M. A. Jafar Mazumder

PNIPAAm-Grafted-Collagen as an Injectable, In Situ Gelling, Bioactive Cell Delivery Scaffold

We synthesized two thermoresponsive, bioactive cell scaffolds by decorating the backbone of type I bovine collagen with linear chains of poly(N-isopropylacrylamide) (PNIPAAm), with the ultimate aim of providing facile delivery via injection and support of retinal pigment epithelial (RPE) cells into the back of the eye for the treatment of retinal degenerative diseases. Both scaffolds displayed rapid, subphysiological phase transition temperatures and were capable of noninvasively delivering a liquid suspension of cells that gels in situ forming a cell-loaded scaffold, theoretically isolating treatment to the injection site. RPE cells demonstrated excellent viability when cultured with the scaffolds, and expulsion of cells arising from temperature-induced PNIPAAm chain collapse was overcome by incorporating a room-temperature incubation period prior to scaffold phase transition. These results indicate the potential of using PNIPAAm-grafted-collagen as a vehicle for the delivery of therapeutic cells to the subretinal space.

Core-Cross-Linked Alginate Microcapsules for Cell Encapsulation

Self-cross-linkable polyelectrolyte pairs comprised of poly(methacrylic acid, sodium salt-co-2-[methacryloyloxy]ethyl acetoacetate) (70:30 mol ratio, A70) and poly-L-lysine are incorporated into CaAlg beads to form either a covalently cross-linked shell or a core-cross-linked bead. In both cases the reactive polyanion is added to a solution of sodium alginate that may contain live cells and dropped into a calcium chloride gelling bath. Subsequent exposure to poly-L-lysine (15-30 kDa) leads to formation of a cross-linked shell, while exposure to lower molecular weight poly-L-lysine (4-15 kDa) leads to formation of an interpenetrating matrix of covalently cross-linked synthetic polymer within the CaAlg template. The resulting spherical composites are resistant to chemical and mechanical stress yet remain cyto-compatible. This approach to cell-encapsulation may be useful for cell immuno-isolation in therapeutic cell transplants.

Cell-adhesive thermogelling PNIPAAm/hyaluronic acid cell delivery hydrogels for potential application as minimally invasive retinal therapeutics †

Copolymers of N-isopropylacrylamide (NIPAAm) and acrylic acid N-hydroxysuccinimide (NAS) were synthesized via free radical polymerization and conjugated with amine-functionalized hyaluronic acid (HA) and cell adhesive RGDS peptides. These novel copolymers were designed to facilitate noninvasive delivery of a liquid suspension of cells into the delicate subretinal space for treatment of retinal degenerative diseases such as age-related macular degeneration (AMD) and diabetic retinopathy. The various synthesized copolymers all displayed subphysiological phase transition temperatures, thereby allowing temperature-induced scaffold formation and subsequent entrapment of transplanted cells within an adhesive support matrix. Successful grafting of HA and RGDS peptides were confirmed with Fourier Transform Infrared (FTIR) spectroscopy and quantified with (1)H Nuclear Magnetic Resonance (NMR) spectroscopy. All copolymers demonstrated excellent compatibility with retinal pigment epithelial (RPE) cells in culture and minimal host response was observed following subcutaneous implantation into hairless SKH1-E mice (strain code 447).

Self-cross-linking polyelectrolyte complexes for therapeutic cell encapsulation.

Self-cross-linking polyelectrolytes are used to strengthen the surface of calcium alginate beads for cell encapsulation. Poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), containing 30 mol % 2-aminoethyl methacrylate, and poly(sodium methacrylate), containing 30 mol % 2-(methacryloyloxy)ethyl acetoacetate, were prepared by radical polymerization. Sequential deposition of these polyelectrolytes on calcium alginate films or beads led to a shell consisting of a covalently cross-linked polyelectrolyte complex that resisted osmotic pressure changes as well as challenges with citrate and high ionic strength. Confocal laser fluorescence microscopy revealed that both polyelectrolytes were concentrated in the outer 7-25 microm of the calcium alginate beads. The thickness of this cross-linked shell increased with exposure time. GPC studies of solutions permeating through analogous flat model membranes showed molecular weight cut-offs between 150 and 200 kg/mol for poly(ethylene glycol), suitable for cell encapsulation. C 2C 12 mouse cells were shown to be viable within calcium alginate capsules coated with the new polyelectrolytes, even though some of the capsules showed fibroid overcoats when implanted in mice due to an immune response.

Development of injectable, resorbable drug-releasing copolymer scaffolds for minimally invasive sustained ophthalmic therapeutics.

Copolymers based on N-isopropylacrylamide (NIPAAm), acrylic acid N-hydroxysuccinimide (NAS) and varying concentrations of acrylic acid (AA) and acryloyloxy dimethyl-γ-butyrolactone (DBA) were synthesized to create thermoresponsive, resorbable copolymers for minimally invasive drug and/or cell delivery to the posterior segment of the eye to combat retinal degenerative diseases. Increasing DBA content was found to decrease both copolymer water content and lower critical solution temperature. The incorporation of NAS provided an amine-reactive site, which can be exploited for facile conjugation of bioactive agents. Proton nuclear magnetic resonance analysis revealed the onset of hydrolysis-dependent opening of the DBA lactone ring, which successfully eradicated copolymer phase transition properties and should allow the gelled polymer to re-hydrate, enter systemic circulation and be cleared from the body without the production of degradation byproducts. Hydrolytic ring opening occurs slowly, with over 85% copolymer mass remaining after 130 days of incubation in 37°C phosphate buffered saline. These slow-degrading copolymers are hypothesized to be ideal delivery vehicles to provide minimally invasive, sustained, localized release of pharmaceuticals within the posterior segment of the eye to combat retinal degenerative diseases.