Document Type

Dissertation

Date of Degree

Fall 2011

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmacy

First Advisor

Aliasger K. Salem

Abstract

Gene therapy and immunotherapy are powerful techniques in the treatment of many life threatening diseases. The major challenge in these therapies is to seek a safe and efficient delivery carrier for gene and antigen materials. Carriers are designed to protect these molecules from degradation, improve their stability and facilitate the delivery of them to the site of action. This research study aims to develop appropriate carriers for small interferencing RNA (siRNA), DNA, antigen and ajuvant respectively. In the case of siRNA, material encompassing mannose, polyethylene glycol (PEG) and polyethylenimine (PEI) was investigated. Two structures were assembled: in one construct, mannose was conjugated to PEI directly (Mannose-PEI-PEG) whilst in a second construct; the mannose was conjugated to PEI via a PEG spacer (PEI-PEG-mannose). Confocal microscopy images suggested a faster escape and release of siRNA into the perinuclear region when siRNA was complexed with mannose-PEG-PEI. Mannosylation and PEGylation generated significant toxicity reduction compared to unmodified PEI alone. Real-time polymerase chain reaction (RT-PCR) results showed a significant decrease on mRNA knockdown when using modified PEIs. It was found that PEI-PEG-mannose was a stronger candidate for siRNA delivery because it displays lower toxicity, higher uptake efficiency and higher relative knockdown efficiencies. In the case of pDNA delivery, dextran was introduced to reduce the toxicity generated by PEI. PEI 2000 was more effective than PEI 800 in condensing DNA and inducing transfection when incorporated with dextran. The toxicity of dextran-PEI was greatly reduced when compared to unmodified PEI. Dextran-PEI was able to generate significantly higher transfection efficiencies than PEI alone in the presence of serum. An improved stability of complexes in serum by dextran-PEI was noticed along with a faster release of complexes to the perinuclear region of cells after endocytosis. These observations help to account for the higher efficiency of dextran-PEI in gene transfer.

In our final study on vaccines, we utilized cationic polyamidoamine (PAMAM) dendrimer polymers to modify biodegradable particles for enhanced delivery of antigens and adjuvants. Vaccines were formulated by loading CpG oligonucleotide (CpG ODN) and ovalbumin (OVA) into biodegradable microparticles. In one group, OVA and CpG were conjugated together and then loaded into the PLGA microparticles. In other groups, CpG was loaded into the particles and OVA bound to the surface and finally particles were prepared that were loaded with OVA and surface modified with cationic PAMAM dendrimers to electrostatically bind CpG ODN. The microparticles were able to provide sustained release of antigen and adjuvants over 14 day's course. The up regulation of CD86 and H2Kb indicated strong activation of DC and therefore strong induction of CD8+ T-cells. MHC II markers were not as significantly affected. Particles loaded with OVA and surface bound CpG ODN ((OVA)-CpG) showed the highest cytotoxic CD8+ T cell response, suggesting that formulation is optimal for vaccine applications.These observations were further supported by IgG1 and IgG2a antibody levels in mice sera.

Pages

xi, 121 pages

Bibliography

Includes bibliographical references (pages 109-121).

Copyright

Copyright 2011 Dahai Jiang

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