以聚乙烯亞胺修飾之多功能雙極性奈米微粒作為 藥物與基因傳輸載體之研究

Abstract

多數疾病的發生都與體內特定蛋白質的變異、缺乏或過量表現有關，因而造成蛋白質功能的異常導致細胞訊息傳遞的調控變異。由於蛋白質藥物在臨床上的應用仍有相當多的限制，因此，近十幾年來所發展出的基因治療（gene therapy）被視為最具革命性的醫療技術。所謂的基因治療主要針對患者之體細胞提供給予特定的基因訊息，由細胞自己本身調控特定的目標蛋白質，以達到治療的目的。其中利用聚合物或脂質所形成的非病毒式載體（non-viral vectors）相較於病毒式基因載體（viral vectors）目前已被認為是較具生物安全性的基因載體。在眾多製作基因載體的材料中，聚乙烯亞胺（PEI）因對pH值的變化具有緩衝能力（pH-buffering），可保護基因不會因酸化而被分解，並可促進內質體破裂而釋放出所包覆的基因，是相當有潛力的材料之一。其中分子量越高(>25kDa)的聚乙烯亞胺可提供較高的基因轉殖效率，但相對也具有較高的生物毒性，使得聚乙烯亞胺在臨床應用上的發展受到限制，因此如何經由修飾聚乙烯亞胺以降低其生物毒性，並提高低分子量聚乙烯亞胺的轉殖效率值得被探討。 本研究是以聚乙烯亞胺為基礎，設計開發低毒性多功能的奈米雙極性微粒作為藥物與基因傳輸之載體。利用EDC為架橋劑將硬脂酸（SA）接枝於聚乙烯亞胺上進行改質，以溶劑發散法（oil-in-water solvent evaporation method）將其製成表面帶正電的聚合雙極性微粒（PEI-SA micelle），並以1H NMR確認其接合的結構。依據動態光散射法測得10k PEI-SA之大小為149.6 ± 1.2 nm，水中表面電位為64.1±1.5 mV。此由自我組裝（self-assemble）所形成的帶正電的雙極性微粒可有效率的結合DNA或siRNA作為基因傳輸載體。相較於聚乙烯亞胺，PEI-SA表現出較低的生物毒性，經由流式細胞儀分析10k PEI-SA/pEGFP複合體可以使62%的細胞具有綠色螢光蛋白表現。經由冷光酵素活性分析可以得到當10k PEI-SA/pLuc重量比達4.5以上時可以有相當良好的轉殖基因表現。結果證明相較於未修飾的聚乙烯亞胺，經由硬脂酸修飾的聚乙烯亞胺雙極性微粒(PEI-SA micelle)除了具有較低的細胞毒性外，亦同時可以表現高轉殖效率。 此外，PEI-SA雙極性微粒可提供物理性的屏障保護DNA或siRNA不受核苷酵素活性的影響而降解。本研究也證明了相較於游離的siRNA，經由PEI-SA雙極性微粒傳輸的siRNA可以達較高的細胞攝取效率（cellular uptake efficiency），且在含血清蛋白的環境下，亦可有良好的穩定性表現。而由10k PEI-SA/siRNA所形成的複合體可以表現良好的基因沈默效率（gene silencing efficiency）。在動物實驗的研究中，以PEI-SA奈米顆粒同時傳輸doxorubicin及VEGF siRNA對於抑制腫瘤細胞生長具有協同作用(synergistic effect)的影響。 此多功能的PEI-SA奈米雙極性微粒可提供一親水性並具有正電荷的外層（shell）及一具疏水性之核心(core)。其中，帶正電荷的外殼可吸附負電荷的DNA及siRNA作為基因之傳輸載體，而具疏水性的核心可用來包覆水溶性較低的化學藥物，形成具多功能的基因與藥物傳輸載體，在基因與化學治療的應用可具有相當的潛力。

The absence or overproduction of a specific protein in the body can lead to a variety of clinical manifestations depending on its structural or functional role. Gene therapy is a method for treatment or prevention of disease by transferring genetic information to the patient’s somatic cells. Non-viral gene carriers composed of biodegradable polymers or lipids have been considered as a safer alternative for gene carriers over viral vectors. Among some of the cationic polymers, polyethyleneimine (PEI) possess high pH-buffering capacity that can provide protection to nucleotides from acidic degradation, and promotes endosomal and lysosomal release. However, it has been reported that cytotoxicity of PEI depends on the molecular weight of the polymer such that high molecular weight (>25kDa) of PEI can elevate the transfection efficiency. Hence modifications of PEI structure for clinical application have been developed in order to reduce the cytotoxicity, or improve the insufficient transfection efficiency of lower molecular weight PEI. In this study, a multi-functional nano-micelle was developed for both drug and gene delivery application. PEI was modified by grafting stearic acid (SA) and formulated to polymeric micelles with positive surface charge to evaluate for gene delivery. The amine group on PEI was crosslinked with the carboxylic group of stearic acid by 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) as linker. PEI-SA micelles were then prepared using oil-in-water (o/w) solvent evaporation method. The success of PEI –SA conjugation structure was confirmed with 1H NMR. The average diameter and zeta potential of 10k PEI-SA determined by photon correlation spectroscopy was 149.6 ± 1.2 nm and 64.1±1.5 mV in water, respectively. Gel retardation assay indicated that these self-assemble positive charge micelles can effectively bind to pDNA and siRNA for gene delivery. PEI-SA micelles exhibited lower cytotoxicity compared to that of PEI only, while flow cytometry analysis revealed 10k PEI-SA/pEGFP complex provided 62% high EGFP expression. Luciferase activity also showed high transfection efficiency of PEI-SA micelles for weight ratio above 4.5 that was comparable to PEI only. These results proved that stearic acid grafted PEI micelles can provide high transfection efficiency comparable to unmodified PEI, and exhibit low cytotoxicity. Furthermore, PEI-SA micelles not only can serve as gene carriers, but also offered physical barriers to protect DNA and siRNA against nuclease digestion. The siRNA delivered by PEI-SA carriers also demonstrated significantly higher cellular uptake efficiency and stability even in the presence of serum proteins than free siRNA. The polyplex formulated by 10k PEI-SA/siRNA also provided excellent post transcriptional gene silencing efficiency, in particular when co-delivered with lysosomotropic agent chloroquine. In the animal model study, the combination effect of co-delivering doxorubicin and VEGF siRNA by PEI-SA nanoparticles showed a promising synergistic effect towards anti-tumor growth. In summary, the amphiphilic structure of PEI-SA micelles can provide advantages for multifunctional tasks; where the hydrophilic shell modified with cationic charges can electrostatically interact with DNA or siRNA, and the hydrophobic core can serve as payloads for hydrophobic drugs, making it truly a promising multifunctional vehicle for both genetic and chemotherapy application.