溫度應答式微胞於橙皮苷元之釋放研究

The studies of thermal response micelle for Hesperetin release

許雅婷(Ya-Ting Hsu)

指導教授 ： 婁世亮

中原大學/工學院/生物醫學工程研究所/碩士(2012年)

https://doi.org/10.6840/cycu201200705

摘要

橙皮苷元已被證實具有抑制發炎反應，因而可被作為粥狀動脈硬化治療使用。使用溫度應答型微胞能夠控制藥物釋放行為，更能延長藥物於體內的循環時間及降低副作用的產生。本研究之目的將製備生物可降解性及溫度應答型微胞，並且探討其特性、生物相容性及藥物釋放能力。藉由溫度應答型片段poly(NIPAAm-co-DMAAm)架接於疏水性主幹Poly(succinimide) (PSI)上，並透過氫氧化鈉水解部分PSI形成具有生物可降解性溫度應答型奈米微胞P(NIPAAm-co-DMAAm)-g-PAsp/PSI。將橙皮苷元透過薄膜透析方式包埋於微胞內，即可得到包埋有橙皮苷元之微胞。使用傅立葉轉換光譜儀、核磁共振光譜儀、熱重分析儀及膠體滲透層析儀分析其特性。另外，使用掃描式電子顯微鏡及動態光散射儀，觀察包埋藥物後微胞型態及粒徑大小的變化。螢光分析結果發現微胞之低臨界微胞濃度為0.057 wt%，且低臨界溶液溫度約為42.6 oC。生物相容性部分，經由溶血分析及細胞毒性試驗結果發現，微胞沒有明顯的溶血及細胞毒性的情況發生，表示此微胞具有良好的生物相容性。微胞對橙皮苷元之包埋率約為20%，包埋有藥物之微胞在TEM與DLS觀察其粒徑大約為332與411.8 nm。包埋藥物之微胞在室溫下可穩定於磷酸鹽緩衝溶液中，但是在高於體溫環境下(42.6 oC)，能夠驅使微胞中的藥物釋放於環境中。包埋有橙皮苷元之微胞在藥物釋放後，能夠降低55%因脂多醣誘發細胞內產生的一氧化氮。此外，微胞在37 oC磷酸緩衝溶液下，7天後微胞降解率約55%。依據上述研究結果，包埋有橙皮苷元之生物降解型溫度應答型微胞在高於體溫的環境中，能夠釋放橙皮苷元，並且顯示能夠抑制一氧化氮生成。因此，包埋有橙皮苷元之生物降解溫度應答型微胞將可作為粥狀動脈硬化臨床治療使用。

關鍵字

溫度應答微胞 ； 橙皮?元 ； 藥物釋放 ； 抗發炎反應 ； 生物降解性 ； 生物相容性

並列摘要

Hesperetin has been demonstrated as an inflammation response suppressor and could be used for atherosclerosis treatment. Using temperature responsive micelles to control drug delivery can prolong circulation time and reduce side effects. The aim of this study was to investigate the chemical synthesis, biocompatibility and in vitro drug delivery response of biodegradable and temperature responsive micelles. The biodegradable and temperature responsive micelles, poly(N-isopropylacryamide-co-N,N-dimethylacrylamide)-graft-
poly(aspartic acid)/poly(succinimide), were synthesized by grafting P(NIPAAm- co-DMAAm) onto poly(succinimide) and followed by partial aminolysis with sodium hydroxide. The anti-inflammation responsive drug, Hesperetin, was encapsulated by micelles using membrane dialysis method to obtain Hesperetin-loaded PND-g-PAsp/PSI nanoparticle. The micelles were characterized by Fourier transform infrared spectroscopy, 1H-NMR, thermo gravimetric analyzer and gel permeation chromatography. The morphology and particle size of micelles was observed by transmission electron microscopy and dynamic light scattering. Fluorescence analysis indicated that the micelles had a low critical micelle concentration of 0.057 wt% in aqueous media. The low critical solution temperature (LCST) of the micelles was about 42.6 °C. The biocompatibility of micelles was confirmed by hemolysis assay and cytotoxicity study indicating there was no significant hemolysis and cell cytotoxicity. After encapsulation, the transmission electron microscopy and dynamic light scattering showed that Hesperetin loaded micelles were regularly spherical in shape with an average diameter approximately 332 and 411.8 nm. The encapsulation efficiency of Hesperetin was about 20%. Hesperetin-loaded micelles were stable in phosphate buffer solution (PBS) at room temperature but could be deformed at 42.6 °C above normal body temperature to trigger the release of Hesperetin. After Hesperetin releasing into the medium, Hesperetin-loaded micelles reduced 55% of nitric oxide generation, which was lipopolysaccharides-induced intracellular reactive oxygen species. In addition, P(NIPAAm-co-DMAAm)-g-PAsp/PSI was stable in PBS at 37 °C, and 55% of micelles could be degraded within 7 days. Based on these results, the present study demonstrated that Hesperetin-loaded P(NIPAAm-co-DMAAm)-g- PAsp/PSI micelles can release Hesperetin above normal body temperature and show greater inhibition of nitric oxide generation. Therefore, Hesperetin-loaded P(NIPAAm-co-DMAAm)-g-PAsp/PSI micelles can be used for atherosclerosis treatment.

並列關鍵字

thermal response micelles ； biocompatibility ； anti-inflammatory response ； drug release ； biodegradable ； Hesperetin

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