Study On Substrate-mediated Gene Delivery Systems Loaded With Non-viral Vector/DNA Complexes

To overcome extracellular barriers in gene therapy, "substrate-mediated delivery" or so-called "solid phase delivery" has been developed through immobilizing DNA in polymeric scaffolds, which sustain the release of naked DNA or vector/DNA complexes as well as support cell growth. The substrate-mediated delivery can localize the gene transfection to a target tissue to avoid adverse side effects, prevent the rapid washing away of the cytokine from the application site, and maintain the DNA in a desirable concentration range for a prolonged time. In substrate-mediated delivery, the gene transfections could be improved through modifications of the vector/DNA complexes and polymer scaffolds.In Charpter 1, the recent progress of non-viral gene vectors, polymer matrices used for substrate-mediated delivery, and the substrate-mediated delivery systems loaded with vector/DNA complexes are reviewed.Chapter 2 focuses on the studies on fast degrading cholic acid functionalized star poly(DL-lactide) (CA-(DLL)n) polymer films loaded with Ca-P/DNA co-precipitates for the substrate-mediated delivery. The in vitro gene transfections of HEK293 cells, Hela cells and NIH 3T3 cells show that the expressions of pGL3-Luc and pEGFP-C1 plasmids could be effectively mediated by the Ca-P/DNA co-precipitates deposited and encapsulated polymer films. The expressions of pGL3-Luc and pEGFP-C1 plasmids indicate that the transfections mediated by the Ca-P/DNA co-precipitates deposited film show comparable gene expression levels as compared with the solution-based transfections. Compared to Ca-P/DNA co-precipitates encapsulated in the conventional linear PDLLA, Ca-P/DNA co-precipitates encapsulated in CA-(DLL)n exhibit a much higher gene expression level because CA-(DLL)n has an accelerated degradation which facilitates the rapid release of Ca-P/DNA co-precipitates to mediate transfection. In addition, the films do not exhibit any additional cytotoxicity to the cells during the transfections, indicating the fast degrading polymer films have great potential in localized gene delivery.Chapter 3 reports the studies on fast degrading cholic acid functionalized star poly(DL-lactide) polymer films loaded with Lipofectamine 2000/DNA co-precipitates for the substrate-mediated delivery. The Lipofectamine 2000/DNA were encapsulated in water soluble PHEA and then deposited on or sandwiched in a fast degrading cholic acid functionalized star poly(DL-lactide) film to mediate cellular transfection. By adding water soluble PHEA which could keep the bioactivity of DNA during film fabrication, the gene expression levels of pGL3-Luc and pEGFP-C1 plasmids in 293T, 3T3 and HeLa cells could be significantly improved for the film-mediated transfections, and the expression levels are comparable to that of the solution-based transfections. During the cellular transfection, the degradation of the polymer films does not show any negative effects on the gene transfection.Chapter 4 is concentrated on twp types of novel complexes for gene delivery, i.e. PAMAM/DNA/Heparin and PAMAM/DNA/Heparin-biotin. To prepare the complexes, Heparin or Heparin-biotin was complexed with PAMAM/DNA via electrostatic interactions. The addition of Heparin and Heparin-biotin results in decreased cytotoxicity. As compared with PAMAM/DNA, PAMAM/DNA/Heparin exhibits an enhanced gene transfection activity because the addition of Heparin results in the decreased positive charge density of the complexes, leading to lowered cytotoxicity. In addition, PAMAM/DNA/Heparin-biotin complexes exhibit much higher cellular uptake into HeLa cells than PAMAM/DNA and PAMAM/DNA/Heparin complexes due to the specific interaction between the biotin moiety and the biotin-specific receptors on HeLa cells.Chapter 5 focuses on the studies on fast degrading cholic acid functionalized star poly(DL-lactide) polymer films loaded with PAMAM/DNA/Heparin or PAMAM/DNA/Heparin-biotin for the substrate-mediated delivery. During film preparation, the PAMAM/DNA/Heparin or PAMAM/DNA/Heparin-biotin complexes were encapsulated in water soluble PHEA and then deposited on or sandwiched in the fast degrading cholic acid functionalized star poly(DL-lactide) to mediate gene transfection. With the presence of water soluble PHEA to protect the activity of DNA during film preparation, the PAMAM/DNA/Heparin-biotin complexes loaded film shows higher gene express levels compared with PAMAM/DNA and PAMAM/DNA/Heparin complexes loaded film in HeLa cells. However the gene express levels in 293T cells mediated by different complexes loaded films do not show an obvious difference, which indicates the introduce of biotin moiety could inprove the gene express level in particular target cells for the film-mediated gene transfection.Chapter 6 discusses the effect of addition of a nuclear localed signal protein (HMGB1) on the solution-based gene transfection. HMGB1 formed complexes with DNA through the affinity of HMGB1 and DNA, and then DNA/HMGB1 complexes were further condensed by PAMAM and biotinylated heparin (HB). The cell uptake experiment shows HB could significantly increase the cell uptake of HeLa cells. And the presence of HMGB1 increases the amount of complexes entered cell nuclei. PAMAM/DNA/HMGB1/HB complex displays significantly increased gene express levels in HeLa,293T, and COS-7 cells. Additionally, the introducution of HMGB1 does not exhibit any extra cytotoxicity to the cells.