Study of calcium phosphate nanoparticles as non-viral vectors for gene delivery system
Date Issued
2004
Date
2004
Author(s)
Chen, I-Hua
DOI
zh-TW
Abstract
摘要
基因治療的重要關鍵在於一個良好且有效的基因遞送系統將具有功能性的基因送至細胞中表現。奈米微粒作為基因遞送載體具有可靶向和於體內長效循環以增加轉染效果的潛力。然而，適當的製程、粒徑大小和低毒性等性質是發展奈米微粒作為基因載體首要課題。本研究目的在於發展磷酸鈣奈米微粒作為非病毒基因載體和探討載體的材料特性，研究以water/AOT/hexane 之w/o 微乳化系統製備磷酸鈣奈米微粒，探討製程條件對粒徑分佈影響和材料性質分析，進而探討製備DNA-磷酸鈣奈米微粒複合物作為非病毒基因載體的適當條件。
研究以DLS 作粒徑分析並以TEM、FE-SEM 觀察奈米微粒型態，DNA-磷酸鈣奈米微粒複合物由TEM 觀察結果為球殼狀結構，且分散性良好，粒徑分佈集中於100nm 附近，平均粒徑隨Wo 值減少而減小。磷酸鈣奈米微粒擁有大於80%包覆基因的效率，且電泳結果證實DNA-磷酸鈣奈米微粒複合物能於DNaseI 環境下保護基因不被水解。由MTT 測試結果DNA-磷酸鈣奈米微粒複合物對細胞活性無負面影響。體外細胞實驗以293T 細胞進行轉染，DNA-磷酸鈣奈米微粒複合物能遞送基因並表現蛋白質，且轉染效果隨時間增加而增加，屬於緩慢釋放性質，符合非病毒載體適當製程、粒徑大小、低毒性、保護基因及基因表現等基本要求。
未來將有潛力經由顆粒表面修飾特定配位體有選擇性的遞送基因至
標的細胞，達到靶向效果，成為新的基因遞送系統選擇。
Abstract
In the post genomic era, gene therapy has become a major part of medical research. One of the major challenges of gene therapy is producing usable and reliable vectors to deliver the therapeutic gene. The vectors used for gene therapy need to deliver the therapeutic genes into the desired target tissues both efficiently and specifically. The aim of this study is producing novel non-viral calcium phosphate nanoparticles as a new vector for gene delivery. Calcium phosphate nanoparticles were prepared by water-in-oil microemulsion method with water to surfactant molar ratio, Wo = 2~10.
In this study, the particles size distribution of nanoparticles was determined by DLS and the morphology of nanoparticles was observed by TEM and FE-SEM. We report the design and synthesis of ultra-low size, highly monodispersed DNA doped calcium phosphate nanoparticles of size around 100 nm in diameter. The structure of DNA -calcium phosphate nanocomplex observed by TEM was displayed shell-like structure. In this study, we used pEGFP as reporter gene. The encapsulating efficiency to encapsulate DNA inside the nanoparticles was greater than 80%. In MTT test, both calcium phosphate nanoparticles and DNA-calcium phosphate nanocomplexes have no negative effect for 293T cells. By gel electrophoresis of free and entrapped pEGFP DNA, the DNA encapsulated inside the nanoparticles were protected from the external DNaseI environment. In vitro transfection studies in 293T cell-line, the DNA-calcium phosphate nanocomplex could be used safely to transfer the encapsulated DNA into the 293T cells and expression green fluorescent protein. The characteristic of DNA-calcium phosphate nanocomplexes to deliver DNA belongs to slow release. The properties of DNA-calcium phosphate nanocomplexes were fit in the requirement of non-viral vectors for gene delivery system.
In the future study, the surface of these nanoparticles can be suitably modified by some ligands to deliver gene to target cells under in vivo condition.
Calcium phosphate nanoparticles have potential to serve as efficient and
alternative DNA carriers for targeted delivery of genes.
Subjects
磷酸鈣
奈米微粒
基因治療
奈米結構
自組裝
nanoparticle
calcium phosphate
gene therapy
self-assemble
Type
thesis
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