膠原蛋白膜之特性解析

Characterization of collagen film

劉佳旻(Liu Chia-min)

指導教授 ： 許明照

臺北醫學大學/藥學院/藥學研究所/碩士(1998年)

摘要

隨著生物技術的進步，天然的生物聚合物逐漸成為臨床上的重要材料；其中，膠原蛋白 具有生物相容性、生物可降解性、無毒性及低過敏反應性應而更增加它的實質應用。一般 膠原蛋白的來源自豬或牛的皮膚或骨骼中純化。而台灣目前，每年屠宰超過一千萬頭豬隻 。然而，其豬皮大都被當作垃圾丟棄或當作皮革材料。我們若能從這些的豬皮中純化出膠 原蛋白，就能使這些打算丟棄的豬皮大幅增加它的經濟價值及應用性。 在本論文中，我 們找尋一個既簡單且有效率的純化type Ⅰ膠原蛋白的方法。首先，將去毛及?肪的豬皮 給予均質化，置於在pH=2的鹽酸溶液中，加入適量的pepsin來去除免疫反應，整個裝置在 20oC作用20小時。隨後調整pH值到鹼性來終止pepsin的活性，放置24小時。同時使膠原蛋 白利用等電點的原理沈析下來。我們將會鑑定，不同批次間所純化出的膠原蛋白間的差異 性及是否具有再現性。 純化出的膠原蛋白將和不同比例的酒精、水及丙二醇混合，來製 造膠原蛋白膜。利用實驗設計法的原理來探討，各溶劑對膠原蛋白膜的特性探討。同時利 用不同碳鏈長度的paraben及不同分子量的巨分子蛋白質來進行穿透實驗，來了解膠原蛋 白膜的基本物理特性。我們採用動態機械性質分析儀來了解膠原蛋白膜所能承受的拉張力 。隨後我們在膠原蛋白膜的處方中加入hydroxyapatita 進行前述的實驗。 首先由S.D.S .電泳及Size exclusion HPLC的結果中，告知我們膠原蛋白的純化方法具有相當的穩定性 及再現性。同時純化的步驟並不繁雜且產率相當高。 從X-ray晶格繞射及電子顯微鏡的 拍攝，我們可以確知hydroxyapatite確能在膠原蛋白膠體中合成，且其結構不會因在膠原 蛋白膠體中而有所改變。然而膠原蛋白膜的張力會因有hydroxyapatite的存在而大幅降低 。應該是hydroxyapatite的存在而破壞膠原蛋白膜的連續性，而使其受到外力時較易斷裂 。由實驗設計之分析結果顯示，不論hydroxyapatite的有無，在膠原蛋白膜的處方中，製 備膠原蛋白膜時添加的水份對於所形成之膠原蛋白膜的張力貢獻最大，有增強的效果。 Parabens 在膠原蛋白膜中的穿透試驗結果顯示，隨著parabens的碳鏈的增加其穿透力隨 之下降，非極性的碳數增加，造成模型藥的整體極性降低，此因為進而降低溶解度。因此 模型藥的穿透力隨著碳鏈的增加而降低。當膠原蛋白膜處方中加入hydroxyapatite時，會 使parabens的穿透力比在不含hydroxyapatite更低，或許是hydroxyapatite會影響模型藥 的穿透路徑之曲折度而使穿透力降低。由實驗設計之分析結果顯示，處方中的三個溶劑( 水、propylene glycol、酒精)對於parabens的穿透貢獻均差不多，均有些微的促進效果 。 蛋白質藥物在膠原蛋白膜中的穿透力，會隨著蛋白質藥物的分子量增加而降低，是由 於分子大小的緣故，同時由實驗結果顯示隨著製備膠原蛋白膜時添加的丙二醇的增加而降 低。相同地膠原蛋白膜中含有hydroxyapatite時，會使模型藥的穿透力降低，或許是hydr oxyapatite影響 蛋白質藥物的穿透路徑之曲折度而使得穿透量降低所致。

關鍵字

膠原蛋白 ； 純化 ； 穿透力 ； 張力

並列摘要

With the advance of biotechnology, the natural biompolymers have bec ome one of the most important biomaterials for clinical applications. Among th em, interest on the use of collagen as biomaterials was increased as a result of its appealing characteristics: biocompatible and biodegradable,less toxic a nd least immunological reactions, obtainable in an appreciable quantity, and p rocessable in a variety of forms. The common source of collagen comes from the bone or the skin of bovine or pig. In Taiwan, morethan 1 million pigs were sl aughtered for food a year. However, most of thepig skin was discarded as trash or was processed as shoe leather. It was thought that the added value of the pig skin could be improved if these discarded pig skins was isolated for colla gen to have practical applications. In this study, an efficient and simple way to isolate type I collagen fromthe pig skin was developed and validated. Firs t of all, the swollen skin was sliced into small pieces and homogenized in HCl solution (pH=2).After stirring for 24 hours at 4 ℃, pepsin was added into th e suspension toeliminate the terminal telopeptide on collagen fibers, which wa s main factorscausing immunological reaction. After deactivation of pepsin by adjustingpH value of the suspension to basic and storage for over night, acidi c-soluble collagen was purified by precipitation method at its pI value. The q uantity and characteristics of so obtained collagen from different batch or fr om different sources was further confirmed to be reproducible. Collagen so obt ained was processed into film in the mixture containing various ratios of wate r,propylene glycol, and ethanol. An experimental mixture design was applied to characterize the effect of individual solventand their interaction on the pro perties of such a collagen film. Scanning Electronic Microscopy (SEM) was used to examine the surface property of collagen film. Penetration studies using a series of parabens with differentalkyl chain length and macromolecules with d ifferent molecular weight wereused to characterize their physical properties. The tensile strength and related characters of collagen films was evaluated as well using DynamicMechanical Analyzer (DMA). The effect of adding Hydroxyapat ite (HA) at a concentration of 10 mM on the physical characters of collagen fi lms was evaluated by the same studies as above. First of all, it was confirmed by SDS electrophoresis and size exclusion HPLC that the process conditions pr oposed in this study was a simple and reproducible way with a high yield to is olate type I collagen from the pig skin. By the examination of X-ray and SEM, it was confirmed that hydroxyapatitewas formed inside the collagen film. Howev er, the tensile strength of collagen films without hydroxyapatite was 4-5 time higher than that with hydroxyapatite. This is probably due to the discontinui ty of film structurecaused by the existence of hydroxyapatite in the collagen films. This results in the easiness of breakage when the force was applied ont o the collagen films. The results of statistical analysis of experimental desi gnrevealed that whether the existence of hydroxyapatite or not, water added du ring the preparation of collagen films was the most significant factor that en hancing the tensile strength of collagen films produced.The flux of four parab ens penetrated through these collagen films with or without hydroxyapatite dec reased with the increase of the alkyl chain length. Nevertheless, the flux pen etrated through collagen film without hydroxyapaptite was about 4 times higher than these films with hydroxyapatitefor the same drug and formulation. The re sults of the statistical analysis of experimental design demonstrated that all of three solvents, water, propylene glycol, and alcohol, which were used to p repare collagen films, were able to increase the permeability of parabens pene trated through thecollagen film produced.The permeability of proteins penetrat ed through the collagen films without hydroxyapatite decreased with the increa se of molecular weight of protein and with the added amount of propylene glyco l in the film formation. There showed a lower flux for the same protein molecu le penetrated through the collagen films with hydroxyapatite than that without hydroxyapatite.

並列關鍵字

Collagen ； Isolation ； Permeability ； Tensile strength