請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/52095
標題: Optimization for the production of liposomal berberine with the comparison of antihepatoma effects, and the apoptotic pathways of berberine solution or liposomal berberine on HepG2 cells
小檗鹼微脂體製程之最適化及小檗鹼溶液與其微脂體抗HepG2細胞生長能力和相關凋亡路徑之比較
作者: 郭旃暥
Guo, Jhan-Yan
關鍵字: Nano-traditional Chinese medicine
奈米中藥
berberine liposome
HepG2 cell
cell apoptosis
鹽酸小檗鹼微脂體
HepG2細胞
細胞凋亡
出版社: 食品暨應用生物科技學系所
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摘要: 奈米中藥是指將中藥有效成分、有效部位、原藥或複方製劑與奈米技術相結合而製造出粒徑小於100 nm的中藥劑型,進而提高生物利用度、控制藥物釋放速率和延長藥物的作用時間來增進治療效果。小檗鹼是中草藥黃連的主要功效成分,具有抗菌、抗病毒、抗發炎、抑制腫瘤生長及誘導癌細胞凋亡的功效。本研究目的是以微脂體包覆鹽酸小檗鹼以提高其對於肝癌細胞的毒殺作用。首先先探討鹽酸小檗鹼微脂體的脂質組成及製備方式對鹽酸小檗鹼微脂體的粒徑、粒徑分佈及鹽酸小檗鹼包覆率的影響,確立鹽酸小檗鹼微脂體的最適化製程。結果顯示1,2-distearoyl-3-sn-phosphatidylcholine(DSPC)及hydrogenated soy phosphatidylcholine(HSPC)兩種脂質不影響鹽酸小檗鹼微脂體的粒徑大小與包覆率,而添加5% PEG可顯著地使包覆率提高3.3- 6.4倍。在方法上,以薄膜水合法搭配濾膜擠壓法可以得到最高的包覆率(14%)的鹽酸小檗鹼微脂體。微脂體包覆鹽酸小檗鹼可以顯著的提高對HepG2細胞的毒殺能力,MTT試驗結果指出鹽酸小檗鹼微脂體對HepG2細胞的生長抑制效果為鹽酸小檗鹼溶液的1.2-2.3倍。由Annexin V/ PI雙染的結果可以發現鹽酸小檗鹼微脂體可以顯著的誘導HepG2細胞產生凋亡,且隨處理時間(24- 72小時)與濃度(0- 20 µg/mL)的增加,凋亡細胞的比例也隨之提高,相對的,鹽酸小檗鹼溶液在同樣的處理條件下,亦有凋亡細胞的出現,但是數量較少。在凋亡路徑之探討方面,鹽酸小檗鹼溶液或鹽酸小檗鹼微脂體能藉由降低粒線體的膜電位,接著依序活化caspase-9及-3,caspase-3的活化使其受質PARP斷裂並失去活性,導致HepG2細胞發生凋亡。此外,外在路徑中的caspase-8並無活化的情形。另外以西方墨點法分析結果顯示pro-caspase-3表現量,兩處理組的pro-caspase-3表現量均出現濃度依賴性,而caspase-3的受質PARP之表現情形在鹽酸小檗鹼濃度5 µg/mL以上,亦有斷裂的情形出現,故鹽酸小檗鹼溶液與其微脂體應是以caspases/mitochondria依賴性的凋亡路徑促使HepG2細胞產生細胞凋亡。而與鹽酸小檗鹼溶液相比,鹽酸小檗鹼微脂體能讓粒線體膜電位下降幅度加大,且caspase-9和caspase-3的活性也都高於鹽酸小檗鹼溶液組。由細胞的攝食情形與微脂體釋出鹽酸小檗鹼的情形證實鹽酸小檗鹼微脂體無法提高HepG2細胞對鹽酸小檗鹼的攝食量,但是藉由微脂體對藥物的保護效果及逐步釋出藥物的特色能延長鹽酸小檗鹼作用於HepG2細胞。歸納以上實驗結果,說明鹽酸小檗鹼微脂體不改變原本鹽酸小檗鹼所誘導的凋亡路徑,可能是利用延長作用時間來增加對HepG2細胞的毒殺效果。
Nano-traditional Chinese medicine (nano-TCM) is a new packaging format hybridizing traditional Chinese medicine's effective substmces/ component (either as a crude drug alone or mixed with other pharmaceutic compounds) with the nanotechnology to produce particle size smaller than 100 nm. Nano-TCM could enhance bioavailability, control drug releasing rate, and extend medicine response time for improving therapeutic purposes. Berberine is the major therapeutic compound from Coptidis Rhizoma Franch. (huanglian). Berberine has been shown to exhibit antimicrobial, antiviral, and anti-inflammatory activities, as well as to inhibit the growth of tumor and to induce apopotosis of cancer cells. The aim of this study is to increase berberine cytotoxicity to cancer cells by encapsulating it within liposome. At first, we evaluated the effects of lipid composition and the preparation methods of berberine liposome on berberine encapsulation efficiency (E.E.) and liposomal particle size; this established the optimal berberine liposome. The results indicated that particle size and E.E. of berberine liposome containing DSPC or HSPC is uninfluenced. However, adding 5 mol% PEG could improve 3.3- 6.4 times of the E.E. of berberine when compared to liposomes without PEG. Among preparation motheds, the thin-film hydration/extrusion motheds provided the highest E.E. of berberine (14%). The cytotoxicity of berberine encapsulated liposome to HepG2 cells showed an increase. MTT test results pointed out that berberine liposome had 1.2-2.3 times of the capability to suppresse the growth of HepG2 cells than that of berberine solution alone. After the addition of berberine liposomes into HepG2 cells, the apoptotic cell percentage, using Annexin V/ PI double staining, significantly increased. The results were regulated in a time-dependent (24-72 hr) and dose-dependent (0- 20 µg/mL) manner. At the same time, berberine solution, without liposome encapsulation, could not induce apoptosis of HepG2 cells. When studying the apoptotic pathway of HepG2 cells induce by berberine solution alone or encapsulated by liposome, the mitochondrial membrane potential decreased, while caspase-3 and caspase-9 activities increased and caspase-8 was unactive. In addition, the expressions of pro-caspase-3 showed a decrease following treatment concentration and the caspase-3 substrate, PARP, was cleaved in HepG2 cells treated with more than 5 µg/mL berberine solution or its liposome. Thus, berberine solution and berberine liposome might induce apoptosis in HepG2 cells through the caspase/mitochondria dependent pathway. But comparing both treatments, there were lower mitochondrial membrane potential, more active of caspase-9 and caspase-3, fewer pro-caspase-3 expressed and more PARP cleaved in HepG2 cells treated with berberine liposome. Otherwise, the results of berberine uptake by HepG2 cells and berberine release from liposome showed that berberine liposome would not enhance berberine uptake of HepG2 cells. Liposome might protect drug and gradually release drug following time, thus prolong treatment time. The results of our study suggest that berberine liposome could extend treatment time to improve the cytotoxicity and apoptosis of HepG2 cells, without altering the apoptotic pathway.
URI: http://hdl.handle.net/11455/52095
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