Tam Le

The effect of RAFT-derived cationic block copolymer structure on gene silencing efficiency

In this work a series of ABA tri-block copolymers was prepared from oligo(ethylene glycol) methyl ether methacrylate (OEGMA(475)) and N,N-dimethylaminoethyl methacrylate (DMAEMA) to investigate the effect of polymer composition on cell viability, siRNA uptake, serum stability and gene silencing. Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization was used as the method of polymer synthesis as this technique allows the preparation of well-defined block copolymers with low polydispersity. Eight block copolymers were prepared by systematically varying the central cationic block (DMAEMA) length from 38 to 192 monomer units and the outer hydrophilic block (OEGMA(475)) from 7 to 69 units. The polymers were characterized using size exclusion chromatography and (1)H NMR. Chinese Hamster Ovary-GFP and Human Embryonic Kidney 293 cells were used to assay cell viability while the efficiency of block copolymers to complex with siRNA was evaluated by agarose gel electrophoresis. The ability of the polymer-siRNA complexes to enter into cells and to silence the targeted reporter gene enhanced green fluorescent protein (EGFP) was measured by using a CHO-GFP silencing assay. The length of the central cationic block appears to be the key structural parameter that has a significant effect on cell viability and gene silencing efficiency with block lengths of 110-120 monomer units being the optimum. The ABA block copolymer architecture is also critical with the outer hydrophilic blocks contributing to serum stability and overall efficiency of the polymer as a delivery system.

Biodegradable and injectable cure-on-demand polyurethane scaffolds for regeneration of articular cartilage.

This paper describes the synthesis and characterization of an injectable methacrylate functionalized urethane-based photopolymerizable prepolymer to form biodegradable hydrogels. The tetramethacrylate prepolymer was based on the reaction between two synthesized compounds, diisocyanato poly(ethylene glycol) and monohydroxy dimethacrylate poly(epsilon-caprolactone) triol. The final prepolymer was hydrated with phosphate-buffered saline (pH 7.4) to yield a biocompatible hydrogel containing up to 86% water. The methacrylate functionalized prepolymer was polymerized using blue light (450 nm) with an initiator, camphorquinone and a photosensitizer, N,N-dimethylaminoethyl methacrylate. The polymer was stable in vitro in culture media over the 28 days tested (1.9% mass loss); in the presence of lipase, around 56% mass loss occurred over the 28 days in vitro. Very little degradation occurred in vivo in rats over the same time period. The polymer was well tolerated with very little capsule formation and a moderate host tissue response. Human chondrocytes, seeded onto Cultispher-S beads, were viable in the tetramethacrylate prepolymer and remained viable during and after polymerization. Chondrocyte-bead-polymer constructs were maintained in static and spinner culture for 8 weeks. During this time, cells remained viable, proliferated and migrated from the beads through the polymer towards the edge of the polymer. New extracellular matrix (ECM) was visualized with Massons trichrome (collagen) and Alcian blue (glycosaminoglycan) staining. Further, the composition of the ECM was typical for articular cartilage with prominent collagen type II and type VI and moderate keratin sulphate, particularly for tissue constructs cultured under dynamic conditions.

High Refractive Index Polysiloxane as Injectable, In Situ Curable Accommodating Intraocular Lens

Functionalised siloxane macromonomers, with properties designed for application as an injectable, in situ curable accommodating intraocular lens (A-IOL), were prepared via re-equilibration of a phenyl group-containing polysiloxane of very high molecular weight with octamethylcyclotetrasiloxane (D₄) and 2,4,6,8-tetra(n-propyl-3-methacrylate)-2,4,6,8-tetramethyl-cyclotetrasiloxane (D₄(AM)) in toluene using trifluoromethanesulfonic acid as a catalyst. Hexaethyldisiloxane was used as an end group to control the molecular weight of the polymer. The generated polymers had a consistency suitable for injection into the empty lens capsule. The polymers contained a low ratio of polymerisable groups so that, in the presence of a photo-initiator, they could be cured on demand in situ within 5 min under irradiation of blue light to form an intraocular lens within the lens capsule. All resulting polysiloxane soft gels had a low elastic modulus and thus should be able to restore accommodation. The pre-cure viscosity and post-cure modulus of the generated polysiloxanes were controlled by the end group and D₄(AM) concentrations respectively in the re-equilibration reactions. The refractive index could be precisely controlled by adjusting the aromatic ratio in the polymer to suit such application as an artificial lens. Lens stretching experiments with both human and non-human primate cadaver lenses of different ages refilled with polysiloxane polymers provided a significant increase in amplitude of accommodation (up to 4 D more than that of the respective natural lens). Both in vitro cytotoxicity study using L929 cell lines and in vivo biocompatibility study in rabbit models demonstrated the non-cytotoxicity and ocular biocompatibility of the polymer.

Glycosylated RAFT polymers with varying PEG linkers produce different siRNA uptake, gene silencing and toxicity profiles

Achieving efficient and targeted delivery of short interfering (siRNA) is an important research challenge to overcome to render highly promising siRNA therapies clinically successful. Challenges exist in designing synthetic carriers for these RNAi constructs that provide protection against serum degradation, extended blood retention times, effective cellular uptake through a variety of uptake mechanisms, endosomal escape, and efficient cargo release. These challenges have resulted in a significant body of research and led to many important findings about the chemical composition and structural layout of the delivery vector for optimal gene silencing. The challenge of targeted delivery vectors remains, and strategies to take advantage of natures self-selective cellular uptake mechanisms for specific organ cells, such as the liver, have enabled researchers to step closer to achieving this goal. In this work, we report the design, synthesis, and biological evaluation of a novel polymeric delivery vector incorporating galactose moieties to target hepatic cells through clathrin-mediated endocytosis at asialoglycoprotein receptors. An investigation into the density of carbohydrate functionality and its distance from the polymer backbone is conducted using reversible addition-fragmentation chain transfer polymerization and postpolymerization modification.