Development of A Method To Quantify The DNA Content in Cationic Peptide-DNA Nanoparticles

Gene therapy has the potential to provide safe and targeted therapies for a variety of diseases. A range of intracellular gene delivery vehicles have been proposed for this purpose. Non-viral vectors are a particularly attractive option and among them cationic peptides have emerged as promising candidates. For the pharmaceutical formulation and application to clinical studies it is necessary to quantify the amount of pDNA condensed with the delivery system. There is a severe deficiency in this area, thus far no methods have been reported specifically for pDNA condensed with cationic peptide to form nanoparticles. The current study seeks to address this and describes the evaluation of a range of disruption agents to extract DNA from nanoparticles formed by condensation with cationic fusogenic peptides RALA and KALA. Only proteinase K exhibited efficient and reproducible results and compatibility with the PicoGreen reagent based quantification assay. Thus we report for the first time a simple and reliable method that can quantify the pDNA content in pDNA cationic peptide nanoparticles

Formulation strategies for the mucosal delivery of HIV vaccines

Vaccination seems to be the key to curtailing the HIV epidemic but all efforts to make a clinically effective vaccine have failed to date. Eliciting high titres of neutralising antibodies at the mucosal portals of viral entry is a key goal in HIV vaccine research. Thus, this thesis specifically focuses at the strategic development and evaluation of women-controlled mucosal vaccine delivery systems for HIV envelope based constructs, H4A, gp140 and FP-A for the purpose of eliciting high antibody titres at the vaginal mucosa. Sustained release rod-insert vaginal rings (RiRs) loaded with gp140, quick release rods containing H4A, FP-A loaded liposomal gels and microneedles in conjunction with mucosal inoculations were evaluated for induction of specific antibodies in animal models (sheep/mice/rabbits). The formulations were evaluated mainly using vaginal delivery with nasal route being used as an auxiliary. However, we found that the nasal route was extremely potent compared to the vaginal route for the induction of antibody responses. In addition, we found that there is a definite rationale for sustained vaginal delivery ofvaccines. FP-A loaded gels showed ability to elicit neutralising antibody at mucosal sites and are hence being scaled to GMP production with a view to progressing them to clinical trials. Storage stability of bovine serum albumin (BSA) loaded RiRs and protein loaded immediate release dosage forms were also evaluated. Protein aggregation was found to be the most important route of degradation of BSA in RiRs, while freeze-drying induced changes predominate in the immediate release dosage forms. Finally, a solvent emulsion diffusion based method was developed for the generation of neutral, anionic and cationic liposomes. Homogenisation time was studied as a process parameter; however this was not able to control the particle size significantly.EThOS - Electronic Theses Online ServiceGBUnited Kingdo