奈米雙性幾丁聚醣載體之增效設計應用於癌症治療之研究

Abstract

非小細胞肺癌是全世界致死率高的癌症之一，現階段的非小細胞肺癌治療方式仍須配合化學藥物治療，然而使用目前的化學藥物治療除了要考量藥物毒性與副作用之外，持續使用同一種藥物容易遭遇到多重抗藥性的問題，反而大幅降低了醫學療效。因此，建構一個具有抑制多重抗藥性的藥物釋放系統是現今不容忽視的非小細胞肺癌治療議題。本研究以設計一可裝載雙藥並具有標靶效果的多功能膠體奈米粒子為主軸來建構一藥物釋放系統。 奈米粒子係由雙性幾丁聚醣以自組裝的方式裝載藥物形成一具有高包覆率、良好生物相容性及生物可降解性的藥物載體；並以自身的親水性與疏水性側鏈，在自組裝的過程中，利用分子間親疏水交互作用包覆/攜帶兩種不同物化性質及醫療功能之藥物分子.另外，在此奈米粒子上以化學修飾特定抗體，使奈米粒子具有標靶的功能，形成核殼結構，進而保護藥物活性。此外，這樣的結構設計，可以使兩種藥物以特定的比例濃度，在相同的時間內到達同一個癌細胞，更容易造成藥物的協同效應來毒殺癌細胞，藉此增加藥物療效以達成減低用藥劑量與副作用的目的。 經過化學修飾後的多功能奈米藥物釋放系統利用電子顯微鏡、動態光散射儀、核磁共振儀、高效液相色譜儀等設備，檢視其物化特性與微觀結構。另外，該奈米粒子在生物環境中 (in vitro) 的行為，包括: 表皮細胞生長因子受器的標靶能力、對多重抗藥性相關蛋白質的抑制力、細胞毒性、藥物的協同效應等，也在細胞培養實驗觀察驗證。 在本研究中，多功能奈米粒子在動物體內的抗癌功效與生物安全性皆在小鼠模組上驗證。根據A549異位腫瘤的抑制效率結果，明顯表現出標靶雙藥奈米粒子的ED50比純藥物具低於兩倍以上。此外，在急毒性試驗當中，也顯示標靶雙藥奈米粒子的生物安全性遠高過純藥物，並揭露其藥物動力特性與動物體分布情況。 根據本研究的結果，證實以雙性幾丁聚醣奈米粒子作為多功能雙藥標靶載體，不僅成功解決不同藥性的藥物包覆運輸的難題，提供一個可以讓兩種藥物產生協同效應的微環境，更具有良好的生物相容性與安全性。此奈米平台將在合併療法 (combination therapy) 當道的抗癌世代中，扮演重要的角色，也將開啟高分子奈米載體應用的新頁。

Non-small cell lung cancer (NSCLC) is one of most fatal cancers with less than 20% five-year survival rate under current clinical statistics, which is complicated by earlier and unpredictable advancement of metastasis, associated with multidrug resistance (MDR). Earlier stage of diagnosis of NSCLC has often inaccessible due to numerous limitations and these result in poor chemotherapeutic efficacy toward those cancer patients. Immunotherapy has recently received great attention in treating NSCLC by prolonging overall survival rate, however, exceptional treatment cost, immunogenic response, and less-to-negligible efficacy to some patients in clinical reports encourages a new therapeutic strategy to be explored for the coming new era of molecular medicine. In a number of clinical reports from ASCO 2016 and 2017, combination chemotherapy has been raised in clinic for a more powerful and efficacious anti-cancer treatment among the many anti-cancer weapons available nowadays. However, under clinical anti-cancer treatment, single dose of each free drug at different time point may give some degree of synergistic effect, but at a risk of additional side effect coupled with additional cost. Among the evolution of combination chemotherapy, synergistic co-loading of drugs in a single nanocarrier has been found to be more interesting in ensuring simultaneous delivery at the site of action where enhanced pharmacokinetic of each drug can be synchronized and giving highest synergistic efficacy. In recent decades, polymer-based nanoparticle is one of key technologies to advance our lives, especially on cancer treatment. The nanoparticles can be functionalized, including drug delivery, imaging, targeting and drug release control to meet different clinical requirements, with less systemic toxicity. However, most of these multifunctional nanoparticles experienced numerous stages of synthetic processes leading to poor drug encapsulation efficiency. It turns to be even worse to co-loading drugs with different physicochemical properties in a single nanocarrier and achieving optimal pharmacokinetic for each drug. Additionally, only few of them provide good biocompatibility and biodegradable property, which is the most important characteristics applied in biomedical field. Thus, establishing a high efficient multifunctionalized polymer-based dual-drug co-delivery nanosystem with excellent biosafety is the main purpose of this work. To reach this goal, this thesis is specifically designed with three focuses: 1. Design and characterize a novel amphiphilic chitosan (CHC)-based single-drug loading nanoparticle on anti-MDR in vitro and in vivo. 2. In vitro inhibition of cancer-stem like cells (CSCs) by dual-drug carrying CHC nanoparticle with synergistic effect. 3. Establishment of multifunctional dual-drug co-delivery CHC nanoparticle on anti-NSCLC xenograft in vivo with synergistic efficiency and biosafety. The first part of the thesis is the targeting controlled release core-shell nanocarriers with the potential to overcome MDR lung cancer, which were prepared based on demethoxycurcumin (DMC) loaded amphiphilic chitosan nanoparticles coated with an anti-EGFR antibody layer. Physico-chemical characterization confirmed the resulting nanocarriers to possess a surface layer of anti-EGFR antibody and an ultimate size suitable for circulating delivery. In vitro drug release revealed extended quasi-Fickian release from the nanocarriers, with the anti-EGFR layer further modulated the release profile. Cell culture experiments using normoxic and MDR hypoxic cells overexpressing EGFR confirmed improved DMC delivery for anti-EGFR coated nanocarriers and revealed that the DMC was delivered to the cytoplasmic region of the cells, forming nano-precipitates in lysosomes and endosomes. The effective endocytosis and targeting of the core-shell nanoparticles resulted in the nanocarriers achieving high-potent cytotoxicity against MDR cells. The therapeutic potential was further confirmed in an A549 xenograft lung tumor mouse model. Both in vitro and in vivo data suggest the anti-EGFR coated core-shell nanocarrier a highly promising vehicle to against hypoxic MDR cancers, especially for non-small cell lung cancer. In tumor tissue, not only cancer cells can against chemotherapy, but CSCs is much magligancy with MDR and renewing properties, which is emerging as a reason for failed treatments. One concept which addresses this root cause of treatment failure is the utilization of nanoparticles to simultaneously deliver dual drugs to cancer cells with synergistic performance, easy to envision-hard to achieve. It is challenging to simultaneously load drugs of highly different physicochemical properties into one nanoparticle, release kinetics may differ between drugs and general requirements for biomedical nanoparticles apply. Here self-assembled nanoparticles of amphiphilic CHC were shown to present nano-to-subnano environments enabling simultaneous loading of hydrophilic and hydrophobic drugs in different molecular domains. This was expanded into a dual-drug nano-delivery system to treat lung CSC. CHC nanoparticles were loaded/chemically modified with the anticancer drug cisplatin and the MDR-suppressing DMC, followed by biofunctionalization with CD133 antibody for enhanced uptake by lung CSC, all in a feasible one-pot preparation. The nanoparticles were especially characterized with drug loading/release behavior. Subsequently the cytotoxicity of single and dual drugs, free in solution or in nanoparticles, was evaluated against lung CSC at different doses. From the dose response at different concentrations the degree of synergy was determined through Chou-Talalay’s Plot. The biofunctionalized nanoparticles promoted synergistic effects between the drugs and were highly effective against MDR lung CSC. The efficacy and feasible one-pot preparation suggested preclinical studies using relevant disease models to be justified. Finally, we developed a protocol for synergistic co-delivery of DMC and the second-line anti-lung cancer drug gemcitabine (dFdC) using anti-EGFR-biofunctionalized, self-assembled, amphiphilic, CHC nanocarriers. The drugs were co-loaded over a range of controllable doses and the effect on in vitro viability of A549 NSCLC was investigated to determine the performance at different drug ratios. After this investigation and in vitro studies of physicochemical properties and drug release, the best performing drug ratio was brought forward to in vitro investigation of pharmacokinetics, biodistribution, histopathological analysis, safety, and efficacy against advanced NSCLC xenograft in a murine model. The results revealed excellent biosafety for the nano-formulated dFdC-DMC-CHC and excellent antitumor efficiency. At maximum synergy, the nanocarrier co-delivered drugs reduced largely the tumor volume and maintained it below the initial volume through the 28 days of observation, i.e. > 100% growth inhibition. The experimental outcomes demonstrate promising therapeutic potential in terms of biosafety and efficacy, suggesting that developments towards clinical translation are justified. In summary, a multifunctional amphiphilic chitosan-based nanoparticle with dual-drug carrying, specific targeting and biosafety properties was produced and applied on anti-NSCLC xenograft successfully with highly efficiency and low toxicity. CHC polymer encapsulated both hydrophilic and hydrophobic drugs with high concentration by self-assemble behavior in facile one-pot synthesis scheme. The core-shell structure protected the drugs activity during circulation in animals with less systemic side effect and targeted on cancerous tissue. CHC nanoparticles disintegrated and degraded in lysosome, and both drugs were released into cytoplasm leading best synergistic effect by anti-MDR and inducing apoptosis. The new generation CHC nanoparticle with all these elements is becoming a promising biomedical platform to treat the most lethal disease, i.e., magligant cancer, which is now forwarding clinical trial investigation from various nano-formulation aspects.