Reducible Polycations As Gene Delivery And Drug Carriers

Polyelectrolytes, nucleic acids, and nanoparticles would be tethered on various surfaces or even scaffolds via layer-by-layer assembly (LBL). The LBL technique would offer spatial and temporal control over the incorporation and release of its compositions. Successful disassembly of multilayered films and release of incorporated DNA from DNA-containing multilayered films under physiological relevant conditions would promote its localized transfection efficiency. Recently, many attentions focused on the disulfide-containing polycations. Disulfide is stable, but degradable in reductive conditions. From the chapter 2 to 4, a detailed study of the disulfide-contained LBL assembly for localized gene delivery was studied.In the chapter 2, we reported the fabrication of multilayered polyelectrolyte films of dendrimer-grafted bioreducible polycations and plasmid DNA by layer-by-layer assembly. Poly(disulfide amine) dendrimers with surface amine groups of generation 0,1,2, and 3 (GO, G1, G2, and G3) have been used as building units. Characterization of coated quartz substrates using UV-vis spectrometry and profilometry demonstrated that plasmid DNA was successfully incorporated into each alternate dendrimer layer. The disassembly of such multilayered films in 2.5 mM glutathione decreased with addition of dendrimer generations. MTT assay revealed that cell cytotoxicity increased with addition of dendrimer generation. Transfection activity of each multilayered film was evaluated in 293T cells using different concentrations of glutathione (0,2.5,5, and 10 mM). Moderate degradation of generation 2 dendrimer/DNA film without reducing agent led to the best transfection efficiency.In the chapter 3 and 4, a novel series of multilayered films composed of bioreducible cationic micelles, formed from amphiphilic disulfide-containing poly(amidoamine)s. and DNA were prepared via layer-by-layer assembly for localized gene delivery. In the chapter 3, a detailed study of the effect of changes in film ionic strength and surface charge density on the localized gene delivery of multilayered films was carried out. The multilayered films constructed from the solutions of micelles and DNA without salt, and with the outermost layer as polyanion showed the best transfection efficiency. Therefore, in the chapter 4, we choose the solution of micelles and DNA without salt, and the outermost layer as polyanion to construct multilayered films. Various grafting degrees (8%,18.6%, and 25.6%) of 1-bromohexadecane was conjugated to poly(disulfide amine)s to form a series of bioreducible cationic micelles. A series of multilayered films were fabricated from such micelles and DNA, and applied in localized gene delivery. The thickness of cationic micelles/DNA films with 10 bilayers increased with increasing the grafting degree of alkyl groups in poly(amidoamine)s. In vitro release, the multilayered films were stable in phosphate-buffered saline (PBS7.4), but reducible in reductive conditions. Multilayered films prepared from bioreducible polycations with 25.6% of alkyl side chains, showed the fastest release of DNA in the presence of 2.5 mM glutathione. pGL-3 encoding luciferase and green fluorescence protein pEGFP-C1 were used to evaluate gene transfection efficiency of the multilayered films. Micelles-based DNA films exhibited reduced cytotoxicity and higher transfection efficiency when compared with control PE1/DNA films. Moreover, an increased hydrophobic alkyl side chains obviously promoted transfection efficiency when 293T cells were cultured on the surface of films.Biodegradable polymers are very promising candidates for the development of drug delivery systems. Introducing acid-sensitive linkage between biodegradable polymers and drug may lead to ideal polymeric drug delivery systems.In the chapter 5, poly(disulfide amine) dendrimers with surface amine groups of generation 2 (G2) were used as drug delivery carriers, and acid-insensitive succinic linkage and acid-sensitive cis-aconityl linkage were incorporated between DOX and G2 to produce DSG2 and DAG2 polymers, respectively. The in vitro release of DOX from DAG2 polymers dependent on the pH conditions, and the release rate increased with the decreased pH, while the DSG2 polymers released little drug at any pH conditions. The cytotoxicity of DAG2 and DSG2 polymers against HepG2 and HeLa cells increased with the increase of polymer concentration, and under the same conditions, the cytotoxicity of DAG2 polymers was higher than that of DSG2 polymers. The cellular uptake behaviors were studied against HepG2 and HeLa cells using confocal laser scanning microscopy (CLSM). The CLSM observation confirmed that under the same conditons, the cellular uptake of DAG2 polymers was higher than that of DSG2 polymers, and the fluorescence in HeLa cells was higher than that in HepG2 cells. The DOX released from DAG2 polymers under high concentrations accumulated in the whole cell and entranced into cell nucleus, while for DSG2 polymers, the fluorescence accumulated especially in the cytoplasm.Cleavable-polycation template is used as a general method for the construction of stable porous multilayered films without crosslinked.In the chapter 6, a novel cleavable-polycation template method was used for the fabrication of porous films. Porous films were fabricated from nonporous layer-by-layer multilayers composed of a blend of positively charged disulfide-containing poly(amidoamine) and poly(allylamine hydrochoride), and negatively charged poly(acrylic acid), followed by removal of cleavable disulfide-containing polycation after incubation in 1 mM DTT solution. The formed porous films were stable in buffer solution at pHs ranging from 7.4 to 1.6, whereas they showed slight changes in pore number and pore size when incubated in PBS buffer at a pH of 10.0.