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

Abstract

The aim of this study was to monitor the expression of secreted protein in differentiated Caco-2 cells after transfection with nanoparticles, in order to improve gene delivery. Based on unmodified chitosan and thiolated chitosan conjugates, nanoparticles with the gene reporter pSEAP (recombinant Secreted Alkaline Phosphatase) were generated at pH 4.0. Transfection studies of thiolated chitosan in Caco-2 cells during the exponential growth phase and differentiation growth phase of the cells led to a 5.0-fold and 2.0-fold increase in protein expression when compared to unmodified chitosan nanoparticles. The mean particle size for both unmodified chitosan and cross-linked thiolated chitosan nanoparticles is 212.2 ± 86 and 113.6 ± 40 nm, respectively. The zeta potential of nanoparticles was determined to be 7.9 ± 0.38 mV for unmodified chitosan nanoparticles and 4.3 ± 0.74 mV for cross-linked thiolated chitosan nanoparticles. Red blood cell lysis evaluation was used to evaluate the membrane damaging properties of unmodified and thiolated chitosan nanoparticles and led to 4.61 ± 0.36% and 2.29 ± 0.25% lysis, respectively. Additionally, cross-linked thiolated chitosan nanoparticles were found to exhibit higher stability toward degradation in gastric juices. Furthermore the reversible effect of thiolated chitosan on barrier properties was monitored by measuring the transepithelial electrical resistance (TEER) and is supported by immunohistochemical staining for the tight junction protein claudin. According to these results cross-linked thiolated chitosan nanoparticles have the potential to be used as a non-viral vector system for gene therapy.

Nanoparticle toxicity by the gastrointestinal route: evidence and knowledge gaps

“…Chitosan derivatives stabilise and improve delivery of therapeutic peptides in the GIT. Several studies have investigated various chitosan NP formulations in Caco-2 cells with similar conclusions (Thanou et al, 2000; Silva et al, 2006; Korjamo et al, 2008; Kowapradit et al, 2008; Kudsiova and Lawrence, 2008; Martien et al, 2008; Sadeghi et al, 2008; Jia et al, 2009; Sonaje et al, 2009; Kowapradit et al, 2010; Saremi et al, 2011; Zheng et al, 2011). Using various cell viability measures, all of these studies documented chitosan particle-mediated decreases in TEER, increases in tight junction permeability, and enhanced absorption without evident adverse effects on the intestinal monolayers.…”

OPTIMIZATION OF STIRRING SPEED AND STIRRING TIME TOWARD NANOPARTICLE SIZE OF CHITOSAN-SIAM CITRUS PEEL (Citrus nobilis L.var Microcarpa) 70% ETHANOL EXTRACT

“…Nanopartikel pada penelitian ini ditujukan untuk pemberian oral. Kitosan dapat membuka kait antar sel pada membran usus secara sementara (Bhardwaj dan Kumar, 2006;Martien et al, 2008) sehingga sangat potensial dalam pembuatan nanopartikel per oral.…”

Spinning Disc Processing Technology: Potential for Large-Scale Manufacture of Chitosan Nanoparticles

“…This was observed whether acetic acid or tartaric acid was used, and could be related to the relatively strong acid strength of both acids. Even at the lowest concentration of 0.1 M, the two acids yielded pH of less than 3.5, which would facilitate complete ionisation of amino groups in the chitosan chains (pK a 6.5 22 ).…”