Nicholas A. D. Burke

Mechanically enhanced microcapsules for cellular gene therapy.

Microcapsules bearing a covalently cross-linked coating have been developed for cellular gene therapy as an improvement on alginate-poly(L-lysine)-alginate (APA) microcapsules that only have ionic cross-linking. In this study, two mutually reactive polyelectrolytes, a polycation (designated C70), poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride-co-2-aminoethyl methacrylate hydrochloride) and a polyanion (designated A70), poly(sodium methacrylate-co-2-(methacryloyloxy)ethyl acetoacetate), were used during the microcapsule fabrication. Ca-alginate beads were sequentially laminated with C70, A70, poly(L-lysine) (PLL), and alginate. The A70 reacts with both C70 and PLL to form a approximately 30 microm thick covalently cross-linked interpenetrating polymer network on the surface of the capsules. Confocal images confirmed the location of the C70/A70/PLL network and the stability of the network after 4 weeks implantation in mice. The mechanical and chemical resistance of the capsules was tested with a stress test where microcapsules were gently shaken in 0.003% EDTA for 15 min. APA capsules disappeared during this treatment, whereas the modified capsules, even those that had been retrieved from mice after 4-weeks implantation, remained intact. Analysis of solutions passing through model flat membranes showed that the molecular weight cut-off of alginate-C70-A70-PLL-alginate is similar to that of alginate-PLL-alginate. Recombinant cells encapsulated in APA and modified capsules were able to secrete luciferase into culture media. The modified capsules were found to capture some components of regular culture media used during preparation, causing an immune reaction in implanted mice, but use of UltraCulture serum-free medium was found to prevent this immune reaction. In vivo biocompatibility of the new capsules was similar to the APA capsules, with no sign of clinical toxicity on complete blood counts and liver function tests. The increased stability of the covalently modified microcapsules coupled with the acceptable biocompatibility and permeability demonstrated their potential for use as immunoisolation devices in gene therapy.

Poly(methyl vinyl ether-alt-maleic acid) polymers for cell encapsulation.

Polyanions based on poly(methyl vinyl ether-alt-maleic acid) were investigated as materials for cell encapsulation. These water-soluble polyanions having molecular masses ranging from 20 to 1980 kDa were prepared by functionalization of poly(methyl vinyl ether-alt-maleic anhydride) with 5-aminofluorescein and/or α-methoxy-ω-amino-poly(ethylene glycol), followed by base hydrolysis of the residual anhydride groups to form the corresponding poly(methyl vinyl ether-alt-sodium maleate). Their potential to replace alginate both in the core and, in particular, the outer shell of calcium alginate-poly(L-lysine)-alginate (APA) capsules was determined using confocal fluorescence microscopy, osmotic pressure tests, permeability studies, protein binding and cell viability assays. These polymers were shown to be able to replace the outer layer of alginate, forming more resilient capsule shells. The resulting capsules showed similar permeability and resistance to bovine serum albumin binding, as well as superior viability for encapsulated cells, when compared to standard APA capsules. In addition, these polymers showed promise for use as functional additives to the capsule cores.

Systematic study of alginate-based microcapsules by micropipette aspiration and confocal fluorescence microscopy

Micropipette aspiration and confocal fluorescence microscopy were used to study the structure and mechanical properties of calcium alginate hydrogel beads (A beads), as well as A beads that were additionally coated with poly-L-lysine (P) and sodium alginate (A) to form, respectively, AP and APA hydrogels. A beads were found to continue curing for up to 500 h during storage in saline, due to residual calcium chloride carried over from the gelling bath. In subsequent saline washes, micropipette aspiration proved to be a sensitive indicator of gel weakening and calcium loss. Aspiration tests were used to compare capsule stiffness before and after citrate extraction of calcium. They showed that the initial gel strength is largely due to the calcium alginate gel cores, while the long term strength is solely due to the poly-L-lysine-alginate polyelectrolyte complex (PEC) shells. Confocal fluorescence microscopy showed that calcium chloride exposure after PLL deposition led to PLL redistribution into the hydrogel bead, resulting in thicker but more diffuse and weaker PEC shells. Adding a final alginate coating to form APA capsules did not significantly change the PEC membrane thickness and stiffness, but did speed the loss of calcium from the bead core.

Tunable hydrogel thin films from reactive synthetic polymers as potential two-dimensional cell scaffolds.

This article describes the formation of cross-linked 10-200-nm-thick polymer hydrogel films by alternating the spin-coating of two mutually reactive polymers from organic solutions, followed by hydrolysis of the resulting multilayer film in aqueous buffer. Poly(methyl vinyl ether-alt-maleic anhydride) (PMM) was deposited from acetonitrile solution, and poly(N-3-aminopropylmethacrylamide-co-N-2-hydroxypropylmethacrylamide) (PAPMx, where x corresponds to the 3-aminopropylmethacrylamide content ranging from 10 to 100%) was deposited from methanol. Multilayer films were formed in up to 20 deposition cycles. The films cross-linked during formation by reaction between the amine groups of PAPMx and the anhydride groups of PMM. The resulting multilayer films were covalently postfunctionalized by exposure to fluoresceinamine, decylamine, d-glucamine, or fluorescently labeled PAPMx solutions prior to the hydrolysis of residual anhydride in aqueous PBS buffer. This allowed tuning the hydrophobicity of the film to give static water contact angles ranging from about 5 to 90°. Increasing the APM content in PAPMx from 10 to 100% led to apparent Youngs moduli from 300 to 700 kPa while retaining sufficient anhydride groups to allow postfunctionalization of the films. This allowed the resulting (PMM/PAPMx) multilayer films to be turned into adhesion-promoting or antifouling surfaces for C2C12 mouse myoblasts and MCF 10A premalignant human mammary epithelial cells.

Preparation and study of multi-responsive polyampholyte copolymers of N-(3-aminopropyl)methacrylamide hydrochloride and acrylic acid

A series of polyampholytes was prepared by free radical batch-copolymerization of N-(3-aminopropyl)methacrylamide hydrochloride (APM) and acrylic acid (AA). The drifts in monomer feed ratio during copolymerization were monitored by 1H-NMR, and used to extract reactivity ratios of 0.68 (APM) and 0.48 (AA). The phase separation in aqueous solutions of copolymers containing between 4 and 90 mol% APM were studied by potentiometric turbidity titration. The obtained isoelectric point (pH(I)) values agree well with theoretical values for weak polyampholytes. The influence of ionic strength on the phase separation during potentiometric turbidity titrations was measured for three different copolymer compositions and the effect on solubility was most pronounced for the stoichiometric (1u2006:u20061 APMu2006:u2006AA) polyampholyte, reflecting the greatest number of electrostatic interactions at pH(I). Aqueous solutions of this stoichiometric polyampholyte showed LCST-type cloud points upon heating, which varied as a function of pH, ionic strength and polymer concentration. The cloud point increased with ionic strength, and showed U-shaped dependence on pH, being constant within ±1.5 pH units of the pH(I). Some non-stoichiometric, high AA-content polyampholytes also were temperature responsive at pH(I), except that the phase transition was of the UCST-type, reflecting the increasing importance of hydrogen bonding on polymer solubility.

Synthetic polycations with controlled charge density and molecular weight as building blocks for biomaterials

Abstract A series of polycations prepared by RAFT copolymerization of N-(3-aminopropyl)methacrylamide hydrochloride (APM) and N-(2-hydroxypropyl)methacrylamide, with molecular weights of 15 and 40 kDa, and APM content of 10–75 mol%, were tested as building blocks for electrostatically assembled hydrogels such as those used for cell encapsulation. Complexation and distribution of these copolymers within anionic calcium alginate gels, as well as cytotoxicity, cell attachment, and cell proliferation on surfaces grafted with the copolymers were found to depend on composition and molecular weight. Copolymers with lower cationic charge density and lower molecular weight showed less cytotoxicity and cell adhesion, and were more mobile within alginate gels. These findings aid in designing improved polyelectrolyte complexes for use as biomaterials.

Synthetic hydrogels formed by thiol–ene crosslinking of vinyl sulfone-functional poly(methyl vinyl ether-alt-maleic acid) with α,ω-dithio-polyethyleneglycol

Polymer hydrogels formed by rapid thiol-ene coupling of macromolecular gel formers can offer access to versatile new matrices. This paper describes the efficient synthesis of cysteamine vinyl sulfone (CVS) trifluoroacetate, and its incorporation into poly(methyl vinyl ether-alt-maleic anhydride) (PMMAn) to form a series of CVS-functionalized poly(methyl vinyl ether-alt-maleic acid) polymers (PMM-CVSx) containing 10 to 30 mol% pendant vinyl sulfone groups. Aqueous mixtures of these PMM-CVS and a dithiol crosslinker, α,ω-dithio-polyethyleneglycol (HS-PEG-SH, Mn = 1 kDa), gelled through crosslinking by Michael addition within seconds to minutes, depending on pH, degree of functionalization, and polymer loading. Gelation efficiency, Youngs modulus, equilibrium swelling and hydrolytic stability are described, and step-wise hydrogel post-functionalization with a small molecule thiol, cysteamine, was demonstrated. Cytocompatibility of these crosslinked hydrogels towards entrapped 3T3 fibroblasts was confirmed using a live/dead fluorescence assay.

Synthetic Polyampholytes as Macromolecular Cryoprotective Agents

A series of polyampholytes based on different molar ratios on N, N-dimethylaminopropyl methacrylamide (DMAPMA), acrylic acid (AA), and optionally, N- tert-butylacrylamide ( t-BuAAm), were prepared by free radical copolymerization, and tested as DMSO-free cryoprotective agents for 3T3 fibroblast cells by using a standard freeze-rethaw protocol. Polybetaines prepared by reaction of DMAPMA homo and copolymers with 1,3-propane sultone were used as additional controls. Results showed strong effects of copolymer composition, molecular weight, polymer and NaCl concentrations, on post-thaw cell viability. Binary (DMAPMA/AA) copolymers showed best post-thaw cell viability of 70% at a 30/70 mol % ratio of DMAPMA/AA, which increased to 90% upon introduction of 9 mol % t-BuAAm while maintaining the 30/70 mol % cation/anion ratio. The use of acrylamide linkages in DMAPMA ensures absence of hydrolytic loss of cationic side chains. These polyampholytes were found to decrease ice crystal size and to form a polymer-rich, ice-free layer around cells, reducing damage from intercellular ice crystals during both freezing and thawing steps. These polyampholytes also dehydrate cells during freezing, which helps protect cells from intracellular ice damage. While cell viability immediately after thawing was high, subsequent culturing revealed poor attachment and long-term viability, which is attributed to residual cell damage from intracellular ice formation. Addition of 2 wt % DMSO or 1% BSA to the polymer-based freeze medium was found to mitigate this damage and result in post-thaw viabilities matching those achieved with 10 wt % DMSO.