Reactive Copolymers Based on N-Vinyl Lactams with Pyridyl Disulfide

Article pubs.acs.org/Macromolecules

Reactive Copolymers Based on N‑Vinyl Lactams with Pyridyl Disulfide Side Groups via RAFT Polymerization and Postmodification via Thiol−Disulfide Exchange Reaction Huan Peng,†,§ Kristin Rübsam,§ Xiaobin Huang,∥ Felix Jakob,§ Marcel Karperien,∥ Ulrich Schwaneberg,‡,§ and Andrij Pich*,†,§ †

Functional and Interactive Polymers, Institute of Technical and Macromolecular Chemistry, and ‡Institute for Biotechnology, RWTH Aachen University, Aachen, Germany § DWI-Leibniz Institute for Interactive Materials e.V., Aachen, Germany ∥ Developmental BioEngineering, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands S Supporting Information *

ABSTRACT: Herein, we report the synthesis of a series of novel pyridyl disulfide (PDS)-functionalized statistical reactive copolymers that enable facile access to complex polymeric architectures through highly selective thiol−disulfide exchange reaction with thiol-containing ligands or proteins. Functional reactive poly(Nvinyl lactam)-based copolymers including poly(N-vinylpyrrolidone-co-pyridyl disulfide ethyl methacrylate) (PVPD), poly(Nvinylpiperidone-co-pyridyl disulfide ethyl methacrylate) (PVPID), and poly(N-vinylcaprolactam-co-pyridyl disulfide ethyl methacrylate) (PVD) with PDS side groups were synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization at 60 °C in anisole with methyl 2-(ethoxycarbonothioylthio)propanoate as chain transfer agent. The PDS contents in the synthesized copolymers were varied from 2 to 10 mol % (as confirmed by systematical characterization with FTIR/Raman and 1H NMR spectroscopy) using well-controlled continuous feeding method. The kinetics study suggested that copolymerizations were less favored with the enlargement of the lactam rings, indicated by lower conversions and larger dispersity indexes (Đ). The PDS-functionalized reactive polymers were amenable to functionalization with a variety of thiol-containing molecules, including 3-mercaptopropionic acid (3M), 2-phenylethanethiol (2P), methyl 3-mercaptopropionate (M3), 2-mercaptoethanol (2M), 2-aminoethanethiol (2A), poly(ethylene glycol) methyl ether thiol (PEG-SH), and enhanced green fluorescent protein (EGFP) via thiol−disulfide exchange reaction under mild conditions, confirmed by 1H NMR and SDS-PAGE. The conversions in all cases were higher than 95%, displaying that the thiol− disulfide exchange reaction to PDS groups with thiol-containing molecules is highly selective and tolerant to different ligands including amine, carboxyl, hydroxyl, phenyl, PEG and even polypeptides, providing a versatile scaffold for facile conjugation of various biological components. The contact angle measurement results and fluorescence microscopy study indicated that the reactive films based on the PDS-functionalized copolymers allowed facile, direct, and environmental-friendly surface engineering of surfaces from aqueous solution suggesting potential application in surface decoration of tissue-engineering scaffolds and medical implants. The initial cell culture experiments with HeLa cells displayed that the unmodified PVPD film was nontoxic and biocompatible while the film modified with PEG (a type of antifouling polymer) showed diminished cell attachment and growth, indicating that elegant engineering of the film surface can meet demands of particular applications.

1. INTRODUCTION The past decades have witnessed significant progress in the fields of functional reactive polymeric materials including films,1−3 brushes,4−6 and bioconjugates due to their essential roles in various applications such as colloidal chemistry,7 theranostics,8 and smart biohybrid materials.9−11 Advances in polymeric biomaterials by attaching reactive synthetic macromolecules with biological components such as proteins,12,13 nucleic acids,14,15 peptides, and polysaccharides have led to the © XXXX American Chemical Society

emergence of new interdisciplinary research frontiers which are closely related to human health.16−20 In many cases, the architectures and properties of the functional building blocks are of crucial importance to the potentials of such polymeric materials. Although conventional free radical polymerization is Received: June 6, 2016 Revised: September 6, 2016

A

DOI: 10.1021/acs.macromol.6b01210 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules Scheme 1. Controlled Synthesis of PDS-Functionalized Poly(vinyl lactam) Copolymers via RAFT Polymerization and Postmodification of the Copolymers and Films with Thiol-Containing Ligands

attracted considerable attention due to the versatility of monomer functionality and metal-free process,43 which is of critical significance for biological applications. Our previous study reported water-soluble reactive copolymers with succinimide side groups via RAFT polymerization for conjugation with a cellulase.44 The biohybrid nanogels based on the functional polymers demonstrated enzymatic activity by hydrolysis of 4-methylumbelliferyl-β-D-cellobioside. Lybaert et al. prepared reactive copolymers based on hydroxypropylmethacrylamide (HPMA) with pyridyl disulfide moieties via RAFT polymerization, which formed polymer−protein conjugates with ovalbumin for vaccine delivery.45 In the present work, the reactive copolymers were designed based on N-vinyl lactams and pyridyl disulfide ethyl methacrylate (PDSM) via RAFT polymerization. PDSM is widely reported in various literature regarding different controlled polymerization techniques.46−49 Because of the highly reactive pyridyl disulfide group and abundant thiol groups in biological components, polymers containing pyridyl disulfide (PDS) groups attracted increasing interests and demonstrated various applications.50−53 Thayumanavan et al. designed water-soluble reactive copolymers based on PEG and PDSM via RAFT polymerization and utilized as versatile drug delivery vehicle.54 Maynard et al. prepared poly(2-hydroxyethyl methacrylate) via atom transfer radical polymerization with pyridyl disulfide end group, which was conjugated directly with the cysteine residues of bovine serum albumin in mild conditions.55 Voit et al. obtained intelligent polymer nanocapsules via surface-initiated RAFT polymerization of diethylaminoethyl methacrylate and PDSM, which displayed controlled permeability and pH-dependent degradability.56 The Nvinyl lactam monomer family contains N-vinylpyrrolidone, Nvinylpiperidone, and N-vinylcaprolactam. Although various functional materials based on poly(N-vinyl lactam) polymers were prepared via conventional methods,57−60 the controlled polymerizations of these interesting monomers were seldom reported until recently. This is mainly due to the strong

versatile for various types of monomers and reaction conditions, the technique lacks control over polymer structure and molecular weight due to the high reactivity of propagating radicals. The development of living radical polymerization (LRP) including reversible addition−fragmentation chain transfer (RAFT) polymerization,21,22 atom-transfer radical polymerization (ATRP), and nitroxide-mediated radical polymerization (NMP) facilitated the production of polymers with well-defined architectures, controlled molecular weight, and narrow dispersity in the past two decades.23−29 These techniques enable access to complex macromolecular structure with site-specific functionality for advanced materials applications.30−33 Controlled synthesis of reactive polymers has aroused widespread concerns in recent years. A variety of macromolecules with specific reactivity to amines, thiols, and other functionalities were developed and found appealing applications in drug delivery,34,35 tissue engineering,36,37 bioimageing, and so on.38,39 Heredia et al. reported copper-mediated ATRP of Nisopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), and poly(ethylene glycol) methacrylate (PEGMA) with narrow dispersity.40 The aminooxy end group of the polymers from the initiators enabled chemoselective oxime formation with levulinyl-modified bovine serum albumin (BSA) to form “smart” polymer conjugates. However, removing of the heavy metal catalysts employed in ATRP remains unresolved challenge which limits the applications of the products. Sunder et al. prepared hyperbranched aliphatic polyethers with controlled molecular weights and narrow molecular weight distribution by ring-opening anionic polymerization of glycidol.41 Hawker and co-workers reported reactive copolymers with succinimide ester groups via nitroxide-mediated polymerization using TEMPO as a stable nitroxide radical.42 The copolymer structure is well-defined with a maximum incorporation of the activated ester monomer of 40%, which was used to attach polyether dendrons to obtain dendritic− linear graft copolymers. Recently, RAFT polymerization B


Article

Macromolecules

and purified as reported in previous literature.66 Deionized water was used for all experiments. Measurements. Nuclear Magnetic Resonance (NMR). 1H NMR and 13C NMR spectra were recorded on a Bruker DPX-400 FT NMR spectrometer at 400 MHz. The chemical shifts reported are all relative to the residual solvent peak. Gel Permeation Chromatography (GPC). GPC was performed on a combined GPC system with a high performance liquid chromatography pump (Bischoff HPLC), a Jasco 2035-plus RI detector, and four MZ-DVB gel columns (30 Å, 100 Å, and 2 × 3000 Å) in series at 30 Å. A solution of DMF containing 1.0 g LiBr L−1 was used as eluent at a flow rate of 1.0 mL min−1. The molecular weights were calculated using a polystyrene (PS) calibration. Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectra (resolution 4 cm−1) were recorded using a Nicolet NEXUS 670 Fourier Transform IR spectrometer. Samples were prepared onto silica plates at room temperature. For each spectrum more than 200 scans were averaged to enhance the signal-to-noise ratio. Raman Spectroscopy. Raman spectra were recorded on Bruker RFS 100/S spectrometer. The laser used was a Nd:YAG at 1064 nm wavelength at a power of 250 mW. On average, 1000 scans were taken at a resolution of 4 cm−1. For sample holding aluminum pans of 2 mm before were used. Software used for data processing was OPIS 4.0. Atomic Force Microscopy (AFM). AFM images were recorded on a Veeco Dimension ICON Nanoscope V scanning probe microscope with tapping mode. Cantilevers (250−300 kHz, 42−100 N/m) were used. The images were analyzed by Picoimage software provided by Agilent. Spectroscopic Ellipsometry. Spectroscopic ellipsometry measurements were performed on Omt Imaging ellipsometer (mm30 series) at 70° incidence angle in a spectral range of 450−800 nm using VisuEl software 3.8. The results were analysis by Scout Software through fitting using a Cauchy model where the polymer film and silica wafer substrate are modeled as a homogeneous material. Static Contact Angle Measurements. The static contact angles of the films were measured with static sessile drop method (G2/DSA II, Krüss GmbH, Germany). Fluorescence Microscopy. The fluorescent film measured with ApoTome 2 fluorescence microscopy (Zeiss, Germany) and analyzed with ApoTome 2 software. Synthesis of Pyridyl Disulfide Ethyl Methacrylate (PDSM). The monomer was synthesized according to previous literature.56 15 g of aldrithiol-2 was dissolved in 75 mL of methanol with 1 mL of glacial acetic acid. Then 2.65 g of 2-mercaptoethanol in 15 mL of methanol solution was added dropwise at room temperature. Afterward, the solvent was evaporated after the reaction mixture was stirred at room temperature for an additional 3 h. The obtained crude product was purified by flash column chromatography using ethyl acetate/hexane as eluent and silica gel as a stationary phase. The polarity of the eluent was increased from 15% to 40% ethyl acetate/n-hexane after the excess aldrithiol-2 came out. The product pyridyl disulfide ethanol was obtained as colorless oil with yield of 52.3%. 1H NMR (in CDCl3): δ [ppm] = 8.46 (aromatic proton ortho-N), 7.55 (aromatic proton metaN), 7.39 (aromatic proton para-N), 7.11 (aromatic proton, orthodisulfide linkage), 5.25 (−CH2CH2OH), 3.77 (−CH2CH2OH), 2.91 (−CH2CH2OH). 13C NMR (in CDCl3): δ [ppm] = 159.14 (aromatic carbon, ortho-disulfide linkage), 149.76 (aromatic carbon ortho-N), 137.48 (aromatic carbon para-N), 119.72−121.86 (aromatic carbon meta-N), 58.32 (−CH2CH2OH), 42.61 (−CH2CH2OH). 4.62 g of pyridyl disulfide ethanol was dissolved in 20 mL of dry dichloromethane with 3 g of trimethylamine in an ice bath, and 10 mL of dichloromethane solution of 2.58 g of methacryloyl chloride was added dropwise into this cold mixture with continuous stirring. The reaction mixture was stirred at room temperature for another 6 h after the addition. Afterward, the reaction mixture was washed with 3 × 50 mL of distilled water and then with 30 mL of brine. The organic layer was collected and dried over anhydrous Na2SO4. The solvent was evaporated to get the crude product as yellow oil. It was purified by column chromatography using silica gel as the stationary phase and a mixture of 25% ethyl acetate/hexane as eluent; yield: 71%. 1H NMR

electron-donating effect from the lactam ring and lack of resonance stabilization, which make the generated polymer radicals highly active and unfavorable side reactions difficult to be suppressed.61 The main progress of controlled polymerization of N-vinyl lactam is the RAFT/MADIX (macromolecular design via the interchange of xanthates) process. Keddie et al. reported synthesis of poly(N-vinylpyrrolidone) with switchable RAFT agent.62 Youk et al. prepared poly(Nvinylpyrrolidone)-b-poly(ε-caprolactone) block copolymers using a dual initiator for RAFT and ring-opening polymerization (ROP) in one spot with low Đ around 1.17.63 Destarac et al. synthesized poly(N-vinylpyrrolidone)-based double hydrophilic block copolymers with controlled architecture via aqueous ambient RAFT/MADIX polymerization.64 However, well-designed functional polymers based on the two different types of monomers were not reported previously, regardless of the interesting properties of both components and potential applications. This could be attributed to the hugely different reactivity of the monomers.65 To enrich the family of reactive functional polymeric materials, in this paper a series of reactive copolymers based on N-vinyl lactams with different compositions of PDS side groups were designed and performed (Scheme 1). The polymerization kinetics and polymer structures were systematically investigated. Results suggested that the well-defined reactive copolymers can be obtained with controlled architectures. The copolymers were easily altered with a variety of thiol-containing molecules or protein under mild conditions, minimizing possible contamination and protein denaturation. The thiol−disulfide exchange reaction of the PDS groups with thiol-containing molecules is highly selective and tolerant to different ligands including amine, carboxyl, hydroxyl, phenyl, PEG, and even polypeptides, providing a versatile scaffold for facile conjugation of various biological components. More importantly, functional films based on these PDS-functionalized copolymers are amenable to facile, direct, and environmentalfriendly surface decoration in aqueous solution with thiolcontaining molecules or proteins. The biocompatible polymeric film can be elegantly decorated by surface engineering with thiol-containing molecules to control cell attachment and growth, indicating promising application in the fields of tissueengineering, medical implants and so on.

2. EXPERIMENTAL SECTION Materials. Aldrithiol-2 (Aldrich, 99%), glacial acetic acid (Aldrich, 99.7%), methacryloyl chloride (Aldrich, 97%), anisole (Aldrich, anhydrous, 99.7%), 1,4-dioxane (Aldrich, anhydrous, 99.7%), 1,3,5trioxane (Aldrich, 99%), methyl 2-bromopropionate (Acros, 99%), potassium ethyl xanthogenate (Aldrich, >98%), 3-mercaptopropionic acid (3M, Aldrich, 99%), 2-phenylethanethiol (2P, Aldrich, 98%), methyl 3-mercaptopropionate (M3, Aldrich, 98%), 2-mercaptoethanol (2M, Aldrich, 99%), 2-aminoethanethiol (2A, Aldrich, 98%), poly(ethylene glycol) methyl ether thiol (PEG-SH, Aldrich, Mn ∼ 800), potassium phosphate buffer (PB buffer, prepared by KH2PO4, K2HPO4, Aldrich), ethanol (VWR, 99.9%), dichloromethane (VWR, 99.9%), sulfate magnesium (VWR, >99%), diethyl ether (VWR, 99.9%), DMSO-d6, chloroform-d1 (CDCl3, Deutero GmbH, 99.8%), and dialysis tubes (MWCO 12−14 kDa, Carl Roth, Germany; MWCO 50 and 100 kDa, SPECTRUM LABORATORIES, USA) were used as received. N-Vinylpyrrolidone (VP, Aldrich, 99%), N-vinylpiperidone (VPI, BASF, 98%), and N-vinylcaprolactam (VCL, Aldrich, 98%) were distilled and recrystallized from hexane before use. 2,2′-Azobis(2methylpropionitrile) (AIBN, Aldrich, 98%) was recrystallized in ethanol before use. Enhanced green fluorescent protein (EGFP with two cysteines containing thiol groups, Mn ∼ 26 kDa) was produced C


Article

Macromolecules (in CDCl3): δ [ppm] = 8.45 (aromatic proton ortho-N), 7.60−7.68 (aromatic proton para-N and meta-N), 7.06 (aromatic proton, orthodisulfide linkage), 6.10 (vinylic proton, cis-ester), 5.56 (vinylic proton, trans-ester), 4.37 (−OCH2CH2S−), 3.07 (−OCH2CH2S−), 1.91 (−CCH2CH3). 13C NMR (in CDCl3): δ [ppm] = 167.44 (−CO), 160.15 (aromatic carbon, ortho-disulfide linkage), 150.09 (aromatic carbon ortho-N), 137.49 (−CHCH2), 136.35 (aromatic carbon para-N), 126.45 (−CH2CH−), 120.18−121.26 (aromatic carbon meta-N), 62.80 (−OCH2CH2S−), 37.84 (−OCH2CH2S−), 18.68 (−CCH2CH3). Synthesis of Methyl 2-(Ethoxycarbonothioylthio)propanoate. The chain transfer agent (CTA) methyl 2-(ethoxycarbonothioylthio)propanoate was performed according to the instructions given by Destarac.67 A solution of methyl 2-bromopropionate (2 g, 11.7 mmol, 1 equiv) in ethanol (15 mL) was stirred in a double-neck flask immersed in an ice bath. Potassium ethyl xanthogenate (2.16 g, 13.4 mmol, 1.1 equiv) was progressively added with a spatula for 45 min. The solution became yellowish. After addition, the solution was mixed for 3 h at room temperature. The formation of potassium bromide (insoluble in ethanol) was observed. The solution was then filtered with a Buchner and concentrated under reduced pressure with a rotary evaporator. Dichloromethane (30 mL) was then added, and the product was extracted in the organic phase after treatment with distilled water (4 × 50 mL). The organic phase was dried over sulfate magnesium overnight in an Erlenmeyer flask. After filtration, dichloromethane was removed in a rotary evaporator. The product was dried under vacuum to yield a yellow bright liquid (2.09 g, yield 91.1%). 1H NMR (in CDCl3): δ [ppm] = 4.59 (−OCH2CH3), 4.35 (−CHCH3), 3.72 (−OCH3), 1.53 (−CHCH3), 1.38 (−OCH2CH3). 13 C NMR (in CDCl3): δ [ppm] = 212.14 (−CS), 171.87 (−CO), 70.46 (−OCH2CH3), 52.75 (−OCH3), 47.42 (−CHCH3), 16.80 (−CHCH3), 13.84 (−OCH2CH3). Polymerization of VP. The polymerization of VP in the presence of aldrithiol-2 (2,2′-dipyridyl disulfide) via conventional free radical polymerization and RAFT polymerization were performed to study the chain transfer effect of pyridyl disulfide (PDS)-containing molecules.48 In the conventional free radical polymerizations, VP (10 g, 90 mmol) and AIBN (30 mg, 0.185 mmol) in 10 mL of 1,4dioxane were divided into five glass tubes with progressively increased concentrations of aldrithiol-2 (0, 0.1, 0.2, 0.3, and 0.4 M). The tubes were sealed with rubber septa and degassed with dry nitrogen for 1 h in ice bath before transferring to preheated oil bath at 60 °C. The tubes were immersed into liquid nitrogen after 18 h to stop the reactions. The polymers were precipitated in large excess amount of diethyl ether in ice bath for five times and dried under vacuum at 40 °C for 48 h before 1H NMR (CHCl3) and GPC analysis. The RAFT polymerizations of VP in the presence of aldrithiol-2 were performed in similar manner. A 10 mL 1,4-dioxane solution of VP (10 g, 90 mmol), 2-(ethoxycarbonothioylthio)propanoate (0.2 g, 1 mmol), and AIBN (30 mg, 0.185 mmol) was divided into five glass tubes equally with varying amount of aldrithiol-2 (0, 0.1, 0.2, 0.3, and 0.4 M). The polymerization, purification, and analysis procedures were carried out similarly as described above. General Polymerization Procedure of N-Vinyl Lactam and PDSM. All polymerizations were preceded under dry nitrogen in anhydrous anisole solution with standard Schlenk techniques, using methyl 2(ethoxycarbonothioylthio)propanoate as chain transfer agent (CTA) and AIBN as initiator. A typical procedure is described below.44,68 2 g (18 mmol) of N-vinylpyrrolidone, 0.042 g (0.21 mmol) of CTA, and 0.02 g of 1,3,5-trioxane (as internal standard for monomer conversion calculation via 1H NMR) were taken in a 25 mL Schlenk flask in 2 mL of anisole, and the solution was degassed by five freeze−pump−thaw cycles. A Schlenk tube charged with 0.094 g (0.37 mmol) of PDSM in 1 mL of anisole was degassed with the same procedure. A small vial containing 0.0058 g (0.035 mmol) of AIBN in 0.5 mL of anisole equipped with septa and gas inlet/outlet was purged with dry nitrogen for 1 h. A small amount of solution was taken out for 1H NMR analysis before the Schlenk flask was transferred to a preheated oil bath at 60 °C. The AIBN solution was fed into the Schlenk flask immediately with a nitrogen prewashed syringe while the PDMS solution was

added continuously with a syringe pump (Harvard Apparatus, Holliston, MA) in 6−8 h. The reaction was carried out at the same temperature under a nitrogen atmosphere until the monomer conversion did not change. Afterward, the reaction was stopped by fast cooling in liquid nitrogen. A small amount of the solution was diluted with CDCl3 and sent for 1H NMR. The copolymers were precipitated and further washed with cold diethyl ether and dried under vacuum for 48 h and then characterized by 1H NMR, FTIR, Raman, and GPC. Here the systems using N-vinylpyrrolidone, Nvinylpiperidone, and N-vinylcaprolactam are named as PVPD, PVPID, and PVD system, respectively. 1 H NMR (in CDCl3): (PVPD) δ [ppm] = 8.57 (aromatic proton ortho-N), 7.78−7.98 (aromatic proton para-N and meta-N), 7.01 (aromatic proton, ortho-disulfide linkage), 3.74 (−OCH2CH2S−, −CHCH3, −OCH2CH3 in end group), 3.64 (−NCHCH2, −OCH3 in end group), 3.19 (−NCH2CH2−), 2.27−2.37 (−OCH2CH2S−, −NCOCH2−), 1.99−2.05 (−NCH2CH2−, −CH2CCH3 in backbone), 1.15−1.68 (−CH2CHN−, −CH2CCH3 in backbone, −OCH2CH3, −COCHCH3 in end group); (PVPID) δ [ppm] = 8.46 (aromatic proton ortho-N), 7.69 (aromatic proton para-N and meta-N), 7.12 (aromatic proton, ortho-disulfide linkage), 4.59 (−CH2CHN− in backbone, −OCH2CH2S−, −CHCH3, −OCH2CH3 in end group), 3.65 (−OCH3 in end group), 3.04 (−NCH2CH2−), 2.21−2.36 (−OCH 2 CH 2 S−, −NCOCH 2 −), 1.42−1.77 (−CH 2 CHN−, −CH2CCH3, −NCH2CH2−, −CH2CCH3 in backbone, −OCH2CH3, −COCHCH3 in end group); (PVD) δ [ppm] = 8.50 (aromatic proton ortho-N), 7.80 (aromatic proton para-N and meta-N), 7.02 (aromatic proton, ortho-disulfide linkage), 4.37 (−CH2CHN− in backbone, −OCH2CH2S−, −CHCH3, −OCH2CH3 in end group), 3.69 (−OCH 3 in end group), 3.19 (−NCH 2 CH 2 −), 2.41−2.46 (−OCH2CH2S−, −NCOCH2CH2−), 1.44−1.75 (−CH2CHN−, −CH 2 CCH 3 , −NCH 2 CH 2 CH 2 −, −CH 2 CCH 3 in backbone, −OCH2CH3, −COCHCH3 in end group). FTIR (on silica plate): (PVPD) 2950 cm−1 (CH2), 1721 cm−1 (OCOCH2CH2S−), 1662 cm−1 (−NCO), 1574 cm−1 (aromatic ring). (PVPID) 2943 cm −1 (−CH 2 −), 1722 cm −1 (O COCH2CH2S−), 1627 cm−1 (−NCO), 1574 cm−1 (aromatic ring). (PVD) 2925 cm−1 (−CH2−), 1725 cm−1 (OCOCH2CH2S−), 1623 cm−1 (−NCO), 1574 cm−1 (aromatic ring). Raman: (PVPD): 3057 cm−1, 1559 cm−1, 960 cm−1 (aromatic ring), 1730 cm−1 (OCOCH2CH2S−), 1667 cm−1 (−NCO), 550 cm−1 (−S−S−). (PVPID) 3050 cm−1, 1557 cm−1, 945 cm−1 (aromatic ring), 1727 cm−1 (OCOCH2CH2S−), 1625 cm−1(−NCO), 550 cm−1 (−S−S−). (PVPID) 3052 cm−1, 1558 cm−1, 947 cm−1 (aromatic ring), 1728 cm−1 (OCOCH2CH2S−), 1629 cm−1 (−NCO), 550 cm−1 (−S−S−). The theoretical number-average molecular weights of the copolymers were calculated with the following equation based on conversions: M n(theory) =

[Monomer]0 × M monomer × S + MCTA [CTA]0

(1)

Here MCTA is the molecular weight of the RAFT agent, Mmonomer is the average molecular weight of the monomers, S is conversion, and [CTA]0 and [Monomer]0 are initial concentrations of chain transfer agent and monomers, respectively. Polymerization Kinetics. A small fraction of the reaction solution was taken out periodically for 1H NMR in CDCl3 to determine the conversion. Meanwhile, a few milligrams of copolymers was precipitated from cold diethyl ether; the dry samples were sent for DMF GPC to determine the molecular weight and dispersity (Đ). The conversion of VP (VPI or VCL) was determined by comparing the integration of vinyl proton peaks with that of the internal standard. Postpolymerization Modification of Copolymers. The postmodifications of the reactive copolymers with small thiol-containing molecules were carried out in DMSO at room temperature. A variety of compounds including 3-mercaptopropionic acid (3M), 2-phenylethanethiol (2P), methyl 3-mercaptopropionate (M3), 2-mercaptoethanol (2M), 2-aminoethanethiol (2A), and PEG-SH were utilized. A D


Article

Macromolecules

Figure 1. (a) Experimental molecular weight (Mw,GPC) and dispersity (Mw/Mn) of poly(N-vinylpyrrolidone) prepared in the presence of varying amount of aldrithiol-2 via conventional free radical polymerization. (b) 1H NMR spectrum of purified poly(N-vinylpyrrolidone) prepared in the presence of 0.4 M aldrithiol-2 via conventional radical polymerization. few drops of glacial acetic acid were added into the PVPD10% (10.65% PDS, Mn,GPC = 6873 Da) in DMSO solution (5 mg/mL) to obtain acidic environment. For PEG-SH and 2A deionized water was added to make the solution transparent. The SH/PDS (pyridyl disulfide) molar ratio was set at 3/2. The reactions proceeded overnight, and the modified polymers were precipitated and further washed with cold diethyl ether or purified with extensive dialysis (to remove PEG-SH) and dried under vacuum at room temperature for 48 h. The dry samples were sent to 1H NMR in DMSO-d6 to quantify the modification efficiency. For postmodification with EGFP, PVPD2% (2.06% PDS, Mn,GPC = 7994 Da) was used, and the reaction was performed in phosphate buffer solutions (pH 7.2) under otherwise the same conditions. Preparation of Functional Copolymer Films and Surface Engineering with Thiol-Containing Molecules. Functional copolymer films were prepared by spin coating (Convac 1001S, Germany) on silica wafer slides with PVPD10% copolymers solution (5 mg/mL in DMSO). The films were dried under vacuum for 2 days to remove solvent thoroughly and characterized with AFM and spectroscopic ellipsometry. Afterward, the films were immersed in the phosphate buffer solutions (pH 7.2) of 3-mercaptopropionic acid (3M), 2mercaptoethanol (2M), 2-aminoethanethiol (2A), and PEG-SH with concentration of 2 mg/mL for 24 h. The obtained films were rinsed with deionized water, dried thoroughly under vacuum, and afterward performed with contact angle measurements or fluorescence microscopy (for EGFP modified film). The functional film modified with 3M is named as PVPD-3M. The other modified films are named in a similar manner. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis. SDS-PAGE samples were prepared by mixing 8 μL of sample solutions and 10 μL of 4xSDS loading buffer. The mixture was heated at 95 °C for 5 min. The volume of 8 mL of the sample was loaded and analyzed using a stacking gel (5% (w/v) acrylamide) and a separating gel (15% (w/v) acrylamide). The gel ran for 90 min at 125 V at room temperature, with subsequent Coomassie Brilliant Blue staining (4 h) and overnight destaining (10% acetic acid).69 Cell Culture on the Surface Modified Polymeric Films. The influence of polymeric films on cell adhesion and growth was investigated using PVPD10% as parent film. The parent film and PEGSH modified film were prepared on a glass slide with spin coating as described above. The films were placed into a 96-well plate tissue culture plate and sterilized by radiation before use. HeLa cells were seeded on the films at a density of 1 × 103 cells/well in Dulbecco’s Modified Eagle Medium (DMEM), containing 10% FBS in a humidified 5% CO2 atmosphere at 37 °C. The culture media was replaced every day. After 1, 4, and 7 days culture, the cells were observed on an inverted light microscope (Nikon Eclipse TE300 equipped with a Nikon Digital Sight DS-L2 camera).

3. RESULTS AND DISCUSSION Polymerization VP in the Presence of PDS-Containing Compound. It was reported in the literature that disulfides can cause chain transfer effect in free radical polymerization.70,71 Wong et al. performed conventional free radical polymerization and RAFT polymerization of methyl methacrylate in the presence of a pyridyl disulfide-containing compound hydroxyethylpyridyl disulfide (HPDS).48 The experimental data suggested that the chain transfer effect of HPDS was so small that in most cases not detectable in both conventional free radical polymerization and RAFT polymerization. In the present work, the free radical polymerization and RAFT polymerization of VP in the presence of a model compound (aldrithiol-2) bearing pyridyl disulfide groups were performed to study the capacity of the PDS group as chain transfer center. 1,4-Dioxane was reported as favorable solvent as anisole for polymerization of N-vinyl lactam monomers.44 It was used instead of anisole in the polymerization of VP in the presence of PDS group-containing compound since the signal of the phenyl group from the trace of anisole may overlap with that of the pyridyl group in 1H NMR spectra. As observed in Figure 1a, the number-average molecular weights (Mw,GPC) and dispersities (Đ) of poly(N-vinylpyrrolidone) homopolymers are comparable, regardless of the presence of aldrithiol-2 with different concentrations. It indicates that the chain transfer effect from the pyridyl disulfide group can be ignored. The theoretical molecular weight Mn,th calculated according to eq 2 is around 99 598−139 437 Da (assuming f around 0.5−0.7), which is actually smaller than the experimental obtained molecular weight (180 000−190 000 Da) (Figure 1a). It supports that no chain transfer reaction happened during the polymerization process, as chain transfer reaction during the polymerization process would be expected to result in polymers with smaller molecular weights than the theoretical molecular weight. M n,th =

[M]0 M m 2f [I]0 (1 − e−kdt )

(2)

Here, [M]0 is the initial concentration of the monomer, Mm is the molecular weight of the monomer, [I]0 is the initial concentration of AIBN, f is the initiator efficiency, setting as 0.5−0.7,48 kd is decomposition rate constant, 7.68 × 10−6 s−1 in 1,4-dioxane at 60 °C,72 and t is reaction time. E


Article

Macromolecules

Figure 2. (a) Kinetics plot of conversion versus time in the RAFT copolymerization of N-vinylpyrrolidone (VP, squares)/N-vinylpiperidone (VPI, circles)/N-vinylcaprolactam (VCL, triangles) and pyridyl disulfide ethyl methacrylate (PDSM) at 60 °C. (b) Kinetic plot of ln([M]0/[M]t) versus time in the RAFT copolymerization of N-vinylpyrrolidone (VP, squares)/N-vinylpiperidone (VPI, circles)/N-vinylcaprolactam (VCL, triangles) at 60 °C. The concentration of PDSM is 0.11 mmol/mL.

Figure 3. (a−c) GPC traces at different conversions for the RAFT polymerization of N-vinylpyrrolidone (VP, a)/N-vinylpiperidone (VPI, b)/Nvinylcaprolactam (VCL, c) and pyridyl disulfide ethyl methacrylate (PDSM) with AIBN in the present of CTA in anisole at 60 °C and target molecular weight is 10 000 Da. (d) Plot of experimental molecular weight from GPC (MGPC) and dispersity index versus theoretical number-average molecular weight (Mth) for RAFT copolymerization of N-vinylpyrrolidone (VP)/N-vinylpiperidone (VPI, circles)/N-vinylcaprolactam (VCL, triangles) and pyridyl disulfide ethyl methacrylate at 60 °C. The dashed line is the ideal situation when MGPC = Mth. The concentration of PDSM is 0.11 mmol/mL.

F


Article

Macromolecules Table 1. Properties of VP/PDSM Copolymers PVPD (Reactions Run for 48 h) PVPD2% PVPD4% PVPD6% PVPD8% PVPD10%

Mtarget/Da

Mtheory/Da

MGPC/Da

Đ

PDSM% (input)

PDSM% (1H NMR)

conv/%

water solubility

10000 10000 10000 10000 10000

8068 7882 7519 7264 7049

7994 7754 7351 7009 6873

1.21 1.23 1.26 1.26 1.29

2 4 6 8 10

2.06 4.11 5.94 7.93 10.65

80.3 78.4 74.7 72.1 69.9

yes yes no no no

Table 2. Properties of VPI/PDSM Copolymers PVPID (Reactions Run for 48 h) PVPID2% PVPID4% PVPID6% PVPID8% PVPID10%

Mtarget/Da

Mtheory/Da

MGPC/Da

Đ

PDSM% (input)

PDSM% (1H NMR)

conv/%

water solubility

10000 10000 10000 10000 10000

6941 6911 6608 6539 6362

6728 6631 6327 6259 5974

1.23 1.25 1.25 1.29 1.32

2 4 6 8 10

2.41 3.94 5.91 7.96 10.44

68.8 68.5 65.4 64.7 62.9

yes no no no no

Table 3. Properties of VCL/PDSM Copolymers PVD (Reactions Run for 48 h) PVD2% PVD4% PVD6% PVD8% PVD10%

Mtarget/Da

Mtheory/Da

MGPC/Da

Đ

PDSM% (input)

PDSM% (1H NMR)

conv/%

water solubility

10000 10000 10000 10000 10000

6088 6019 5666 5225 4990

5844 5679 5306 5068 4753

1.28 1.29 1.32 1.32 1.35

2 4 6 8 10

2.14 3.97 6.07 8.15 10.17

60.1 59.4 55.8 51.3 48.9

no no no no no

The GPC traces of the polymers in Figure S7a and 1H NMR spectrum in Figure 1b further verify the absence of significant chain transfer reactions in the polymerizations, as indicated by the similar molecular weight distribution and absence of signals from pyridyl group (7.1−8.5 ppm) after purification. Furthermore, no obvious influence of aldrithiol-2 concentration on the molecular weight of poly(N-vinylpyrrolidone) polymers prepared via RAFT polymerization was observed in Figure S7b. It excludes the possibility of significant unfavorable chain transfer effect of the pyridyl disulfide group in RAFT mediated polymerization. Investigation of Polymerization Kinetics. The copolymerizations of N-vinyl lactam monomers and pyridyl disulfide ethyl methacrylate are very interesting reactions to study the influence of steric hindrance on polymerization kinetics. Additionally, such copolymers possess intriguing potential applications due to the temperature-responsiveness properties of poly(N-vinylpiperidone) and poly(N-vinylcaprolactam). Furthermore, the even distributions of the pyridyl disulfide side groups of the statistical copolymers can lead to functional polymeric materials like nanogels/microgels and membranes with well-distributed reactive sites. As reported before, polymerization conditions including solvent type, reaction temperature, and ratio of [CTA]/ [initiator] are of great significance to the reaction process as well as properties of obtained polymers.73 Considering previous RAFT polymerization conditions,44 here all the polymerizations were performed in anisole with [CTA]:[AIBN] ratio 6:1. The temperature was set at 60 °C to avoid possible facture of the disulfide bond. The molar feed ratio of PDSM was 2% while the target molecular weight was set as 10 000 Da. The time-dependent monomer conversions of the three polymerization systems are demonstrated in Figure 2a. As expected, monomer conversions decreased from 80.3% to 68.8% to 60.1% with the enlargement of the lactam ring. Obviously, the increased steric hindrance may disfavor the propagation process, resulting shorter propagating radical

chains. It may also make the reinitiation process more difficult to happen since the leaving group radicals need more energy to react with monomer species with higher steric hindrance. However, the monomer conversions for the three systems were quite high and all above 60%, indicating relatively high chain transfer efficiency of the RAFT agent. The first-order kinetics plots of the three polymerization systems in Figure 2b clearly show that polymerizations proceeded in a controlled manner. Although the induction period at the very initial stage was reported in some RAFT polymerizations,74 this period could be observed in none of the present systems. This could be attributed to the relatively fast initiation speed and high polymerization rate, which may contribute to the relatively high conversions. Moreover, the polymerization rates revealed from the slopes of the plots decreased by 47% and 31% from PVPD to PVPID and from PVPID to PVD system, respectively. This difference may be explained by the steric hindrance mainly originating from the lactam ring. Considering the propagating radical chains are important intermediates, which participate in both propagation and pre-equilibrium processes, estimation of their steric energy from the three systems may help better understanding of the polymerization kinetics. Since the PDSM monomer was fed slowly into the N-vinyl lactam solutions, here it is reasonable to assume the initial radicals as short homo poly(vinyllactam) chains. The steric hindrance energy of PVP4, PVPI4, and PVCL4 calculated from Chem 3D Ultra software (Cambridgesoft, USA) is 156.14, 2937.29, and 5646.79 kcal/ mol, respectively. This data may support the hypothesis that steric hindrance energy increase greatly from 5- to 6-membered ring (∼18-fold) but slightly (∼1-fold) from the 6- to 7membered analogue. It also helps explaining the decreased polymerization rate of the three systems. Furthermore, the GPC traces at different time intervals presented in Figure 3a−c were symmetric and unimodal. Combined with the low Đ of the three polymerization runs and linear increase of the experimentally obtained molecular weight from GPC with G


Article

Macromolecules

Figure 4. (a) FT-IR spectra of PVPD serial copolymers (PDSM molar feed ratios are 2%, 4%, 6%, 8%, and 10%, respectively; the intensities at 1662 cm−1 were normalized). (b) Peak height ratio of CO from PDSM and VP versus molar feed ratio of PDSM and VP.

Figure 5. (a) Raman spectra of PVPD serial copolymers (PDSM molar feed ratios are 2%, 4%, 6%, 8%, and 10%, respectively; the intensities at 1662 cm−1 were normalized). (b) 1H NMR spectra of PVPD serial copolymers (PDSM molar feed ratios are 2%, 4%, 6%, 8%, and 10%, respectively; signals at 3.19 ppm were normalized).

monomers are much more reactive than N-vinyl lactam monomers. Simple batch polymerization of the two monomers resulted in very low yield and uncontrolled polymer architecture indicating composition drift happened. To avoid composition drift and obtain random copolymers with statistical distribution of PDSM in the polymer chain, the more reactive monomer was fed continuously with a syringe pump. As shown in Tables 1−3, the polymer compositions are well controlled with the PDSM ratios quite close to the feed ratios. Although previous work by Arzamendi and van Doremeale et al. suggested that copolymer composition control in seeded semicontinuous emulsion copolymerization required an addition profile of the more reactive monomer to get constant composition during the polymerization,74,75 the conditions in the present polymerizations are different. The polymerizations were performed in anhydrous anisole, and chain transfer agents were used to control the polymerization process. The more active monomer PDSM was fed into the Nvinyl lactam solution in 6−8 h to make copolymer compositions similar to the formulations. The Đ of copolymers increases with increased amount of PDSM in the copolymers.

theoretical molecular weight observed in Figure 3d, it can be concluded the polymerizations run in a controlled manner. Chemical Structure of Copolymers by FTIR/Raman and NMR Spectroscopy. A series of PVPD copolymers were synthesized with increased PDSM feed ratios of 2%, 4%, 6%, 8%, and 10%, named as PVPD2%, PVPD4%, PVPD6%, PVPD8%, and PVPD10%, respectively. PVPID and PVD copolymers were synthesized and named in a similar manner. All the polymerizations were carried out at 60 °C, keeping the [CTA]/[AIBN] molar ratio as 6 and target molecular weight as 10 000 Da. The results are concluded in Tables 1, 2, and 3. As discussed above, the steric hindrance energy increased with the enlargement of the lactam ring, which may decrease the reactivity of the monomers. This helps explaining the decreased monomer conversions from the PVPD to PVD system (PVPD, 80.3%−69.9%; PVPID, 68.8%−62.9%; PVD, 60.1%−48.9%). It is worth to mention that N-vinyl lactams and PDSM belong to two different classes of monomers with extremely different reactivity. It is reported that the reactivity ratio of methyl methacrylate/N-vinylpyrrolidone in bulk polymerization at 50 °C was 940,65 which means methacrylate H


Article

Macromolecules

Figure 6. 1H NMR of PVPD10% modified with 3-mercaptopropionic acid (3M, a) and methyl 3-mercaptopropionate (M3, b). The x-axis represents chemical shift.

1662 cm−1 (−CO from VP) were normalized first and the peak height ratio values were collected, which were found to increase linearly with the molar feed ratio of PDSM and N-VP as shown in Figure 4b. Furthermore, one can observe the gradually increased peak intensities at 1574 cm−1 (the inset image), which belong to pyridyl groups, indicative progressive increased pyridyl disulfide side groups in corresponding copolymers. Analogous results could be obtained from the FTIR spectroscopy of the other two systems (Figure S12), displaying control character in synthesis of series of copolymers. For Raman spectrum as shown in Figure 5 and Figure S13, the characteristic signals of the copolymers increased accordingly in a similar manner. Analogously, as observed in 1H NMR spectra from Figure 5b and Figure S11,

Considering the huge reactivity difference between the two monomers, it is easy to understand that the PDSM droplet may lead to slight composition drift in its surrounding area, which was weaken in a while due to stirring and diffusion. This monomer reactivity variation favored the increase of dispersity. To have a complete investigation of the polymer structures, FTIR and Raman spectroscopy were utilized. Figure 4 demonstrates the FTIR spectra of PVPD copolymers. The characteristic peaks at 1721, 1662, and 1574 cm−1 are assigned to −CO from PDSM, N-VP, and the pyridyl group from PDSM. Since the PDSM feed ratio increases progressively, the characteristic peaks of −CO from PDSM should be intensified accordingly, as demonstrated from the inset of Figure 4a. To further confirm this, the intensities at I


Article

Macromolecules the signals around 8.5 ppm were intensified progressively, indicating that the pyridyl groups increased accordingly. Combined with the FTIR/Raman and NMR analysis results, it can be safely concluded that the PDS components were increased near proportionally to the PDSM feeding molar ratio. This further suggests that the polymerizations ran in a controlled manner. The copolymer gradually lost water solubility in water with the increased amount of PDSM due to its hydrophobicity. Meanwhile, the copolymer hydrophilicity decreased with the enlargement of the lactam ring. Since VP is most hydrophilic among the three monomers, the water solubility of PVPD copolymers is much better than the others. It is reasonable that PVPD4% is still water-soluble while none of PVD copolymers are. However, the hydrophilicity of the copolymer could be adjustable via introducing new hydrophilic components or postmodification of the PDS group with thiol-containing molecules. Facile Decoration of PDS-Functionalized Copolymer with Different Ligands. The pyridyl disulfide (PDS) groups were widely utilized for the reaction with thiol groups to form disulfide linkage over a broad pH range (3−7.5). To investigate whether the PDS functional groups in the reactive copolymer are amenable to derivatization via thiol−disulfide exchange reaction, PVPD10% was used for facile modification with various thiol-containing molecules including 3-mercaptopropionic acid (3M), methyl 3-mercaptopropionate (M3), 2mercaptoethanol (2M), 2-aminoethanethiol (2A), 2-phenylethanethiol (2P), and poly(ethylene glycol) methyl ether thiol (PEG-SH) at a ligand-SH/PDS molar ratio of 3/2 via stirring at room temperature overnight. As observed from the 1H NMR spectra in Figure 6, the signals from PDS groups almost vanish while new peaks of conjugated ligands appear accordingly. Considering that the hydrophobicity/hydrophilicity of 3mercaptopropionic acid and methyl 3-mercaptopropionate are quite different, it indicates that the substitution reaction is quite efficient and irrelevant to the lipophilicity of the ligands. This can be further confirmed by the postmodification with hydrophobic 2-phenylethanethiol and other hydrophilic ligands as observed from the 1H NMR spectra in Figure S14. Although other reactive groups like acryloyl groups can also be utilized as scaffold for thiol-containing ligand conjugation, the conversions of such reactions still need to be improved. The conversions of PDS groups in present system are all higher than 95% under mild conditions, displaying great advantage over other systems. Thiol−disulfide exchange reaction involving the pyridyl disulfide group is appealing for bioconjugation of functional polymers due to the mild conditions and versatility for immobilization of various peptides and proteins.76 After successful optimization of the postmodification conditions, enhanced green fluorescent protein (EGFP) as representative protein was used to prepare polymer−protein conjugates. The water-soluble polymer PVPD2% was utilized to make the reaction proceed in aqueous solution instead of organic solvent to avoid denaturation of the protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to confirm the successful conjugation as demonstrated in Figure 7. Upon conjugation to PVPD2%, an increase in molecular weight of EGFP was observed in lane 2 compared with free EGFP in lane 3. The polymer in lane 3 did not stain and thus has no influence on the localization of the protein. As indicated from the SDS-PAGE results, the polymer−protein conjugates possess a molecular weight of around 38 kDa, which

Figure 7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lane (1) and (5) standard protein ladder, (2) PVPD2%−EGFP, (3) EGFP, and (4) PVPD2% the same concentration as in lane 2.

is approximately 12 kDa larger than that of EGFP. According to data in Table 1, it can be calculated that each polymer chain of PVPD2% has 1.44 PDS groups in average. In theory, the molecular weight of the polymer−protein conjugates should be around 11.8 kDa when all the PDS groups were reacted, which is quite close to the SDS-PAGE results. It further verifies the successful conjugation. These results safely confirm that PDSfunctionalized copolymers are amenable to facile derivatization with thiol-containing ligands, highly selective, and tolerant to most functional groups including phenyl, hydroxyl, amino, carboxyl, amine, PEG, and protein. Surface Engineering of PDS-Functionalized Polymeric Films. After successful postmodification of the PDS-functionalized polymers with different ligands, we tried to fabricate functional polymer films by spin-coating of organic copolymer solutions on solid substrates. For the fabrication of thin films we selected nonwater-soluble copolymer (PVPD10%) to make sure that no dissolution of polymer chains in aqueous solutions will take place. The polymeric film demonstrated smooth surface as confirmed by the AFM high and phase images presented in Figure 8. The film was scratched to measure its thickness by scanning the cross section with AFM. As displayed in Figures 8c and 8d, the polymer film was around 35 nm thick. This data is in good accordance with the ellipsometry result, which detected the film thickness as 34 nm. The obtained thin polymer films were treated with water-soluble thiol-containing molecules in aqueous solutions. These films should demonstrate different surface properties compared with the parent copolymer film, which was proved by the static contact angle measurement results shown in Figure 9. Remarkably, the films prepared with PVPD−3M and PVPD−PEG became significantly more hydrophilic with contact angles reduction by 12.2° and 19.5°, respectively, depending on the nature of conjugated ligands. However, only small variation of contact angles for PVPD−2M and PVPD−2A films was observed under otherwise the same conditions, indicating similar hydrophilicity of the ligands. The PVPD−EGFP film was characterized with fluorescence microscopy. It is clearly observed that the film is fluorescent compared with the parent film in Figure 10. The above results indicate that the PDS-functionalized polymeric films allow efficient and versatile surface engineering with thiolcontaining ligands in aqueous solution under mild conditions. This could be attributed to the highly reactivity of the PDS J


Article

Macromolecules

Figure 8. (a) Height and (b) phase images of the polymer film measured by AFM. (c) Height image of cross section of the scratch on the polymer film. (d) Height profile of lines 1, 2, and 3 in image c. The scale bar is 2 μm.

Figure 10. (a) Fluorescence microscopy images of the parent film prepared with copolymer PVPD10% and (b) film modified with EGFP.

Cell Culture Study on Functional Films. Considering that the surface properties of the polymeric films have significant influence on the in vivo performance of medical devices and scaffolds, cell culture experiments using HeLa cells were performed to study cell attachment and growth activities on the surface of parent and modified films. As observed in Figure 11, the parent PVPD film (Figure 11a) can well support cell attachment and growth like the tissue culture plate (Figure 11c) while the PEG (a type of antifouling polymer) modified films (Figure 11b) displayed diminished cell adhesion and growth compared with the parent film as well as the tissue

Figure 9. Static contact angle measurement results of functional films prepared via modified copolymers.

groups with thiol groups via thiol−disulfide exchange reaction and the hydrophilic property of these groups, making them readily accessible on the surface in aqueous solution. K


Article

Macromolecules

Figure 11. Images of HeLa cells cultured on the unmodified and modified PVPD10% films for 1, 4, and 7 days: (a) unmodified PVPD10% film, (b) PEG-SH modified film, and (c) tissue culture plate (control sample). The scale bar is 1000 μm.

potential in developing diverse multifunctional bioactive polymeric materials for various applications including tissue engineering, medical treatment, and diagnostic molecular biology.

culture plate. This preliminary experiment suggests that the PVPD parent film is nontoxic and biocompatible and can be elegantly engineered on the surface to control the cell growth activity, meeting demands of a particular application. These PDS-functionalized polymers offer a new strategy to anchor varying ligands and even proteins on synthetic biocompatible polymers for potential applications in various fields including tissue engineering, medical treatment, and diagnostic molecular biology.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b01210. Synthetic characterization, FTIR, Raman, polymer composition, and characterization data (PDF)

4. CONCLUSION In summary, a series of reactive statistical copolymers based on N-vinyl lactams (N-vinylpyrrolidone, N-vinylcaprolactam, and N-vinylpiperidone) and pyridyl disulfide ethyl methacrylate (PDSM) were synthesized via RAFT polymerization at 60 °C in anisole. The polymerizations proceed in controlled manner as confirmed by the low Đ, linear relationship between conversion and molecular weight, and symmetrical unimodal GPC traces. The experimental data indicated that steric hindrance originating from the N-vinyl lactam monomers is a main factor influencing copolymerization with PDSM as the enlargement of the lactam ring made the monomer conversions and controllability of the polymerization decreased. The PDSfunctionalized copolymers enable facile access to complex polymer architectures through highly selective thiol−disulfide exchange reaction with thiol-containing ligands including phenyl, hydroxyl, amino, carboxyl, amine, PEG, and protein under extremely mild conditions. The biocompatible thin polymer film prepared using water nonsoluble PDS-functionalized copolymer can be elegantly decorated by surface engineering with thiol-containing molecules to control cell attachment and growth activity. The parent polymer film can well support cell attachment and growth while the film modified with PEG (a type of antifouling polymer) exhibited diminished cell adhesion and growth. We believe the biocompatible PDS-functionalized copolymers have great

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.P.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS H.P. thanks China Scholarship Council for financial support. A.P. thanks Volkswagen Foundation and Deutsche Forschungsgemeinschaft (DFG) with Collaborative Research Center SFB 985 “Functional Microgels and Microgel Systems” for financial support.

■

REFERENCES

(1) Kasai, Y.; Tamai, Y.; Ohkita, H.; Benten, H.; Ito, S. Ultrafast Singlet Fission in a Push−Pull Low-Bandgap Polymer Film. J. Am. Chem. Soc. 2015, 137, 15980−15983. (2) Lu, X.; Hlaing, H.; Nam, C.; Yager, K.; Black, C.; Ocko, M. Molecular Orientation and Performance of Nanoimprinted PolymerBased Blend Thin Film Solar Cells. Chem. Mater. 2015, 27, 60−66. (3) Li, Z.; Ma, G.; Ge, R.; Qin, F.; Dong, X.; Meng, W.; Liu, T.; Tong, J.; Jiang, F.; Zhou, Y.; Li, K.; Min, X.; Huo, K.; Zhou, Y. Free-

L


Article

Macromolecules Standing Conducting Polymer Films for High-Performance Energy Devices. Angew. Chem., Int. Ed. 2016, 55, 979−982. (4) Zhou, F.; Zheng, Z.; Yu, B.; Liu, W.; Huck, W. Multicomponent Polymer Brushes. J. Am. Chem. Soc. 2006, 128, 16253−16258. (5) Steenackers, M.; Gigler, A.; Zhang, N.; Deubel, F.; Seifert, M.; Hess, L.; Lim, C.; Loh, K.; Garrido, J.; Jordan, R.; Stutzmann, M.; Sharp, I. Polymer Brushes on Graphene. J. Am. Chem. Soc. 2011, 133, 10490−10498. (6) Poelma, J. E.; Fors, B. P.; Meyers, G. F.; Kramer, J. W.; Hawker, C. J. Fabrication of Complex Three-Dimensional Polymer Brush Nanostructures through Light-Mediated Living Radical Polymerization. Angew. Chem. 2013, 125, 6982−6986. (7) Feng, L.; Laderman, B.; Sacanna, S.; Chaikin, P. Re-entrant solidification in polymer−colloid mixtures as a consequence of competing entropic and enthalpic attractions. Nat. Mater. 2015, 14, 61−65. (8) Bigall, N.; Wilhelm, C.; Beoutis, M.; Hernandez, M.; Khan, A.; Giannini, C.; Ferrer, A.; Mezzenga, R.; Materia, M.; Garcia, M.; Gazeau, F.; Bittner, A.; Manna, L.; Pellegrino, T. Colloidal Ordered Assemblies in a Polymer ShellA Novel Type of Magnetic Nanobeads for Theranostic Applications. Chem. Mater. 2013, 25, 1055−1062. (9) Boyer, C.; Bulmus, V.; Liu, J.; Davis, T.; Stenzel, M.; BarnerKowollik, C. Well-Defined Protein−Polymer Conjugates via in Situ RAFT Polymerization. J. Am. Chem. Soc. 2007, 129, 7145−7154. (10) Danial, M.; Tran, C.; Jolliffe, K.; Perrier, S. Thermal Gating in Lipid Membranes Using Thermoresponsive Cyclic Peptide−Polymer Conjugates. J. Am. Chem. Soc. 2014, 136, 8018−8026. (11) Cobo, I.; Li, M.; Sumerlin, B.; Perrier, S. Smart hybrid materials by conjugation of responsive polymers to biomacromolecules. Nat. Mater. 2015, 14, 143−159. (12) O’Day, E.; Lin, E.; Maynard, H. Therapeutic Protein−Polymer Conjugates: Advancing Beyond PEGylation. J. Am. Chem. Soc. 2014, 136, 14323−14332. (13) Lele, B.; Murata, H.; Matyjaszewski, K.; Russell, A. Synthesis of Uniform Protein−Polymer Conjugates. Biomacromolecules 2005, 6, 3380−3387. (14) Gaylord, B.; Heeger, A.; Bazan, G. Proc. DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 10954−10957. (15) Ho, H.-A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Doré, K.; Boudreau, D.; Leclerc, M. Colorimetric and Fluorometric Detection of Nucleic Acids Using Cationic Polythiophene Derivatives. Angew. Chem. 2002, 114, 1618−1621. (16) Kühnle, H.; Börner, H. G. Biotransformation on Polymer− Peptide Conjugates: A Versatile Tool to Trigger Microstructure Formation. Angew. Chem., Int. Ed. 2009, 48, 6431−6434. (17) Zhang, Q.; Li, M.; Zhu, C.; Nurumbetov, G.; Li, Z.; Wilson, P.; Kempe, K.; Haddleton, D. Well-Defined Protein/Peptide−Polymer Conjugates by Aqueous Cu-LRP: Synthesis and Controlled SelfAssembly. J. Am. Chem. Soc. 2015, 137, 9344−9353. (18) Mukherjee, B.; Karmakar, S.; Hossain, C.; Bhattacharya, S. Peptides, Proteins and Peptide/Protein-Polymer Conjugates as Drug Delivery System. Protein Pept. Lett. 2014, 21, 1121−1128. (19) Ge, J.; Neofytou, E.; Lei, J.; Beygui, R. E.; Zare, R. N. Protein− Polymer Hybrid Nanoparticles for Drug Delivery. Small 2012, 8, 3573−3578. (20) Larson, N.; Ghandehari, H. Polymeric Conjugates for Drug Delivery. Chem. Mater. 2012, 24, 840−853. (21) Keddie, D.; Moad, G.; Rizzardo, E.; Thang, S. RAFT Agent Design and Synthesis. Macromolecules 2012, 45, 5321−5342. (22) Benaglia, M.; Chiefari, J.; Chong, Y.; Moad, G.; Rizzardo, E.; Thang, S. Universal (Switchable) RAFT Agents. J. Am. Chem. Soc. 2009, 131, 6914−6915. (23) Simakova, A.; Averick, S.; Konkolewicz, D.; Matyjaszewski, K. Aqueous ARGET ATRP. Macromolecules 2012, 45, 6371−6379. (24) Li, W.; Matyjaszewski, K. Star Polymers via Cross-Linking Amphiphilic Macroinitiators by AGET ATRP in Aqueous Media. J. Am. Chem. Soc. 2009, 131, 10378−10379.

(25) Gao, H.; Matyjaszewski, K. Synthesis of Molecular Brushes by “Grafting onto” Method: Combination of ATRP and Click Reactions. J. Am. Chem. Soc. 2007, 129, 6633−6639. (26) Sciannamea, V.; Jérôme, R.; Detrembleur, C. In-Situ NitroxideMediated Radical Polymerization (NMP) Processes: Their Understanding and Optimization. Chem. Rev. 2008, 108, 1104−1126. (27) Nicolas, J.; Guillaneuf, Y.; Lefay, C.; Bertin, D.; Gigmes, D.; Charleux, B. Nitroxide-mediated polymerization. Prog. Polym. Sci. 2013, 38, 63−235. (28) Zetterlund, P.; Kagawa, Y.; Okubo, M. Controlled/Living Radical Polymerization in Dispersed Systems. Chem. Rev. 2008, 108, 3747−3794. (29) Zetterlund, P.; Thickett, S.; Perrier, S.; Bourgeat-Lami, E.; Lansalot, M. Controlled/Living Radical Polymerization in Dispersed Systems: An Update. Chem. Rev. 2015, 115, 9745−9800. (30) Hauser, C.; Jagielski, N.; Heller, J.; Hinderberger, D.; Spiess, H.; Lieberwirth, I.; Weiss, C.; Landfester, K. Structure Formation in Metal Complex/Polymer Hybrid Nanomaterials Prepared by Miniemulsion. Langmuir 2011, 27, 12859−12868. (31) Tam, K.; Wyn-Jones, E. Insights on polymer surfactant complex structures during the binding of surfactants to polymers as measured by equilibrium and structural techniques. Chem. Soc. Rev. 2006, 35, 693−709. (32) Gregory, A.; Stenzel, M. Complex polymer architectures via RAFT polymerization: From fundamental process to extending the scope using click chemistry and nature’s building blocks. Prog. Polym. Sci. 2012, 37, 38−105. (33) Akkarachaneeyakorn, K.; Li, M.; Harris, J.; Davis, S.; Mann, S. Bioactive Hybrid Organogels Based on Miniemulsion Synthesis of Morphologically Complex Polymer/Surfactant/Calcium Phosphate Nanostructures. Chem. Mater. 2014, 26, 5965−5972. (34) Allen, T.; Cullis, P. Drug delivery systems: entering the mainstream. Science 2004, 303, 1818−1822. (35) Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P. Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery. Chem. Soc. Rev. 2013, 42, 1147−1235. (36) Matricardi, P.; Di Meo, C.; Coviello, T.; Hennink, W.; Alhaique, F. Interpenetrating Polymer Networks polysaccharide hydrogels for drug delivery and tissue engineering. Adv. Drug Delivery Rev. 2013, 65, 1172−1187. (37) Liu, X.; Holzwarth, J. M.; Ma, P. X. Functionalized Synthetic Biodegradable Polymer Scaffolds for Tissue Engineering. Macromol. Biosci. 2012, 12, 911−919. (38) Kim, J.; Park, K.; Nam, H.; Lee, S.; Kim, K.; Kwon, I. Polymers for bioimaging. Prog. Polym. Sci. 2007, 32, 1031−1053. (39) Zhao, W.; Jin, X.; Cong, Y.; Liu, Y.; Fu, J. Degradable natural polymer hydrogels for articular cartilage tissue engineering. J. Chem. Technol. Biotechnol. 2013, 88, 327−339. (40) Heredia, K.; Tolstyka, Z.; Maynard, H. Aminooxy EndFunctionalized Polymers Synthesized by ATRP for Chemoselective Conjugation to Proteins. Macromolecules 2007, 40, 4772−4779. (41) Sunder, A.; Hanselmann, R.; Frey, H.; Muelhaupt, R. Controlled Synthesis of Hyperbranched Polyglycerols by Ring-Opening Multibranching Polymerization. Macromolecules 1999, 32, 4240−4246. (42) Desai, A.; Atkinson, N.; Rivera, F.; Devonport, W.; Rees, I.; Branz, S. E.; Hawker, C. J. Hybrid dendritic−linear graft copolymers: Steric considerations in “coupling to” approach. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 1033−1044. (43) Muthukrishnan, S.; Pan, E.; Stenzel, M.; Barner-Kowollik, C.; Davis, T.; Lewis, D.; Barner, L. Ambient Temperature RAFT Polymerization of Acrylic Acid Initiated with Ultraviolet Radiation in Aqueous Solution. Macromolecules 2007, 40, 2978−2980. (44) Peng, H.; Kather, M.; Rübsam, K.; Jakob, F.; Schwaneberg, U.; Pich, A. Water-Soluble Reactive Copolymers Based on Cyclic NVinylamides with Succinimide Side Groups for Bioconjugation with Proteins. Macromolecules 2015, 48, 4256−4268. M


Article

Macromolecules (45) Lybaert, L.; Vanparijs, N.; Fierens, K.; Schuijs, M.; Nuhn, L.; Lambrecht, B.; De Geest, B. A Generic Polymer−Protein Ligation Strategy for Vaccine Delivery. Biomacromolecules 2016, 17, 874−881. (46) Wang, Y.; Hong, C.; Pan, C. Galactose-Based Amphiphilic Block Copolymers: Synthesis, Micellization, and Bioapplication. Biomacromolecules 2013, 14, 1444−1451. (47) Huang, P.; Liu, J.; Wang, W.; Li, C.; Zhou, J.; Wang, X.; Deng, L.; Kong, D.; Liu, J.; Dong, A. Zwitterionic Nanoparticles Constructed with Well-Defined Reduction-Responsive Shell and pH-Sensitive Core for “Spatiotemporally Pinpointed” Drug Delivery. ACS Appl. Mater. Interfaces 2014, 6, 14631−14643. (48) Wong, L.; Boyer, C.; Jia, Z.; Zareie, H.; Davis, T.; Bulmus, V. Synthesis of versatile thiol-reactive polymer scaffolds via RAFT polymerization. Biomacromolecules 2008, 9, 1934−1944. (49) Ghosh, S.; Basu, S.; Thayumanavan, S. Simultaneous and Reversible Functionalization of Copolymers for Biological Applications. Macromolecules 2006, 39, 5595−5597. (50) Grover, G.; Lee, J.; Matsumoto, N.; Maynard, H. Aminooxy and Pyridyl Disulfide Telechelic Poly(poly(ethylene glycol) acrylate) by RAFT Polymerization. Macromolecules 2012, 45, 4958−4965. (51) Vázquez-Dorbatt, V.; Tolstyka, Z.; Chang, C.; Maynard, H. Synthesis of a Pyridyl Disulfide End-Functionalized Glycopolymer for Conjugation to Biomolecules and Patterning on Gold Surfaces. Biomacromolecules 2009, 10, 2207−2212. (52) Vanparijs, N.; Maji, S.; Voorhaar, L.; Laplace, D.; Zhang, Q.; Shi, Y.; Hennink, W.; Hoogenboom, R.; De Geest, B. Polymer-protein conjugation via a ‘grafting to’ approach − a comparative study of the performance of protein-reactive RAFT chain transfer agents. Polym. Chem. 2015, 6, 5602−5614. (53) Yang, S.; Choi, J.; Palanikumar, L.; Choi, E.; Lee, J.; Kim, J.; Choi, I.; Ryu, J. Cytocompatible in situ cross-linking of degradable LbL films based on thiol−exchange reaction. Chem. Sci. 2015, 6, 4698− 4703. (54) Ryu, J.; Chacko, R.; Jiwpanich, S.; Bickerton, S.; Babu, R.; Thayumanavan, S. Self-Cross-Linked Polymer Nanogels: A Versatile Nanoscopic Drug Delivery Platform. J. Am. Chem. Soc. 2010, 132, 17227−17235. (55) Bontempo, D.; Heredia, K.; Fish, B.; Maynard, H. CysteineReactive Polymers Synthesized by Atom Transfer Radical Polymerization for Conjugation to Proteins. J. Am. Chem. Soc. 2004, 126, 15372−15373. (56) Huang, X.; Appelhans, D.; Formanek, P.; Simon, F.; Voit, B. Tailored Synthesis of Intelligent Polymer Nanocapsules: An Investigation of Controlled Permeability and pH-Dependent Degradability. ACS Nano 2012, 6, 9718−9726. (57) Tijink, M.; Janssen, J.; Timmer, M.; Austen, J.; Aldenhoff, Y.; Kooman, J.; Koole, L.; Damoiseaux, J.; van Oerle, R.; Henskens, Y.; Stamatialis, D. Development of novel membranes for blood purification therapies based on copolymers of N-vinylpyrrolidone and n-butylmethacrylate. J. Mater. Chem. B 2013, 1, 6066−6077. (58) Spulber, M.; Baumann, P.; Saxer, S.; Pieles, U.; Meier, W.; Bruns, N. Poly(N-vinylpyrrolidone)-Poly(dimethylsiloxane)-Based Polymersome Nanoreactors for Laccase-Catalyzed Biotransformations. Biomacromolecules 2014, 15, 1469−1475. (59) Bronstein, L.; Kostylev, M.; Tsvetkova, I.; Tomaszewski, J.; Stein, B.; Makhaeva, E.; Okhapkin, I.; Khokhlov, A. Core−Shell Nanostructures from Single Poly(N-vinylcaprolactam) Macromolecules: Stabilization and Visualization. Langmuir 2005, 21, 2652−2655. (60) Liang, X.; Kozlovskaya, V.; Chen, Y.; Zavgorodnya, O.; Kharlampieva, E. Thermosensitive multilayer hydrogels of poly(Nvinylcaprolactam) as nanothin films and shaped capsules. Chem. Mater. 2012, 24, 3707−3719. (61) Nakabayashi, K.; Mori, H. Recent progress in controlled radical polymerization of N-vinyl monomers. Eur. Polym. J. 2013, 49, 2808− 2838. (62) Stace, S.; Moad, G.; Fellows, C.; Keddie, D. The effect of Zgroup modification on the RAFT polymerization of N-vinylpyrrolidone controlled by “switchable” N-pyridyl-functional dithiocarbamates. Polym. Chem. 2015, 6, 7119−7126.

(63) Kuang, H.; Yu, Y.; Shin, S.; Kim, J.; Youk, J. One-Pot Synthesis of Poly(N-vinylpyrrolidone)-b-poly(ε-caprolactone) Block Copolymers Using a Dual Initiator for RAFT Polymerization and ROP. Macromolecules 2013, 46, 1291−1295. (64) Guinaudeau, A.; Coutelier, O.; Sandeau, A.; Mazieres, S.; Thi, H.; Le Drogo, V.; Wilson, D.; Destarac, M. Facile Access to Poly(Nvinylpyrrolidone)-Based Double Hydrophilic Block Copolymers by Aqueous Ambient RAFT/MADIX Polymerization. Macromolecules 2014, 47, 41−50. (65) Greenley, R. Z. Recalculation of Some Reactivity Ratios. J. Macromol. Sci., Chem. 1980, 14, 445−515. (66) Noor, M.; Dworeck, T.; Schenk, A.; Shinde, P.; Fioroni, M.; Schwaneberg, U. Polymersome surface decoration by an EGFP fusion protein employing Cecropin A as peptide “anchor. J. Biotechnol. 2012, 157, 31−37. (67) Beija, M.; Marty, J. D.; Destarac, M. Thermoresponsive poly(Nvinyl caprolactam)-coated gold nanoparticles: sharp reversible response and easy tenability. Chem. Commun. 2011, 47, 2826−2828. (68) Peng, H.; Xu, W.; Pich, A. Temperature and pH dual-responsive poly(vinyl lactam) copolymers functionalized with amine side groups via RAFT polymerization. Polym. Chem. 2016, 7, 5011−5022. (69) Laemmli, U. K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 1970, 227, 680− 685. (70) Perrier, S.; Takolpuckdee, P. Macromolecular design via reversible addition−fragmentation chain transfer (RAFT)/xanthates (MADIX) polymerization. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 5347−5393. (71) Suzuki, S.; Kikuchi, K.; Suzuki, A.; Okaya, T.; Nomura, M. Effect of chain transfer agents on the kinetics and mechanism of particle nucleation in the emulsion polymerization of vinyl pivalate. Colloid Polym. Sci. 2007, 285, 523−534. (72) Van Hook, J. P.; Tobolsky, A. V. The Thermal Decomposition of 2,2′-Azo-bis-isobutyronitrile. J. Am. Chem. Soc. 1958, 80 (779−782), 779. (73) Mori, H.; Matsuyama, M.; Sutoh, K.; Endo, T. RAFT Polymerization of Acrylamide Derivatives Containing l-Phenylalanine Moiety. Macromolecules 2006, 39, 4351−4360. (74) Arzamendi, G.; Asua, J. M. Monomer addition policies for copolymer composition control in semicontinuous emulsion copolymerization. J. Appl. Polym. Sci. 1989, 38, 2019−2036. (75) Van Doremaele, G. H. J.; Schoonbrood, H. A. S.; Kurja, J.; German, A. L. Copolymer composition control by means of semicontinuous emulsion copolymerization. J. Appl. Polym. Sci. 1992, 45, 957−966. (76) van der Vlies, A. J.; O’Neil, C. P.; Hasegawa, U.; Hammond, N.; Hubbell, J. A. Synthesis of Pyridyl Disulfide-Functionalized Nanoparticles for Conjugating Thiol-Containing Small Molecules, Peptides, and Proteins. Bioconjugate Chem. 2010, 21, 653−662.

N


Reactive Copolymers Based on N-Vinyl Lactams with Pyridyl Disulfide

Recommend Documents