Study On Vector/pDNA And Vector/shRNA Complexes Loaded Films And Microspheres For Localized Gene Delivery

Gene therapy involves gene expression mediated byDNAand gene silencing mediated by RNA interference. For both DNA delivery and RNA delivery, the gene delivery systems which can effectively deliver gene into target cells are of critical importance to achieve success of gene therphy. In gene delivery, to overcome extracellular barriers, controlled release technology has been adapted to the gene delivery. This technology is known as "localized gene delivery" or "substrate-mediated delivery". Through immobilizing DNA in polymeric scaffolds which sustain the release of nakedDNAor vector/DNA complexes as well as support cell growth, theDNAdelivery can be localized to a target tissue with a minimized adverse side effect, and the localDNAconcentration can be maintained in a desirable concentration range with a sustained release pattern.In Chapter 1,a recent progress of non-viral gene vectors, polymer matrices used for substrate-mediated delivery, substrate-mediated delivery systems loaded with vector/DNA complexes gene vectors, and several approcahes to realize the targeted gene delivery is reviewed.In Chapter 2 focuses on Ca-P/DNA co-precipitates deposited and encapsulated polymer films for substrate-mediated pDNA delivery. The polymer films were composed of fast degrading functionalized star poly(DL-lactide) CA-PDLLA and block copolymer poly(L-lactide)-poly(ethylene glycol)-poly(L-lactide) (PLLA-PEG-PLLA).The in vitro gene transfections of pGL3-Luc and pEGFP-C1 plasmids in HEK293T cells mediated by different films were studied. The effect of presence of a water soluble polymer, poly-α,β-[N-(2-hydroxyethyl)-L-aspartamide],on the transfection activity of the film-mediated transfection was studied. The gene expressions could be effectively mediated by the Ca-P/DNA co-precipitates deposited and encapsulated polymer films. During the cellular transfection, the fast degrading films could rapidly release Ca-P/DNA co-precipitates to mediate gene transfection, and the degradation products did not show any negative effects on the gene expression. The Ca-P/DNA co-precipitates deposited and encapsulated films showed promising application in substrate-mediated gene delivery.Chapter 3 is concentrated on Ca-P/DNA co-precipitates encapsulated polymer microspheres for localized pDNA delivery. Ca-P/DNA co-precipitates encapsulated microspheres were fabricated by a convenient "ultrasonic dispersion method". To prepare the microspheres, the Ca-P/DNA co-precipitates together with a water soluble polymer poly-α,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA) were encapsulated in a polymer film which was mainly composed of fast degrading cholic acid functionalized star poly(DL-lactide), and then the film was immersed in ethanol and ultrasonicated to afford the microspheres. Contrary to other conventional methods, the ultrasonic dispersion method did not involve the use of toxic organic solvents, and was easy to control. TheDNArelease from the film and the microspheres was measured. The in vitro gene transfections of pGL3-Luc and pEGFP-C1 plasmids in HEK293T cells mediated by the Ca-P/DNA co-precipitates encapsulated microspheres were studied. Through being encapsulated in polymer microspheres, Ca-P/DNA co-precipitates exhibited an enhanced gene transfection activity.The expressions of pGL3-Luc and pEGFP-C1 in HEK293T cells could be effectively mediated by the Ca-P/DNA co-precipitates encapsulated microspheres. In addition, the microspheres did not exhibit any additional cytotoxicity to the cells during the transfection.Chapter 4 reports the Lipofectamine 2000/shRNA complexes deposited and encapsulated polymer films for substrate-mediated shRNA delivery. We have successfully constructed and identified the psiRNA, which could transcript shRNA in the nuclei.The porous polymer films were composed of fast-degrading CA-PDLLA and block copolymer PLLA-PEG-PLLA. In order to keep the activity of shRNA, Lipofectamine 2000/shRNA lipoplexes were encapsulated by the water soluble polymer poly-α,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA), and then deposited on or encapsulated in the porous polymer films. The films could successfully mediate the shRNA expression which caused the silence of the luciferase expression in Hela-Luc cells in vitro.Compared with the films without PHEA, the presence of PHEA resulted in a higher transfection efficiency for Lipofectamine 2000/shRNA lipoplexes loaded films, indicating the presence of PHEA played an important role in keeping the bioactivity of shRNA and improving the transfection efficiency.Chapter 5 focues on two types of novel complexes for gene delivery, i.e. PAMAM/shRNA/Heparin and PAMAM/shRNA/Heparin-biotin. To prepare the complexes, Heparin or Heparin-biotin was complexed with PAMAM/shRNA via electrostatic interactions. Since the nagetive charge of Heparin and Heparin-Biotin could shield the excessive positive charge of PAMAM/shRNA lipoplexes, which led to a decreased cytotoxicity, PAMAM/shRNA/Heparin and PAMAM/shRNA/Heparin-biotin exhibitd enhanced gene transfection activities. In addition, due to the specific interaction between the biotin moiety and the biotin-specific receptors on Hela-Luc cells, PAMAM/shRNA/Heparin-biotin complexes exhibited a higher cellular uptake into HeLa-Luc cells and an enhanced gene silence effect.