Bombardment of intact cells and tissue with DNA-coated microprojectiles represents a novel approach to the genetic transformation of biological material. In this thesis, a gas propulsion particle gun is developed for such a purpose. The particle gun utilises gas dynamics to control particle velocity and spread, thereby enabling optimisation of transformation efficiencies for varying types of target material. The dynamics associated with particle acceleration are shown to relate to transient, shock tube flow. Measurements from schlieren high-speed video photographs of the shock structure of an underexpanded jet demonstrate good agreement with empirical correlations of previous researchers; pitot tube measurements of the nozzle exit Mach number are made and shown to be in good agreement with theoretical contact surface velocities. A novel optical particle velocimeter is used to measure particle time-offlight between axial locations in the system's target chamber, providing a consistent, distance-averaged measurement of particle velocities. Particle velocities are measured for a range of system pressure ratios and driver gases. Variation in particle velocities is seen to be similar to theoretical variation in contact surface velocities. Analytical theory is used to predict small gas-particle velocity lag. Particle velocities with helium as a driver gas are shown to be considerably higher than those with air. High speed video recording of chalk particles exiting the nozzle is used to visualise the spatial and temporal variation in particle spread as a function of nozzle pressure ratio. This technique demonstrates that particle spread increases with increasing nozzle pressure ratio, as gas dynamics theory indicates. Conditions for bombardment of maize suspension cells are experimentally optimised. Significant rates of transient genetic expression are achieved with both air and helium as driver gases. High levels of transient genetic expression are also found with bombardment of maize coleoptiles and the leaves of various dicot species. Transformation efficiencies for bombardment of HL60 human leukaemia cells show dramatic increases over efficiencies seen with conventional techniques not involving cell bombardment. For other cell types the gas propulsion device described here appears to give rates of transient genetic expression similar to those reported for commercial systems using microprojectiles. Other data for the performance of such systems are too limited at present to allow comparisons of controllability and reproducibility of bombardment efficiency.

Thesis (Ph. D.)--Cornell University, March, 2008. / Vita. Includes bibliographical references (leaves 376-419).

Due to recent advances in molecular biology and genomic research, numerous diseases have been given their genetic identities for which gene therapy may be a possible prescription. Gradually, the development of viral and non-viral vectors to translocate genes has become a bottleneck. For non-viral vectors, polyethylenimine (PEI) is considered as a potential vector candidate for gene delivery because of its ability to compact DNA and its intrinsic pH buffering capacity. PEI and its derivates have been widely tested in both in-vitro and in-vivo gene transfection experiments. The progress is limited due to the lack of a better understanding of the intracellular mechanism. So far, their cytotoxicity is relatively high and gene transfection efficiency is low. This study was designed to modify PEI and optimize its cytotoxicity and gene transfection efficiency. / During the complexes formation, both LLS and zeta-potential were used to follow the process. The results showed that most of anionic DNA are complexed by cationic PEI-based polymers when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ∼3, but the gene transfection reaches the highest efficiency when N:P ∼10. When N:P > 3, there exist two population of PEI chains in the solution mixture: bound to DNA and free in the solution. The bound PEI chains condense and protect DNA. Our current study confirms that it is those free PEI chains that play a vital role in promoting the gene transfection. Our preliminary data shows that the promotion mainly occurs in the intracellular space. The detailed mechanism is still lacking at this moment. Nevertheless, our finding leads to a totally different way in the development of non-viral vectors. / Further, we grafted PEI with polyethylene glycol (PEG), respectively via a reductive disulfide -S-S- and a non-degradable -C-C- bond to form two copolymer vectors. A comparative study shows that the polyplexes formed between the two copolymers and DNA are more stable than that formed between unmodified PEI and DNA under the physiological condition, presumably because the grated PEG chains form a protective hydrophilic shell on the PEI/DNA polyplexes. However, PEGylation reduces the internalization of the copolymer/DNA polyplexes in in-vitro experiments. For the two copolymer vectors, PEG-SS-PEI is 2-8 times more effective than its counterpart (PEG-CC-PEI) in the gene transfection, presumably due to the cleavage of the grafted PEG chains inside the reductive cytosol, which promotes the release and translocation of DNA. Our results demonstrate that using the disulfide as a linker is a promising approach to overcome the PEGylation dilemma in the development of low cytotoxic and high efficient non-viral polymeric vectors. / It has been known that short PEI chains are less toxic, but long chains are more effective in gene transfection. Therefore, we decide to use the disulfide bond (-S-S-) to extend short PEI chains to increase efficiency and also utilize the reductive cytosol environment to cleave such extended PEI chains to reduce their cytotoxicity inside the cell. Laser light scattering (LLS) was used to in-situ monitor the linking reaction between short PEI chains (M w = 2000 g/mol) and dithiobis(succinimidyl propionate) (DSP). The molar mass and crosslinking degree of the extended PEI chains was controlled by either the amounts or the adding rate of DSP. A comparative study of two linked PEI samples (PEI-7K-L and PEI-400K-L, respectively with M w = 6.5 x 103 and 3.8 x 10 5 g/mol) reveals that cytotoxicity and gene transfection efficiency of such extended PEI chains are related to the chain length and structure. Namely, PEI-7K-L with an extended chain structure is less cytotoxic and 2--10 times more effective in the gene transfection than the "golden standard" (PEI25K) and the widely used commercial vector, Lipofectamine 2000RTM. Comparatively, PEI-400K-L with a spherical microgel structure is ineffective in spite of its non-toxicity. Our study clearly demonstrates that a proper control of the chain length and structure is important. / by Deng, Rui. / Adviser: Chi Wu. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Yue, Yanan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 62-63). / Abstract also in Chinese. / ABSTRACT (Chinese) --- p.i / ABSTRACT --- p.iii / CONTENT --- p.v / ACKNOWLEDGMENT --- p.vii / ABBREVIATIONS --- p.viii / Chapter CHAPTER 1 --- Introduction and Background / Chapter 1.1 --- Methods of Gene Delivery --- p.1 / Chapter 1.1.1 --- Viral Delivery Systems --- p.2 / Chapter 1.1.2 --- Non-Viral Delivery Systems --- p.3 / Chapter 1.2 --- The Gene-delivery Problems --- p.7 / Chapter 1.2.1 --- Extracellular Barriers --- p.8 / Chapter 1.2.2 --- Intracellular Barriers --- p.10 / Chapter 1.3 --- Polymer-Mediated Systems for Gene Delivery --- p.13 / Chapter 1.3.1 --- Polyethylenimine (PEI)-Based Vectors --- p.13 / Chapter 1.3.2 --- Cyclodextrin-Based Vectors --- p.15 / Chapter 1.4 --- Objective and Main Achievements --- p.16 / Chapter 1.5 --- References --- p.18 / Chapter CHAPTER 2 --- Effect of Free Polyethylenimine-Mediated Polycations on Gene Delivery: Fundamentals and Vital Factors / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Experimental Section --- p.25 / Chapter 2.3 --- Results and Discussions --- p.29 / Chapter 2.3.1 --- Fundamentals --- p.29 / Chapter 2.3.2 --- Vital Factors for the Efficacy of Free Chains --- p.37 / Chapter 2.4 --- Conclusions --- p.42 / Chapter 2.5 --- References --- p.42 / Chapter CHAPTER 3 --- Effect of Free Polyethylenimine-Mediated Polycations on Gene Delivery: Mechanistic Study / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Experimental Sections --- p.46 / Chapter 3.3 --- Results and Discussion / Chapter 3.3.1 --- Potential Effect of Free PEI Chains on Cellular Uptake --- p.49 / Chapter 3.3.2 --- Potential Effect of Free PEI Chains on Endolysosomal Release --- p.51 / Chapter 3.3.3 --- Exploration on Proton Sponge Hypothesis --- p.53 / Chapter 3.3.4 --- Interactions of PEI-based Polycations and Phospholipid Membranes --- p.55 / Chapter 3.4 --- Conclusions --- p.61 / Chapter 3.5 --- References --- p.62

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Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 112-133).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 152-169).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.