Methods of genetic engineering were applied to the design and biosynthesis of three extracellular matrix protein analogues constructed from identical elastin- and fibronectin-derived repeating units but characterized by different molecular weights in the range of 14,000 to 59,000. Expression levels were enhanced by the serendipitous choice of an N-terminal fusion sequence such that gram-scale syntheses were achieved for each protein. Purification protocols were developed that resulted in proteins of high purity and correct sequence, as determined by amino acid analysis, NMR spectroscopy, and lower critical solution temperature (LCST). Glutaraldehyde was shown to insolubilize the otherwise soluble proteins in a concentration-dependent manner. Tensile moduli of cross-linked protein films were measured and found to be inversely related to the molecular weights of the engineered proteins, which in each case corresponds to the theoretical molecular weight between cross-links. At the highest cross-link density (lowest molecular weight) the elastic modulus was similar to that of native elastin.

The present investigation focuses on the synthesis and application of a cross-linking agent that is compatible with the solubility characteristics of chitosan. A water-soluble, blocked-diisocyanate was prepared as a bisulfite adduct to 1,6-hexamethylene diisocyanate, which proved to be stable for several weeks in aqueous, acidic chitosan solutions at room temperature. Thermal cross-linking of chitosan as cast, dried films was investigated by varying the NCO/NH(2) ratio from 0.0 to 1.2. Spectroscopic (IR), thermal (TGA), swelling, and structural (WAXD) studies indicated that chitosan was cross-linked in a concentration-dependent manner under mild thermal conditions: 60 degrees C for 24 h. Cross-linking inefficiency was concluded to be due to lack of mobility of the reacting species in the solid state. In a preliminary study, the enzymatic degradation with Chitinase (E. C. 3.2.1.14) from Streptomyces griseus was found to be the greatest for non-crosslinked chitosan, followed by chitin, and then by cross-linked samples.

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Using alternating layers of charged polymers, we have constructed reactive thin films by incorporating enzymes, specifically alkaline phosphatase (AP) and glucose oxidase (GOD), into multilayers of a polycation, branched polyethylenimine (PEI), and a polyanion, poly(styrenesulfonate) (PSS), supported on a glass substrate. Experiments using a quartz crystal microbalance (QCM) demonstrated that the films grew sequentially on a solid support, while X-ray photoelectron spectroscopy (XPS) further confirmed the growth and deposition of successive enzyme layers. For both enzymes, the reactive films demonstrated increased activity with the successive number of deposited enzyme layers. In hybrid films, consisting of alternating layers of AP and GOD, both enzymes retained activities similar to those of their corresponding films of either enzyme alone. The effect of elevated temperature was also investigated for these reactive films. Increased thermal stability was found to be associated with the increase in the number of deposited enzyme layers.