The absence of CpG in plasmid DNA-chitosan polyplexes enhances transfection efficiencies and reduces inflammatory responses in murine lungs.
Chitosan polyplexes containing plasmid DNA (pDNA) have significant potential for pulmonary gene delivery applications. However, prior to using chitosan/pDNA polyplexes (CSpp) in clinical applications, their potential cytotoxicity needs to be investigated. In this study, we formulated 200-400 nm CSpp with amine to phosphate (N/P) ratios that ranged from 1 to 100. We compared two types of plasmids within CSpp: pDNA that was free of CpG sequences (CpG(-)) and pDNA that contained CpG sequences (CpG(+)). Both forms of CSpp showed low cytotoxicity when cultured with A549 and HEK293 cell lines in vitro. CSpp(CpG(-)) generated higher luciferase expression both in vitro, for A549 cells, and in vivo, compared with CSpp(CpG(+)). In addition, CSpp(CpG(-)) elicited milder inflammatory responses in mice one day subsequent to nasal instillation, as determined by proinflammatory cytokine levels within the bronchoalveolar lavage fluid. Our findings suggest that to achieve optimal gene expression with minimal cytotoxicity, inflammation, and oxidative stress, the N/P ratios and CpG sequences in the pDNA of CSpp need to be considered. These findings will inform the preclinical safety assessments of CSpp in pulmonary gene delivery systems.
Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, ‡Department of Occupational and Environmental Health, College of Public Health, and §Department of Radiation Oncology, Carver College of Medicine, University of Iowa , Iowa City, Iowa 52242, United States.
Amaraporn
A
Wongrakpanich
Andrea
A
Adamcakova-Dodd
Wei
W
Xie
Vijaya B
VB
Joshi
Kranti A
KA
Mapuskar
Sean M
SM
Geary
Douglas R
DR
Spitz
Peter S
PS
Thorne
Aliasger K
AK
Salem
D000281:Administration, Intranasal; D000818:Animals; D017209:Apoptosis; D048271:Chitosan / Q000737:chemistry* / Q000378:metabolism; D018899:CpG Islands; D016207:Cytokines / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D018014:Gene Transfer Techniques; D057809:HEK293 Cells; D006801:Humans; D008081:Liposomes; D008156:Luciferases / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids / Q000008:administration & dosage*; D011014:Pneumonia / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D014162:Transfection / Q000379:methods*
D016207:Cytokines; D008081:Liposomes; D004247:DNA; D048271:Chitosan; D008156:Luciferases