幾丁聚醣和聚丙烯酸樹酯作為遞送基因及抗癌藥物之奈米複合體的研究

Abstract

近年來，奈米技術的發展對製藥工業是一個重大突破。因為在奈米的範圍，材料的物理性會有所改變，例如表面積、電性及磁力。特別是在醫藥方面，又以具有可生物分解性、生物可相容性的高分子聚合物，所形成的奈米載體，在遞送抗癌藥物或基因最具優勢。 在本研究中，主要是以低分子量幾丁聚醣(Chitosan)、去乙醯去聚合幾丁聚醣(DADPCS)以及聚丙烯酸樹酯(EudragitR RS 100)作為遞送藥物或基因的載體。在幾丁聚醣的部分，先進行了去乙醯去聚合的化學反應得到去乙醯去聚合幾丁聚醣(DADPCS)，並以傅立葉轉換紅外線光譜儀(FTIR)、膠體滲透層析儀(GPC)確認其化學結構與分子量。再來是利用帶正電荷的聚合物如低分子量幾丁聚醣、去乙醯去聚合幾丁聚醣以及聚丙烯酸樹酯與帶負電荷的質體DNA進行聚電解質複合反應(polyelectrolyte complexation)形成奈米複合體；並針對此奈米複合體進行物性探討，包括粒徑大小分析、圖二色偏光儀、凝膠阻滯分析、安定性試驗、紅血球凝集試驗以及電子顯微鏡影像拍攝，再利用流式細胞儀分析三種高分子聚合物對於綠色螢光基因(pEGFP-N1)轉染至HepG2、MDA-MB-468以及MCF-7三種癌細胞的效率。 另一方面，則再加入硫酸葡聚醣(Dextran Sulfate)一個帶有負電荷的高分子聚合物，搭配聚丙烯酸樹酯(EudragitR RS 100)或去乙醯去聚合幾丁聚醣(DADPCS)兩個帶正電的高分子聚合物，利用聚電解質複合反應(polyelectrolyte complexation)包覆抗癌藥物杜薩魯比辛(Doxorubicin)。並針對其奈米複合體作物性探討，包括粒徑大小分析、安定性試驗、紅血球凝集試驗以及電子顯微鏡影像拍攝，並以連續式多功能微孔盤偵測系統分析藥物的包覆率。另外，也進行了奈米複合體在pH4.0與pH7.4的體外釋放試驗(In vitro drug release study)，確認奈米複合體中藥物杜薩魯比辛(Doxorubicin)的釋放模式。最後進行細胞存活率試驗(MTT Assay)，了解藥物與含藥奈米複合體對MDA-MB-468和MCF-7的毒殺效果。 在第一部分的實驗結果顯示，低分子量幾丁聚醣、去乙醯去聚合幾丁聚醣以及聚丙烯酸樹酯奈米複合體，在重量比7:1和20:1粒徑大小約在150-350 nm，帶正電，能形成穩定的粒子並且能保護質體DNA，不受核酸分解酶分解；從圖二色光譜可得知，質體DNA在構形上也不會受製備過程影響，最後以流式細胞儀分析轉染試驗，可以發現在HepG2細胞株，其轉染綠色螢光蛋白的能力為去乙醯去聚合幾丁聚醣>低分子量幾丁聚醣>聚丙烯酸樹酯；而在MDA-MB-468和MCF-7其轉染綠色螢光蛋白的能力為聚丙烯酸樹酯>去乙醯去聚合幾丁聚醣>低分子量幾丁聚醣。在第二部分以硫酸葡聚醣、去乙醯去聚合幾丁聚醣以及聚丙烯酸樹酯藉由正負電荷進行包覆杜薩魯比辛，所形成的奈米複合體粒徑約在100至200 nm之間且呈現良好的安定性，在紅血球凝集試驗，奈米複合體帶越強的正電，越容易造成紅血球凝集。在藥物體外釋放方面，在pH 4.0時的奈米複合體的杜薩魯比辛釋放較pH 7.4迅速，而與杜薩魯比辛藥物相比，可以發現所製備的奈米複合體，皆釋放較慢，是緩釋劑型。細胞存活率實驗結果顯示，在MDA-MB-468細胞株，各奈米複合體的毒殺效果並未優於杜薩魯比辛，但在MCF-7細胞株則是呈現相反的結果，MCF-7細胞株對於杜薩魯比辛藥物敏感性較差是由於有P-glycoprotein在細胞膜表面，而帶正電的含藥奈米複合體，可以黏附到帶負電的細胞膜，不受到外排蛋白所影響，能夠確實可以遞送杜薩魯比辛到作用部位，產生毒殺效果。

In the past few years, the development of nanotechnology is a major breakthrough in pharmaceutical industry. Due to nano-scale, the physical characteristic of material was changed including the surface area, electricity and magnetism. Especially in medical application, the biodegradable and biocompatible nanocarriers showed great advantages in cancer or gene therapies. The main topic of this research was focused on chitosan (LCS), deacetylated depolymerized chitosan (DADPCS) and EudragitR RS 100 (ERS). Chitosan was deacetylated and depolymerized. The chemical structure and molecular weight of DADPCS were confirmed by Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC). We used LCS, DADPCS and ERS polymer to complex with pEGFP plasmid to form nanocomplexes via polyelctrolyte complexation method and the physical properties of nanocomplexes were characterized. Furthermore, we used flow cytometer to evaluate the tranfection efficiency of green fluorescent protein in three cancer cell lines (e.g., HepG2, MDA-MB-468 and MCF-7). On the other hand, we used the DADPCS and ERS polymers to loaded anti-cancer drug doxorubicin (DOX) and dextran sulfate was used as ionic crosslinker. The nanocomplex was formed and the physical properties were characterized. The in vitro drug release from nanocomplex was performed in pH 4.0 and pH 7.4 media. Finally, the cell viability of MDA-MB-468 and MCF-7 was investigated in free drug and nanocomplexes. In the first part, the particle size of three nanocomplexes in weight ratios 7/1 and 20/1 were around 150-350 nm. The nanocomplex had good binding affinity with plasmid DNA and protected plasmid DNA from DNase I degradation. There was no conformation change in plasmid DNA after loaded in nanocomplex. The tranfection efficiency in HepG2 was in the order of DADPCS > LCS > ERS but in MDA-MB-468 and MCF-7 cell lines were ERS > DADPCS > LCS. In the second part, the particle size of DOX-DSNP, DADPCS-DOX-DSNP and DOX-DS-ERSNP were around 100-200 nm. Three nanocomplexes showed good stability at 4oC. The nanocomplex containing more positive charge caused more RBC agglutination. In vitro drug release study, the drug release in pH 4.0 release medium was faster than in pH 7.4 release medium. The drug release from DOX-DSNP, DADPCS-DOX-DSNP and DOX-DS-ERSNP showed much slower than Doxorubicin. In MTT assay, DOX-DSNP, DOX-DS-ERSNP and DADPCS-DOX-DSNP showed lower cell viability than doxorubicin in MDA-MB-468 but oppsite result in MCF-7. Doxorubicin was less sensitive to MCF-7 due to p-glyconprotein in the cancer cell membrane. The positive nanocomplex adhered to negative cell membrane in order to deliver doxorubicin into target cells and cause cell death.