- 學位論文
創新標靶奈米脂質包覆氯化鉑技術與光動力組合治療在胰臟癌治療之研究
Novel targeted delivery of Lipid-Platinum-Chloride nanoparticles combined with photodynamic therapy applied in pancreatic cancer
摘要
胰臟癌是一個高度惡性的癌症，約90%的病人無法以手術根除治療，大多以化學治療為主，整體而言，五年的存活率低於5%。目前順鉑已是常用於治療惡性腫瘤的化療藥物，但其毒性高，造成副作用相對較高。尤其是腎毒性及神經毒性，順鉑 (cisplatin) 在常溫下難溶於水，導致順鉑在臨床用藥劑量及效果有限。為了使其能溶於水中並發揮出最大效用，使用化學合成的方式，改變順鉑的化性，再利用微脂體包覆，將其成為奈米粒子，再結合標靶治療，標靶物質選用茴香酰胺，平均粒徑為21.6 ± 1.0 nm，膜位電荷為31.0 ± 1.1 mV，藥載量為71.0 ± 10.3% wt，因茴香酰胺可與癌細胞膜表面的sigma 受器結合，能將藥物準確送達至腫瘤位置產生作用並降低原有順鉑之毒性，所以能提高治療劑量。這個方法稱作Lipid-Platinum-Chloride nanoparticles。LPC奈米粒子 這項技術能克服順鉑在藥物傳遞量較低的問題，且適用於難溶解於水的疏水性藥物。光動力療法，是一種治療癌症的新方式，他的原理是先以光感物質標定腫瘤細胞，等光感物質附著在癌組織細胞上，再以特定波長的光照射癌組織，當光與光感物質發生光化學作用，使光感物質產生細胞毒性，進而殺死癌細胞。本研究以光動力結合LPC奈米粒子治療胰臟癌惡性腫瘤，選用人類胰臟癌細胞PANC-1、MIAPaCa-2，分別於體外及動物進行研究探討。於體外實驗以LPC奈米粒子及cisplatin比較細胞毒殺效益，我們發現在濃度6、12及18 uM 的LPC奈米粒子，於MIAPaCa-2可提高60%癌細胞毒殺率 (p<0.01)；在PANC-1則無顯著差異 (p=0.53)。進一步動物實驗則分為5組為PBS、CDDP、LPC、PDT+CDDP、PDT+LPC治療組 (n=5) 治療週期為22天，於每週的第7天以尾靜脈方式給藥，共給藥3次，劑量為3 mg Pt/kg。結合光動力治療組，則是於第1天施以光動力治療，第2天注射CDDP或LPC，PDT+CDDP組在治療週期同上，結果發現唯有PDT+LPC組，只須在光動力治療後注射1次LPC奈米粒子，於PANC-1腫瘤生長率下降71.5 %；MIAPaCa-2下降71.3 %，能有效抑制腫瘤生長。PANC-1 五組動物之腫瘤生長率與PBS對照組相比，PDT+LPC組腫瘤生長率下降71.5 % (p<0.01)、LPC組下降63 % (p<0.01)、PDT+CDDP 組下降45 % (p<0.01)、CDDP組下降 30.8 % (p<0.01)；MIAPaCa-2 五組動物之腫瘤生長率與PBS對照組相比，PDT+LPC組腫瘤生長率下降71.3 % (p<0.01)、 LPC組下降54.2 % (p<0.01)、PDT+CDDP組下降45.5 % (p<0.01)、CDDP組下降16.5 % (p<0.01)，經統計分析後(p<0.01)具有顯著差異，因LPC奈米粒子為小分子藥物且結合標靶及光動力治療下，毒殺腫瘤細胞效果增加，能有效抑制腫瘤生長，未來運用在臨床治療上具有正面的發展性。
並列摘要
Cisplatin (CDDP) has been commonly used as a chemotherapeutic drug and limited due to high toxicity as well as the side effects. The CDDP has poor solubility in water and to take advantage of this a new method was performed in this study to load CDDP in nanoparticles via reverse microemulsion technique and termed as Lipid-Pt-Cl nanoparticles (LPC NPs). LPC NPs were enhance with anisamide to target over-expressing sigma receptor tumor cells. Western blot analysis confirmed that sigma receptor were highly expressed by PANC-1 and MIAPaCa-2 cells. The particle size measured was 21.6 ± 1.5 nm with zeta potential of 31.0 ± 1.1 mV and the drug loading capacity of 71.0 ± 10.3% wt. In vitro study in PANC-1 & MIAPaCa-2 cells showed that the cell viability decreased with CDDP encapsulated in LPC NPs which has a lower dosage as compared with free CDDP. This result indicating LPC NPs exhibit the anticancer efficacy of cisplatin to a lower dose than using free CDDP alone. In vivo study, we used BALB/c Nude mice to create animal model distributed as 5 groups (n=5) such as: PBS, CDDP, LPC, PDT+CDDP and PDT+LPC at a dose of 3mg Pt/kg for CDDP and LPC. The photosensitizer used was photosan at a dose of 2mg/kg for photodynamic therapy (PDT). All of the treatments observed for 23 days. The results showed LPC can inhibit tumor growth for both of cell lines. PANC-1 tumor growth rate compared with control PBS group: PDT+LPC group decrease 71.5 % (p<0.01), LPC group decrease 63 % (p<0.01), PDT+CDDP group decrease 45 % (p<0.01), CDDP group decrease 30.8 % (p<0.01); MIAPaCa-2 tumor growth rate compared with control PBS group: PDT+LPC group decrease 71.3 % (p<0.01), LPC group decrease 54.2 % (p<0.01), PDT+CDDP group decrease 45.5 % (p<0.01), CDDP group decrease 16.5 % (p<0.01). Despite inhibit the tumor growth with LPC alone, when it combined with PDT a better result found as the tumor size significantly decreased. In addition, the LPC alone need injected 3 times (0, 7, 14 days), but combined treatment just one times. All results were analysed with Sigma Plot software with significant value (p<0.01).Therefore, LPC have a potential as enhancing anticancer drug for chemotherapy. Because it’s small size, high drug loading and was enhanced with target ligand of anisamide. Importantly in combination with PDT the therapeutic effect have shown to be more effective in treating pancreatic cancer, PANC-1 and MIAPaCa-2 cells.
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