PH Amplified Exponential Growth Multilayers As An Approach For Construction Of Ultrathin Coatings Of DNA And Oligo-Peptide

With the development and interdisciplines of protein technology, gene technology, tissue regeneration medicine and pharmacy, advanced drug controlled release coatings applied in drug/medical devices becomes an important issue of biomedical implants and tissue regenerative materials. This thesis explores to establish a facile method to rapidly construct multilayers embedded with gene or oligo-peptide via the synergetic action of pH tunable charge density and diffusivity of the weak natural polyelectrolytes.The exponential growth behaviors of multilayers of the diffusible poly-L-lysine (PLL) with hyaluronic acid (HA) or DNA at different pH are investigated. The data indicate that pH controlled assembly can enhance the exponential growth behavior of PLL/HA and PLL/DNA multilayers, and realize the rapid fabrication of biomacromolecule multilayers. As HA is a weak polyelectrolyte and DNA is a strong polyelectrolyte, the characteristic growth behavior is different between PLL/HA and PLL/DNA multilayers constructed via pH controlled assembly. PLL/HA exponential growth is amplified by decreasing pH of HA solution or increasing pH of PLL alone or simultaneously. In PLL/DNA systems, the pH of PLL solution exerts a dominant role in the multilayer growth while that of DNA has little effect. The rapid growth of multilayer embedded with DNA can be achieved at high pH (pH 9.5 and 10.0). The growth behavior characteristic indicates that the change of charge density of weak polyelectrolyte is the main effect to influence the pH amplified exponential growth of the multilayers.Based on the pH effect on the exponential growth behavior of PLL/DNA multilayer, rapid fabrication of gene coating is established. PLL/DNA 10.0/4.6 film (PLL and DNA are deposited at pH 10.0 and pH 4.6, respectively) could reach 121.2μg/cm² within 7.5 bilayers. The N/P ratios (molar ratio of free amino groups in polycation to phosphate groups in DNA) within the multilayers are proved to be elevated by increasing either the pH difference between the two deposition solutions or the pH value of the outmost deposition solution. The N/P ratios of PLL/DNA 9.5/5.0 and 10.0/4.6 are respectively 6.3 and 9.8 within 7.5 bilayers. The PLL/DNA multilayers are degraded as exposed to trypsin solution and the mean diameters of the degraded DNA complexes decrease when the N/P ratio increases. The diameter of DNA complexes degraded from PLL/DNA 10.0/4.6 film reaches 151 nm. Relatively higher transfection level is achieved for plasmid degraded from the PLL/plasmid 9.5/5.0 and 10.0/4.6 films, and the transfection efficiency are 10.43% and 9.67%, respectively, while the transfection efficiency in PLL/plasmid 7.2/7.2 multilayer is 0.18%. The film with more DNA and higher N/P ratio might provide a new method to enhance transfection in localized gene delivery system.Based on the enhancement effect of pH on the diffusion behavior of polypeptide, coating systems incorporated with a trans-activating transcriptional factor (TAT) peptide are established. The result indicates that TAT could diffuse throughout the exponentially growing PLL/HA film. The amount of peptide embedded within multilayer could be adjusted not only by the multilayer thickness but also by the multilayer assembly pH and TAT loading pH. The density of TAT loaded into the (PLL/HA 9.5/2.9)₅ film can reach 25.0μg/cm² when TAT loading pH is 9.5. Compared with direct layer-by-layer assembly of TAT and HA, the postdiffusion of TAT into (PLL/HA 9.5/2.9)5 film can be loaded much more peptide and realize the sustained release of oligo-peptide. The cell culture results indicate that the TAT embedded within the film maintains the ability to traverse across the Hep G2 cell membrane. The functionalized (PLL/HA 9.5/2.9)₅ TAT 9.5 film is more efficient than the 10-fold more amount of free TAT peptide in the TAT uptake test. The postdiffusion of oligo-peptide within an exponential growth multilayer can serve as an effective approach for localized and sustained peptide delivery.