Insulin bound to chiral polymer with N-acetyl-D-glucosaminyl units. Lacks of mitogenic activity on rat aorta smooth muscle cell proliferation.

Insulin was covalently attached to two terpolymers of N-(2-hydroxypropyl) methacrylamide, N-methacryloyldiglycine and a) R-(-)-1-methyl-2-methacryloylamidoethyl 2-acetamido-2-deoxy-beta-D-glucopyranoside or b) S-(+)-1-methyl-2-methacryloylamidoethyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The mitogenic effect of both conjugates on vascular smooth muscle cell proliferation was investigated. The results indicated that insulin bound to both carriers with pendant N-acetylglucosaminyl groups possesses hypoglycemic activity but not the mitogenic effect of native insulin. This study shows that for these insulin conjugates, the effect does not depend on the steric configuration of the sugar-containing monomer units incorporated in the terpolymer. A hypothesis is developed that some competition is taking place between N-acetylglucosaminyl groups on the polymeric insulin carrier and the same moieties in the insulin receptor expressed on the surface of smooth muscle cells leading to a lack of mitogenic activity.

Phase transition parameters of potential thermosensitive drug release systems based on polymers of N-alkylmethacrylamides.

The phase separation and its thermohysteresis in dilute aqueous solutions of polymeric components of potential drug release systems (homopolymers and copolymers of N-isopropylacrylamide, N-isopropylmethacrylamide, N-propylmethacrylamide, N-sec-butylmethacrylamide, and N-(2-hydroxypropyl)methacrylamide) was studied, both on heating and cooling. Plots of light transmittance vs temperature were constructed and the parameters characterizing them were correlated with polymer structures. Qualitative information was obtained on the rate of formation of the concentrated phase on heating and its disappearance on cooling. Attention has been drawn to the improper identification of the cloud-point temperature, measured at an arbitrary concentration, with the lower critical solution temperature (LCST) as is frequently found in papers dealing with biomedical applications of thermosensitive polymers.

Conjugates of insulin with copolymers of N-(2-hydroxypropyl) methacrylamide: effects on smooth muscle cell proliferation.

The hypothesis that an elevated plasma insulin level contributes to an increase in coronary heart disease has led to studies of the mitogenic effect of native insulin and its conjugates on smooth muscle cells (SMC). In this study, insulin was covalently attached to two water-soluble polymers containing N-(2-hydroxypropyl)methacrylamide using the mixed anhydride method. The first polymer was a copolymer of N-(2-hydroxypropyl)methacrylamide and N-methacryloyldiglycine. The second one was a terpolymer of two of the above-given monomers and R-(-)-1-methyl-2-methacryloylamidoethyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. Insulin conjugates were isolated and characterized, and the mitogenic effect on SMC was investigated. The results showed that only conjugates of insulin and terpolymers bearing pendant N-acetyl-glucosamine groups do not have a mitogenic effect on SMC while maintaining the hypoglycemic activity of insulin. This finding suggests that some inter- or intramolecular interactions of coupled insulin with the sugar moiety(ies) attached to the polymer backbone contribute to the observed effects.

SYNTHETIC POLYMER MATRICES FOR NEURAL CELL TRANSPLANTATION.

This study proposes a strategy to promote the integration of a neural graft into the host brain tissue. It involves the attachment of donor cells to a polymeric matrix, and the implantation of this cell-polymer matrix. We have synthesized hydrogels based on N-(2-hydroxypropyl)-methacrylamide (HPMA) to produce highly porous matrices. As preliminary steps, we have examined: 1) The response of the brain tissue to the implantation of PHPMA/collagen hydrogels; 2) adhesion, growth, differentiation, and viability of embryonic neuronal cells, and embryonal carcinoma-derived neurons seeded onto PHPMA substrates containing hexosamine residues (glucosamine and N-acetylglucosamine), and after entrapment of cells within the hydrogels. Histological analysis seven wk after implantation showed the tolerance of PHPMA hydrogels, and the penetration of host cells into the pore structures. However, cellular ingrowth requires the presence of collagen, and is dependent upon porosity. In vitro data showed that PHPMA substrates supported neuronal cell attachment and neuritic growth, but the biocompatibility of the substrate was enhanced after incorporation of N-acetylglucosamine into the hydrogel. The data also showed the feasibility of entrapping cells into the polymer matrices, and that these "cellular" hydrogel matrices could be maintained in vitro with preservation of cell viability and differentiation. These findings suggest that PHPMA-based hydrogels can serve as carriers for neural transplant, and as a support to guide tissue ingrowth and organization.

Enzymatic degradation and immunogenic properties of derivatized dextrans.

Dextran T40 was modified with carboxylic, benzylamide and benzylamine-sulphonated groups (samples 1-10). The polymers were incubated with dextranase and the decrease of molecular weight was determined by high performance size exclusion chromatography. It was shown that the higher the substitution of dextrans, the lower their degradability. Modification of dextran with benzylamine and benzylamine-sulphonated groups appeared to hinder the formation of the enzyme-substrate complex more than the same quantity of carboxylic groups. The immunogenicity of one of the modified dextrans, containing 54% of carboxylic groups and 19.5% of benzylamine-sulphonated groups, was determined after subcutaneous and intravenous administration into Balb/c mice. The antibody titre was very low even if administered in complete Freund's adjuvant and did not depend on the injected dose. On the average, the titres of antibodies were lower by five orders of magnitude compared to bovine gamma globulin.

Effect of galactose on interaction of N-(2-hydroxypropyl)methacrylamide copolymers with hepatoma cells in culture: preliminary application to an anticancer agent, daunomycin.

A series of copolymers were prepared containing 1,2:3,4-di-O-isopropylidene-6-O-methacryloyl-alpha-D-galactopyranose (0 to 99 mol %), methacryoyltyrosinamide and N-(2-hydroxypropyl)methacrylamide (99 to 0 mol %). The effect of galactose content on interaction with hepatoma cells in vitro was studied. Increased galactose content caused increased accumulation of N-(2-hydroxypropyl)methacrylamide copolymers by two human hepatoma cell lines (Hep G2 and SAH), but accumulation by rat and mouse hepatoma (HTC and NCTC) was not galactose dependent. Accumulation of N-(2-hydroxypropyl)methacrylamide copolymers by Hep G2 was shown to be an active process, being inhibited by low temperature and by the metabolic inhibitor 2,4-dinitrophenol. Addition of N-acetylgalactosamine and polymer-galactose to the incubation medium resulted in a concentration-dependent inhibition of accumulation of galactose-containing polymers. Addition of fucose or galactose was without effect at the concentrations used. Polymers bearing galactosamine or fucosylamine residues and, in addition, daunomycin were evaluated for cytotoxicity against Hep G2 and SAH. N-(2-Hydroxypropyl)methacrylamide copolymer-bound daunomycin produced a dose-dependent inhibition of DNA synthesis (measured by incorporation of [3H]thymidine), and the galactose-containing polymer showed greatest inhibition.

The removal of residuals and oligomers from poly(2-hydroxyethylmethacrylate).

Poly(2-hydroxyethyl methacrylate) gels obtained by the cross-linking polymerization using four different free-radical initiators were washed with water. Chromatographically, the eluate appeared to be a mixture of low-molecular-weight compounds and of a small amount of the high-molecular-weight component. The UV and IR absorption spectra of compounds present in the eluate were compared with those of model compounds that were assumed to exist in the gel as impurities after the polymerization (monomers and oligomers of hydroxyethyl methacrylate, decomposition products of initiators). Time dependences of the removal of impurities from the gels by washing were measured. Most of the impurities were washed out within a few hours. In addition to the assumed impurities, the eluate was found to contain an unidentified compound that was still washed out after several months. Intracutaneous applications of this compound did not produce local irritation of the tested tissue.