Volga Bulmus

Bioapplications of RAFT Polymerization

A living radical polymerization (LRP) is a free radical polymerization that aims at displaying living character, (i.e., does not terminate or transfer and is able to continue polymerization once the initial feed is exhausted by addition of more monomer). However, termination reactions are inherent to a radical process, and modern LRP techniques seek to minimize such reactions, therefore providing control over the molecular weight and the molecular weight distribution of a polymeric material. In addition, the better LRP techniques incorporate many of the desirable features of traditional free radical polymerization, such as compatibility with a wide range of monomers, tolerance of many functionalities, and facile reaction conditions. The control of molecular weight and molecular weight distribution has enabled access to complex architectures and site specific functionality that were previously impossible to achieve via traditional free radical polymerizations. These LRPs are classified in three different subgroups: (1) stable free-radical polymerization such as nitroxide mediated polymerization (NMP),1,2 (2) degenerative transfer polymerization, such as iodine transfer polymerization (ITP and RITP),3,4 single electron transfer-degenerative transfer living radical polymerization(SET-DTLRP),5,6reversibleaddition-fragmentation chain transfer (RAFT),7,8 and macromolecular design via the interchange of xanthates (MADIX)9,10 polymerization, and (3) metal mediated catalyzed polymerization, such as atom transfer radical polymerization (ATRP),11-14 single electron transfer-living radical polymerization (SET-LRP),15 and organotellurium mediated living radical polymrization16-19 Among the existing LRP techniques, RAFT and MADIX are probably the most versatile processes, as they are tolerant * E-mail: T.P.D., T.Davis@UNSW.edu.au; S.P., S.Perrier@ chem.usyd.edu.au. † Centre for Advanced Macromolecular Design (CAMD), School of Chemical Sciences & Engineering, UNSW. ‡ Centre for Advanced Macromolecular Design (CAMD), School of Biotechnology & Biomolecular Sciences, UNSW. § The University of Sydney. Chem. Rev. 2009, 109, 5402–5436 5402

Really smart bioconjugates of smart polymers and receptor proteins

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Acid-Labile Core Cross-Linked Micelles for pH-Triggered Release of Antitumor Drugs

Micelles of a model amphiphilic block copolymer, poly(hydroxyethyl acrylate)-block-poly(n-butyl acrylate) (PHEA-b-PBA), synthesized via the RAFT polymerization were cross-linked by copolymerization of a degradable cross-linker from the living RAFT-end groups of PBA chains, yielding a cross-linked core without affecting significantly the original micelle size. The cross-linker incorporation into the micelles was evidenced via physicochemical analysis of the copolymer unimers formed upon acidic cleavage of the cross-linked micelles. High doxorubicin loading capacities (60 wt %) were obtained. Hydrolysis of less than half of the cross-links in the core was found to be sufficient to release doxorubicin faster at acidic pH compared to neutral pH. The system represents the first example of core-cross-linked micelles that can be destabilized (potentially both above and below CMC) by the pH-dependent cleavage of the cross-links and the subsequent polarity change in the core to enable the release of hydrophobic drugs entrapped inside the micelle.

Efficient Usage of Thiocarbonates for Both the Production and the Biofunctionalization of Polymers

End group modification of polymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization was accomplished by conversion of trithiocarbonate into reactive functions able to conjugate easily with biomolecules or bioactive functionality. Polymers were prepared by RAFT, and subsequent aminolysis led to sulfhydryl-terminated polymers that reacted in situ with an excess of dithiopyridyl disulfide to yield pyridyl disulfide-terminated macromolecules or in the presence of ene to yield functional polymers. In the first route, the pyridyl disulfide end groups allowed coupling with oligonucleotide and peptide. The second approach exploited thiol-ene chemistry to couple polymers and model compounds such as carbohydrate and biotin with high yield.

One-Pot Conversion of RAFT-Generated Multifunctional Block Copolymers of HPMA to Doxorubicin Conjugated Acid- and Reductant-Sensitive Crosslinked Micelles

N-(2-Hydroxypropyl)methacrylamide (HPMA) containing polymers that are widely used as anticancer drug carriers. We have synthesized new amphiphilic block copolymers of HPMA with a functional monomer 2-(2-pyridyldisulfide)ethylmethacrylate (PDSM) via reversible addition-fragmentation chain transfer (RAFT) polymerization. In a one-pot reaction, the versatility of PDS groups on poly(PDSM)- b-poly(HPMA) was used to conjugate an anticancer drug, doxorubicin (DOX), and also simultaneously crosslink the micellar assemblies via acid-cleavable hydrazone bonds and reducible disulfide bonds. DOX-conjugated crosslinked micelles with an average diameter of approximately 60 nm were observed to be formed in aqueous medium. Disintegration of the micelles into unimers in the presence of a disulfide reducing agent confirmed the crosslinking via disulfide bonds. While the release of DOX from the crosslinked micelles at pH 5.0 was faster compared to the release at pH 7.4, a high proportion of released DOX was found to retain the original active structure. Overall results demonstrate the simplicity and the versatility of the poly(PDSM)- b-poly(HPMA) system, which are potentially important in the design of new generation of polymer therapeutics.

The stabilization and bio-functionalization of iron oxide nanoparticles using heterotelechelic polymers

Iron oxide nanoparticles (IONPs) are important tools for nanobiotechnology applications. However, aqueous instability and non-specific biodistribution problems limit the applications of IONPs. Considering this, α-phosphonic acid, ω-dithiopyridine functionalized polymers were synthesized via the reversible addition–fragmentation chain transfer (RAFT) polymerization and used for stabilizing and biofunctionalizing IONPs. A new trithiocarbonate RAFT agent bearing dimethyl phosphonate group was utilized in the synthesis of well-defined telechelic polymers of styrene, oligoethylene glycol acrylate (OEG-A) and N-isopropylacrylamide (NIPAAm). IONPs were grafted with α-phosphonic acid, ω-dithiopyridine functionalized poly(OEG-A) through the α-chain end of the polymer, as evidenced by FTIR-ATR, XPS and zeta potential measurements. Using TGA results, the grafting density of the polymer chains was calculated between 0.12 and 0.23 chains/nm2 particle depending on the molecular weight of the polymer. DLS measurements indicated that the particles grafted with poly(OEG-A) larger than 10 000 g/mol were stable in water for several days and the mean diameter of the particles was between 40 and 130 nm depending on the molecular weight of the polymer. Moreover, particles stabilized with poly(OEG-A) with a Mn = 62 000 g/mol were stable in phosphate buffer (pH 6.5, 0.1 M) containing varying concentrations of BSA. Polymer-stabilized IONPs were successfully functionalized with two different peptides, i.e. reduced glutathione as a model peptide and NGR motif as a tumor-targeting peptide through the ω-dithiopyridine functionality of the polymer, as measured by XPS and zeta potential analysis. Poly(OEG-A)-stabilized IONPs were also found to be resistant to protein adsorption.

Synthesis of versatile thiol-reactive polymer scaffolds via RAFT polymerization

Well-defined polymer scaffolds convertible to (multi)functional polymer structures via selective and efficient modifications potentially provide an easy, versatile, and useful approach for a wide variety of applications. Considering this, a homopolymer scaffold, poly(pyridyldisulfide ethylmethacrylate) (poly(PDSM)), having pendant groups selectively reactive with thiols, was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Soluble polymers with controlled molecular weights and narrow PDIs were generated efficiently. The versatility of the scaffold to generate random co- and ter-polymers combining multiple functionalities with controlled-composition was shown by separate and simultaneous conjugation of different mercapto-compounds, including a tripeptide in one-step. Conversion of water-insoluble scaffold to peptide-containing water-soluble copolymers was observed to yield nanometer-size particles with narrow polydispersity. The overall results suggest that the well-defined PDSM homopolymer scaffold generated via RAFT polymerization can be a versatile building block for generation of new structures having potential for drug delivery applications via a straightforward synthetic approach.

Anti-fouling magnetic nanoparticles for siRNA delivery

Iron oxide nanoparticles (IONPs), with a diameter of 8 nm, have been coated with two different polymers, i.e. poly(oligoethylene glycol) methyl ether acrylate (P(OEG-A)) and poly(dimethylaminoethyl acrylate) (P(DMAEA)). The polymers were attached to the nanoparticle surface using two different strategies, with the aim of creating an internal layer of P(DMAEA) and an outer shell of P(OEG-A). The subsequent polymer-stabilized IONPs were characterized using ATR, XPS and TGA, proving the presence of polymers on the IONP surfaces with a grafting density ranging from 0.05 to 0.22 chain per nm2. High grafting densities were demonstrated when the two homopolymers were assembled on the surfaces of the IONPs simultaneously. The polymer composition at the surfaces of the IONPs could be controlled by manipulating the feed ratio P(OEG-A)–P(DMAEA) present in solution. These hybrid organic–inorganic particles (70–150 nm) proved to be stable in both water and 50 vol% fetal bovine serum (FBS). In addition, zeta-potential measurements confirmed that P(OEG-A) chains effectively mask the positive charge originating from P(DMAEA) thereby limiting protein adsorption on these particles. Hybrid nanoparticles were exploited for the complexation of siRNA, thereby generating IONP siRNA nano-carriers with anti-fouling P(OEG-A) shells. The transfection efficiency was measured using human neuroblastoma SHEP cells both in the presence and in the absence of a magnetic field in FBS. The transfection efficiency was determined by both fluorescence microscopy and flow cytometry. Cytotoxicity studies revealed that the IONP carriers were non-toxic to SHEP cells. In addition, studies on the proton transverse relaxation enhancement properties of these stabilized IONPs indicated high relaxivities (∼160 s−1 per mM of Fe).

In Vitro Cytotoxicity of RAFT Polymers

The RAFT technique has been increasingly used to generate polymers for potential biological applications. However, to-date, the toxicity of the RAFT-polymers has received limited attention. In this study, the in vitro cytotoxicity of three different, RAFT-synthesized, water-soluble polymers was investigated using three different adherent cell lines via CellTiter-Blue cell viability and the cytosolic enzyme lactate dehydrogenase (LDH) cytotoxicity assays. In brief, P(OEG-A) and P(OEG-MA) samples bearing omega-dithiobenzoate or omega-trithiocarbonate end groups and varying P(HPMA) samples bearing omega-dithiobenzoate, omega-trithiocarbonate, or non-RAFT end groups, were investigated using Chinese hamster ovary cells (CHO-K1), mouse macrophage cells (Raw264.7), and mouse fibroblast cells (NIH3T3). Any changes in the morphology of the cells after treatment with polymers were monitored via microscopy. The cytotoxicity of the polymers after treatment with metabolic liver enzymes was also evaluated. The average viability of CHO-K1 and NIH3T3 cells treated with dithiobenzoate- and trithiocarbonate-ended OEG-based polymers (1000 microM) for 24 h was close to 100%. The RAW264.7 cells were slightly more sensitive when incubated with dithiobenzoate-ended polymers (cell viability above 73%) for 24 h. The viability of the cells after 3 days of incubation with the polymers either slightly decreased or showed no change with respect to the viabilities obtained after 1 day of incubation. Analyses of cell morphology and cell membrane integrity via microscopy and a LDH assay confirmed the cell viability results obtained via CellTiter-Blue Assay. Unexpectedly, dithiobenzoate-ended P(HPMA) (at 1000 microM) exhibited high cytotoxicity after 24 h with all three cells lines. Further investigation of various P(HPMA) samples revealed that trithiocarbonate-ended and HPMA-capped P(HPMA)s at the same concentration were nontoxic over the same period of time. Also, dithiobenzoate-ended P(HPMA) at low concentrations (< or = 200 microM) can be tolerated by the cells tested.

Modified PMMA monosize microbeads for glucose oxidase immobilization

Glucose oxidase (GOD) was immobilized onto modified polymethylmethacrylate (PMMA) microspheres by covalent bonding. Monosize PMMA microbeads with 1.5 μm diameter were produced by dispersion polymerization of methylmethacrylate by using polyvinyl alcohol as a stabilizer. Hydroxyl groups on the microbeads were first converted to aldehyde groups by periodate oxidation. Three amino compounds, namely ammonium hydroxide, ethylene diamine and hexamethylene diamine were incorporated through the aldehyde groups. Then, GOD molecules were immobilized through the spacer-arms by using glutaraldehyde. The highest amount of immobilization and activity were obtained in which hexamethylene diamine was used as the spacer-arm with 14 atom length, and were 2.1 mg g−1 polymer and 129 IU g−1 polymer, respectively. The optimal conditions for GOD immobilization were obtained as follows: pH, 6.0; temperature, 30 °C; immobilization time, 60 min; and GOD initial concentration, 0.10 mg ml−1. The optimal conditions for the GOD-immobilized PMMA microbeads were at pH 6.0 and at a temperature of 30 °C. The Km and Vmax values of the GOD-immobilized PMMA microbeads were, 13.79 mM and 26.31 mM min−1 calculated by non-linear regression, respectively.