Application of positively-charged lipid nanoparticles complexed with oligonucleotides in gene therapy

• Main Authors: Jo-Chieh Tung, 董若傑
• Other Authors: 張富雄
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/87596235559030037880

碩士 === 臺灣大學 === 生物化學暨分子生物學研究所 === 98 === Gene therapy is the procedure that a gene is delivered into cells, and subsequently expresses appropriately to compensate for the dysfunctional gene. And a gene needs to be repressed when its abnormal overexpression has resulted in a disease. Presently, the nucleic acids used to inhibit protein expression are antisense oligodeoxynucleotides (ODN) and RNAi, both of which could block translation to suppress abnormal gene-expression. Another type of gene therapy is gene correction which revises the wrong nucleotides to recover the coding sequence of the targeted protein. Viral and non-viral vector have been used to deliver nucleic acid materials. Considering to stability, the non-viral vectors is more prevalent. 3β-[N-(2-guanidinoethyl)carbamoyl]cholesterol (GEC-Chol) is a cationic lipid based on cholesterol (Chol). We mixed GEC-Chol and Chol with molar ratio 1：1 and investigated the applications of the GEC-Chol/Chol cationic nanomicelles carrying ODN in gene therapy. To obtain the most appropriate condition, we measured the size, zeta potential, encapsulation and delivery efficiency of GEC-Chol/Chol cationic nanomicelles with different N/P ratio. We found that the delivery efficiency and encapsulation of oligonucleotides was highest when the N/P ratio was 3. In addition, the cytotoxicity of GEC-Chol/Chol cationic nanomicelles was low which suggested that GEC-Chol/Chol cationic nanomicelles could be used to carry more oligonucleotides in gene therapy. Moreover, we found that GEC-Chol/Chol cationic nanomicelles are fine vectors for antisense ODN delivery by observing the cell viability and protein level in the cancer cells which antisense oligodeoxynucleotides of c-myc and bcl-2 were delivered to. Comparing to the commercial transfection reagent Lipofectamine2000, GEC-Chol/Chol cationic nanomicelles also showed higher efficiency to correct aberrant sequence of enhanced green fluorescent protein (EGFP). In summary, GEC-Chol/Chol cationic nanomicelle is a potential tool for gene therapy owing to its high efficiency of ODN delivery and low toxicity to cells in vitro. Drugs and specific targeting molecules can be capsulated or embedded into this vector to execute their therapeutic functions. However, the in vivo test of intratumor injection of antisense oligodeoxynucleotides carried by the GEC-Chol/Chol cationic nanomicelles is still at the initiation stage. In vivo applications of such nanomicelle needs further study.