開發同時具有化學/光熱治療功能之複合型奈米 藥物載體作為癌症治療平台

Abstract

近年來，治療癌症的方法日新月異，其中又以設計、合成奈米載體結合多種治療藥物為一大熱門領域。本研究旨於開發具有多功能之藥物載體，並將靜電吸附力及其他物理作用力應用於奈米藥物載體的製備和藥物的控制與釋放。首先，將聚乙烯二胺樹枝狀分子(PAMAM Dendrimer)交聯成表面帶正電的奈米粒子(PAMAM dendrimer nanocluster, DNC)，當作載體模板(template)。將帶負電的光敏感藥物靛氰綠(Indocyanine green, ICG)、具有酸鹼應答特性的聚丙烯酸(polyacrylic acid, PAA)、帶正電的化療藥物艾黴素(Doxorubicin, DOX)裝載於DNC上，形成初步的載體(I:D NPs)。再來，藉由共價鍵結將聚乙二醇(polyethylene glycol, PEG)及抗HER2蛋白的抗體(anti-HER2 antibody, HER2ab)固定於I:D NPs上，用以提高生物相容性及標靶專一性，形成最終的載體(I:D-P/H NPs)。此奈米藥物載體(I:D-P/H NPs)具有高載藥量的特性，其ICG和DOX的藥物承載量(drug loading content, DLC)分別為54.48±1.03 %和19.13±0.44 %，藥物承載效率(encapsulation efficiency, EE)分別為 81.92±3.36 % 和 82.11±0.02 %。當環境酸鹼值從pH 7.4降為pH 5.0時，會使藥物與載體之間的靜電吸附力下降，因此裝載的藥物會被釋出。在近紅外雷射光(808 nm)的照射下，會造成ICG表面不可逆的電荷性質改變，因此會大幅促進I:D-P/H NPs的釋藥量。利用流式細胞儀(flow cytometry)可得知I:D-P/H NPs對於高HER2蛋白質表現量的細胞株(SKOV3)有專一標靶特性。此裝載藥物的載體會被運送至酸性胞器進行藥物釋放，進而產生細胞毒性。當細胞在近紅外雷射光的照射下，I:D-P/H NPs會產生光熱效應(photothermal effect)，藉由高溫毒殺細胞，以及提升藥物釋放量，增強細胞毒性。綜合以上，我們合成出具有新穎性、標靶特性、高載藥量且可同時進行光熱治療及化學治療的奈米藥物載體。未來臨床研究方面，由於此具有高載藥量特性的奈米載體能夠克服傳統上直接將小分子藥物送入體內造成的許多缺點，因此在治療癌症上具有更廣泛的應用潛力。

An advanced design of nanoparticle formulation as a potential combined therapy for cancer treatment has gained tremendous attentions. In this study, electrostatic/physical interaction was applied for nanoparticle formulation. The as-prepared cross-linked PAMAM dendrimer nanocluster (DNC) served as a central template for the nanoparticle formulation. A considerable amount of Indocyanine green (ICG, a negatively charged photothermal agent), polyacrylic acid (PAA, a pH-responsive polymer), and Doxorubicin (DOX, a positively charged chemo-drug) were adsorbed sequentially to form the multi-functional nanoparticle. The surface of ICG:DOX nanoparticles (I:D NPs) was further modified with anti-HER2 antibody (HER2ab) and polyethylene glycol (PEG) through covalent linkage. The resulting nanoparticle complex (I:D-P/H NPs) exhibited high drug loading capacity. The drug loading content (DLC) of ICG and DOX was 54.48±1.03 % and 19.13±0.44 % (w/w) with the corresponding encapsulation efficiency (EE) of 81.92±3.36 % and 82.11±0.02 %, respectively. Encapsulated drug can be released when the environmental pH changed from 7.4 to 5. The release of DOX (pH-sensitive drug) was largely improved by NIR (808 nm) laser irradiation. Flow cytometry analysis showed that I:D-P/H NPs are specifically targeting to HER2-positive cell lines (e.g. SKOV3). The I:D-P/H NPs are expected to be trafficked to acid organelles and further releasing DOX to kill the cell. The cytotoxicity assay revealed that once the treated cells were irradiated with NIR laser, the therapeutic efficacy was substantially improved, presumably due to the enhancement of drug release, at least partially, resulting from the photothermal effect of ICG. In conclusion, a novel and high drug-loading agent that can specifically target to HER2-positive cell was prepared. This multi-functional nano-complex is potentially useful for a wild-range clinical study.