可注射式雙性幾丁聚醣奈米凝膠之設計與特性分析及其生醫應用

Abstract

在定點長時效藥物釋放系統中，特別是可注射式攜藥凝膠系統，在全球已經引起了學界及業界各人士的注意。然而，在凝膠藥物釋放系統中，經常面臨一個材料上的問題，釋藥初期的爆發性釋放(burst release)，此現象限制了凝膠系統在生醫領域上之應用。為了能有效的抑制初期爆發性釋放並控制凝膠藥物釋放系統的釋放特性，我們設計並研發出由奈米粒子所組合而成的奈米生醫凝膠，此凝膠利用奈米粒子乘載藥物達到減緩初期爆發性釋放的現象，並針對奈米生醫凝膠之材料特性及生醫應用進行研究。此篇論文主題主要著重於三個項目，分別是奈米生醫凝膠之合成及特性分析，奈米生醫凝膠對於青光眼動物模型之醫療成效評估及具高度酸鹼靈敏性之奈米生醫凝膠的設計與研究。 於本論文中第一部分之研究，採用經過改質的雙性幾丁聚醣高分子(carboxymethyl hexanoyl chitosan, CHC)做為奈米生醫凝膠之主材料，並結合了甘油及磷酸甘油酯，共同組裝成一種由奈米粒子所構成的生醫凝膠。雙性幾丁聚醣高分子是由本實驗室先前研究所得，在水溶液環境下可自組裝成奈米粒子，除了具有良好的生物相容性外，也被廣泛的應用於各種藥物包覆及釋放之研究。在第一部分中，除了針對奈米生醫凝膠之流變特性、電顯微結構、藥物釋放特性及細胞毒性進行分析。並採用乙琥胺(ethosuximide, ESM)-一種癲癇用藥-做為模擬藥物，長期抑制癲癇鼠所產生的棘徐波(spike wave discharges, SWDs)，以證明此藥物凝膠系統具有確切的治療效果。此外，利用核磁共振顯影技術(Magnetic Resonance Imaging, MRI)追蹤奈米生醫凝膠在動物體中降解之速率及情況。此部分研究成功地研發出一種具有生醫應用潛力之奈米生醫凝膠藥物釋放平台，可廣泛的應用於慢性病及癌症之治療。 而在第二部分中，主要著重於研究奈米生醫凝膠的藥物釋放特性與流變特性及青光眼(Glaucoma)動物模型之建立與治療。為了研發具有長時效藥物釋放特性之奈米生醫凝膠，凝膠內添加了氯化苯二甲羥銨(Benzalkonium chloride, BAK)做為抗菌劑及穩定藥物功能，並針對添加BAK的奈米生醫凝膠做了流變學、藥物釋放特性及細胞毒性等研究。在動物實驗模型上，採用紐西蘭大白兔，藉由注射曲安奈德(triamcinolone acetonide, TA)誘發高眼壓症狀，以模擬青光眼疾病。之後再經由下眼瞼注射方式，將乘載有拉坦前列腺素(Latanoprost)之凝膠注入兔子眼球下進行長時間的藥物釋放並觀察兔子眼壓變化，也針對眼睛附近的組織進行切片分析。此研究除了更進一步考慮到注射式長時效藥物釋放之注射及保存問題，更將此奈米生醫凝膠藥物釋放平台推向新的疾病應用及更深入的生物學探討。 除了針對親疏水雙性幾丁聚醣所構成的注射式生醫凝膠進行研究外，更利用化學方法進行雙性幾丁聚醣奈米粒子的改質，以增加其功能性，例如：溫度敏感性或酸鹼敏感性。在第三部分，我們採用丙烯酸單體(acrylic acid monomer，AA)進行聚合反應(polymerization)，合成聚丙烯酸(poly(acrylic acid), PAA)。同時PAA與雙性幾丁聚醣高分子進行共價鍵連結，合成高度酸鹼敏感性之奈米凝膠(CHC-PAA nanogel)，不需多步驟反應，在未來應用上將可減少藥物及材料的損失。PAA具有羧酸基(carboxylic group)，在不同的pH值環境下將呈現不同的帶電特性，使CHC-PAA nanogel更具有酸鹼敏感性。不同的pH值下，CHC-PAA奈米凝膠將有顯著的粒徑變化，此變化將造成奈米凝膠之藥物釋放速率明顯的變化，達到藥物傳輸及控制釋放之目的。此研究之目的為拓展生醫凝膠之功能性，以增加其生物醫療之應用領域。 總結上敘所做之研究，此可注射式生醫凝膠可乘載多種藥物進行定點且長時效的藥物釋放，並於藥物釋放完畢後不需開刀取出，可經由生物降解(Biodegradation)代謝出體外。並大幅減少傳統幾丁聚醣凝膠於釋放初期會發生的突爆式釋放。此外，此生醫凝膠展現出良好的生物相容性(Biocompatibility)，有利於此凝膠運用在各種的慢性疾病及癌症治療上。雙性幾丁聚醣奈米粒子除了經由電性中和的方法，配製出具有注射功能的生醫凝膠外，更可進一步以化學方法修飾其奈米粒子，設計出具有多功能性的奈米凝膠，提升此材料的應用價值，可因應不同疾病或治療方法的需求進行設計，以符合臨床或病人所需。

Sustained drug release system has been received greatest attention in the world, especially injectable hydrogel drug delivery system. However, burst release in the initial stage of drug release limits the biomedical application in the hydrogel system. In order to efficiently suppress and control the drug release property in the initial drug release, we have developed a hydrogel composed of nanoparticles, the nanoparticles can reduce the release rate in the initial stage of drug release, and investigated the material properties and its biomedical applications. Therefore, this thesis is focused on three topics: 1. Design and characterization of a novel amphiphilc chitosan nanocapsule-based thermo-gelling hydrogel (CHC gel). 2. In-vitro characterization and in-vivo glaucoma treatment of a shear reversible injectable hydrogel. 3. Design and investigation of a highly pH-sensitive CHC-based nanogel for biomedical applications. The first part of this thesis is to employ a modified amphiphilic chitosan polymer as a key material for fabricating hydrogel, and combine with glycerol and β-glycerophasphate to prepare a thermo-gelling hydrogel for biomedical use. The amphiphilic chitosan (carboxymethyl-hexanoyl chitosan, CHC) was successfully synthesized in this lab. The CHC demonstrated self-assembly into nanocapsules about 200 nm in size in aqueous environment, and the capsules demonstrated good drug loading properties. The hydrogel in this study was composed of CHC nanocapsules and named CHC gel. The CHC gel was investigated its rheological property, electron microscope image, drug release property and cytotoixicity. The potential as a depot drug delivery system was demonstrated in vivo through the therapeutic effect of ethosuximide (ESM) loaded CHC gel, suppressing spike wave discharges (SWDs) in Long Evan rat model. Simultaneously, the clearance of gels from the site of administration was monitored by using non-invasively MRI. Finally, this study developed a potential material for injectable depot gel for drug delivery. To develop an injectable and long-term drug release hydrogel system, the CHC gel was merged with benzalkonium chloride (BAK) as biocide and stabilizer for protecting drugs. The BAK-containing CHC gel (CHC-BAK gel) was investigated the effects caused by BAK through rheological study, drug release property and cytotoxicity. New Zealand rabbit was employed as animal model and induced an abnormally high IOP (intraocular pressure) by intravitreal injection of triamcinolone acetonide (TA), a kind of synthetic corticosteroid. Then, we injected a latanoprost loaded BAK-CHC gel under rabbit’s eye ball for sustained release and monitored the IOP variation. In this study, the injection ability and drug preservation of injectable hydrogel system for drug delivery were investigated and discussed further. The CHC drug delivery hydrogel system was progressed in a new therapeutics and thorough study in biology. In addition to prepare an injectable biogel for sustained release, CHC nanoparticle was modified with functional groups by chemical synthesis for increasing functions, such as thermosensitivity and pH sensitivity, and improving its value in biomedical applications. In the final part of this thesis, Acrylic acid (AA) monomer was employed as the group improving the function of CHC polymer. AA monomers were grafted on the CHC by the chemical bond between the protonated amine on CHC and the double bond of AA monomers. The macroradicals can react with the double bond of AA monomers and act as the active site to initiate chain propagation of the monomers and form poly(acrylic acid). CHC-PAA nanogel presents higher pH-responsive behavior in terms of size or volume than existing reports in the literature and are aimed to use for drug delivery application. The nanogel also presents low cell cytotoxicity for cancerous cells (MCF-7) and normal cells (BCE), which indicates the nanogel can be design as a new biomaterial for cellular-based therapeutics. In summary, this injectable hydrogel prepared by amphiphilic chitosan and other chemicals can be loaded with multi-drugs and process in situ sustained drug release. The hydrogel can be degraded naturally without any additional invasive surgery. Burst release in the initial release of the hydrogel also conspicuously decreased because of its microstructure. Besides, the hydrogel exhibits good biocompatibility and low cell cytotoxicity, which improve the potential for using in various biomedical applications. Moreover, the amphiphilic chitosan polymer can be modified with some specific groups for improvig the functions, and be used in different biomedical applications, such as oral drug delivery.