Preparation of surface modified protein nanoparticles by introduction of sulfhydryl groups

The objective of the present study was to establish several methods for the introduction of thiol groups onto the surface of human serum albumin (HSA) nanoparticles. Besides the epsilon-amino groups of lysine, the carboxyl groups of asparaginic and glutaminic acid, and the carbonyl groups of the cross-linker glutaraldehyde, sulfhydryl groups are possible targets for the covalent linkage of drugs to particle surfaces. In principle, the thiol groups were introduced by the reaction with dithiotreitol (DDT) or 2-iminothiolane, by quenching reactive aldehyde residues with cystaminiumdichloride or by coupling L-cysteine and cystaminiumdichloride by the aqueous carbodiimide reaction. The resulting nanoparticulate systems were characterised concerning the number of available sulfhydryl groups, particle size and particle density. It was shown, that by variation of the reaction conditions, e.g., the concentration of the coupling reagent or the sulfhydryl containing component as well as the reaction time, the proposed methods enabled the preparation of HSA nanoparticles with a well defined surface characteristic. Stability studies showed that the introduced thiol groups were relatively stable and lost their reactivity with a half-life of 28.2 days independently of the method used for the sulfhydryl group introduction. Besides the quantification of free sulfhydryl groups the covalent attachment of cystaminiumdichloride by the carbodiimide reaction was used to calculate the amount of free carboxyl groups on the surface of the nanoparticles. The toxicity of the modified nanoparticles was evaluated in cell culture experiments.