title: Synthesis and evaluation of N-(2,3-dihydroxypropyl)-PEIs as efficient vectors for nucleic acids.
abstract: Branched polyethylenimine (bPEI, 25 kDa) has been widely used as an efficient delivery vector for nucleic acids in vitro. However, its charge-associated toxicity has limited its in vivo applications. In an attempt to control its toxicity, it was reacted with varying amounts of glycidol (2,3-epoxy-1-propanol) to obtain a small series of hydrophilic polymers, 2,3-dihydroxypropyl-grafted-polyethylenimines (DHP-g-P). The resulting polymers were characterized by (1)H-NMR and subjected to interaction with negatively charged pDNA, which yielded complexes in the size range of ~171-190 nm with a zeta potential of ∼+33-39 mV. Acid-base titration revealed no effect of substitution on the buffering capacity of the modified polymers. Grafting of 2,3-dihydroxypropyl groups on bPEI significantly improved the cell viability (i.e. almost non-toxic) as well as the DNA release properties of these modified polymers compared to native bPEI. Formation of a relatively loose DHP-g-P25/pDNA complex (the best working system in terms of transfection efficiency) resulted in the efficient nuclear release of pDNA for transcription, a prerequisite for efficient transfection. Subsequently, upon evaluation of their ability to transfer nucleic acids in vitro, the DHP-g-P/pDNA complexes exhibited higher gene transfection efficiency with one of the formulations, DHP-g-P25/DNA complex, displaying ~2.7 folds higher GFP expression than bPEI and ~2.3-3.5 folds higher than the selected commercial transfection reagents used in this study. Further to quantify the extent of GFP positive cells, FACS analysis was performed, which revealed DHP-g-P25/DNA mediated gene expression in ~51% cells outcompeting bPEI, Superfect™, Fugene™ and Lipofectamine™. Sequential delivery of GFP-specific siRNA resulted in ~78% suppression of the target gene compared to ~49% achieved by Fugene™. All these results demonstrate the potential of these polymers for in vivo gene delivery.
authors.affiliation: CSIR-Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi-110007, India.
authors.forename: Sushil K
authors.initials: SK
authors.lastname: Tripathi
authors.forename: Santosh
authors.initials: S
authors.lastname: Yadav
authors.forename: Kailash C
authors.initials: KC
authors.lastname: Gupta
authors.forename: Pradeep
authors.initials: P
authors.lastname: Kumar
mesh_terms: D000818:Animals; D002021:Buffers; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D005453:Fluorescence; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D018613:Microscopy, Confocal; D023282:Nuclease Protection Assays; D009696:Nucleic Acids / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011094:Polyethyleneimine / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D055672:Static Electricity; D014162:Transfection
chemical_list: D002021:Buffers; D009696:Nucleic Acids; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D004247:DNA; D003850:Deoxyribonuclease I