生醫材料於藥物傳遞系統及組織工程之應用

The application of biomaterials on drug delivery system and tissue engineering

陳瑩真(Ying-Chen Chen)

指導教授 ： 許明照

臺北醫學大學/藥學院/藥學系(碩博士班)/博士(2015年)

摘要

第一部分:
藥物 losartan 和 alendronate 的低生體可用率可能由於通過吸收部位的時間極短或在非吸收部位的藥物釋放，胃滯留藥物傳遞系統可延長吸收窗狹窄藥物在胃部停留的時間，並在抵達吸收部位前緩慢釋放以提升藥物生體可用率。本研究使劑型迅速懸浮於胃液上方以避免胃排空，或/和讓劑型在短時間內膨脹避免通過胃幽門離開胃部的方式來延長胃滯留時間；幾丁聚醣和羥乙基纖維素因具有生體相容性和可降解性以及多樣性物化性質而廣泛應用於製劑學研究。
首先使用幾丁聚醣、羥乙基纖維素和碳酸氫鈉，以不同比例混合製備 losartan胃滯留藥物傳遞系統，高分子量幾丁聚醣處方組別由於黏度過高無法有效導水進入，故膨脹和漂浮效果不如低分子量幾丁聚醣處方組；添加碳酸氫鈉可改善漂浮能力，使錠片一分鐘內即懸浮；混有相同比例的幾丁聚醣和羥乙基纖維素以及添加碳酸氫鈉的 losartan 胃滯留藥物傳遞系統，在模擬胃液中可懸浮、錠片直徑膨脹兩倍以上並緩慢地釋放藥物。
另一方面將幾丁聚醣分別溶在醋酸、琥珀酸、檸檬酸、乳酸或蘋果酸，加入不同比例羥乙基纖維素後冷凍乾燥製備alendronate胃滯留藥物傳遞系統，發現隨著酸液解離常數 (pKa) 的下降，膨脹速率相對增加，低含水量及硬度的處方處可促進膨脹速率增加；羥乙基纖維素雖未對膨脹程度和懸浮能力貢獻，但其加入可穩定錠劑結構。在最佳處方中添加 alendronate後錠片膨脹，藥物皆能緩慢釋出，而釋藥機轉主要由藥物擴散與膠體膨脹緩解效用所管控。
總而言之，胃滯留能力、錠片機械強度和釋放速率會受到幾丁聚醣分子量、共溶酸性溶液、羥乙基纖維素和碳酸氫納添加量及壓錠力的影響，此外胃腸生理學亦是胃滯留藥物傳遞系統的考量因子，隨著可增加生體可用率之胃滯留藥物傳遞系統藥品與日俱增的上市，將提供胃滯留藥物傳遞系統持續發展的動力與臨床使用的應用價值。

第二部分:
鑒於骨關節炎患者數目持續的增加以及尚未有理想治療方式的出現，實有其必要性和急迫性以組織工程方式製備軟骨替代物以增進人類健康福祉。本研究旨在發展一新穎去細胞方法，期望在去除宿主細胞降低免疫排斥風險的同時，亦能保留對細胞貼附生長有益的胞外間質。比較多種酸液 (醋酸、甲酸、過氧化醋酸、蘋果酸、琥珀酸和檸檬酸) 的去細胞效果後，發現以甲酸處理豬軟骨組織兩小時，可有效移除宿主細胞且保留多數的胞外間質，並製備出高度親水性和生物相容性的多孔三維空間支架；相較於未經處理的豬軟骨組織，當豬軟骨組織浸泡於甲酸後，DNA 含量減低至 4.10±0.03 %, 且無任何對葡萄胺聚糖或膠原蛋白的顯著性傷害，組織染色影像亦確認此結果；在細胞存活率和活體試驗沒有顯著性細胞死亡或動物發炎反應的發生，證實該支架的生物相容性；接者將人類軟骨細胞種入該去細胞之胞外間質支架後，發現軟骨細胞可大量在此支架上貼附增生，並持續分泌胞外間質如葡萄胺聚糖和第二型膠原蛋白，維持軟骨細胞分化特性；進一步觀察此支架不論生長因子添加與否，可誘導生長其中的人類骨髓幹細胞分化為軟骨細胞，且分泌軟骨相關胞外間質。該天然去細胞之胞外間質支架提供軟骨細胞生長的原始環境且保留細胞生長所需物質，因此可有效促使軟骨細胞增生和分泌軟骨胞外間質有利新組織生成，並成功誘導幹細胞分化為軟骨細胞；本研究提供一簡便、低成本和有效的去除豬軟骨組織細胞方法，可製備出一良好仿生支架供軟骨組織再生之用。

關鍵字

胃滯留藥物傳遞系統 ； 幾丁聚醣 ； 組織工程 ； 軟骨

並列摘要

Part I:
The low bioavailability (BA) of losartan and alendronate may be due to a short transit period in the proximal gastrointestinal tract or non-absorbing site release. Gastroretentive drug delivery system (GRDDS) can improve the controlled delivery of drug that has a narrow absorption window by continuously releasing the drug for a prolonged period of time before it reaches its absorption site, thus increasing BA. In this study, gastric retention was achieved by floating to prevent gastric emptying, and/or swelling to limit emptying through the pyloric sphincter. Chitosan (CS) and hydroxyethyl cellulose (HEC) have been used in pharmaceutical development due to their biocompatible, biodegradable, and versatile physiochemical property.
First of all, we developed GRDDS for losartan by mixing CS, HEC, and sodium bicarbonate (SB) to improve gastric retention. Formulations with high molecular weight CS had poorer swelling and floating abilities than those with low molecular weight CS owing to the retardation of water inward progression by its high viscosity. SB improved the floating capacity and initiated floating within 1 min. The optimal losartan GRDDS was formulated with SB and an equivalent ratio of HEC and CS. The tablets floated in simulated gastric fluid (SGF) and exhibited a swelling ratio greater than 200 % with a sustained release profile.
Besides, GRDDS for alendronate was prepared by dissolving CS in acetic, lactic, succinic, malic, or citric acids. Then different amounts of HEC were added and freeze-dried. There is a negative correlation between pKa of acid and the swelling rate of CS-salt. The low water content and hardness caused high swelling rate. Although HEC did not contribute to swelling and floating, it helped maintain structural integrity. The optimal formulations incorporated with alendronate in SGF showed sustained release patterns which were controlled by drug diffusion and gel swelling relaxation.
Overall, the gastroretentive ability, mechanical integrity and release rate can be controlled by the type of CS, countering acids with CS, addition of HEC and SB, and compression force. Besides, the stomach anatomy and physiology also affect the development of GRDDS. The growing launch list of GRDDS products used for enhancing BA makes the continued development of GRDDS a clinically relevant and important effort.

Part II:
Given the growing number of arthritis patients and the limitations of current treatments, there is a great urgency to explore cartilage substitutes by tissue engineering. In this study, we developed a novel decellularization method for menisci to prepare acellular extracellular matrix (ECM) scaffolds with minimal adverse effects on the original ECM. Among all the acid treatments (acetic, formic, peracetic, malic, succinic, and citric acids), formic acid treatment for 2 hr removed most of the cells and preserved the highest ECM contents in the decellularized porcine menisci. Compared to fresh porcine menisci, the content of DNA decreased to 4.10±0.03 %, and there was no significant damage on glycosaminoglycan (GAG) or collagen. Histological staining also confirmed the presence of the ECM and the absence of cellularity. In addition, a highly hydrophilic scaffold with 3-dimensional interconnected porous structure was fabricated from the decellularized menisci tissue. No significant in vitro cytotoxicity and inflammatory response of rats were observed, indicating the biocompatibility of the scaffold. In vitro cytotoxicity and in vivo implantation conducted in rats were to assess the biological response of the scaffolds. Human chondrocytes showed enhanced cell proliferation and synthesis of chondrocyte ECM including type II collagen and GAG when cultured in this acellular scaffold. Moreover, the scaffold effectively supported chondrogenesis of human bone marrow-derived mesenchymal stem cells regardless the addition of growth factors. The acellular ECM scaffold provided a native environment for cells with diverse physiological functions to promote cell proliferation and new tissue formation. This study reported a simple, low cost, and effective way to prepare decellularized meniscus tissue and demonstrated its potential as scaffolds to support cartilage repair.

並列關鍵字

Gastroretentive drug delivery system ； chitosan ； tissue engineering ； cartilage

參考文獻

Part I:

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