Role of sulfhydryl groups in transfection? A case study with chitosan-NAC nanoparticles.
This study investigated the use of chitosan-N-acetylcysteine (NAC) as a non-viral gene carrier. In particular, we aimed to elucidate whether the advantage of thiolation was more pronounced in the stabilization of particles or in the effect of nonspecific sulfhydryl reduction of the target cells. Low-viscosity chitosan was modified by covalent binding of NAC. The resulting conjugate displayed 1.35 mM SH/g polymer. Particles produced via self-assembly of chitosan conjugate and pDNA had a mean particle size of 113.7 nm and a positive zeta-potential. Sulfhydryl group content on the particle surface was investigated by Ellman's test and papain reactivation assay, with the result of about 100 nM SH groups/mL nanoparticle suspension. An oxidation step was performed to stabilize polyplexes via disulfide bonds. The enhanced stability of oxidized particles against both polyanion heparin and alkaline pH was proven by a gel retardation assay. The stabilization was demonstrated to be reversible by treatment with glutathione. Further, the effect of immobilized SH groups and of supplementation with free NAC on transfection efficacy on Caco-2 cells was investigated. The expression of the transgene was raised 2.5-fold and 10-fold with nonoxidized thiomer polyplexes in comparison to polyplexes of unmodified chitosan and oxidized chitosan-NAC, respectively. The impact of sulfhydryl reduction on transfection was assessed via thiol group inactivation with 5,5'-dithiobis-(2-nitrobenzoic acid) (DNTB). This inactivation resulted in a decrease of transfection efficacy. In conclusion, chitosan-NAC conjugate was demonstrated to be beneficial for transfection, either for stabilization via disulfide bonds or for raising the expression of transgene via shifting the redox potential of the target cells.
Loretz
Brigitta
B
Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University Innsbruck, 6020 Innsbruck, Austria.
Thaler
Marlene
M
Bernkop-Schnürch
Andreas
A
D000111:Acetylcysteine / Q000008:administration & dosage / Q000737:chemistry*; D018938:Caco-2 Cells; D002470:Cell Survival / Q000187:drug effects; D048271:Chitosan / Q000008:administration & dosage / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000008:administration & dosage / Q000737:chemistry* / Q000648:ultrastructure; D010206:Papain / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014162:Transfection / Q000379:methods*
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D048271:Chitosan; D004228:Dithionitrobenzoic Acid; D007770:L-Lactate Dehydrogenase; D010206:Papain; D000111:Acetylcysteine