Formulation of a peptide nucleic acid based nucleic acid delivery construct.
Gene delivery biomaterials need to be designed to efficiently achieve nuclear delivery of plasmid DNA. Polycations have been used to package DNA and other nucleic acids within submicrometer-sized particles, offering protection from shear-induced or enzymatic degradation. However, cytotoxicity issues coupled with limited in vivo transfection efficiencies minimize the effectiveness of this approach. In an effort to improve upon existing technologies aimed at delivering nucleic acids, an alternative approach to DNA packaging was explored. Peptide nucleic acids (PNAs) were used to directly functionalize DNA with poly(ethylene glycol) (PEG) chains that provide a steric layer and inhibit multimolecular aggregation during complexation. DNA prePEGylation by this strategy was predicted to enable the formation of more homogeneous and efficiently packaged polyplexes. In this work, DNA-PNA-peptide-PEG (DP3) conjugates were synthesized and self-assembled with 25 kDa poly(ethylenimine) (PEI). Complexes with small standard deviations and average diameters ranging 30-50 nm were created, with minimal dependence of complex size on N/P ratio (PEI amines to DNA phosphates). Furthermore, PEI-DNA interactions were altered by the derivatization strategy, resulting in tighter compaction of the PEI-DP3 complexes in comparison to PEI-DNA complexes. Transfection experiments in Chinese hamster ovary (CHO) cells revealed comparable transfection efficiencies but reduced cytotoxicities of the PEI-DP3 complexes relative to PEI-DNA complexes. The enhanced cellular activities of the PEI-DP3 complexes were maintained following the removal of free PEI from the PEI-DP3 formulations, whereas the cellular activity of the conventional PEI-DNA formulations was reduced by free PEI removal. These findings suggest that DNA prePEGylation by the PNA-based strategy might provide a way to circumvent cytotoxicity and formulation issues related to the use of PEI for in vivo gene delivery.
Millili
Peter G
PG
Department of Chemical Engineering, Department of Biological Sciences, Department of Chemistry and Biochemistry, and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19716, and Department of Pharmaceutical Research, Development, Merck Research Laboratories, West Point, Pennsylvania 19486.
Yin
Daniel H
DH
Fan
Haihong
H
Naik
Ulhas P
UP
Sullivan
Millicent O
MO
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D003412:Cricetulus; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D010316:Particle Size; D020135:Peptide Nucleic Acids / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011092:Polyethylene Glycols / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013499:Surface Properties; D014162:Transfection / Q000379:methods*
D020135:Peptide Nucleic Acids; D011092:Polyethylene Glycols; D011094:Polyethyleneimine; D004247:DNA