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DNA-LPEI complexes encapsulated in LTP nanospheres as a non-viral gene therapy vector

Ditto, Andrew J
http://rave.ohiolink.edu/etdc/view?acc_num=akron1165596983

Abstract Details

2006, Master of Science in Engineering, University of Akron, Biomedical Engineering.
Viruses are uniquely equipped to insert their genome into cells, which is due partly to their ability to transport across cellular membranes and to escape from endosomes. The primary disadvantage of using viruses for gene therapy is that they elicit severe immune responses. Therefore, we have formulated nanospheres by blending chitosan grafted with polyethylene glycol (PEG-g-CHN) and linear poly-ethylenimine (LPEI) conjugated to DNA with L-tyrosine-polyphosphate (LTP). PEG-g-CHN enhances nanosphere biocompatibility, since PEG has been shown to prevent protein adsorption. LPEI protects DNA from shearing during nanosphere formation, provides a route of escape from endosomes, and enhances transfection. Previous studies show that LTP degrades within 7 days, which provides an appropriate time frame for intracellular release. These nanospheres are prepared by sonication of oil and water emulsion and solvent evaporation. Scanning electron microscopy and laser light scattering (LLS) show a nanosphere diameter distribution between 200 to 600 nm. Also, LLS shows that nanospheres are completely degraded after 7 days. A sustained release over 7 days of DNA-LPEI from nanospheres is revealed in gel electrophoresis and transfection studies. Furthermore, the viability of the nanospheres is shown to be comparable to DNA alone from a LIVE/DEAD® cell assay. In addition, confocal microscopy reveals nanosphere uptake within human fibroblasts. Ultimately, a controllable transfection of human fibroblasts over 11 days by nanospheres is shown from X-gal staining. This controllable transfection is sustained longer than DNA-LPEI and DNA-FuGENE 6, a commercial transfection reagent. Therefore, our nanosphere formulations enhance transfection compared to FuGENE 6 and DNA alone, exhibit a controlled release and transfection, and can be used as a non-viral vector that overcomes the barriers of gene therapy.
Yang Yun (Advisor)
141 p.
Engineering, Biomedical
nanospheres; gene therapy; non-viral vectors; gene vectors; sustained release; DNA loaded nanospheres; endocytosed nanospheres; DNA-LPEI

Recommended Citations

Citations

  • Ditto, A. J. (2006). DNA-LPEI complexes encapsulated in LTP nanospheres as a non-viral gene therapy vector [Master's thesis, University of Akron]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=akron1165596983

    APA Style (7th edition)

  • Ditto, Andrew. DNA-LPEI complexes encapsulated in LTP nanospheres as a non-viral gene therapy vector. 2006. University of Akron, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=akron1165596983.

    MLA Style (8th edition)

  • Ditto, Andrew. "DNA-LPEI complexes encapsulated in LTP nanospheres as a non-viral gene therapy vector." Master's thesis, University of Akron, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=akron1165596983

    Chicago Manual of Style (17th edition)

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© 2006, all rights reserved.
This open access ETD is published by University of Akron and OhioLINK.