具影像對比功能正電荷奈米微粒作為蛋白質藥物載體之研究

Abstract

研究指出許多疾病起因於體內特定蛋白質的缺乏或表現異常，導致蛋白質的變異進而影響細胞訊息傳遞及調控的功能。近年來有許多研究致力於以基因轉殖技術治療蛋白質表現變異所產生的疾病，但基因治療的風險及成效至今仍無法預測，因此利用蛋白質藥物直接對細胞進行調控以達治療目的之研究仍有很大的發展潛力。由於蛋白質容易被生理環境因子破壞，故在臨床上的應用仍有許多限制。為克服蛋白質藥物傳輸之限制，設計一奈米載體做為蛋白質傳輸之媒介被認為可增進蛋白質之細胞攝入效率及保護蛋白質免於環境因子的破壞。除了增進蛋白質傳輸效率，如何使蛋白質藥物在攝入細胞後能避免因內質體或溶酶體酸化而分解為設計蛋白質載體之重要考慮因素。聚乙烯亞胺(polyethyleneimaine, PEI)具有對pH變化時的緩衝能力(pH-buffering)，可保護蛋白質藥物不因酸化環境而分解，並可促使內質體及溶酶體的破裂而使蛋白質藥物釋放至細胞質中，但PEI存在細胞毒性，且其毒性程度隨著分子量增加而提升，因此在設計PEI相關之奈米載體時，選擇低分子量之PEI以降低毒性的同時是否能提高蛋白質承載，及傳輸效率之議題值得被深入研究。 本研究主要是發展同時具有蛋白質藥物傳輸以及醫學影像探針特性之多功能奈米載體，利用表面修飾聚乙烯亞胺使奈米載體具有帶正電之特性，並研究此奈米載體於蛋白質傳輸之成效。以去溶解法(desolvation method)製備明膠奈米粒子，並以EDC做為架橋劑將低分子量之正電荷支鏈型聚乙烯亞胺修飾於明膠奈米粒子表面。為使明膠奈米粒子具備細胞示蹤之功能，將紅色螢光探針Rhodamine B isothiocyanate(RITC)接合於明膠奈米粒子中，並將螯合劑DTPA接枝於明膠—聚乙烯亞胺奈米粒子之表面，螯合釓離子(Gadolinium, Gd)作為磁振造影T1-weighted影像之對比劑。 實驗結果顯示本研究所製備之正電荷明膠—聚乙烯亞胺奈米粒子之尺寸約150nm，於中性環境下之表面電位約為60mV。在穿透式電子顯微鏡(TEM)及原子力顯微鏡(AFM)的影像可觀察到奈米粒子之型態為球型，且尺寸大小符合動態光散射法所測得之尺寸。細胞毒性之研究結果顯示細胞與明膠—聚乙烯亞胺奈米粒子共同培養後之細胞存活率與控制組無顯著差異，證明本研究所製備之奈米載體具有低細胞毒性之特質。經由流式細胞儀分析結果及螢光顯微鏡影像可驗證帶正電荷之明膠—聚乙烯亞胺奈米粒子相較於未修飾之明膠奈米粒子具有更有效率的細胞攝入率。此外，研究結果顯示明膠—聚乙烯亞胺奈米粒子具有良好的蛋白質承載能力，相較於游離之蛋白質，其良好細胞及組織間之傳輸情形藉由流式細胞儀分析及組織切片螢光影像得到驗證。 另一方面，將本研究所發展之奈米載體作為多樣化影像對比功能之應用，可從螢光顯微影像及共軛焦雷射顯微影像證明其於細胞間之示蹤能力，可幫助追蹤奈米粒子於細胞間的分布以及蛋白質傳輸及釋放情形。在磁振影像中也可證明以DTPA螯合釓離子之明膠—聚乙烯亞胺奈米粒子具備T1-weighted之對比能力。此外奈米粒子於腫瘤組織之間的累積情形也可藉由磁振造影觀察，以上結果可說明本研究所發展之正電性之多功能明膠奈米粒子具有做為醫學影像探針之潛力。 以聚乙烯亞胺修飾明膠奈米粒子做為正電性之奈米載體傳輸系統在蛋白質藥物傳輸及診斷用影像探針之應用皆顯示了相當程度的優勢，其中粒子表面之帶正電特性不但具有蛋白質藥物承載能力，且能促使承載蛋白質之奈米粒子進入細胞並有效地從內質體內釋放，避免蛋白質被分解破壞，同時此奈米粒子顯示低細胞毒性及影像示蹤之特性，有助於診斷性治療功能方面應用之發展。

It has been reported that the absence of specific protein or the disorder of protein expression which induce the malfunction of signal transduction or regulation in cells may cause various clinical syndromes. Although gene therapy is the strategy for the cure of diseases by transferring genetic information into the targeting cells to regulate the subsequent protein expressions, so far the risk and effects of gene transfection method are not predictable. Hence the protein delivering into cells to directly regulate the mechanism of cells has also been investigated for the treatment of the corresponding diseases. Owing to the inefficiency of free protein transportation by the environmental degradation, the design of protein carrier is required to enhance the intracellular delivery efficiency of protein and prevent protein degradation by environmental factors. Beside the effectient delivery of protein into specific cells, the prevention of protein degradation in endosomal or lysosomal digestion is the critical issue in nano-scale protein delivery system. The cationic polymer polyethyleneimine (PEI) has been demonstrated possessing high pH-buffering capacity, providing the protection to biomolecules from acidic degradation, and promoting the release from endosome or lysosome. Nevertheless, the cytotoxicity of PEI exists by the increasing molecular weight. There is an urgent need for developing PEI associated nanocarrier system for clinical application with improved protein delivery efficiency and less the cytotoxicity. In this study, the multi-functional nanoparticle-based protein delivery system was developed for the applications of both protein drug delivery and medical imaging probe. With the surface modification of lower molecular weight PEI, the formulated cationic nanoparticles were evaluated for the intracellular protein delivery. The gelatin nanoparticles were prepared by modified desolvation method and grafted with 1.8 kD branched PEI by cross-linking the amino group of PEI and the carboxyl group of gelatin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). For the intracellular tracking application, the fluorescent molecules Rhodamine B isothiocyanate(RITC) were conjugated with gelatin nanoparticles (GR-PEI NPs) by EDC cross-linking. The chelator diethylene triamine pentaacetic acid (DTPA) was conjugated on the surface of gelatin-PEI nanoparticles for chelating gadolinium (Gd), the MRI T1-weighted imaging contrast agent. The average diameter of formulated RITC labeled gelatin-PEI nanoparticles was near 150nm, and zeta potential was about +60mV in neutral condition. The TEM and AFM images showed the spherical morphology of formulated nanoparticles and indicated nano-scale size correlated to the hydrodynamic size measurement. The GR-PEI NPs showed highly stability against the variation of temperature and pH value, and there was no significant variation between the cell viability of control cells and GR-PEI NP treated cells, indicating the low cytotoxicity of GR-PEI NPs. The data of flow cytometry and fluorescent microscopic images revealed that the cellular uptake of GR-PEI NPs was more efficient than that of GR NPs in several types of cells, suggesting the cell binding ability of cationic nanoparticles. Moreover, the outstanding protein loading capacity of GR-PEI NPs was obtained compared with negatively charged GR NPs. The intracellular and intra-tumor protein delivery efficiency of GR-PEI NPs was demonstrated by flow cytometry and fluorescent images of histological sections, indicating the effect protein transportation ability of GR-PEI NPs. For the multi-modalities of imaging application, the images from fluorescent microscope and confocal laser microscope showed the distribution of nanoparticles and the release profile of proteins, suggesting the intracellular tracking ability of GR-PEI NPs. The MRI phantom image demonstrated the T1-weighted contrast ability of GR-PEI NPs. The accumulation of Gd conjugated GR-PEI NPs in solid tumor was also evaluated by the MRI T1-weighted images. The above results indicate the function of image probe has been achieved. In summary, the cationic nanocarrier system which provides advantages for multifunctional tasks, including delivery of protein drugs and imaging probe for diagnostic applications, has been established by designing the gelatin nanoparticles with surface modification of polycationic PEI followed the conjugation of imaging agents. The characteristics of low cytotoxicity, outstanding protein loading ability, high efficiency of intracellular protein delivery, and imaging tracing ability demonstrate that the formulation designed in this study is a promising multifunctional vehicle for the theranostic applications.