奈米顆粒及化學改質對藥物載體材料之物理性質與生物醫學特性影響研究

Abstract

本研究主要是研究缺鈣氫氧基磷灰石(Ca-deficient hydroxyapatite, CDHA) 奈米顆粒對於PMMA與幾丁聚醣高分子物理性質及藥物釋放行為的影響。首先，PMMA-CDHA 的流變行為與 CDHA 形貌與濃度的關聯性如下：增加CDHA 濃度會使shear-thinning 行為變得顯著，代表奈米顆粒間以凡得瓦力與機械接觸構成交互作用使流變行為偏離牛頓流體，同時，複合材料中奈米顆粒的堆積效率與流變降服強度均與奈米顆粒長寬比有高度相關。至於以CDHA奈米顆粒做為牛血清蛋白載體時，其攜藥奈米載體的合成方式(原位製程與非原位製程)對牛血清蛋白攜帶量及後續蛋白質釋放行為的影響如下：對這兩種製程而言，奈米載體之牛血清蛋白攜帶量均隨合成環境pH值增加而減少，在pH值超過8時，相對原位製程而言，透過非原位製程所合成的奈米載體具有較大的蛋白質攜帶量，然而，其也明顯地對應一個突放(bursting release)的藥物釋放行為，這個突放行為主要歸因於牛血清蛋白分子的脫附(desorption)；相反地，透過原位製程所合成的奈米載體則表現出較和緩的釋放行為，原因為透過原位製程使得牛血清蛋白分子與奈米載體產生較強的交互作用，這樣的解釋可以透過高解析度穿透式電子顯微鏡(HR-TEM)來證明﹔另一方面，在低pH值環境下以原位製程所合成的奈米載體，則表現出兩階段釋放行為，第一階段突放是來自於牛血清蛋白分子的脫附，第二階段的釋放是來自於奈米顆粒c軸(長軸)溶解時所釋放出的蛋白質分子。另外，CDHA奈米顆粒對於攜藥幾丁聚醣複合薄膜藥物釋放行為的影響如下： CDHA奈米顆粒的添加量與複合材料合成方式均會對擴散機制造成影響，以原位合成所製備的複合薄膜，其擴散機制由擴散控制機制朝向膨潤控制機制變化。 為了進一步調控奈米粒子與幾丁聚醣高分子間之作用力，並擴展幾丁聚醣的生醫適應性使其可包覆雙性藥物分子及DNA等會在酸性環境中變性的生物分子，本研究成功地合成一種無細胞毒性的雙性幾丁聚醣(NOCHC)，其上之己醯基對於細胞存活率並無顯著負面影響，本材料因有高度的親水性及部份疏水基所造成的靜電遮蔽效用，使得纖維母細胞非常不易貼附，然而，己醯基的進一步增加似乎有助於纖維母細胞的貼附﹔另外，己醯基的導入對於吸水率產生了非常顯著的影響，其在低溼度時會使水接合位置增加，在飽和狀態時則會增加分子間的空間使得大量的自由水可以存在，這個現象，也造成了NOCHC的酸鹼敏感性大於NOCC(N,O-carboxymethyl chitosan)﹔而且，疏水性的己醯基會影響水分子的移動使材料的保水能力提升﹔另外，相較於NOCC 與未改質的幾丁聚醣，NOCHC可以包覆較多的雙性藥物Ibuprofen，然而， NOCHC 因膨潤度大所以導致過於快速的藥物釋放，為了改善此特性，本研究使用疏水性的O-hexanoyl chitosan (OHC)微球來分散於NOCHC基底中，可以達到程序式釋放的目的﹔另一方面，CDHA 奈米顆粒的混掺亦可調控此二者的藥物釋放行為，CDHA奈米顆粒對於此二幾丁聚醣衍生物藥物釋放行為的影響如下：CDHA 對於NOCHC 有類似交聯的效應，可減緩釋放速度並使擴散機制改變，然而，CDHA則會破壞OHC的疏水結構使水份容易進入材料內部，加速降解導致釋放速度增加。

Ca-deficient hydroxyapatite (CDHA) nanoparticles were incorporated with poly(methyl methacrylate) (PMMA), bovine serum albumin (BSA) and chitosan-based materials to explore the influence of filler-filler interaction, nanocarrier-protein interaction and filler-polymer interaction on the physical properties and drug release behaviors of the bioactive nanocomposites. Rheological behaviors of the PMMA-CDHA melting suspensions were systematically investigated in terms of solid loading and aspect ratio of the CDHA nanoparticles. It was found that an increase in solid contents causes pronounced shear-thinning behavior. This result suggests that a strong interaction, including van der Waals attraction and mechanical interlocking, between the CDHA nanoparticles makes the nanocomposite mixture more non-Newtonian. In addition, the packing efficiency and yield strength in the suspension were strongly influenced by the aspect ratio of nanoparticles. For the BSA-incorporated CDHA nanocarrier, the amount of BSA uptake by CDHA decreases with increasing pH. Besides, the release profile showed a bursting behavior for the nano-carrier prepared via the ex-situ process, which is probably due to the desorption of BSA molecules. In contrast, for the sample synthesized via the in-situ process at a higher pH level, a slower release profile without bursting behavior due to the dissolution of the BSA-incorporated CDHA crystal as seen from high solution TEM that indicates different extent of interaction between BSA and CDHA. In addition, the effect of CDHA nanofiller on drug release kinetics of chitosan-CDHA nanocomposite monolithic membrane was investigated. It demonstrates that the drug diffusion mechanism is altered by the CDHA-chitosan interaction which is strongly influenced by both the synthesis process and the concentration of the CDHA nanofiller in the membrane. In order to encapsulate the partially hydrophobic agents to prevent the agent from denature in acid environment, a novel carboxymethyl-hexanoyl (NOCHC) hydrogel with amphiphatic nature and pH-sensitivity was synthesized. The cell viability results indicate that carboxymethyl-hexanoyl chitosan did not show any sign of cytotoxicity in vitro. The degree of hexanoyl substitution did not affect the number of vital cells. In addition, the amount of fibroblast with spread out morphology increased with degree of the hexanoyl substitution. On the other hand, the hexanoyl substitution affected significantly the water-absorption ability by altering the number of water-binding sites and the state of water under low humidity and the fully-swollen state, respectively. Moreover, the presence of hydrophobic hexanoyl substitution retarded significantly water mobility during deswelling, causing better water-retention ability. Besides, compared with that of pristine chitosan and N,O-carboxymethyl chitosan (NOCC), the encapsulation efficiency of ibuprofen (IBU, partially hydrophobic agent) was significantly enhanced with the incorporation of the hexanoyl group. However, the bursting release of IBU was less prominent for the NOCHC samples with high degree of carboxymethyl substitution. Moreover, the pH-sensitivity was more pronounced for NOCHC than NOCC. In order to regulate the bursting release behavior of the NOCHC hydrogel in a highly swollen state, the release kinetics of microsphere-embedded hydrogel prepared by NOCHC and O-hexanoyl chitosan (OHC) was further investigated by incorporating various amounts of CDHA nanoparticles. It was found that the release duration of IBU from NOCHC was prolonged with the amounts of CDHA. On the contrary, the release duration of IBU from OHC (hydrophobic phase) was shortened through increasing the CDHA amount, which is due to the hydrophilic nature of the CDHA nanoparticles, destroying the intermolecular hydrophobic interaction and accelerating OHC degradation.