磁性奈米微胞載體結合化療藥物(SN-38)與血管內皮生長因子小干擾RNA(VEGF siRNA)作為大腸癌顯影及治療之評估

Abstract

近來磁性奈米載體在生醫方面有許多應用，像是藥物傳輸、磁振造影MRI顯影以及磁熱治療，而常見的磁性奈米可經由修飾或其他高分子材料包覆並結合標靶及化療藥物達到同時顯影和治療的效果。而大腸癌是常見的癌症之一，大部分大腸癌的治療方式以外科治療為主，但常合併化學治療以達到更好的治療效果，而在化學治療方面則會使用化療藥物和標靶藥物合併治療。大腸癌在臨床上使用的第一線藥物為CPT-11，而SN-38則是為CPT-11的活性代謝物，其活性優於CPT-11的100-1000倍，另外除了化療藥物在臨床上還會配合標靶藥物Avastin (Bevacizumab)，它是對抗血管內皮生長因子VEGF的單株抗體可有效抑制多種癌症細胞株的生長，而且和化學治療合用有加乘效果，Avastin 目前也已經獲得美國FDA核准可以合併化療使用於轉移性大腸癌第一線治療的適應症，但Avastin和化療藥物需分開給藥，故在使用上較為不方便。 為了有更良好的大腸癌治療，高分子奈米藥物載體被廣泛運用在藥物傳輸系統來治療癌症，且基因治療與標靶治療也於近年來有許多的研究發展，且奈米載體可藉由增強通透性與延遲效應(EPR effects)進而增加藥物在腫瘤的累積，此外核糖核酸干擾技術(RNAi)抑制特定基因之表現，對於惡性腫瘤有可以抑制其增長的成果。因此，本篇碩士論文將利用帶正電之高分子pDMAEMA-b-PCL並混和mPEG-PCL形成穩定的奈米微胞，作為磁性粒子及化療藥物SN-38的載體，並利用奈米微胞帶正電的特性攜帶具負電之VEGF siRNA，增加其治療效果。在物理性質方面，粒徑大小約為200-250nm，且化療藥物SN-38包覆率也可達成六成以上，而在顯影方面，此載體是具有磁振造影顯影功能，另外在細胞實驗中，細胞毒性與轉染效率皆具有對人類大腸癌細胞(LS174T)有治療能力，因此希望能將這載體應用於大腸癌治療，使其具有同時具有顯影和治療的功能。

Magnetic nanoparticles actually play important roles as negative MRI contrast agent in T2-weighted imaging, thus several magnetic nanoparticles formulations have been approved for uses in image and drug delivery simultaneously. For efficient colon cancer therapy, the combine agents were proven activity in clinical applications such as irinotecan and avastin. SN-38 (7-Ethyl-10-Hydroxycamptothecin) derived from irinotecan hydrochloride (CPT-11) which had 100-1000 times more active than irinotecan; avastin is monoclonal antibody that could inhibit vascular endothelial growth factor (VEGF). The combination of SN-38 and VEGF siRNA may enhance the antitumor effect, thus magnetic micelles as VEGF siRNA and SN-38 carriers have been developed for magnetic resonance imaging and colon cancer therapy. Small interfering RNA (siRNA) is a double-stranded RNA with short base pairs that can be unstable and degraded by enzyme in environments full of serum or plasma proteins, low transfection efficiency may even happened. To solve this problem, siRNA can be conjugated with PEG (siRNA-PEG) to improve its stability and to prolong its blood circulation time after intravenous administration. PEG can also protect nanoparticles from fast blood stream removal by reticulo-endothelial system (RES). In this study, PDMA-b-PCL mixed mPEG-PCL micelles with a positive charge were synthesized as USPIO, SN-38, and VEGF siRNA nano-carrier. The magnetic micelles loaded with SN38 were then labeled with siRNA-PEG via charge neutralization and used Enhanced Permeability and Retention (EPR) effect to accumulate in tumor. Characterization of micelles, the size and zeta-potential of magnetic micelles loaded SN-38 with siRNA-PEG binding were approximately 222.1 nm and 21.2 mV, although PDMA-b-PCL polymer had high positive charge but RBC hemolysis and protein aggregations would not occur. In addition, with the existence of protonated amino groups, PDMA-b-PCL mixed mPEG-PCL micelles would cause pH-dependent endosomal disruption. The MTT cytotoxicity assay demonstrated that micelles encapsulated with USPIO and SN-38 would not affect the cytotoxicity of SN-38 compared with free drugs. In vivo study, the PDMA-b-PCL mixed with mPEG-PCL micelles were used due to its stability and lower zeta-potential, but it’s not influenced the siRNA-PEG binding efficiently. This results obtained show that magnetic micelles could be developed as VEGF siRNA and SN-38 carrier and innovated micelles have both imaging and therapeutic capabilities for colon cancer.