鬼臼毒素二元醇质体体外抗宫颈HPV感染的研究
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摘要
目的研究鬼臼毒素二元醇质体(POD-BE)体外抗宫颈人乳头瘤病毒(HPV)感染的效果。方法采用超声注入法制备POD-BE,采用透析法测定POD-BE包封率(EE%),观察POD-BE形态学、外观性状及稳定性,运用CCK-8法和流式细胞术(FCM)分别检测POD-BE和POD处理后永生化人宫颈上皮细胞(H8细胞)的增殖和凋亡,以透射电镜和激光共聚焦显微镜(LCSM)分别观察细胞的形态变化及药物在细胞的分布。结果制备的POD-BE混悬液电镜下为类球状小囊泡,平均粒径(84.6±9.2)nm,Zeta电位(-27.2±4.2)mV。保存在4℃冰箱内的POD-BE样本,6个月内仍保持半透明、质地均匀外观,未见沉淀及药物结晶析出。3000 r/min离心20 min未发现分层现象。而保存在室温下的样本,24 h未见任何变化,3~6个月时出现少许混浊,震荡后样本恢复均一外观,未见药物结晶析出。POD-BE组和POD组均呈剂量和时间依赖性地抑制H8细胞的增殖。在相同浓度同一时间条件下,POD-BE组对H8细胞的增殖抑制效果明显强于POD组(P<0.01)。POD-BE组12、24、48 h的半数抑制浓度(IC50)分别为782、595、97μg/L,而POD组的IC50则分别为1966、1570、758μg/L;空白BE组对细胞无明显毒性。与空白对照组相比,POD-BE组和POD组均能明显诱导H8细胞凋亡(P<0.01);然而POD-BE组诱导凋亡的效果则显著强于POD组(P<0.01)。10μg/L的POD-BE和POD分别处理H8细胞24 h后,透射电镜下显示:H8细胞被药物处理后其染色质高度凝聚、边缘化,形成凋亡小体。H8细胞分别被POD-BE和POD处理24 h后,两组的荧光均表现出聚集于细胞核的趋势,且POD-BE组较POD组荧光信号强度显著增强,而空白BE组和空白对照组未见有阳性荧光信号表达。结论 POD-BE具备理想的理化性质,POD-BE较POD对H8细胞具有更强的增殖抑制和凋亡诱导效果。
Objective To research effect by podophyllotoxin binary ethosomes(POD-BE) in external anti-cervical human papilloma virus(HPV) infection. Methods POD-BE was prepared by ultrasonic injection method, and encapsulation efficiency(EE%) of POD-BE was detected by dialysis method. Observation was made on morphology, external properties and stability of POD-BE. CCK-8 method and flow cytometry method(FCM) were applied to detect POD-BE and multiplication and apoptosis of immortalized human cervical epithelial cell(H8 cell) after POD treatment. Transmission electron microscope and laser scanning confocal microscope(LCSM) were applied to respectively observe morphologic change and drug distribution of cell. Results Prepared POD-BE suspension showed globular small vesicae under electron microscope, with mean grain diameter as(84.6±9.2) mm and Zeta electric potential as(-27.2±4.2) m V. POD-BE samples in 4℃ refrigerator showed semitransparent and balanced appearance without sediment and drug crystal separation. No stratification appeared after 20 min of centrifugation by 3000 r/min. Samples in indoor temperature showed no changes after 24 h, while there was a few suspension in 3~6 months without drug crystal separation after shock. Both POD-BE group and POD group showed H8 cell proliferation inhibition with dosage and time dependence. Under the condition of same concentration, POD-BE group showed stronger proliferation inhibition effect on H8 cell than POD group(P<0.01). POD-BE group had median inhibitory concentration(IC50) in 12, 24 and 48 h respectively as 782, 595 and 97 μg/L, which were respectively 1966, 1570 and 758 μg/L in POD group. Blank BE group showed no obvious cytotoxicity. Comparing with blank control group, POD-BE group and POD group showed obvious induction on H8 apoptosis(P<0.01), and POD-BE group had stronger apoptosis induction effect than POD group(P<0.01). After H8 treated by POD-BE and POD respectively by 10 μg/L for 24 h, transmission electron microscope showed high chromatin condensation, marginalization and formed apoptotic body in H8 cell. After 24 h treatment by POD-BE and POD in H8 cell, both groups showed fluorescence focused on cell nucleus, and POD-BE group had obviously stronger fluorescence signal than POD group, while there was no positive fluorescence signal expression in blank BE group and blank control group. Conclusion Due to its ideal physicochemical property, POD-BE provides more stronger proliferation inhibition and apoptosis induction effects on H8 cell than POD.
Objective To research effect by podophyllotoxin binary ethosomes(POD-BE) in external anti-cervical human papilloma virus(HPV) infection. Methods POD-BE was prepared by ultrasonic injection method, and encapsulation efficiency(EE%) of POD-BE was detected by dialysis method. Observation was made on morphology, external properties and stability of POD-BE. CCK-8 method and flow cytometry method(FCM) were applied to detect POD-BE and multiplication and apoptosis of immortalized human cervical epithelial cell(H8 cell) after POD treatment. Transmission electron microscope and laser scanning confocal microscope(LCSM) were applied to respectively observe morphologic change and drug distribution of cell. Results Prepared POD-BE suspension showed globular small vesicae under electron microscope, with mean grain diameter as(84.6±9.2) mm and Zeta electric potential as(-27.2±4.2) m V. POD-BE samples in 4℃ refrigerator showed semitransparent and balanced appearance without sediment and drug crystal separation. No stratification appeared after 20 min of centrifugation by 3000 r/min. Samples in indoor temperature showed no changes after 24 h, while there was a few suspension in 3~6 months without drug crystal separation after shock. Both POD-BE group and POD group showed H8 cell proliferation inhibition with dosage and time dependence. Under the condition of same concentration, POD-BE group showed stronger proliferation inhibition effect on H8 cell than POD group(P<0.01). POD-BE group had median inhibitory concentration(IC50) in 12, 24 and 48 h respectively as 782, 595 and 97 μg/L, which were respectively 1966, 1570 and 758 μg/L in POD group. Blank BE group showed no obvious cytotoxicity. Comparing with blank control group, POD-BE group and POD group showed obvious induction on H8 apoptosis(P<0.01), and POD-BE group had stronger apoptosis induction effect than POD group(P<0.01). After H8 treated by POD-BE and POD respectively by 10 μg/L for 24 h, transmission electron microscope showed high chromatin condensation, marginalization and formed apoptotic body in H8 cell. After 24 h treatment by POD-BE and POD in H8 cell, both groups showed fluorescence focused on cell nucleus, and POD-BE group had obviously stronger fluorescence signal than POD group, while there was no positive fluorescence signal expression in blank BE group and blank control group. Conclusion Due to its ideal physicochemical property, POD-BE provides more stronger proliferation inhibition and apoptosis induction effects on H8 cell than POD.
引文
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[8]曾抗,张三泉,江彬彬,等.脂质体鬼臼毒素混悬液在大鼠表皮及真皮的分布规律初探.中华皮肤科杂志,2003,36(6):329-331.
[9]谢方明,曾抗,陈志良,等.鬼臼毒素固体脂质纳米粒凝胶治疗复发性尖锐湿疣的随机双盲对照研究.南方医科大学学报,2007,27(5):657-659.
[10]Meng S,Chen Z,Yang L,et al.Enhanced transdermal bioavailability of testosterone propionate via surfactant-modified ethosomes.Int J Nanomedicine,2013(8):3051-3060.
[11]Li G,Fan C,Li X,et al.Preparation and in vitro evaluation of tacrolimus-loaded ethosomes.Scientific World Journal,2012(2012):874053.
[12]Zhang JP,Wei YH,Zhou Y,et al.Ethosomes,binary ethosomes and transfersomes of terbinafine hydrochloride:a comparative study.Arch Pharm Res,2012,35(1):109-117.
[2]Goindi S,Dhatt B,Kaur A.Ethosomes-based topical delivery system of antihistaminic drug for treatment of skin allergies.J Microencapsul,2014,31(7):716-724.
[3]朱晓亮,曾抗,李国锋,等.脂质体鬼臼毒素混悬液的制备及包封率测定.中国医院药学杂志,2005,25(6):569-570.
[4]王军,何文.酮洛芬二元醇质体凝胶的研制及其质量考察.中国药师,2012,15(6):780-782.
[5]肖顺汉,姚健,刘明华,等.皂角刺总黄酮诱导HCT116细胞凋亡的透射电镜观察.泸州医学院学报,2010,33(2):116-118.
[6]Godin B,Touitou E.Erythromycin ethosomal systems:physicochemical characterization and enhanced antibacterial activity.Current Drug Delivery,2005,2(3):269-275.
[7]于燕燕,赵继会,冯年平,等.鬼臼毒素醇质体的体外经皮渗透特性研究.中草药,2012,43(1):74-77.
[8]曾抗,张三泉,江彬彬,等.脂质体鬼臼毒素混悬液在大鼠表皮及真皮的分布规律初探.中华皮肤科杂志,2003,36(6):329-331.
[9]谢方明,曾抗,陈志良,等.鬼臼毒素固体脂质纳米粒凝胶治疗复发性尖锐湿疣的随机双盲对照研究.南方医科大学学报,2007,27(5):657-659.
[10]Meng S,Chen Z,Yang L,et al.Enhanced transdermal bioavailability of testosterone propionate via surfactant-modified ethosomes.Int J Nanomedicine,2013(8):3051-3060.
[11]Li G,Fan C,Li X,et al.Preparation and in vitro evaluation of tacrolimus-loaded ethosomes.Scientific World Journal,2012(2012):874053.
[12]Zhang JP,Wei YH,Zhou Y,et al.Ethosomes,binary ethosomes and transfersomes of terbinafine hydrochloride:a comparative study.Arch Pharm Res,2012,35(1):109-117.