Cytocompatible gel formation of chitosan-glycerol phosphate solutions supplemented with hydroxyl ethyl cellulose is due to the presence of glyoxal.

To deliver and retain viable repair cells in a surgically prepared cartilage lesion, we previously developed an adhesive in situ-gelling cell carrier by suspending cells in a solution of hydroxyethyl cellulose (HEC), which was then mixed with chitosan-glycerol phosphate to form a chitosan-GP/HEC gel. The purpose of this study was to elucidate the mechanism of gelation to maximally control gel time and viability of encapsulated cells. We analyzed the role of osmolality, pH, gelation temperature, gel shrinkage, and HEC. A chitosan-GP solution at pH 6.8 with cytocompatible osmotic pressure (419 mOsm/kg) was achieved by lowering disodium GP concentration from 370 to 135 mM. This solution was still thermogelling but only at 73 degrees C. We next discovered that glyoxal, a common additive in ether cellulose manufacturing, was responsible for chitosan gelation. Monolayer cells survived and proliferated in up to 1 mM of glyoxal, however only a very narrow range of glyoxal concentration in chitosan-GP/HEC, 0.1-0.15 mM, permitted gel formation, cell survival, and cell proliferation. Chitosan gels containing HEC required slightly less glyoxal to solidify. Chitosan-GP/HEC loaded with viable chondrocytes formed an adhesive seal with ex vivo mosaic arthroplasty defects from sheep knee joints. In mosaic arthroplasty defects of live sheep, bleeding occurred beneath part of the hydrogel carrier, and the gel was cleared after 1 month in vivo. These data indicate that chitosan-GP/HEC is suitable as an adhesive and injectable delivery vehicle for clinical orthopedic applications involving single use treatments that guide acute cartilage repair processes.

A novel non-toxic camptothecin formulation for cancer chemotherapy.

The use of a novel injectable biocompatible and biodegradable camptothecin-polymer implant for sustained intra-tumoral release of high concentrations of camptothecin is described. The drug delivery vehicle is an in situ thermogelling formulation, which is based on the natural biopolymer chitosan. This formulation, containing homogeneously dispersed camptothecin, was implanted intra-tumorally into a sub-cutaneous mouse tumor model (RIF-l). The effectiveness of treatment was measured in terms of tumor growth delay (TGD). Animals treated with the polymer implants containing camptothecin had significantly longer TGDs compared to untreated animals as well as to animals treated systemically with camptothecin by intra-peritoneal injection with no evidence of toxicity in terms of loss of body weight. The results indicate that this novel biodegradable polymer implant is an effective vehicle for the sustained intra-tumoral delivery of camptothecin which might also be suitable to deliver other insoluble anti-cancer drugs such as taxol.

A validated 1H NMR method for the determination of the degree of deacetylation of chitosan.

A method for the determination of the degree of deacetylation (DDA) of chitosan by 1H NMR spectroscopy has been formally validated. Chitosans with DDA ranging from 48 to 100% have been used for the validation. The method is found to be simple, rapid and more precise than other known techniques like IR or titration for %DDA measurements. The precision, ruggedness, robustness, specificity, stability and accuracy of the technique are discussed in this paper.

Analogs of reaction intermediates identify a unique substrate binding site in Candida rugosa lipase.

The structures of Candida rugosa lipase-inhibitor complexes demonstrate that the scissile fatty acyl chain is bound in a narrow, hydrophobic tunnel which is unique among lipases studied to date. Modeling of triglyceride binding suggests that the bound lipid must adopt a "tuning fork" conformation. The complexes, analogs of tetrahedral intermediates of the acylation and deacylation steps of the reaction pathway, localize the components of the oxyanion hole and define the stereochemistry of ester hydrolysis. Comparison with other lipases suggests that the positioning of the scissile fatty acyl chain and ester bond and the stereochemistry of hydrolysis are the same in all lipases which share the alpha/beta-hydrolase fold.