A new method of controlled grafting modification of chitosan via nitroxide-mediated polymerization using chitosan-TEMPO macroinitiator

The controlled graft modification of chitosan has first been achieved by nitroxide-mediated polymerization using chitosan-TEMPO macroinitiator. Chitosan-TEMPO macroinitiator was obtained from the (60)Co gamma-ray irradiation of N-phthaloylchitosan and 4-hydroxy-TEMPO in DMF under argon atmosphere. The graft copolymers were characterized by (1)H nuclear magnetic resonance ((1)H NMR), Fourier transform infrared spectrometer (FT-IR), X-ray powder diffractometer (XRD) and high performance particle sizer (HPPS). The results indicate that the graft copolymers were successfully synthesized and that the graft polymerization was well controlled by the nitroxide-mediated process. The size distribution of chitosan-g-polystyrene in benzene is very narrow, which may be associated with the "well-defined" polystyrene (PSt) onto chitosan from nitroxide-mediated polymerization. This work provides a new method to prepare chitosan grafting copolymers with controlled molecular weights and "well-defined" structures.     

Bioengineering functional copolymers. XXI. Synthesis of a novel end carboxyl-trithiocarbonate functionalized poly(maleic anhydride) and its interaction with cancer cells

A novel carboxyl-trithiocarbonate functionalized polymer with a highly selective antitumor activity was synthesized by a reversible addition-fragmentation chain transfer (RAFT) polymerization of maleic anhydride (MA) with benzoyl peroxide as an initiator and S-1-dodecyl-S-(α,α'-dimethyl-α″-acetic acid)trithiocarbonate as a RAFT agent with the aim to design and synthesize an effective anticancer agent with minimum side effects. The structure, molecular weights and composition of synthesized polymers were investigated by (1)H ((13)C) NMR, MALDI-TOF-MS and GPC analyzes. It was demonstrated that RAFT polymerization of MA was accompanied by a partially controlled decarboxylation of anhydride units and the formation of conjugated double bond fragments in backbone macromolecular chains. The mechanism of interaction of pristine RAFT agent and PMA-RAFT polymer with cancer (HeLa human cervix carcinoma) and normal (L929 Fibroblast) cells was investigated by using a combination of chemical, biochemical, statistical, spectroscopic (SEM and fluorescence inverted microscope) and real-time analysis (RTCA) methods. PMA-RAFT exhibited higher and selective cytotoxicity, apoptotic and necrotic effects toward HeLa cells at relatively low concentrations (around 7.5-75μgmL(-1), IC(50)=11.183μgmL(-1)) and toward Fibroblast cells at high concentrations (IC(50)>100μgmL(-1)). The observed highly selective antitumor activity render PMA-RAFT polymers as promising candidates for the utilization in cancer chemotherapy.     

Chemoenzymatic syntheses of amylose-grafted chitin and chitosan

Amylose-grafted chitin and chitosan were synthesized by chemoenzymatic methods according to the following reaction manners. First, maltoheptaose was introduced to chitosan by a reductive amination using sodium cyanotrihydroborate in a mixed solvent of 1.0 mol/L aqueous acetic acid and methanol at room temperature to produce a maltoheptaose-grafted chitosan (1). The functionality of maltoheptaose to chitosan in 1 depended on reaction time. The phosphorylase-catalyzed enzymatic polymerization of R-D-glucose 1-phosphate was then performed from 1 to obtain amylose-grafted chitosan (2). Maltoheptaose-grafted chitin (3) was synthesized by N-acetylation of 1 using acetic anhydride in a mixed solvent of aqueous acetic acid and methanol. Then, synthesis of amylose-grafted chitin (4) was performed by the phosphorylase-catalyzed enzymatic polymerization under conditions the same as those for 2. The average DPs of amylose graft chains in 2 and 4 depended on the feed ratios of R-D-glucose 1-phosphate to maltoheptaose primers in 1 and 3.     

Preparation, characterization, and in vitro drug release behavior of biodegradable chitosan-graft-poly(1, 4-dioxan-2-one) copolymer

A novel copolymer of chitosan-g-poly(p-dioxanone) (CGP) was synthesized in bulk by ring-opening polymerization of p-dioxanone (PDO) initiated by the hydroxyl group or amino group of chitosan using SnOct₂ as catalyst. The chemical structure was determined by ¹H NMR. It was found that the feed ratio of chitosan to PDO had a great effect on the degree of polymerization (DP) and the substitution (DS) of PDO. The thermal stability and crystallization behavior of graft copolymer CGP were closely related to the values of DP and DS. When the resulting copolymer was used as Ibuprofen carrier, the release rate of Ibuprofen decreased compared with that of pure chitosan carrier. The drug release behavior was also influenced by the structure of graft copolymers.     

Degradable thermoresponsive nanogels for protein encapsulation and controlled release

Reversible addition-fragmentation chain transfer (RAFT) polymerization technique was used for the fabrication of stable core cross-linked micelles (CCL) with thermoresponsive and degradable cores. Well-defined poly(2-methacryloyloxyethyl phosphorylcholine), poly(MPC) macroRAFT agent, was first synthesized with narrow molecular weight distribution via the RAFT process. These CCL micelles (termed as nanogels) with hydrophilic poly(MPC) shell and thermoresponsive core consisting of poly(methoxydiethylene glycol methacrylate) (poly(MeODEGM) and poly(2-aminoethyl methacrylamide hydrochloride) (poly(AEMA) were then obtained in a one-pot process by RAFT polymerization in the presence of an acid degradable cross-linker. These acid degradable nanogels were efficiently synthesized with tunable sizes and low polydispersities. The encapsulation efficiencies of the nanogels with different proteins such as insulin, BSA, and β-galactosidase were studied and found to be dependent of the cross-linker concentration, size of protein, and the cationic character of the nanogels imparted by the presence of AEMA in the core. The thermoresponsive nature of the synthesized nanogels plays a vital role in protein encapsulation: the hydrophilic core and shell of the nanogels at low temperature allow easy diffusion of the proteins inside out and, with an increase in temperature, the core becomes hydrophobic and the nanogels are easily separated out with entrapped protein. The release profile of insulin from nanogels at low pH was studied and results were analyzed using bicinchoninic assay (BCA). Controlled release of protein was observed over 48 h.     

Synthesis and property of chitosan graft copolymer by RAFT polymerization with tosylic acid-chitosan complex

pH- and thermo-sensitive (1→4)-2-amino-2-deoxy-β-d-glucan (i.e. chitosan) graft copolymer was prepared by reversible addition fragmentation chain transfer polymerizations of N-isopropylacrylamide with 4-methylbenzenesulfonic acid (i.e. tosylic acid)-chitosan complex. The polymerization was controlled well, and the amino group of chitosan could be deprotected easily and mildly with 15% Tris solution. The model aldehyde vanillin was conjugated with amino group of chitosan-g-PNIPAM via Schiff base bond (Loading efficiency, LE=77.6 mg/g), and the drug release could be controlled with temperature and pH. This property may promote the chitosan graft copolymer to be used in the field of "smart" drug delivery.     

Synthesis of temperature-responsive heterobifunctional block copolymers of poly(ethylene glycol) and poly(N-isopropylacrylamide)

Heterobifunctional block copolymers of poly(ethylene glycol) (PEG) and poly(N-isopropylacrylamide) (PNIPAM) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of NIPAM using a macromolecular trithiocarbonate PEG-based chain transfer agent. The polymerization showed all the expected features of living radical polymerization and allowed the synthesis of copolymers with different lengths of the PNIPAM block. The synthesized block copolymers contained a carboxylic acid group from L-lysine at the focal point and a trithiocarbonate group at the terminus of the PNIPAM block. The trithiocarbonate functionality was converted into a thiol group and used for conjugation of biotin to the end of the PNIPAM block. The copolymers exhibited temperature-dependent association behavior in aqueous solution with a phase transition of approximately 32 degrees C. The described heterobifunctional block copolymers show promise for surface modifications with the potential for stimulus-controlled surface presentation of ligands attached to the terminus of the PNIPAM block.     

Folate-conjugated thermoresponsive block copolymers: highly efficient conjugation and solution self-assembly

A combination of controlled radical polymerization and azide-alkyne click chemistry was employed to prepare temperature-responsive block copolymer micelles conjugated with biological ligands with potential for active targeting of cancer tissues. Block copolymers of N-isopropylacrylamide (NIPAM) and N,N-dimethylacrylamide (DMA) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization with an azido chain transfer agent (CTA). Pseudo-first-order kinetics and linear molecular weight dependence on conversion were observed for the RAFT polymerizations. CuI-catalyzed coupling with propargyl folate resulted in folic acid residues being efficiently conjugated to the alpha-azido chain ends of the homo and block copolymers. Temperature-induced self-assembly resulted in aggregates capable of controlled release of a model hydrophobic drug. CuI-catalyzed azide-alkyne cycloaddition has proven superior to conventional methods for conjugation of biological ligands to macromolecules, and the general strategy presented herein can potentially be extended to the preparation of folate-functionalized assemblies with other stimuli susceptibility (e.g., pH) for therapeutic and imaging applications.     

Facile, controlled, room-temperature RAFT polymerization of N-isopropylacrylamide

Poly(N-isopropyl acrylamide) is a thermoresponsive polymer that has been widely investigated for drug delivery. Herein, we report conditions facilitating the controlled, room-temperature RAFT polymerization of N-isopropylacrylamide (NIPAM). The key to success is the appropriate choice of both a suitable RAFT chain transfer agent (CTA) and initiating species. We show that the use of 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid, a trithiocarbonate RAFT CTA, in conjunction with the room-temperature azo initiator 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), in DMF, at 25 degrees C, yields conditions leading to NIPAM homopolymerizations which bear all of the characteristics of a controlled/"living" polymerization. We also demonstrate facile size exclusion chromatographic analysis of PNIPAM samples in DMF at 60 degrees C, directly on aliquots withdrawn during the polymerizations, which avoids the problems previously reported in the literature.     

One-pot synthesis of chitosan-g-(PEO-PLLA-PEO) via "click" chemistry and "SET-NRC" reaction

For the development of biocompatible and degradable biomaterials, a kind of well-defined graft copolymer consisting of chitosan back-bone and amphiphilic PEO-PLLA-PEO branch chains was synthesized by Cu(0) catalyzed one-pot strategy combining "click" chemistry and single electron transfer-nitroxide radical coupling (SET-NRC) reaction. First, the precursors of 6-azide-N-phthaloyl-chitosan, TEMPO-PEO-alkyne and mPEO-PLLA-Br were designed and produced. Then, the one-pot coupling reactions between these precursors were performed in the presence of nanosized Cu and PMDETA. The efficiencies of the coupling reactions were greater than 90% determined by the FTIR and ESR spectra. The structure of graft copolymer with 43% of the grafting ratio was confirmed by the spectral analysis. This work provided a route to prepare chitosan graft copolymer.     

Facile synthetic procedure for omega, primary amine functionalization directly in water for subsequent fluorescent labeling and potential bioconjugation of RAFT-synthesized (co)polymers

We describe a facile method to amine functionalize and subsequently fluorescently label polymethacrylamides synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. RAFT-generated poly(N-(2-hydroxypropyl) methacrylamide-b-N-[3-(dimethylamino)propyl] methacrylamide) (poly(HPMA-b-DMAPMA)), a water soluble biocompatible polymer, is first converted to a polymeric thiol and functionalized with a primary amine through a disulfide exchange reaction with cystamine and subsequently reacted with the amine-functionalized fluorescent dye, 6-(fluorescein-5-carboxamido)hexanoic acid, succinimidyl ester (5-SFX). Poly(HPMA258-b-DMAPMA13) (Mn = 39 700 g/mol, Mw/Mn = 1.06), previously synthesized by RAFT polymerization, was used to demonstrate this facile labeling method. The problem with labeling the omega-terminal chain end of a RAFT-synthesized polymethacrylamide is that the reduced end yields a tertiary thiol with low reactivity. The key to labeling poly(HPMA-b-DMAPMA) is to first reduce the dithioester chain end with a strong reducing agent such as NaBH4, and then functionalize the tertiary polymeric thiol with a primary amine through a disulfide exchange reaction with dihydrochloride cystamine. We show that the disulfide exchange reaction is efficient and that the amine-functionalized poly(HPMA-b-DMAPMA) can be easily labeled with the fluorescent dye, 5-SFX. This concept is proven by using a ninhydrin assay to detect primary amines and UV-vis spectroscopy to measure the degree of conjugation.     

Backbone degradable multiblock N-(2-hydroxypropyl)methacrylamide copolymer conjugates via reversible addition-fragmentation chain transfer polymerization and thiol-ene coupling reaction

Telechelic water-soluble HPMA copolymers and HPMA copolymer-doxorubicin (DOX) conjugates have been synthesized by RAFT polymerization mediated by a new bifunctional chain transfer agent (CTA) that contains an enzymatically degradable oligopeptide sequence. Postpolymerization aminolysis followed by chain extension with a bis-maleimide resulted in linear high molecular weight multiblock HPMA copolymer conjugates. These polymers are enzymatically degradable; in addition to releasing the drug (DOX), the degradation of the polymer backbone resulted in products with molecular weights similar to the starting material and below the renal threshold. The new multiblock HPMA copolymers hold potential as new carriers of anticancer drugs.     

Synthesis of well-defined amphiphilic block copolymers having phospholipid polymer sequences as a novel biocompatible polymer micelle reagent

To realize safer and effective drug administration, novel well-defined and biocompatible amphiphilic block copolymers containing phospholipid polymer sequences were synthesized. At first, the homopolymer of 2-methacryloyloxyethylphosphorylcholine (MPC) was synthesized in water by reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization. The "living" polymerization was confirmed by the fact that the number-average molecular weight increased linearly with monomer conversion while the molecular weight distribution remained narrow independent of the conversion. The poly(MPC) thus prepared is end-capped with a dithioester moiety. Using the dithioester-capped poly(MPC) as a macro chain transfer agent, AB diblock copolymers of MPC and n-butyl methacrylate (BMA) were synthesized. Associative properties of the amphiphilic block copolymer (pMPC(m)-BMA(n)) with varying poly(BMA) block lengths were investigated using NMR, fluorescence probe, static light scattering (SLS), and quasi-elastic light scattering (QELS) techniques. Proton NMR data in D2O indicated highly restricted motions of the n-butyl moieties, arising from hydrophobic associations of poly(BMA) blocks. Fluorescence spectra of N-phenyl-1-naphthylamine indicated that the probes were solubilized in the polymer micelles in water. The formation of polymer micelles comprising a core with poly(BMA) blocks and shell with hydrophilic poly(MPC) blocks was suggested by SLS and QELS data. The size and mass of the micelle increased with increasing poly(BMA) block length. With an expectation of a pharmaceutical application of pMPC(m)-BMA(n), solubilization of a poorly water-soluble anticancer agent, paclitaxel (PTX), was investigated. PTX dissolved well in aqueous solutions of pMPC(m)-BMA(n) as compared with pure water, implying that PTX is incorporated into the hydrophobic core of the polymer micelle. Since excellent biocompatible poly(MPC) sequences form an outer shell of the micelle, pMPC(m)-BMA(n) may find application as a promising reagent to make a good formulation with a hydrophobic drug.     

Preparation of chitosan-g-polycaprolactone copolymers through ring-opening polymerization of epsilon-caprolactone onto phthaloyl-protected chitosan

The new biodegradable chitosan graft copolymer, chitosan-g-polycaprolactone, was synthesized by the ring-opening graft copolymerization of epsilon-caprolactone onto phthaloyl-protected chitosan (PHCS) at the hydroxyl group in the presence of tin(II) 2-ethylhexanoate catalyst via a protection-graft-deprotection procedure. Toluene acted as a swelling agent in this heterogeneous system. The grafting reactions were conducted with various PHCS/monomer/toluene feed ratios to obtain chitosan-g-polycaprolactone copolymers with various polycaprolactone contents. The chemical structure of the chitosan-g-polycaprolactone was characterized by Fourier transform infrared and one- and two-dimensional NMR spectroscopy. After deprotection, the phthaloyl group was removed and the amino group was regenerated. Thus the obtained chitosan-g-polycaprolactone was an amphoteric hybrid with a large amount of free amino groups and hydrophobic polycaprolactone side chains. Some properties of the final product were also investigated, such as crystallinity, thermal property, and solubility.     

Synthesis of various glycopolymer architectures via RAFT polymerization: from block copolymers to stars

Well-defined linear poly(acryloyl glucosamine) (PAGA) exhibiting molar masses ranging from 3 to 120 K and low polydispersities have been prepared via reversible addition-fragmentation chain transfer polymerization (RAFT) in aqueous solution without recourse to protecting group chemistry. The livingness of the process was further demonstrated by successfully chain-extending one of these polymers with N-isopropylacrylamide affording narrow dispersed thermosensitive diblocks. This strategy of polymerization was finally extended to the preparation of glycopolymer stars from Z designed non-water-soluble trifunctional RAFT agent. After the growth of very short blocks of poly(hydroxyethyl acrylate) ((-)DP(n)(branch) = 10), AGA was polymerized in aqueous solution in a controlled manner affording well-defined 3-arm glycopolymer stars.     

Photophysics of a dithioester RAFT polymerization agent and the acenaphthenyl model light-harvesting chromophore.

The spectroscopic and photophysical properties of a typical dithiobenzoate reversible addition-fragmentation chain transfer (RAFT) polymerization agent alone in solution, as a quencher of electronically excited acenaphthene in solution, and in an acenaphthene donor-dithiobenzoate acceptor dichromophore have been investigated. The RAFT agent is an effective quencher of photo-excited acenaphthene. Energy wastage in the dichromophore occurs by F?rster resonant electronic energy transfer from the S1 state of acenaphthene to the S2 state of the dithioester, which subsequently fragments.     

Synthesis and characterization of thermo- and pH-responsive double-hydrophilic diblock copolypeptides

Synthesis of novel double-hydrophilic diblock copolypeptides (BCPs), poly(l-glutamic acid)-block-poly(N-isopropylacrylamide) (PLGnPNm), and their thermoresponsive properties in aqueous solutions at different pH values are described. The diblock copolypeptides were synthesized by a combination of ring-opening polymerization (ROP) of gamma-benzyl-l-glutamate N-carboxyanhydrides (BLG-NCA) and reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide (NiPAM). A new class of RAFT agents (CTA-2 and CTA-3) with amino-functional groups was designed for this purpose. Two different strategies, i.e., macrochain transfer agent (CTA) and macroinitiator routes, were utilized and compared on the control of the chemical structures of the resulting BCPs. Their block ratios and lengths are broadly varied (n = 21-600 and m =180-442). Their thermally switchable aggregation behaviors in aqueous solutions were investigated at the microscopic level by 1H NMR spectroscopy and at the macroscopic level by turbidity measurements using UV/vis spectroscopy. The latter was also utilized for their lower critical aggregation temperature (LCAT) determination. The effects of block lengths and ratios as well as solution pH values on the collapse of NiPAM chain and aggregation process of BCPs were examined. This aggregation process was also followed by dynamic light scattering (DLS) measurements, and the thermally induced aggregate structures were investigated by transmission electron microscopy (TEM).     

Direct, controlled synthesis of the nonimmunogenic, hydrophilic polymer, poly(N-(2-hydroxypropyl)methacrylamide) via RAFT in aqueous media

Poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) is a nonimmunogenic, neutral-hydrophilic polymer currently employed in the delivery of anticancer drugs. Herein, we report conditions that facilitate the direct, controlled RAFT polymerization of HPMA in aqueous media. We demonstrate that the use of 4-cyanopentanoic acid dithiobenzoate and 4,4'-azobis(4-cyanopentanoic acid) as the chain transfer agent (CTA) and initiating species, respectively, in the presence of an acetic acid buffer solution at 70 degrees C is a suitable condition leading to controlled polymerization. The "living" nature of these polymerizations is demonstrated via chain-extension of an HPMA macroCTA to yield the corresponding poly(HPMA-b-HPMA) "homopolymer".     

Surface hydrophilic modification with well-defined glycopolymer for protein imprinting matrix

Well-defined lactose-containing glycopolymer has been synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization with (4-cyanopentanoic acid)-4- dithiobenozoate (CAD) as chain transfer agent. The glycopolymer was introduced onto the exterior surfaces of the bovine serum albumin (BSA) imprinted polymer beads by grafting copolymerization with methyl methacrylate and ethylene glycol dimethacrylate. After alcoholysis, the hydrophilic lactose residues of glycopolymer will stretched on the surface of the MIP beads and then the hydrophilicity of the surface will be enhanced. Rebinding test shows that the glycopolymer hydrophilic modified BSA imprinted polymer presents higher performance selectivity than that of unmodified one, which means that the hydrophobic-hydrophilic balance of the imprinted polymer surface is in favor of the improvement of specific recognition property of the material.     

Preparation of chitosan derivative with polyethylene glycol side chains for porous structure without specific processing technique

The graft copolymer, chitosan-g-polyethylene glycol (PEG), was prepared through graft polymerization of PEG chains to chitosan due to the esterification reaction between PEG and 6-O-succinate-N-phthaloyl-chitosan (PHCSSA). The graft copolymer with porous structure was observed from scanning electron micrographs. It is a potential method to combine chitosan with the hydrophilic synthetic polymers. The graft reaction was carried out in homogeneous system and yielded copolymers with high grafting content. FTIR, NMR, XRD, DSC, spectrofluorophotometer and SEM were detected to characterize the copolymer.