雙胜肽修飾之奈米載體應用於抗藥性小細胞肺癌的藥物治療

Abstract

多年來，惡性腫瘤位居台灣十大死因之首，根據2016年衛生福利部統計，顯示氣管、支氣管和肺癌造成的死亡率最高。其中，小細胞肺癌（small cell lung carcinoma, SCLC）約佔肺癌的11~20%，為最具侵略性的肺癌，且有高復發率之特性以及較差的病人預後。目前臨床上的首選化療複方用藥為etoposide（ETP）及platinum類藥物。雖然病患起初對首選藥物的反應性很好；然而，大部分病患會於一年之內，對化療藥物產生抗藥性，而使腫瘤復發，因此目前針對具有抗藥性之小細胞肺癌，仍亟需發展新的治療策略。 本篇研究以聚乳酸−甘醇酸（poly(lactide-co-glycolide), PLGA）作為藥物載體的骨架，於上接枝聚乙二醇二胺（poly(ethylene glycol) bis(amine), PEG-diamine），然後將對小細胞肺癌細胞具標靶能力的胜肽配體antagonist G（AG）及有助於載體穿膜的TAT胜肽接枝於PLGA-PEG上，並分析此兩種胜肽修飾對於小細胞肺癌治療之助益。在本研究中使用兩種藥物：ETP和siPIK3CA，ETP用於抑制第二型拓樸異構酶（DNA topoisomerase II），而siPIK3CA可以專一性阻斷PIK3CA基因表現及PI3K p110-α蛋白生成，藉此抑制腫瘤細胞的增生。另外，從母群H69小細胞肺癌細胞中，篩選出CD133(+)細胞，作為具ETP抗藥性之癌幹細胞模型。 本實驗使用的奈米顆粒，皆是以溶媒揮發法製備而得，包覆ETP及siPIK3CA奈米顆粒之粒徑大小，分別落於201.0±1.9~206.5±0.7 nm及155.3±12.4~169.2±11.2 nm；表面電荷皆為正電，分別為36.3±2.4~39.6±1.0 mV及31.0±2.7~34.1±1.4 mV；而藥品包覆率可達61.1±6.0%及58.6±5.0%以上。於4˚Ｃ水中及冷凍乾燥後回溶於水之4週安定性試驗中，顯示奈米顆粒之粒徑皆可維持，並無明顯凝集現象。此外，在體外釋放試驗中，可知於pH 4.0環境下的奈米載體釋放藥物速度，較pH 7.4環境下為快。 除了對胜肽修飾的奈米顆粒進行物化性質分析之外，亦利用流式細胞儀針對G-protein coupled receptor（GPCR）高表現的母群、CD133(+) H69小細胞肺癌細胞以及GPCR低表現的A549非小細胞肺癌細胞，進行兩小時的細胞吞噬實驗。實驗結果顯示，於母群及CD133(+) H69細胞中，具AG、TAT或雙胜肽修飾之奈米顆粒皆比未修飾之奈米載體，具有更佳的細胞吞噬能力（母群細胞：85.7±2.1%、76.3±4.1%、78.3±0.3% vs. 65.3±2.7%；CD133(+)細胞：46.5±2.2%、56.7±2.2%、51.7±2.3% vs. 32.5±0.8%）；於A549細胞中，經AG修飾與未修飾之奈米載體的細胞吞噬能力，並無顯著差異（69.4±2.2% vs. 73.7±1.3%），可知AG具有GPCR標的能力；而經TAT修飾之奈米載體，雖可以增加載體進入細胞效率，但TAT並無細胞選擇性。 此外，本研究分析單獨給予ETP或siPIK3CA之奈米載體以及合併投予兩種藥物之奈米載體，對於H69細胞造成的毒殺性。從實驗結果可得知，在不具抗藥性之母群H69細胞，經AG修飾之奈米顆粒有最佳的細胞毒殺性（ETP奈米載體：IC50為19.8±0.8 μg/mL；siPIK3CA奈米載體：IC50為152.3±8.3 ng/mL），而TAT的效用較不顯著；相對的，於具抗藥性之CD133(+) H69細胞中，TAT顯現其可以克服多重抗藥性的優勢，結果發現雙胜肽AG及TAT修飾之奈米顆粒，具有最大細胞毒殺能力（ETP奈米載體：IC50為15.0±2.9 μg/mL；siPIK3CA奈米載體：IC50為169.7±17.1 ng/mL）。 於合併給藥之結果中，顯示加上siPIK3CA之輔佐，合併給藥比單獨給予相同ETP濃度之包覆ETP奈米顆粒，有著更高的細胞毒性。對於母群H69細胞而言，以經AG修飾之奈米劑型共同給藥，有較高的細胞毒性；而對於CD133(+) H69細胞，則以雙胜肽修飾之奈米劑型最佳，其次為AG修飾之奈米劑型。由上述結果可知，合併投予包覆ETP及siPIK3CA之雙胜肽修飾奈米顆粒，將有潛力作為針對抗藥性小細胞肺癌的治療方法。

Cancer is ranked first in the top ten causes of death in Taiwan. According to statistics from the Ministry of Health and Welfare, it was revealed tracheal, bronchial and lung cancers resulted in the highest mortality. Small cell lung carcinoma (SCLC) is a highly aggressive form of malignancy with rapid recurrence and poor prognosis, accounting for 11~20% of all lung cancers. Nowadays first-line combination chemotherapy for SCLC is etoposide (ETP) and platinum-based. Patients respond well to the combination chemotherapy at the beginning, but tumors relapse in less than one year because of drug resistance. Therefore, novel therapeutic strategies against drug-resistant SCLC are urgently required. In our study, poly(lactide-co-glycolide) (PLGA) acted as the main polymer and was conjugated with poly(ethylene glycol) bis(amine) (PEG-diamine). In addition, we used SCLC targeting ligand (antagonist G (AG) peptide) and cell penetrating peptide TAT to modify PLGA-PEG. We produced dual peptide-modified PLGA-PEG nanoparticles (NPs) consisting of ETP and PIK3CA small interfering RNA (siPIK3CA). The ETP was applied to deactivate DNA topoisomerase II in nuclei. The siPIK3CA can selectively inhibit expression of PIK3CA gene, encoding phosphoinositide 3-kinase (PI3K) p110-α protein, to block SCLC cell proliferation. In our experiment, CD133(+) H69 SCLC cells were sorted from parent H69 cells, serving as ETP-resistant SCLC cell model. All NPs were prepared with solvent evaporation method. The particle sizes of ETP and siRNA loaded NPs were 201.0±1.9~206.5±0.7 nm and 155.3±12.4~169.2±11.2 nm. The zeta potentials of ETP and siRNA loaded NPs were 36.3±2.4~39.6±1.0 mV and 31±2.7~34.1±1.4 mV. The encapsulation efficiencies (EE) were above 61.1±6.0% and 58.6±5.0%, respectively. These formulations exhibited good stability at 4˚C and after lyophilized for 28 days. Furthermore, in the in vitro release study, both drugs released faster from NPs in pH 4.0 than pH 7.4. Cellular uptake study for 2 hours was applied in parent and CD133(+) H69 cells with G-protein coupled receptor (GPCR) high expression and A549 cells with GPCR low expression by flow cytometry. It was suggested AG or TAT and dual peptide-modified NPs had better cellular uptake ability than unmodified NPs in H69 cells (parent cells: 85.7±2.1%, 76.3±4.1%, 78.3±0.3% vs. 65.3±2.7% ; CD133(+) cells: 46.5±2.2%, 56.7±2.2%, 51.7±2.3% vs. 32.5±0.8%). However, there was no significance of cellular uptake ability between AG and unmodified NPs in A549 cells (69.4±2.2% vs. 73.7±1.3%). Therefore, it was indicated that AG could be the GPCR targeting ligand. Additionally, TAT-modified NPs improved cellular uptake, no matter which kinds of cells. It was implied TAT had no cellular selectivity. In addition, we also analyzed cytotoxicity of ETP and siRNA loaded NPs individually and in combination treatment. The results showed drug loaded PLGA-PEG-AG NPs were better than PLGA-PEG NPs on parent H69 cells (IC50 of AG-NPs-ETP = 19.8±0.8 μg/mL；IC50 of AG-NPs-siRNA = 152.3±8.3 ng/mL), while dual peptide-modified NPs were the best formulation in CD133(+) H69 cells (IC50 of A/T-NPs-ETP = 15.0±2.9 μg/mL ; IC50 of A/T-NPs-siRNA = 169.7±17.1 ng/mL). TAT modification could overcome multidrug resistance, as shown in drug-resistant CD133(+) H69 cells, but not in drug-sensitive parent H69 cells. Furthermore, it was revealed combination treatments with the aid of siPIK3CA had more cytotoxicity than ETP loaded NPs alone. From the results form combination treatments, AG-NPs were better formulations in parent H69 cells, while A/T-NPs showed the best cytotoxicity in CD133(+) H69 cells. In conclusion, co-delivery of ETP and siPIK3CA with dual peptide-modified NPs could be a potential approach for improving SCLC therapy in the future.