Thiolated chitosan nanoparticles: transfection study in the Caco-2 differentiated cellculture

Abstract

The aim of this study was to monitor the expression of secreted protein in differentiatedCaco-2 cells after transfection with nanoparticles, in order to improve gene delivery. Basedon unmodified chitosan and thiolated chitosan conjugates, nanoparticles with the genereporter pSEAP (recombinant Secreted Alkaline Phosphatase) were generated at pH 4.0.Transfection studies of thiolated chitosan in Caco-2 cells during the exponential growthphase and differentiation growth phase of the cells led to a 5.0-fold and 2.0-fold increase inprotein expression when compared to unmodified chitosan nanoparticles. The mean particlesize for both unmodified chitosan and cross-linked thiolated chitosan nanoparticles is212.2 ± 86 and 113.6 ± 40 nm, respectively. The zeta potential of nanoparticles was determined to be7.9 ± 0.38 mV for unmodified chitosan nanoparticles and4.3 ± 0.74 mVfor cross-linked thiolated chitosan nanoparticles. Red blood cell lysis evaluation was used to evaluatethe membrane damaging properties of unmodified and thiolated chitosan nanoparticles and led to4.61 ± 0.36% and 2.29 ± 0.25% lysis, respectively. Additionally, cross-linked thiolated chitosan nanoparticles were found toexhibit higher stability toward degradation in gastric juices. Furthermore the reversibleeffect of thiolated chitosan on barrier properties was monitored by measuring thetransepithelial electrical resistance (TEER) and is supported by immunohistochemicalstaining for the tight junction protein claudin. According to these results cross-linkedthiolated chitosan nanoparticles have the potential to be used as a non-viral vector systemfor gene therapy.