In vitro cytotoxicity testing of non-thiolated and thiolated chitosan nanoparticles for oral gene delivery

Abstract

A comparative cytotoxicity study with various chitosan/pDNA nanoparticles was performed in order to evaluate the influence of thiolation, surface charge and size. In particular, the impact of pH changes as encountered along gastrointestinal tract on zeta potential and subsequently toxicity was investigated. For this purpose chitosan and chitosan-N-acetylcysteine nanoparticles of different polymer:pDNA ratios were prepared and characterised by their physicochemical properties. As endpoints to assess cytotoxicity in Caco-2 cells LDH and MTT assay were utilized. The reduction of transepithelial electrical resistance (TEER) induced by nanoparticle treatment was measured and toxic effects on erythrocytes were evaluated to complete the toxicity profile. Size of particles and amount of bound thiol groups slightly affected toxicity. In contrast a high impact and correlation was found for zeta potential and cytotoxicity. While anionic and neutral nanoparticles causeda minor membrane damage and slightly altered mitochondrial activity, cationic nanoparticles caused severe cytotoxic effects. TEER monitoring indicated sub lethal toxicity for neutral nanoparticles by a reversible resistance reduction of up to a third from initial value depending on the concentration of nanoparticles. Cationic particles evinced also drawbacks in erythrocytes assays by causing agglutination. In conclusion, our results showed evidence that zeta potential is the key feature that contributes most to the toxicity of (thiolated) chitosan/DNA nanoparticles.