膠體幾丁聚醣藥物載體之設計與特性分析結合奈米療法於血管平滑肌細胞遷移相關疾病之研究

Abstract

於臨床治療心血管相關疾病，經常面臨一個複雜性的副作用，血管再阻塞，起因於受損傷血管內之平滑肌細胞增生之影響。即使常見之臨床治療方針例如腔內冠狀成形術(PTCA)，切除，或是植入心導管已被廣泛地採用，然而於治療後期的血管再狹窄之風險依舊很高。 因此採用此治療方針時，安全性評估相對也十分重要。基於這些原因，許多藥廠或研究學者投入大量精力欲研發出新型治療策略。因此，奈米技術之應用於開發心血管相關疾病，也許是一種具吸引力與前瞻性的一門科學研究。此篇論文主題主要專注於三個項目，分別是載藥平臺研究，抑制血管平滑肌細胞增生之藥物選擇以及塗布藥物之高分子探討。 於本論文第一部分之研究，以中草藥成分，厚朴酚與聚乙烯吡咯烷酮作用形成內核，外層以改質後之幾丁聚醣為殼層屏障，共同組裝成一核-殼結構之奈米粒子。殼層選用之雙性幾丁聚醣是過去由本實驗室研發所得，具有良好之生物相容性、亦被廣泛使用於各式包藥載體應用。此特殊劑型是由厚朴酚與聚乙烯吡咯烷酮形成內核加以利用雙性幾丁聚醣包覆，共同組裝形成厚朴酚-幾丁聚醣之奈米粒子。實驗主要評估此劑型之藥物釋放曲線與體外試驗控制性抑制平滑肌細胞遷移效果。結果顯示此劑型與未經修飾之原型藥物比較，具有緩慢釋放、傑出的細胞吞噬效果、良好的抑制平滑肌細遷移效果。此研究成功地研發出一具潛力之奈米粒子，同時提供奈米粒子於心血管細胞內之生物療效探討與治療策略。 薑黃抽出物由於其優秀的生物活性與安全性，不論於食品或藥學領域中已被大量使用。同時，早期研究已驗證薑黃抽出物具有多重作用活性，包括抗增生、抗遷移、抗氧化與抗發炎等作用。然而於臨床應用中遇到一棘手之問題，溶解度不佳。因此，同樣使用實驗室研發之具有良好之生物相容性之雙性幾丁聚醣當藥物載體，透過載體本身之特性自組裝形成藥物奈米粒子。選用之候選藥物-去甲氧基薑黃素為薑黃抽出物的其中一種成分，並且沒有任何體外研究試驗證實此藥物具有抗血管再狹窄之作用。綜合以上論述，以去甲氧基薑黃素當候選藥物，透過雙性幾丁聚醣自組裝之特性，架構組成另一種具潛力之奈米藥物劑型。本實驗評估此劑型與細胞間之交互作用與控制平滑肌細胞毒殺效果，劑型之基礎物化特性例如粒子形態、表面電位、包覆率、體外藥物釋放以及藥物於細胞內分布。結果顯示與未經修飾之原型藥物相比，此劑型具有良好的細胞吞噬效果以及增強細胞毒殺與抑制平滑肌細胞遷移效力。此實驗運用此奈米藥物劑型改善藥物治療效力，同時開啟一種具潛力之細胞內藥物輸送策略。 第三部分主要著重於以雙性幾丁聚醣塗佈於不銹鋼平板上，使用電沉積的簡單方式，研究表面奈米起伏變化對於平滑肌細胞與內皮細胞遷移與生長之影響。兩種不同的表面奈米起伏，以原子力顯微鏡分析之數據界定出尖端起伏與踭坦起伏。評估此兩種奈米起伏表面形態變化對於平滑肌細胞與內皮細胞之貼覆性、生長狀況以及細胞骨架之影響。此研究成功地證明奈米起伏表面可以容易地控制與改善治療性能。

In the clinical treatment of cardiovascular disease (CVD), a complicate physiological symptom has been reported, where the vessel occluded itself again, as a result of exaggerated proliferation of vascular smooth muscle cells (VSMCs). Although the common therapeutic strategies such as percutaneous transluminal coronary angioplasty (PTCA), atherectomy, and stenting have been largely utilized, the increasing risk of late thrombosis causing acute coronary diseases resulting from these therapeutic strategies is need for a comprehensive safety consideration. For these reasons, many pharmaceutical companies and researchers put extensive effort to develop a new therapeutic strategy. Nanotechnology is an attractive and prospective technology to providing a research proposal for the development of CVD treatment. Therefore, this study is focused on integrating therapeutic platform, antiproliferative agent, and colloidal chitosan-based carrier to synergize a potential strategy for the treatment of CVD The first part of this thesis (Chapter 4) is employed a core-shell nanoparticle with a structural arrangement of magnolol-polyvinylpyrrolidone (PVP) core and chitosan shell. The shell was virtually constructed using a new type of amphiphilic chitosan-based hydrogel, i.e., carboxymethyl-hexanoyl chitosan (CHC). The highly biocompatible amphiphilic chitosan, which was previously developed in this lab, was employed as a drug carrier. A magnolol-polyvinylpyrrolidone (PVP) core phase was prepared, followed encapsulating by an amphiphilic carboxymethyl-hexanoyl chitosan (CHC) shell to form a magnolol-loaded core-shell hydrogel nanoparticles (termed Magnolol-CHC nanoparticles). The resulting Magnolol-CHC nanoparticles were employed for evaluation of drug release and controlled cytotoxic inhibition of VSMCs migration in vitro. A sustained release of the magnolol from the nanoparticles was determined. The Magnolol-CHC nanoparticles exhibited outstanding cellular uptake efficiency and under a cytotoxic evaluation, an increased anti-proliferative effect and effective inhibition of VSMC migration as a result of efficient intracellular delivery of the encapsulated magnolol in comparison to free magnolol was achieved. We then envision a potential intracellular medication strategy with improved biological and therapeutic efficacy using the Magnolol-CHC nanoparticles illustrated in this work. Curcuminoids are recognized for their broad spectrum of biological activities and safety in foods and pharmaceuticals. Although previous reports suggested that curcuminoids are a class of effective antiproliferative, antimigratory, antioxidant, and anti-inflammatory agent; however, poor bioavailability has hampered the desired therapeutic use of curcuminoids in a number of clinical trials. To overcome those hurdles, a dual-ligand modification rendered the resulting modified chitosan (CHC) to be highly dissoluble in aqueous solution of neutral pH, biocompatible, and is capable of self-assembling to form well-defined nanocapsules upon encapsulating many water-insoluble substances in aqueous solutions. In the second parts (Chapter 5), a highly potential drug, demethoxycurcumin which is one of the most potent derivatives of the curcuminoids, was employed.Unfortunately, there has no relevant reports on investigating demethoxycurcumin as an antirestenotic agent in vitro. Combining these reasons, a potential formulation via a low-dose sustained elution of demethoxycurcumin (DMC), through a self-assembled amphiphilic carbomethyl-hexanol chitosan (CHC) nanomatrix was constructed. Manipulating the cellular internalization and controlled cytotoxic effect of DMC-CHC nanoparticles over the VSMCs was elucidated. The DMC-CHC nanoparticles, which were systematically characterized in terms of structural morphology, surface potential, encapsulation efficiency, and DMC nanocrystallite distribution, exhibited rapid cellular uptake efficiency and considerably improved cytotoxic potency by 2.8 times compared to the free DMC. Under a cytotoxic evaluation, an improved antiproliferative effect and effective inhibition of VSMCs migration as a result of highly-efficient intracellular delivery of the encapsulated DMC in comparison to free DMC was achieved, which also confirmed with a subsequent protein analysis. Cellular drug release and distribution of DMC after internalization into VSMCs was experimentally determined. This work may open a potential intracellular medicinal strategy with improved biological and therapeutic efficacy using the DMC-CHC nanoparticles illustrated in this work. After this potential formulation being extensively explored, a facile nano-topographical control over a stainless steel surface via an electrophoretic deposition of colloidal amphiphilic chitosan for preferential growth, proliferation, or migration of vascular smooth muscle cells (VSMCs) and endothelial cells (HUVECs) was evaluated (Chapter 6). Atomic force microscopy (AFM) revealed that the colloidal surface illustrated a deposition time-dependent nano-topographical evolution, wherein two different nano-topographic textures indexed by “Kurtosis” (Rkur) value were easily designed, which hereinafter, termed as “sharp” (i.e., high peak-to-valley texture) surface, CHC-1 and “flat” (i.e., low peak-to-valley texture) surface, CHC-5. Cellular behavior of VSMCs and HUVECs, on both surfaces demonstrated topographically-dependent morphogenesis, adherent responses, and biochemical properties in comparison with bare stainless steel. This work highlighted a promising development of such a nano-topographic biofunctionalized surface upon which a cell-specific therapeutic strategy can be easily manipulated with improved therapeutic performance.