幾丁聚醣及竹碳複合顆粒之生物相容性及溶液吸附性質研究

The Composite Pellet of Chitosan and Bamboo Charcoal： The Study of Biocompatibility and Adsorption Capability

徐志良(Chih-Liang Shyu)

指導教授 ： 謝明發

中原大學/工學院/生物醫學工程學系/碩士(2008年)

https://doi.org/10.6840/cycu200900335

摘要

摘要
本研究的目的是製備一種用於血液淨化的竹碳顆粒材料，其粒徑範圍為 2-5 毫米。本研究對竹碳顆粒進行物理/化學性質鑑定、生物相容性評估與溶液吸附性質測量。藉由評估以上實驗結果，進一步探討竹碳材料於血液淨化應用的可行性。研究內容分為四部份：(一) 利用超音波洗淨法與透析法洗淨竹碳粉末，比較兩種清洗方法對材料之細胞毒性影響，(二) 以幾丁聚醣為造粒包覆劑，利用酸鹼中和法製備出幾丁聚醣包覆竹碳粉末的顆粒材料，其性質鑑定包括傅立葉紅外線光譜儀分析、掃描式電子顯微鏡觀察、孔洞及表面積分析儀與熱重分析儀分析，(三) 溶血測試量測、瓊脂擴散間接毒性測試、巨噬細胞發炎反應、細胞存活率與免疫細胞激素釋放，(四) 竹碳粉末及竹碳顆粒進行溶液吸附實驗，被吸附物質分別是白蛋白 (Albumin) 及苯丙氨酸 (L-phenylalanine)，以及竹碳顆粒對降血脂藥辛伐它汀 (Simvastatin) 的吸附及釋放實驗。在清洗方法中，(1) 竹碳原料粉末的 pH 值在 10~12，經過透析純化 3-5 天，pH 值降至 7.0~7.2，而以超音波震盪 6-8 小時 pH 值降至 7.0~7.4，(2) 幾丁聚醣包覆之竹碳顆粒粒徑 2-5 毫米，其傅立葉紅外線光譜圖在 1647 cm-1 (amide I band, C=O) 及 1542 cm-1 (amide II band, NH) 有明顯的峰值，為幾丁聚醣特殊官能基團。由掃描式電子顯微鏡觀察看到幾丁聚醣確實包覆竹碳粉末，由孔洞與比表面積分析儀與熱重分析儀可知竹碳的比表面積為 583.68~626.50 m2/g，幾丁聚醣包覆量為 1.16~2.52 %，(3) 竹碳顆粒的發炎反應 (NO O.D/mg protein < 0.029 )、細胞存活率 (大於 95 %)、細胞毒性 (Zone index = 1)、溶血指數 (溶血率小於 2 %) 與細胞激素釋放的評估 (與控制組比較，p 值大於 0.05 沒有統計上的顯著差異) 皆屬於 ISO 10993 系列規範的正常範圍，(4) 溶液吸附性質評估，竹碳粉末與竹碳顆粒對親脂性苯丙氨酸的吸附量較高，濃度由 0.5 mg/mL 增加到 3.0 mg/mL，吸附量由 6.0 mg/g 增加至 34.6 mg/g。竹碳顆粒對辛伐它汀的吸附與釋放行為中，隨著溫度由 27 oC 上升至 55 oC，其吸附量由 57.83 μg/g 增加為 129.81 μg/g，但釋放率皆小於 4 %。以上研究結果顯示，竹碳材料經過清洗，包覆幾丁聚醣後仍具生物相容性質，因此在血液淨化應用方面具有發展潛力。

關鍵詞：竹碳顆粒、生物相容性 、幾丁聚醣、巨噬細胞

關鍵字

巨噬細胞 ； 生物相容性 ； 竹碳顆粒 ； 幾丁聚醣

並列摘要

Abstract
The purpose of this research is to prepare a kind of bamboo charcoal pellet for use in blood purification. Its physical/chemical property, biocompatibility and solution adsorptive property were characterized. The experiments were divided into four parts. In the first part, ultrasonic cleaning and dialysis was used to wash bamboo charcoal powder. The influence of two washing methods on cell toxicity was compared. In the second part, spherical bamboo charcoal particles produced in NaOH-methanol solution were encapsulated in a chitosan matrix. The structure of the composite material was characterized by Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), pore size and surface area analysis and thermogravimetric analysis (TGA). In the third part, hemolysis, agar diffusion, NO and MTT assay of macrophage, cell survival rate and the amount of released cytokines were studied. The fourth part of the study involved the evaluation of the solution adsorptive property of bamboo charcoal particle and pellet for albumin and L-phenylalanine. Finally, the adsorption and release behavior of the antihypercholesterolemic agent (Simvastatin) on bamboo charcoal pellet was also observed. The results indicated several matters. First, it was found that before cleaning, the pH value of bamboo charcoal particle was 10~12. After washing, bamboo charcoal particle thru dialysis for 3-5 days, its pH value decreased to 7.0~7.2. If washed by ultrasonic cleaning for 6-8 hours, its pH value decreases to 7.0~7.4. Second, the chitosan-encapsulated bamboo charcoal pellet size ranges from 2-5 mm and there exist two characteristic peaks of chitosan at 1647 cm-1 (amide I band, C=O) and 1542 cm-1 (amide II band, NH). Chitosan-encapsulated bamboo charcoal particle was further observed using SEM. Its specific surface area was found to be in the range of 590.85~601.57 m2/g. The TGA analysis showed a coverage extent of around 1.16~2.52 %. Third, in terms of biocompatibility, the bamboo charcoal pellet does not cause macrophage activation (NO O.D/mg protein < 0.029). The cell survival rate (greater than 95 %), cell toxicity of agar diffusion (zone index was 1), hemolysis and released cytokines from macrophage, which showed no significance compared with control group, all conformed to ISO 10993 series. Fourth, evaluation of the solution adsorptive capacity of bamboo charcoal particle and pellet showed that the adsorption of L-phenylalanine was better than albumin. If the concentration increases from 0.5 mg/mL to 3.0 mg/mL, the adsorption in turn increases from 6.0 mg/g to 34.6 mg/g. In the adsorption and release behavior of Simvastatin on bamboo charcoal pellet, its adsorbability increases from 57.83 μg/g to 129.81 μg/g if the temperature rises from 27 oC to 55 oC. However, release rate of bamboo charcoal pellet is less than 4 %.The above-mentioned results prove that bamboo charcoal material remains biocompatible after through washing and chitosan-encapsulation. Hence, chitosan-encapsulated bamboo charcoal pellet is a promising microencapsulation material for blood purification.

Key words: bamboo charcoal pellet, chitosan, biocompatibility, macrophage

並列關鍵字

biocompatibility ； chitosan ； bamboo charcoal pellet ； macrophage

參考文獻

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