應用於腫瘤治療與克服多重抗藥性之口服脂質藥物傳輸系統設計與特性研究

Abstract

多重抗藥性被認為是導致癌症化學治療失敗最主要的原因，因為它可以讓腫瘤細胞對多種結構與功能相異的化療藥物同時產生排斥性。近十年來有許多研究利用奈米科技開發出各式具有抑制多重抗藥性能力的藥物載體，這些載體相對於傳統藥物劑型還顯示出更優越的特性，例如：結合功能性材料、更好的療效與較低的副作用。但是離理想的藥物載體仍然有許多值得努力的地方，像是較低的親水性藥物包覆率、過高的漏藥率與缺乏優秀的標靶專一性。為了改善這些問題，本論文結合有機材料與奈米科技，設計與製備具有克服多重抗藥性的奈米載體。第一部分我們將雙性高分子與三酸甘油酯奈米粒子結合，利用聚醣高分子與磷脂質形成的反式微胞包覆親水性藥物阿黴素，接著外部再以三酸甘油酯殼層包覆反式微胞以賦予此奈米藥物載體良好的穩定性與克服P-glycoprotein調控之多重抗藥性的能力。結果顯示聚醣高分子的使用提升了載體包覆率2.75倍，並大幅降低漏藥率。在細胞內藥物可先藉由載體經胞吞作用帶入溶酶体後，載體再於酵素作用下分解釋放而毒殺癌細胞，因而順利避開P-glycoprotein的作用。 對癌症初期病患來說口服給藥是最經濟且可兼顧生活素質的治療方式。但是P-glycoprotein不僅僅存在於腫瘤組織的癌細胞上，還過度表現於腸道上皮細胞的表面以排除外來病原體，也限制了許多抗癌藥物的口服療效。因此第二部分裡我們應用脂質載體可克服P-glycoprotein的能力來提升阿黴素的生體利用率。載體的生化特性與在腸胃道吸收的途徑都經由體外細胞模型與動物實驗的測試詳細了解。結果發現載體在腸胃道主要經由小腸內的培氏斑以胞吞作用吸收進入淋巴系統，與阿黴素以自由擴散經上皮細胞吸收的途徑相異，而且經此途徑可提升其生體利用率達八倍，且可降低對小腸組織的傷害。為了進一步提升載體的生體利用率並賦予其對腫瘤的標靶特異性，在第三部份載體結合了多階段連續標靶策略，也就是利用多種配體連續標靶藥物傳送途徑上的多種細胞，如：(1) RGD胜肽可標靶培氏斑的M細胞標靶提升吸收率；(2) LyP-1胜肽對表面過度表現p32蛋白的腫瘤細胞與腫瘤淋巴細胞具有特異性的標靶能力，對於難以治療的高轉移型腫瘤具有良好的治療效果。此外我們還發現在多階段連續標靶研究中，不同種類標靶物質的濃度比例可以決定載體的標靶與治療效率。這個結果發現若控制多種標靶物質的比例達到最佳化後可以獲取最佳療效與較低的副作用。 近年來單藥物療法在臨床癌症治療上常引發一些問題，例如腫瘤的抗藥性抑制藥物累積量與高藥物劑量引起的副作用，因此近年來新興趨勢轉向研究合併治療法，也就是同時以兩種以上藥物來治療以產生協同治療效應。為了讓載體能同時包覆兩種不同特性藥物，在第四部份我們將載體設計成中空結構，利用三酸甘油酯外殼層與中間水相內核分別包覆疏水性藥物紫杉醇與親水性藥物阿黴素。有鑑於過去合併治療法常無法調控多種藥物到達作用處的時間與濃度，此載體可藉由滲透壓改變外殼層厚度與水相內核體積，進而調整包覆的親疏水性藥物比例，而且三酸甘油酯外殼層厚度可影響其酵素分解速率，因而有不同的藥物釋放曲線。此外量子點在本篇有兩種用途：(1)與DOX結合作為螢光共振能量轉移指示劑，藉由螢光訊號的改變可傳遞出即時的藥物釋放訊號。(2)作為氧化治療劑，誘發癌細胞內部的氧化應力來殺死癌細胞。在癌細胞毒性測試中證實，化療與氧化治療的合併治療法可產生更強的協同效應抑制癌細胞生長。未來期望能結合多功能材料與標靶物質成為下一世代同時具備即時診斷與治療的新型藥物載體。

Multidrug resistance (MDR) is regarded as being responsible for failure in over 90% of chemotherapy because it enables tumor cells to resist to structurally and mechanistically unrelated chemotherapeutic agents. In the past decades, numerous studies have developed various drug carriers equipped with ability of inhibiting MDR, whereby superior features, including multiple functionalities, controllable release behavior and lower systematic toxicity, are also included. However, many challenges, such as poor encapsulation efficiency of hydrophilic drugs, high drug leakage and lack of specific targeting, in designing optimal drug carriers. To address these problems, a MDR-conquerable platform combined polymer micelle and lipid nanoparticle (termed D-PL/TG NPs) was newly designed and characterization of this formulation was investigated in part I. Doxorubicin, encapsulated in the polysaccharide-lecithin reverse micelle, was chosen as a model drug because its potency is seriously undermined by MDR. The D-PL/TG NPs could be internalized into cancer cells via clathrin-mediated endocytic pathway and release doxorubicin within the lysosomes, therefore bypassing the exclusion of P-gp. Moreover, by virtue of the incorporation of polysaccharide (amphiphilic carboxymethyl-hexanoyl chitosan), encapsulation efficiency and encapsulation stability of the nanocarrier was significantly improved. Oral administration has been recognized as the most convenient and safest modality in treating chronic diseases and early stage of malignancy. However, the efflux pump, P-glycoprotein, also exists in intestinal epithelial cell to resist exogenous pathogen, resulting in limited bioavailability of chemotherapeutic drugs. To evaluate the potential of the D-PL/TG NPs in oral administration, the physicochemical features and transport mechanism of the developed nancarrier were investigated using in vitro (Caco-2 monolayers) and in vivo animal model (Balb/c mice) in part II. In the animal study, the results of intestinal absorption assay suggest that the D-PL/TG NPs were preferentially absorbed through the specialized membranous epithelial cells (M cells) of Peyer's patches, by which the absorbed nanoparticles could enter into blood circulation via lymphatic system and bypass first pass metabolism. Consequently, it resulted in a significant improvement in bioavailability of the D-PL/TG NPs, which was 8-fold higher than that of free DOX. To further improve bioavailability of the nanocarrier and endow it with specific targeting to tumors, a multistage continuous targeting strategy was incorporated by conjugating with two targeting peptides: (i) RGD peptide for targeting to β1 integrin of M cells (ii) LyP-1 peptide for targeting to p32 receptor of MDA-MB-231 cells. In vitro permeability in human follicle-associated epithelium model and cytotoxicity against MDA-MB-231 cells indicated that targeting efficacy became better with the increased concentration of the targeting peptides. Moreover, the results of in vivo biodistribution and therapeutic efficiency reveal that the molar ratio of the peptides could determine the targeting antitumor efficacy of the nanocarrier. Single chemotherapeutic treatment was limited by biological and physicochemical hurdles in clinic, such as low accessibility to tumor tissues and multi-drug resistance phenomenon. Therefore, emerging trends in developing combination therapy, to co-administrate two or more pharmacologically active agents simultaneously or a combination of different therapeutic modalities, have become more exuberant. In the part IV, a hollow structure nanocarrier has been synthesized to simultaneously encapsulate multiple drugs with different properties: hydrophobic drug (paclitaxel) and hydrophilic drug (doxorubicin) were encapsulated in the triglyceride shell and aqueous phase core, respectively. Moreover, drug-loaded amount and sequential release of the nanocarrier can be controlled by tailoring the thickness of triglyceride shell and volume of aqueous phase core using induced osmotic pressure. QD not only acted as a Forster resonance energy transfer indicator for real-time monitoring executive stage of the carrier by coupling with doxorubicin but also induced intracellular oxidative stress to kill cancer cells. The results indicate that the performance of chemotherapeutic drugs (DOX and PTX) functioned with oxidative stress against MCF-7/ADR cells synergistically, leading to better therapeutic efficiency. The versatile delivery platform developed in this thesis may offer a potential avenue for cancer treatment by collaborating with chemotherapeutic therapy, oxidation therapy and real-time imaging, and promise a great therapeutic efficacy in clinical practice after further improvement.