Background: Ischemic heart disease remains the number one killer in the developed world. A protein transduction peptide specific for the heart capable of efficiently delivering agents of therapeutic potential at the time of injury, would be of immense public health significance. This work was undertaken to identify peptide(s) able to transduce heart tissue in vivo in a tissue-specific manner. Biopanning was performed in cell culture followed by in vivo with an M13 phage peptide display library. Using the heart-specific peptide, we delivered nemo-binding domain peptide in a murine infarct model to test if NF-kB inhibition can reduce infarct size.
Methods and Results: A cardiomyoblast cell line, H9C2, was incubated with M13 twelve amino acid phage peptide display library. Internalized phage was recovered, amplified and subjected to a total of three rounds of in vivo biopanning where infectious, internalized phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). This peptide was synthesized either fluorescently labeled, biotinylated, or in combination with a NEMO-binding peptide (NBD), an inhibitor of the inducible NF-kappa B Kinase (IKK). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was not observed with a cationic transduction domain. CTP-NBD was able to inhibit NF-kB activation in cell culture in a dose-dependent fashion. When administered to mice in a murine infarct model, CTP-NBD showed a trend towards smaller infarct size, which did not reach statistical significance.
Conclusions: Biopanning using a peptide phage display library identified a peptide, termed CTP, able to transduce cardiomyoblast cell line in vitro, and heart tissue in vivo, efficiently and specifically. Administration of CTP-NBD to post-infarct mice showed a trend towards reduction of infarct size. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12022009-155853 |
| Date | 27 January 2010 |
| Creators | Zahid, Maliha |
| Contributors | Eleanor Feingold, Robert Ferrell, Charles F. McTiernan, Paul d. Robbins |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-12022009-155853/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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