| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 96 === The efficient gene delivery system is important both in therapeutic and laboratory research. The cationic materials can interact with negatively charged nucleic acids and assemble into several hundred nanometers in diameter complexes though the ionic interaction. However, there are several complications, for example, low delivery efficiency and poor stability, when applying these materials to in vivo gene delivery. In order to maintain the size of complexes and enhance the stability in vivo, previous studies have developed a strategy by using the highly hydrophilic polymer, polyethylene glycol (PEG), to modify the surface of particles hence improve the circulation time and stability.
GEC-Chol (2-guanidino-ethyl-carbamic acid-cholesterol) is a cholesterol-based cationic lipid. Previous studies reveal that, when GEC-Chol is mixed with cholesterol, they assemble into 100 nm particles. These particles are highly efficient in linear DNA delivery in in vitro experiments. In this study, we used FITC-labeled oligonucleotides as tracers and found that when N/P ratio of ODN and lipid was 3, the high delivery efficiency was observed. But if we added 10 % PEG-DOPE into the particle formulation, the delivery efficiency decreased dramatically. To solve the conflict between stability and efficiency, we bring up a strategy, post insertion, as a resolution. Briefly, we inserted PEG-modified lipids, PEG-DOPE, to the complexes after the particle of ODNs and cationic lipids formed, expected to reduce the particle size and increase the stability for in vivo applications. The result revealed that post-insertion particle had smaller particle size and higher stability. Using fluorescence microscopy and flow cytometry, we could observe that even lipid particles containing 10 % PEG-DOPE still retained the high delivery efficiency of FITC-labeled oligonucleotides in vitro. In order to detected particles in vivo, we add high quantum yield nanoparticles, quantum dots (QDs), to indicate the distribution of post-insertion particles. After analysis by confocol microscopy and agarose gel, it demonstrated that addition of QDs did not affect oligonucleotide encapsulation. This kind of particles did not induce inflammation in vivo and had high biocompatibility.
In this study, post-insertion particles containing 10 % PEG-DOPE have high delivery efficiency, and they do not induce obvious immune responses in vivo. Furthermore, if we elevate the PEG-DOPE ratio to 50 %, the nonspecific binding decreases evidently. The long and the short of it is that post-insertion particles are more suitable to be the carrier of nucleic acid drug in vivo than previous. In the future, we can add drugs or specific targeting molecules as tools for specific therapy and imaging in vivo.
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