Two light-sensitive, cross-linkable polyethylenimines, P25M and B-PC-PEI, were synthesized for the capture and controlled release of nucleic acids. P25M consists of a polyethylenimine (PEI) to electrostatically condense nucleic acids, a terminal methacrylamide group to be used in conjunction with a polymerization initiator to entrap nucleic acids, and a 2-nitrobenzyl group to release the nucleic acids from the entrapped polymer/nucleic acid complexes (polyplexes) with exposure at 365 nm. P25M was cleaved by 4 min of 365 nm light exposure and condensed plasmid DNA (pDNA) at polymer nitrogen to nucleic acid phosphate (N/P) ratios ≥4. Four different polymerizing initiators or catalysts—ammonium persulfate, VA-044, eosin y, and Grubbs' 2nd generation catalyst—were used to entrap the pDNA within the polyplex, and all caused some degree of pDNA retention, as measured by loss of pDNA mobility in agarose gels. Exposure at 365 nm, however, never restored pDNA mobility. It was determined, through use of unmodified PEI/pDNA polyplexes, that all polymerization conditions led to cross-linking of pDNA to itself or to PEI, thus inhibiting light-activated release. Investigations into using "click chemistry" reactants as cross-linking domains (in place of the terminal methacrylamide group) also proved to be incompatible with pDNA mobility. To avoid such potential side reactions, B-PC-PEI was synthesized to have a PEI domain, a 2-nitrobenzyl domain, and a biotin domain. As a cross-linking domain, biotin non-covalently binds to streptavidin, thus eliminating the need for chemical reactants to mediate entrapment. B-PC-PEI was cleaved by 5 min of exposure at 365 nm and condensed pDNA at N/P ratios ≥8. Light exposure successfully triggered release of B-PC-PEI/nucleic acid polyplexes that were tethered to streptavidin-coated solid supports. Reverse transfection of bovine aortic endothelial cells resulted in significantly increased transfection efficiency after light exposure for polyplexes made with B-PC-PEI, compared to polyplexes made with a non-photolabile biotinylated-PEI. The ability to control transfection with light exposure will have many potential applications ranging from controlled and selective nucleic acid delivery to elucidation of transfection mechanisms to high-throughput studies where controlled release of metered doses of nucleic acids is essential.
|Advisor:||Diamond, Scott L.|
|School:||University of Pennsylvania|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 68/07, Dissertation Abstracts International|
|Subjects:||Biomedical research, Chemical engineering|
|Keywords:||Nucleic acid delivery, Photolabile, Polyethylenimines|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be