Evaluation of the biological properties of soluble chitosan and chitosan microspheres

Chitosan is the N-deacetylated product of chitin, a naturally occurring polymer which has been used extensively to prepare microspheres for oral and intra-nasal delivery. The chitosan polymer has also been proposed as a soluble carrier for parenteral drug delivery. Here the in vitro biocompatibility of seven soluble chitosan polymers (different salts and a chitosan derivative; glycol chitosan, different molecular weights and varying degrees of deacetylation) and chitosan microspheres loaded with horseradish peroxidase (HRP), was systematically studied by evaluating cytotoxicity towards B16F10 cells and ability to lyse rat erythrocytes. Cytotoxicity towards B16F10 was concentration-dependent and varied according to the salt used and polymer molecular weight. Chitosan hydrochloride was most toxic having an IC50 of 0.21 +/- 0.04 mg/ml, only fourfold less toxic than the cationic reference polymer: poly-L-lysine (IC,, of 0.05 +/- 0.01 mg/ml). The ranking of cytotoxicity was: chitosan hydrochloride > chitosan hydroglutamate > glycol chirosan > chitosan hydrolactate. Polymers of higher molecular weight of each type were most toxic. Glutaraldehyde-crosslinked chitosan microspheres were cytotoxic at all concentrations used. In the lysis assay, release of haemoglobin occurred on exposure to all soluble chitosans in a time-dependent manner. After 24 h, 100% lysis was conferred by most soluble chitosans (1 mu g/ml-3 mg/ml), and even after 1 h, chitosan hydroglutamate caused 60-80% lysis. Microspheres were considerably less lyric than the soluble polymer. Scanning electron microscopy showed changes in B16F10 and red cell morphology consistent with the observed chitosan toxicity. Whilst HRP entrapped in glutaraldehyde crosslinked chitosan microspheres retained enzyme activity, this was a reduced 10-fold compared to an equivalent amount of native HRP. (C) 1997 Elsevier Science B.V.