寡核酸包覆型多層聚電解質載體於基因轉殖之應用

Abstract

以單股核酸(5’-C10A20-3’)塗佈於組氨酸修飾之聚丙烯胺與去氧核醣核酸所構成之陽離子型複合粒子上，製備出可應用於基因轉殖之陰離子型複合載體。藉由粒徑電位分析儀得知該粒子於單股核酸對組氨酸修飾之聚丙烯胺比為1.5時，其表面電位為-27毫伏特，且此時的粒徑約為100奈米。並且，此載體能於人類子宮頸癌細胞株(HeLa)內呈現相當高的轉殖效率與相對低的毒性，同時，亦能防止血球凝集與血清抑制轉殖效率的發生。同時本研究也發現其轉殖效率在將所使用的單股核酸組成更替或是替換為其他種陰離子型聚電解質後便受到顯著的抑制，顯示該載體非常有可能是透過某種特殊的載體─受器的交互作用才能被細胞吞噬進而表現出轉殖效率。 為了確定該載體與細胞之間的作用方式，我們接著於研究的第二部分鑑定其載體之內化機制，透過改變塗佈於載體最外層之單股核酸的組成、分子量以及序列，我們發現其轉殖效率乃被載體上單股核酸中的腺苷酸之鹼基所調控。進一步的，透過添加相同的單股核酸以及於載體上再包覆第二層互補單股核酸，顯示該粒子與細胞膜上的腺苷酸受器有顯著的關聯性，而透過使用該受器之拮抗劑確實能使得轉殖效率有顯著的降低，此結果顯示該載體乃是先透過與細胞膜上的腺苷酸受器作用被細胞所吞噬進而表現出基因轉殖效率。此外，我們也發現影響其載體轉殖效率之細胞內化路徑為caveolae-mediated pathway，顯示載體在與細胞膜上的腺苷酸受器作用後，是經由caveolae-mediated pathway被細胞內化吞噬入細胞內，進而再將所攜帶之去氧核醣核酸釋放被細胞表現。 接著，於研究的第三部分，我們著手將使用單股核酸塗佈於陽離子型載體的技術擴大應用到其他種的陽離子型載體，以證實該技術之延伸應用性。於此，我們選擇市面上已被公認為最有效之轉殖載體商品，聚乙烯亞胺，用以製備陽離子型載體。藉由粒徑電位分析儀得知，所製備的載體於單股核酸聚乙烯亞胺比為9時，其表面電位為-50毫伏特，且此時的粒徑約為100~170奈米。且相較於未經包覆的聚乙烯亞胺載體，該載體更能有效防止血球凝集，且不會因單股核酸加入而導致聚乙烯亞胺載體崩解。於研究的第一部分，我們發現透過單股核酸包覆後的轉殖載體，其轉殖效率將有其顯著增加，此結果除了在人類子宮頸癌細胞株，於人類胚胎腎臟細胞株(HEK 293)、人類肝臟細胞株(HepG2)以及人類皮膚纖維母細胞株(Hs68)皆可被觀察到，特別是Hs68，其轉殖效率增加了四十倍之多。因此，第三部分之研究將選擇以Hs68作為檢驗載體效率之細胞株。相較於未經包覆之聚乙烯亞胺載體，載體經包覆後同樣能抵抗血球凝集。並且發現載體於細胞株(Hs68)以及生物體細胞(fibroblast-like synoviocytes)其轉殖效率有顯著的增加，同時並不會增加細胞毒性。此外，包覆單股核酸於聚乙烯亞胺載體外同樣也能夠抵抗血清吸附於載體表面抑制轉殖效率之影響。最後，透過於天竺鼠膝關節轉殖，我們發現該載體確實能於動物實驗中獲得比未經包覆的聚乙烯亞胺載體更為有效的轉殖效率。 總結，本研究開發出一種新型的轉殖載體，運用靜電吸引力包覆技術將特殊單股核酸包覆於聚電解質型的轉殖載體上，進而促進載體轉殖效率，而使用特殊的單股核酸包覆陽離子型載體之技術亦能使得載體應用於各種轉殖與治療時更為安全且有效。

Ternary nanoparticles with negatively-charged surface were prepared by coating single-stranded oligonucleotides (5’-C10A20-3’) on histidine-conjugated polyallylamine (PAA-HIS)/DNA complexes for gene delivery. Characterization of PAA-HIS/DNA/oligonucleotide complexes demonstrated that nanoparticles possessed the negative surface charge -27 mV and size of around 100 nm when the molar ratio of oligonucleotide/PAA-HIS exceeded 1.5. The negatively charged oligonucleotide-coated PAA-HIS/DNA complexes could be entirely internalized by the living HeLa cells to exhibit high gene expression with low cytotoxicity and the resistance against erythrocyte agglutination and serum inhibition. Since the gene expression of PAA-HIS/DNA complexes was significantly inhibited by coating other polyanions and oligonucleotides, the ternary PAA-HIS/DNA/deoxyadenosine-rich oligonucleotide complexes were uptaken by specific receptor-mediated process. In order to classify the fate of PAA-HIS/DNA/oligonucleotide, the internalization mechanism of the ternary nanoparticles was confirmed in the study of part II. Characterization of the constitution effect of the topmost layer of PAA-HIS/DNA/oligonucleotide complexes demonstrated that transfection efficiency was regulated by the nitrogenous base of adenosine on the nanoparticles. The results also showed ternary complexes were uptaken into cells and subsequently expressed genes by interacted with adenosine receptors on cellular membrane. Furthermore, the uptake of ternary complexes by caveolae-mediated pathway showed to influence the transfection efficiency. Furthermore, in the study of part III, the application of techneuqe of assembling oligonucleotides (5’-C10A20-3’) with binay polyethylene imine (PEI)/DNA complexes used to prepare a ternary anioic gene delivery system. Characterization of PEI/DNA/oligonucleotide complexes demonstrated that nanoparticles possessed the negative surface charge -50 mV and size of around 100-170 nm when the molar ratio of oligonucleotide/PEI exceeded 9. Compared with PEI/DNA complexes, PEI/DNA/oligonucleotide complexes could completely resistant against erythrocyte agglutination because of anionic surface charge and would not disassociate during the process of forming ternary complexes. In the study of part I, the increase of transfection efficiency was also observed in the other in vitro cell line system such as HEK 293, HepG2, and Hs68. Therefore, in the study of part III, Hs68 were used to be the model cell line for the transfection efficiency experiment. The oligonucleotide-coated PEI/DNA complexes exhibited high gene expression with low cytotoxicity in the living Hs68 cells (cell line) and fibroblast-like synoviocytes (primary cells), respectively. Additionally, the deposition of a layer of oligonucleotide onto the binary PEI/DNA complexes could prevent serum inhibition during transfection process. Moreover, the ternary PEI/DNA/oligonucleotide complexes showed higher gene expression than PEI/DNA complexes in the knee of guinea pig. Therefore, in this study, we developed a novel gene delivery carrier which coating oligonucleotides by electrostatic attraction to increase the transection efficiency. The technology of coating specific oligonucleotides on cationic binary DNA complexes could facilitate the use of nanoparticles for safe and efficient gene delivery and eventual therapy.