Transfection of mEpo gene to intestinal epitheliumin vivomediated by oral delivery of chitosan-DNA nanoparticles

Abstract

AIM:To prepare the chitosan-pmEpo nanoparticles and to study their ability for transcellular and paracellular transport across intestinal epithelia by oral administration. METHODS:ICR mice were fed with recombinant plasmid AAV-tetO-CMV-mEpo (containing mEpo gene) or pCMVβ (containing LacZ gene), whether it was wrapped by chitosan or no. Its size and shape were observed by transmission electron microscopy. Agarose gel electrophoresis was used to assess the efficiency of encapsulation and stability against nuclease digestion. Before and after oral treatmant, blood samples were collected by retro-orbital puncture, and hematocrits were used to show the physiological effect of mEpo. RESULTS:Chitosan was able to successfully wrap the plasmid and to protect it from DNase degradation. Transmission electron microscopy showed that freshly prepared particles were approximately 70-150 nm in size and fairly spherical. Three days after fed the chitosan-pCMVβ complex was fed, the mice were killed and most of the stomach and 30% of the small intestine were stained. Hematocrit was not modified in naive and 'naked' mEpo-fed mice, a rapid increase of hematocrit was observed during the first 4 days of treatment in chitosan-mEpo-fed animals, reaching 60.9±1.2% (P<0.01), and sustained for a week. The second feed (6 days after the first feed) was still able to promote a second hematocrit increase in chitosan-mEpo-fed animals, reaching 65.9±1.4% (P<0.01), while the second hematocrit increase did not appear in the 'naked' mEpo-second-fed mice. CONCLUSION:Oral chitosan-DNA nanoparticles can efficiently deliver genes to enterocytes, and may be used as a useful tool for gene transfer.

Chitosan–plasmid nanoparticle formulations for IM and SC delivery of recombinant FGF-2 and PDGF-BB or generation of antibodies

“…Other in vivo studies report appreciable protein expression using chitosan as condensing carrier for plasmid delivery. Chitosans with various MW and DDA have been successfully used for systemic, 26 IM, 27 intratracheal, [28][29][30] oral, 31 topical, 32 and direct intestinal 15 administration of plasmid DNA. In most of these studies, reporter genes such as luciferase or EGFP were used.…”

Improved stomach selectivity of gene expression following microinstillation of plasmid DNA onto the gastric serosal surface in mice

“…However, there is great concern about safety because these procedures require physical force against organs; consequently, the continuous and repetitive administration of pDNA is limited. However, although pDNA complexes with chitosan [13], N-acetylated chitosan [14] and montmorillonite [15] were studied for oral gene delivery, the oral route has many hampers reducing transfection efficiency such as a low pH, high concentrations of digestive fluid, and rapid turnover of epithelial cells, etc.…”

Highly stomach-selective gene transfer following gastric serosal surface instillation of naked plasmid DNA in rats

“…13 However, there is great concern about safety because these procedures require physical force against organs; consequently, the continuous and repetitive administration of pDNA is limited. However, although pDNA complexes with chitosan, 14 N-acetylated chitosan 15 and montmorillonite 16 were studied for oral gene delivery, the oral route has many factors reducing transfection efficiency such as gastric contents, low pH, high concentration of digestive fluid, and rapid turnover of epithelial cells, etc. In fact, transgene expression was not observed in the stomach following oral administration with nanoparticles-in-microsphere in rats.…”