maximum separation. The product fractions were combined and solvent removed under reduced pressure to yield pure sticky clear gel glycomonomer. 1 H NMR (300.17 MHz, CDCl3), δ (ppm): 6.51 (dd, 1H, 1, 2J1, 1′ = 1.4 Hz), 6.19 (dd, 1H, 3, 3J3, 1,1′ = 10.4 Hz), 5.88 (dd, 1H, 2, 2J1′,1 = 1.4 Hz), 4.64 (dd, 1H, 6, J = 7.9, 3J6,5 = 2.6 Hz), 4.53 (dd, 1H, 8, 2J8,8′ = C

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Scheme 1. Synthetic Pathway of the Degradable Glycopolymers, i.e., Poly(ε-caprolactone)-b-poly[(1-O-acryloyl-β-Dfructopyranose)-co-(5,6-benzo-2-methylene-1,3-dioxepane)] (PCL-b-P[1-O-AFru-co-BMDO]), Followed by Self-Assembly to Degradable Micelles

the dissolved gas was removed. The flask was then immersed in an oil bath preheated to 90 °C and left to react for 24 h. The polymerization was stopped by immersing the tube into an ice bath and introduced to air. The crude sample was diluted in CHCl3 and precipitated for 3 times into chilled methanol and isolated by centrifugation before drying in vacuo to obtain pure statistical glycopolymer. Real-Time 1H and 1H−13C HSQC NMR Monitoring of RAFT Statistical Copolymerization Kinetics. All the kinetic experiments including 1H and 1H−13C HSQC NMR spectra respectively were recorded on a Bruker Avance III 400 MHz spectrometer equipped with an ultrashielded magnet and BBFO probe. Tetramethylsilane (TMS) was used as internal standard. 1H NMR data were acquired with 8 scans with a relaxation delay of 10 s. Meanwhile, 2D NMR data were acquired with 1 scan and 2048 points in t2, and the number of increments for t1 was 128. Below is a typical procedure for the online

monitoring of RAFT statistical copolymerization kinetics via NMR spectroscopy. The statistical copolymerization was performed under a similar procedure as described previously, unless for the substitution of anhydrous anisole as the polymerization solvent to toluene-d8. Upon the addition of BMDO monomer into the reaction mixture in the glovebox, the reaction mixture was transferred into a J-Young NMR tube and sealed under N2 and taken out of the glovebox. The solution was deoxygenated via 3 cycles of freeze−pump−thaw. The NMR tube was then subjected to in situ online NMR kinetics experiment carried out on a Bruker Avance III 400 MHz spectrometer equipped with an ultrashielded magnet and BBFO probe. Tetramethylsilane (TMS) was used as internal standard. The magnet was preheated to 90 °C before the tube was injected and the reaction started. Polymerization kinetics were monitored and quantified by comparing the integral ratio of D

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Figure 1. 1H NMR spectra (400.13 MHz, toluene-d8) of the online in situ kinetics experiment of RAFT statistical copolymerization of 1-O-AiPrFru and BMDO monomers with [1-O-AiPrFru]0:[BMDO]0:[CTA]:[VAZO88] = 65:35:1:0.1 at 90 °C for 15.4 h. polymeric to monomeric peaks accordingly with respect to time. The polymerization was quenched by immersing the tube into an ice bath and exposing it to air. The crude sample was purified and glycopolymer isolated according to the similar procedure described previously. Synthesis of Block Copolymer Poly(ε-caprolactone)-block-poly[(1-O-AiPrFru)-co-(BMDO)]. 1-O-Acryloyl-2,3:4,5-di-O-isopropylideneβ-D-fructopyranose (1-O-AiPrFru) (0.307 g, 0.976 mmol), poly(εcaprolactone) (PCL)106, MnTheo = 12 084 Da (0.057 g, 0.00610 mmol) as macro-RAFT agent, and 1,1′-azobis(cyclohexanocarbonitrile) (VAZO88, 0.3 × 10−3 g, 1.22 × 10−3 mmol) were added into a previously flame-dried Schlenk flask equipped with a magnetic stirrer bar. The Schlenk flask used was previously evacuated and refilled with N2 gas for 3 cycles on the Schlenk line upon drying by flaming with a carbon monoxide flame torch. Following, the flask was evacuated

before transferring it into the glovebox. 5,6-Benzo-2-methylene-1,3dioxepane (BMDO, 0.0396 g, 0.244 mmol) was added into the flask upon the addition of 0.600 mL of anhydrous Anisole to give a[1-OAiPrFru]0:[BMDO]0 [macroRAFT]:[VAZO88] = 80:20:1.0:0.1 and [M] = 2.0 M. The tube was sealed with a glass stopper and deoxygenated by 3 cycles of freeze−pump−thaw until all the dissolved gas was removed. The flask was then immersed in an oil bath preheated to 90 °C for 24 h. Following, the flask was immersed into an ice bath and exposed to air in order to stop the polymerization. The crude sample was precipitated for 3 times into chilled methanol and isolated by centrifugation before drying in vacuo to obtain pure block copolymer subjected toward characterization. Removal of Isopropylidene Protecting Groups. The removal of the isopropylidene protecting groups from poly(ε-caprolactone)-b-poly[(1-O-acryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose)-coE

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Figure 2. Plot of (a) monomer conversion and (b) total monomer conversion vs time for copolymerization of 1-O-AiPrFru and BMDO at different feed ratios [1-O-AiPrFru]0:[BMDO]0 = 80:20 (◆) and 65:35 (○) monitored via in situ NMR experiments for 19 h at 90 °C. (c) SEC traces (DMAC eluent, cal. to PMMA standard) of a series of statistical glycopolymers synthesized via RAFT polymerization with varying comonomer feed ratios, i.e. [1-O-AiPrFru]0:[BMDO]0 = [100]:[0] (black line); [80]:[20] (blue line); [75]:[25] (green line); [65]:[35] (red line).



(5,6-benzo-2-methylene-1,3-dioxepane)] block copolymer is described below. This deprotection approach was applied to all the other glycopolymers bearing the isopropylidene protected fructose carbohydrate pendants. A typical deprotection for glycopolymers synthesized via ring-opening RAFT copolymerization is described as following. The 0.080 g of glycopolymer obtained from previous copolymerization(s) was dissolved into 1.50 mL of 9:1% v/v TFA/H2O in a vial equipped with a magnetic stirrer bar. The mixture was left stirring at room temperature for 30 min. Dialysis was then performed on the glycopolymer solution against deionized water for 48 h through a cellulose membrane MWCO 12,000. The deprotected glycopolymer was then lyophilized to yield a colorless low density solid. Self-Assembly of Block Copolymers. First, 4.0 mg of poly(εcaprolactone)-b-poly[(1-O-acryloyl-β- D -fructopyranose)-co-(5,6benzo-2-methylene-1,3-dioxepane)] was added to a glass vial equipped with a magnetic stirrer bar. 2.0 mL of DMSO was added into the vial and mixed until all the glycopolymer was dissolved. The vial was capped with a rubber septum and placed in a water bath heated to 20 °C. The temperature was maintained constant throughout the entire period of micellization. 2.0 mL of DI water was added dropwise via a syringe pump with the flow rate of 0.20 mL/h with the solution left stirring for 10 h. The micellar solution was then dialyzed against DI water for 2 days to remove the organic phase. The size of micelles formed was measured by DLS measurement to obtain the hydrodynamic volume (DH), followed by TEM to investigate the shape and size of the micelle in the dry state. Enzymatic Degradation of Nanoparticles. In a typical degradation experiment, 2.0 mg of lipase Pseudomonas sp. (≥22 units/mg solid) was dissolved in 1.0 mL of 0.10 M phosphate buffer solution prepared at pH 7.0. 0.50 mL of the enzyme solution was added into 0.50 mL of micellar solution (0.60 mg·mL−1) and incubated at 37 °C for 23 h (unless stated otherwise). Enzymatic degradation of the micelles was observed by DLS of the solution. Online in situ DLS measurements were also carried out to follow the disintegration of micelles by continuously measuring for 90 min (1 min per measurement with no delay between measurements) at 37 °C. Finally, the solution was lyophilized and degradation products isolated by extraction with DMAC and analyzed by size exclusion chromatography.

RESULTS AND DISCUSSION Polymer Synthesis. The multiple insertion of main chain ester backbones was introduced by RAFT-controlled radical ring-opening polymerization of BMDO monomer to yield welldefined macromolecular architecture (Scheme 1). The CKA comonomer, i.e., 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) is comprised of a 7-membered ring, an acetal and vinyl functional group, and was prepared following a prescribed procedure previously conducted by Landfester and co-workers34 with slight modifications. Dehydration reaction from ortho-benzene dimethanol by bromodimethyl acetaldehyde under inert atmosphere yielded the bromide derivative 5,6benzo-2-(bromomethyl)-1,3-dioxepane as a crystalline solid upon recrystallization from cyclohexane at 82% yield. BMDO monomer was subsequently synthesized via reduction reaction of this analogue under dry and inert atmosphere. This is a critical step due to BMDO monomer being highly susceptible to hydrolysis by protonation in the presence of acid and water molecules. Precipitation in diethyl ether gave the desired product as suspended oil, thus requiring monomer purification by distillation under reduced pressure to obtain BMDO monomer as white crystalline solid at 85% purity. The final product was characterized by 1H and 13C NMR spectroscopy (Figure S2−S3, Supporting Information). In order to examine the reactivity of both comonomers and the versatility of RAFT polymerization in controlling rROP, a series of statistical copolymerizations with varying comonomer feed ratios was carried out. It is noteworthy that the acetal framework of BMDO monomer is highly electrophilic, thus making it extremely susceptible toward hydrolysis via nucleophilic attack, leading to protonation of the vinyl bond generating an undesired byproduct with a ring-opened structure that can no longer be polymerized.13,25,38 Shown in Figure 1 is an overlay of a series of 1H NMR spectra acquired from the F

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Table 1. Synthesis of Statistical Glycopolymers Based on 1-O-AiPrFru and BMDO Monomers via RAFT Polymerization and the Solubility of the Resulting Copolymers at a Monomer Concentration of 1.0 mg·mL−1 a SECb

monomer feed

solubility

entry

[1-O-AiPrFru]

[BMDO]

Mn (Da)

Đ

water

DMSO

phosphate buffer

1 2 3 4

100 80 75 65

0 20 25 35

9700 6800 10 300 8000

1.21 1.14 1.35 1.14

yes yes yes yes

yes yes yes yes

yes yes yes yes

c

d

Each polymerization was carried out at 90 °C for 24 h, in the presence of VAZO88 as the initiator and BHCT CTA; [M]0:[BHCT]:[VAZO88] = [100]:[1]:[0.1]. bSEC analyses were obtained by using DMAc as the mobile phase, calibrated to PMMA standard. cConversion is >90%; d Conversion is ∼40% a

Figure 3. TEM images of (a) the self-assembly of statistical glycopolymers P(1-O-AFru)50-co-P(BMDO)3 forming nanoparticles with the mean diameter of 85 nm. (b) Higher concentration solution formed aggregation at 1.0 mg·mL−1.

susceptible to side reactions. A number of different studies in the literature have also reported similar findings supporting this outcome where reversible chain-transfer catalyzed polymerization (RTCP) techniques19,20 and free radical polymerization39,40 were employed. Four polymers with BMDO feed ratio from f BMDO = 0 to 35 mol % were isolated after 24 h and purified. The resulting glycopolymers exhibit narrow dispersities (Đ) within 1.14−1.35 (Table 1). It is important to note that the presence of traces of water can result in the hydrolysis of BMDO monomer, generating byproducts with the structure depicted in Figure S7(a), Supporting Information. Therefore, the incorporation of ester units on copolymer backbone was further confirmed by the integration of the 2 protons peak adjacent to the ester functional group at δ = 5.06−5.4 ppm (“4”). The resulting polymers were subsequently deprotected, and the removal of the isopropylidene groups was confirmed by NMR. The resulting copolymers were tested in regards to their water-solubility as the hydrophobic BMDO may lower the hydrophilicity of the glycopolymer. The solubility of these glycopolymers was therefore tested using three different polar solvents, such as pure water, dimethyl sulfoxide (DMSO) and phosphate buffer solution, depicting good solubility with up to 35 mol % of BMDO in the monomer feed (Table 1, entry 4). Despite the apparent water-solubility, the hydrophobic BMDO clearly introduced amphiphilicity to the polymer as it is evident from the TEM images as shown in Figure 3, parts a and b, using poly[(1-O-AFru)50-co-(BMDO)3] (Table 1, entry 3). This behavior suggested that the glycopolymers formed reorganized and underwent aggregation to form dense hydrophobic domains surrounded by a corona of swollen loops formed by the hydrophilic parts of the copolymer. Synthesis of Block Copolymers and Self-Assembly. Although the statistical copolymers were already capable of nanoparticle formation, the combination with a hydrophobic

statistical copolymerization with the monomer feed ratio of [1O-AiPrFru]0:[BMDO]0 = [65]:[35]. Each polymerization was conducted under dry and inert environment in the presence of 1,1′-azobis(cyclohexanecarbonitrile) (VAZO88) as the initiator and benzyl 2-hydroxyethyl carbonotrithioate (BHCT) as the chain transfer agent (CTA). A detailed study of the copolymerization kinetics was performed and monitored via in situ NMR experiment where toluene-d8 was used as the reaction solvent and to lock the NMR signal. NMR spectra were recorded for 19 h via 1H and 2D 1H−13C HSQC NMR spectroscopy. A gradual decrease in both the monomer concentrations is evident from the loss of intensity of the characteristic peaks of the monomers, i.e., the acrylate peaks (CH2CH) of 1-O-AiPrFru monomer labeled “F1”, “F2”, and “F3” at δ = 6.3−5.3 ppm, followed by the vinyl peak of BMDO monomer labeled “B1” (CH2) at δ = 3.7 ppm (Figure 1(bottom)). The radical ring-opening polymerization of BMDO monomer was clearly observed from the decrease of the vinyl bond signal (CH2) at δ = 3.7 ppm (labeled “B1”), followed by the appearance of the broad polymer peak in the range of δ = 5.06−5.4 ppm labeled “4” (Figure 1 (top)) in the spectra. The conversion of BMDO monomer throughout the polymerization was monitored and quantified based on the integration of the vinyl bond (“B1”). The conversion for both monomers over 19 h was recorded for feed ratios of f BMDO = 20 mol % and f BMDO = 35 mol % (Figure 2a), respectively, revealing the significantly slower consumption of BMDO. The amount of BMDO in the initial feed did not show a major impact on the total monomer conversion and polymerization rate (Figure 2b) with 1-OAiPrFru reaching almost complete conversion while the conversion of BMDO levels off at 40%. It seems that the increasing concentration of BMDO increases the rate of polymerization. However, this might solely be the results of various factors such as impurities as the BMDO monomer is G

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these block copolymers lie within 1.18−1.40, exhibiting a good control over molecular weight distribution by RAFT polymerization. The higher dispersity value block copolymers PCL106-bP[(1-O-AiPrFru)69-co-(BMDO)9] (Đ = 1.29) indicated the presence of a minor branching on the backbone due to backbiting leading to the formation of side chains.18 This is also evident from the SEC chromatogram displaying monomodal molecular weight distribution with a higher molecular weight shoulder depicted as dashed line in Figure 5. The deprotection of block copolymers was performed under acidic environment in order to remove the isopropylidene protecting groups prior to self-assembly forming micelles. The elimination of the protecting groups is noticeably marked by the disappearance of the isopropylidene peaks between δ = 1.2−1.6 ppm, accompanied by a change in solubility. It should be noted here that SEC analysis of the deprotected block copolymers is not available due to the disparate solubility of both blocks. NMR analysis however suggests that the BMDO esters were not cleaved during hydrolysis in acidic conditions, indicated by the presence of the peak at δ = 5.0−5.3 ppm which belongs to the 2 protons adjacent to the ester bond corresponding to the aliphatic polyester structure. It is widely established that well-defined amphiphilic block copolymers are capable of undergoing self-assembly forming nanoparticles with various morphologies in aqueous solution as a consequence to the unfavorable interaction between the hydrophobic segments and the surrounding aqueous environment. According to the protocol introduced by Eisenberg and co-workers, the micelles were prepared by initially dissolving the block copolymer in a suitable organic solvent. Micellization was induced with the dropwise addition of deionized water.41,42 The sizes and morphologies of micelles prepared from different block copolymers (Table 2) were determined by transmission electron microscopy (TEM), indicating ellipsoidal (egg) shaped micelles for M-1 with an average size of 60 × 143 nm (W × H) (Figure 6a). Meanwhile, the micelles formed by M-2 provided a mixture of both ellipsoidal and spherical micelles with an average width and height of 69 and 125 nm (W × H) (Figure 6b) consecutively. In comparison, M-3 micelles are greater in size at 86 × 232 nm as a result of the longer hydrophilic segment and were observed to be unstable in aqueous solution, which is expected for micelles with such length ratio (Figure S8, Supporting Information). The high crystallinity of PCL as the core-forming block led to the formation of egg-shaped micelles with a clear core−shell structure due to crystallization.43−46 Dynamic light scattering (DLS) provides a supporting evidence of the hydrodynamic diameter (DH) of the micelles in aqueous solution to be approximately 134 nm (by intensity) for M-1, 106 nm for M-2, and 269 nm for M-3. A first indication of the stability of M-2 micelles was depicted by the enduring hydrodynamic diameter as recorded by DLS after isolation and redissolving in aqueous solution. The sizes observed by TEM and DLS are in similar orders of magnitude, but they cannot be directly compared due to the nonspherical shape. Enzymatic Degradation. Randomly distributed ester bonds were incorporated into the shell-forming block of the micelles to induce biodegradability. Intracellular lysosomal enzymes are known to promote hydrolysis toward hydrolytically labile polymeric backbones within the cells.47,48 Numerous studies have been carried out to demonstrate and mimic the biodegradation of these polymers, such as polyesters, polyanhydrides,49 polypeptides50 etc. via in vitro and in vivo pathways. Bearing this in mind, we designed our micelles to be

polymer block that will enable the self-assembly into core−shell structures will ensure potentially high drug loading capacity for a hydrophobic drug. Following the statistical copolymerization of 1-O-AiPrFru and BMDO monomer, which displayed good control under RAFT conditions, a series of amphiphilic welldefined PCLx-b-P[(1-O-AiPrFru)y-co-(BMDO)z] block copolymers were synthesized. The block copolymers with varying chain lengths and comonomers compositions were synthesized via chain extension of PCL macro-RAFT agent with two different number of repeating units (DPn = 72, MnObs. = 8.50 kDa, Mn,SEC = 5.0 kDa, Đ = 1.48 and DPn = 106, MnObs. = 12.3 kDa, Mn,SEC = 17.0 kDa, Đ = 1.12). PCL was synthesized via organo-catalyzed ring-opening polymerization of ε-caprolactone, initiated by alcohol functionalized BHCT RAFT agent in the presence of methanesulfonic acid as the catalyst. The block copolymerization was carried out under similar condition to the previously conducted statistical copolymerization of 1-OAiPrFru and BMDO monomer with the substitution of BHCT RAFT agent to PCL macro-RAFT agent. The incorporation of BMDO comonomer in the feed ( f BMDO) varied between 0 and 20 mol %. The molecular structure and 1H NMR of a representative block copolymer sample is shown in Figure 4 before and after

Figure 4. 1H NMR spectra (300.17 MHz) of (a) poly(εcaprolactone)-b-poly[(1-O-AiPrFru)-co-(BMDO)] (in CDCl3) and (b) poly(ε-caprolactone)-b-poly[(1-O-AFru)-co-(5,6-benzo-2-methylene-1,3-dioxepane)] (in DMSO-d6) after deprotection of the sugar moieties showing complete disappearance of the isopropylidene protecting groups.

deprotection of the sugar moieties. The incorporation of ester backbone obtained from a successful radical ring-opening polymerization is marked by the appearance of the peak at δ = 5.0−5.3 ppm, a characteristic peak of the two protons adjacent to the ester bond (“4”) on the glycopolymer backbone. A summary of the physical characterization and size measurements is presented in Table 2. SEC (Figure 5) depicts a clear shift in molecular weight of the PCL macro-RAFT agent to the higher molecular weight region, indicating successful chain extension forming block copolymers. The dispersities (Đ) of H

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Table 2. Characterization and Analysis of a Series of Block Copolymers of Three Different Chain Lengths PCLx-b-P[(1-OAiPrFru)y-co-(BMDO)z] and the Micelle Sizes and Morphologies Formed by Self-Assembly in DMSO Followed by the Dropwise Addition of Water NMRa

SECb

DLSc

TEMc

sample

block copolymer

MnTheo. (Da)

Mn (Da)

Đ

DHd (nm)

PdI

sizee (nm)

morph.

M-1 M-2 M-3

PCL72-b-P(1-O-AiPrFru)88 PCL106-b-P[(1-O-AiPrFru)69-co-(BMDO)9] PCL106-b-P[(1-O-AiPrFru)150-co-(BMDO)24]

31 700 32 400 60 500

16 400 16 000 14 000

1.18 1.29 1.38

134 106 269

0.178 0.085 0.160

60 × 143 69 × 125 86 × 232

ellipsoidal ellipsoidal and spherical ellipsoidal

a Theoretical molecular weight based on monomer conversion (1H NMR, CDCl3). bSEC analyses were obtained by using THF as the mobile phase (calibrated with PMMA standards). cThe micelles were formed after deprotection of the isopropylidene protecting groups forming PCLx-b-P[(1-OAFru)y-co-(BMDO)z]. dThe hydrodynamic volume (DH) of the nanoparticles as measured by dynamic light scattering (DLS) measurement based on intensity mean. eTEM data obtained by measuring the sizes of negatively stained nanoparticles recorded as width × height.

degradation was also performed on micelles bearing only glycopolymers in the corona, but no BMDO (M-1). As represented in Figure 7b, it was observed that the absence of degradable ester linkages of BMDO units in the micelle shell led to rate retardation and the inability to undergo complete degradation. During the first 23.5 h, no degradation was observed as the hydrodynamic diameter remained around 100 nm. A previous study conducted by Wu et al. reported the biodegradation of poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) core−shell polymeric nanoparticles with an essentially nondegradable corona forming block.52 In this study, it was observed that the biodegradation of the micelles is mainly determined by the enzyme concentration, commencing in a one-by-one fashion. Other studies reporting similar behavior can also be found in the literature.53,54 The mechanism was suggested to involve adsorption of enzymes onto the core of the nanoparticles in order for enzymatic hydrolysis of the PCL chains to take place.55−57 Upon isolation of the degradation products of M-2, SEC chromatograms reveal the disappearance of the original glycopolymer peak at 16 000 Da (Figure S11, Supporting Information) and the generation of lower molecular weight products detected at 1200 Da. Further characterization of the degradation products was not pursued in this study, however previous studies reported the generation of alcohols and carboxylic acids as byproducts of ester group hydrolysis.20 Therefore, the incorporation of ester backbones in the design of glycopolymeric micelles has been shown to provide an enhanced and complete biodegradation profile into fragments of free chains that are no longer detected via dynamic light scattering method and confirmed by SEC. Cytotoxicity. Cytotoxicity studies were performed to investigate the biocompatibility of the micelles M-1 and M-2 and their degradation products, M-1 degraded and M-2 degraded, (after incubation with enzymes for 2 d 22 h and 23 h respectively) against MDA-MB-231 (breast cancer) and HS27 (healthy human fibroblast) cell lines (Figure 8).58 The in vitro cell proliferation assay revealed no toxicity of the micelles and degradation products in both media over the concentration range up to 0.20 mg·mL−1 with the cell viabilities of ≥95%. However, the presence of enzyme that was still active in the solution containing degradation products used for in vitro studies caused a significant decrease in cell viability at a high polymer concentration (0.20 mg·mL−1) due to cytotoxic effects (Figure S13, Supporting Information). Therefore, the enzymatic degradation solution was heated to 85 °C for 15 min after the incubation period as a heat-deactivation method to ensure no cytotoxic effects caused by the enzyme.

Figure 5. SEC chromatograms of the chain extension of poly(εcaprolactone) macro-RAFT agent with 1-O-AiPrFru and BMDO monomers via RAFT polymerization in anhydrous anisole at 90 °C for 24 h. [1-O-AiPrFru] 0 :[BMDO] 0 :[PCL]:[VAZO88] = (- - -) 80:20:1:0.1, MnTheo.= 32 400 Da and (···) 160:40:1:0.1, MnTheo. = 60 500 Da (THF eluent, calibrated to PMMA standard).

biologically reducible generating the known degradation products of PCL and short glycopolymer strands that should be approximately 6−8 repeating units in length. To evaluate the dissociation of the micelles under physiological conditions, two different micellar solutions based on PCL106-b-P[(1-O-AFru)69co-(BMDO)9] (M-2) and PCL72-b-P(1-O-AFru)88 (M-1) block copolymers, respectively, were prepared and subjected to enzymatic degradation in the presence of commercially available enzyme Lipase from Pseudomonas sp. The average concentration of serum lipase found in healthy adults lies in the range 30−190 units·L−1.51 In a typical degradation experiment, 500 μL of the enzyme solution (2.0 mg·mL−1 in 0.10 M PBS buffer, pH 7.0) was added to 500 μL of micelle solution (0.70 mg·mL−1) and incubated at 37 °C for 23 h. The degradation period was extended up to almost 3 days for M-1 micelles due to the slow degradation rate. The enzymatic degradation profile of the micelles was monitored via online in situ DLS measurement (Figure 7). Figure 7a reveals complete degradation of the entire structure of the micelles comprising BMDO units (M-2). This is noticeably evident from the disappearance of the original micelle peak at 73 nm after 1 h, leaving only the residual enzyme peak at 9 nm. It was observed that the micelles were fully degraded by lipase Pseudomonas sp. enzyme within the first 10 min, indicated by the disappearance of the micelle signal at 73 nm. The chains reorganized forming fragments with hydrodynamic diameters at 18 nm (4 min, −·) and 8 nm (6 min, −··) and 2 nm (10 min, red line) (Figure S9(a,b), Supporting Information). As a control experiment, enzymatic I

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Figure 6. TEM images of (a) PCL72-b-P(1-O-AFru)88 block copolymers forming ellipsoidal shaped micelles and of (c) PCL106-b-P[(1-O-AFru)69-co(BMDO)9] block copolymers forming a mixture of spherical and ellipsoidal shaped micelles. DLS histogram of (b) PCL72-b-P(1-O-AFru)88 and (d) PCL106-b-P[(1-O-AFru)69-co-(BMDO)9] block copolymers micelles indicating the hydrodynamic volume (DH) of micelles in aqueous solution based on intensity mean.

Figure 7. Enzymatic degradation of the micelles of (a) PCL106-b-P[(1-O-AFru)69-co-(BMDO)9] and (b) PCL72-b-P(1-O-AFru)88 block copolymers in number-based hydrodynamic diameter (DH) of the micelles recorded via in situ DLS measurement during enzymatic degradation. Blank micelle: Micelle solution before enzyme addition; t0: after addition of enzyme.

Figure 8. Cytotoxicity studies of glycopolymeric micelles M-1 and M-2 and their degradation products after incubation with lipase Pseudomonas sp. (M-1 degraded and M-2 degraded) using SRB assays against (a) HS27 and (b) MDA-MB-231 cell lines at different concentrations after 48 h incubation period (values expressed as the percentage of nontreated cells as controls).



CONCLUSION

biodegradable drug delivery platform that can be tailored to achieve a high degree of main chain scission by enzymatic attack in a short period of time. The feasibility of the RAFT process to control the polymerization was depicted from the

By utilizing RAFT to control radical ring-opening polymerization, we have demonstrated the synthesis of a completely J

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(19) Huang, J.; Gil, R.; Matyjaszewski, K. Polymer 2005, 46, 11698. (20) Siegwart, D. J.; Bencherif, S. A.; Srinivasan, A.; Hollinger, J. O.; Matyjaszewski, K. J. Biomed. Mater. Res., Part A 2008, 87A, 345. (21) Yuan, J.-Y.; Pan, C.-Y.; Tang, B. Z. Macromolecules 2001, 34, 211. (22) Smith, Q.; Huang, J.; Matyjaszewski, K.; Loo, Y.-L. Macromolecules 2005, 38, 5581. (23) Lutz, J.-F.; Andrieu, J.; Ü zgün, S.; Rudolph, C.; Agarwal, S. Macromolecules 2007, 40, 8540. (24) Yuan, J.-Y.; Pan, C.-Y. Eur. Polym. J. 2002, 38, 1565. (25) Kobben, S.; Ethirajan, A.; Junkers, T. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 1633. (26) He, T.; Zou, Y.-F.; Pan, C.-Y. Polym. J. 2002, 34, 138. (27) Paulusse, J. M.; Amir, R. J.; Evans, R. A.; Hawker, C. J. J. Am. Chem. Soc. 2009, 131, 9805. (28) d’Ayala, G. G.; Malinconico, M.; Laurienzo, P.; Tardy, A.; Guillaneuf, Y.; Lansalot, M.; D’Agosto, F.; Charleux, B. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 104. (29) Bell, C. A.; Hedir, G. G.; O’Reilly, R. K.; Dove, A. P. Polym. Chem. 2015, 6, 7447. (30) Hedir, G. G.; Bell, C. A.; O’Reilly, R. K.; Dove, A. P. Biomacromolecules 2015, 16, 2049. (31) Gregory, A.; Stenzel, M. H. Prog. Polym. Sci. 2012, 37, 38. (32) Decker, C. G.; Maynard, H. D. Eur. Polym. J. 2015, 65, 305. (33) Xiao, N.; Liang, H.; Lu, J. Soft Matter 2011, 7, 10834. (34) Siebert, J. M.; Baumann, D.; Zeller, A.; Mailänder, V.; Landfester, K. Macromol. Biosci. 2012, 12, 165. (35) Couffin, A.; Delcroix, D.; Martín-Vaca, B.; Bourissou, D.; Navarro, C. Macromolecules 2013, 46, 4354. (36) Brady, R. F. Carbohydr. Res. 1970, 15, 35. (37) Scarano, W.; de Souza, P.; Stenzel, M. H. Biomater. Sci. 2015, 3, 163. (38) Wu, Z.; Stanley, R. R.; Pittman, C. U. J. Org. Chem. 1999, 64, 8386. (39) Ren, L.; Agarwal, S. Macromol. Chem. Phys. 2007, 208, 245. (40) Agarwal, S.; Ren, L.; Kissel, T.; Bege, N. Macromol. Chem. Phys. 2010, 211, 905. (41) Zhang, L.; Eisenberg, A. Polym. Adv. Technol. 1998, 9, 677. (42) Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168. (43) Legros, C.; De Pauw-Gillet, M.-C.; Tam, K. C.; Taton, D.; Lecommandoux, S. Soft Matter 2015, 11, 3354. (44) Massey, J. A.; Temple, K.; Cao, L.; Rharbi, Y.; Raez, J.; Winnik, M. A.; Manners, I. J. Am. Chem. Soc. 2000, 122, 11577. (45) Molev, G.; Lu, Y.; Kim, K. S.; Majdalani, I. C.; Guerin, G.; Petrov, S.; Walker, G.; Manners, I.; Winnik, M. A. Macromolecules 2014, 47, 2604. (46) Hsiao, M.-S.; Yusoff, S. F. M.; Winnik, M. A.; Manners, I. Macromolecules 2014, 47, 2361. (47) Kopeček, J. Biomaterials 1984, 5, 19. (48) Liederer, B. M.; Borchardt, R. T. J. Pharm. Sci. 2006, 95, 1177. (49) Mathiowitz, E.; Jacob, J. S.; Jong, Y. S.; Carino, G. P.; Chickering, D. E.; Chaturvedi, P.; Santos, C. A.; Vijayaraghavan, K.; Montgomery, S.; Bassett, M. Nature 1997, 386, 410. (50) Duncan, R.; Cable, H.; Lloyd, J.; Rejmanová, P.; Kopecek, J. Makromol. Chem. 1983, 184, 1997. (51) Burtis, C. A.; Ashwood, E. R. Tietz Fundamentals of Clinical Chemistry, 4th ed.; Saunders: Philadelphia, PA, 1996; p 803. (52) Gan, Z.; Jim, T. F.; Li, M.; Yuer, Z.; Wang, S.; Wu, C. Macromolecules 1999, 32, 590. (53) Lam, H.; Gong, X.; Wu, C. J. Phys. Chem. B 2007, 111, 1531. (54) Chawla, J. S.; Amiji, M. M. Int. J. Pharm. 2002, 249, 127. (55) Wu, C.; Jim, T.; Gan, Z.; Zhao, Y.; Wang, S. Polymer 2000, 41, 3593. (56) Zhao, Y.; Hu, T.; Lv, Z.; Wang, S.; Wu, C. J. Polym. Sci., Part B: Polym. Phys. 1999, 37, 3288. (57) Kim, S.; Shi, Y.; Kim, J. Y.; Park, K.; Cheng, J.-X. Expert Opin. Drug Delivery 2010, 7, 49. (58) Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112.

formation of well-defined and controlled statistical glycopolymers and block copolymers as proven by NMR spectroscopy and SEC with narrow molecular weight distributions. The ratio of BMDO incorporation was able to be finely tuned to attain different degrees of cleavable ester bonds while maintaining the amphiphilic nature of the final block copolymers and integrity of the micelles product of self-assembly. We have also confirmed the biocompatibility of the materials and degradation products to be nontoxic via in vitro cell proliferation assay against healthy human fibroblast (HS27) and breast cancer (MDA-MB-231) cell lines.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b00266. NMR spectra, TEM image of the micelles and DLS and SEC of the degradation profile (PDF)



AUTHOR INFORMATION

Corresponding Author

*(M.H.S.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors would like to acknowledge Dr. Krzysztof P. Babiuch for the initial help regarding the monomer syntheses and preliminary studies.



REFERENCES

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DOI: 10.1021/acs.macromol.6b00266 Macromolecules XXXX, XXX, XXX−XXX