【摘要】：目的本研究以鹽酸川芎嗪（ligustrazine hydrochloride，LTH）為抗腫瘤模型藥，先將其制成脂質(zhì)體（liposome，LP），，進行處方、工藝考察；再將脂質(zhì)體包入微囊（microcapsules，MC），制備成載藥脂質(zhì)體微囊（liposome-in-microcapsules，LIM）。以人肝癌細胞（HepG2）和人臍靜脈細胞（HUVECs）為細胞模型，考察鹽酸川芎嗪脂質(zhì)體對兩種細胞株的細胞毒性。 方法 1.鹽酸川芎嗪脂質(zhì)體（LTH-LP）的制備及其理化性質(zhì)考察采用硫酸銨梯度法制備LTP-LP，以包封率為指標(biāo)，采用單因素實驗和正交設(shè)計實驗優(yōu)化處方、工藝條件；并用透析法測定包封率，激光粒度及Zeta電位分析儀測粒徑和電位，采用體外透析釋放實驗考察LTH-LP的體外釋放性等理化性質(zhì)，以包封率為指標(biāo)測定不同存儲條件下LTP-LP的穩(wěn)定性。 2.鹽酸川芎嗪脂質(zhì)體細胞毒性考察采用MTT法考察LTH-LP混懸液、B-LP混懸液和LTH溶液對人肝癌HepG2細胞、人臍靜脈內(nèi)皮HUVECs細胞的增殖抑制作用。 3.鹽酸川芎嗪載脂質(zhì)體微囊（LTH-LIM）的制備及其理化性質(zhì)考察采用乳化交聯(lián)法制備LTH-LIM，以載藥量和包封率為篩選指標(biāo)，采用單因素實驗法研究殼聚糖溶液濃度、殼聚糖溶液與LTH-LP混懸液體積比、乳化劑比例和乳化劑濃度、固化劑比例等因素對LTH-LIM的載藥量和包封率的影響。采用掃描電鏡、DSC對其進行表征；采用體外透析釋放實驗考察LTH-LIM的體外釋性。 結(jié)果 1.鹽酸川芎嗪脂質(zhì)體的制備及其理化性質(zhì)考察采用優(yōu)化后的硫酸銨梯度法制備LTH-LP，通過單因素法和正交設(shè)計實驗篩選出最佳處方：大豆磷脂:膽固醇為2:1，十八胺2.00mg，45℃減壓旋蒸為均勻薄膜；硫酸銨溶液濃度為200mmol·L-1，孵化溫度60℃。通過理化性質(zhì)表征測得平均包封率最高可達82.4%，平均粒徑為173.4nm，Zeta電位為11.5mV；透射電鏡觀察，LTH-LP外觀圓整，粒徑分布較均勻；體外溶出實驗結(jié)果表明：LTH-LP中的LTH藥物釋放減慢且較完全，具有一定的緩釋效果。LTH-LP在4℃充氮氣條件下較穩(wěn)定。 2.鹽酸川芎嗪脂質(zhì)體細胞毒性考察細胞毒性實驗結(jié)果顯示：在處理時間48h后，LTP-LP對人臍靜脈內(nèi)皮HUVECs的細胞抑制作用強于人肝癌HepG2細胞的增殖抑制作用（p＜0.05）。 3.鹽酸川芎嗪載脂質(zhì)體微囊的制備及其理化性質(zhì)考察LTH-LIM制備方法為殼聚糖乳化-交聯(lián)法。最佳處方為：二甲基硅油為油相；乳化劑吐溫-80和司盤-80比例為1:1；殼聚糖冰乙酸溶液濃度為2%；固化劑25%的戊二醛用量為0.6%；水相組成比例為殼聚糖冰乙酸溶液與脂質(zhì)體混懸液比例為1:1。測得LTH-LIM的載藥量在2.925%～3.702%之間，包封率為45%～60%之間，各因素對載藥量影響較大。LTH-LP外觀圓整，粒徑分布均勻，多分布于4～8μm；DSC差熱掃描結(jié)果顯示，LTH-LP包被入微囊中；體外釋放實驗結(jié)果表明，LTH-LIM釋放較慢但不完全。 結(jié)論本研究制得的鹽酸川芎嗪脂質(zhì)體包封率高且粒徑均勻，電位穩(wěn)定，體外釋放緩慢且較完全，4℃充氮氣條件下較穩(wěn)定，LTH-LP對人臍靜脈內(nèi)皮HUVECs的細胞抑制作用強于人肝癌HepG2細胞的增殖抑制作用（p＜0.05）；鹽酸川芎嗪載脂質(zhì)體微囊載藥量較高，外觀圓整，粒徑符合實驗要求，體外釋放緩慢但不完全，最高累積釋放量僅可達35%。
[Abstract]:Objective To study the anti - tumor model of tetramethylpyrazine hydrochloride ( LTH ) and prepare liposome ( liposome , LP ) .
The liposome was encapsulated into microcapsules ( MC ) and liposome - in - liposome ( LIM ) was prepared . The cytotoxicity of tetramethylpyrazine hydrochloride liposome on two cell lines was investigated by using human hepatoma cells ( HepG2 ) and human umbilical vein cells ( HUVEC ) as cellular models .

method

1 . Preparation and physical and chemical properties of tetramethylpyrazine hydrochloride liposome ( LTH - LP ) were investigated by ammonium sulfate gradient method .
The particle size and potential of LTH - LP were determined by dialysis method . The physical and chemical properties of LTH - LP were investigated by in vitro dialysis and release experiments . The stability of LTP - LP was determined by encapsulation efficiency .

2 . The effects of LTH - LP suspension , B - LP suspension and LTH solution on the proliferation of human hepatocellular carcinoma HepG2 cells and human umbilical vein endothelial cells were investigated by MTT assay .

3 . The preparation and physical and chemical properties of LTH - LIM were investigated by emulsifying cross - linking method . The effects of concentration of chitosan solution , the ratio of chitosan solution and LTH - LP suspension , the ratio of emulsifier and the concentration of emulsifier and the ratio of curing agent on the drug loading and encapsulation efficiency of LTH - LIM were studied by means of single factor experiment .
In vitro dialysis release experiment was used to investigate the in vitro release of LTH - LIM .

Results

1 . The preparation and physical and chemical properties of tetramethylpyrazine hydrochloride liposome were investigated . LTH - LP was prepared by the optimized ammonium sulfate gradient method . The optimal prescription was selected by single factor method and orthogonal design experiment : soybean phospholipids : cholesterol 2 : 1 , octadecylamine 2.00 mg , and vacuum evaporation at 45 鈩

本文編號：2137125