CN110339371A - 靶向CD133载sPD1微泡的制备方法及在制备抑制宫颈癌的药物上的应用 - Google Patents
靶向CD133载sPD1微泡的制备方法及在制备抑制宫颈癌的药物上的应用 Download PDFInfo
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Abstract
本发明涉及一种超声联合靶向CD133载sPD1微泡的制备方法,具体是将生物素化脂质微泡滴加PE标记链酶亲和素孵育,加入再加入生物素化的CD133抗体,经孵育后离心得到靶向CD133微泡。将所得的靶向CD133微泡和sPD1质粒室温下孵育后,再用PBS冲洗,离心,即可得到靶向CD133载sPD1脂质微泡。本发明将靶向CD133载sPD1脂质微泡应用于制备抑制宫颈癌移植瘤的药物上,结果表明靶向CD133载sPD1脂质微泡具体更高的抑瘤效果,靶向CD133载sPD1脂质微泡的体积抑制率和体重抑制率分别为78.01%和72.25%。
Description
技术领域
本发明涉及一种脂质微泡,具体为超声联合靶向CD133载sPD1微泡,并将其应用于制备治疗宫颈癌。
背景技术
宫颈癌是全世界女性最常见的妇科恶性肿瘤之一,发病率仅次于乳腺癌高居妇科恶性肿瘤第二位,发病率高且逐渐趋于年轻化,严重危害女性健康。宫颈癌主要是由于人乳头瘤病毒(HPV)感染造成,目前认为高危型HPV持续感染、机体的免疫功能下降及宫颈癌局部微环境之间的相互作用,促进宫颈癌的发生发展。当前治疗方法包括手术切除、放疗、化疗。手术是治疗宫颈癌的主要方法之一,虽然手术能暂时切除病灶,但肿瘤细胞具有很强的侵袭性和转移性,病灶并不能完全切除。辅助化疗在临床上被广泛用于术前和术后治疗,能够有效地降低肿瘤分期、减少宫旁浸润,为手术提供了良好的条件,但化疗的靶向性差,易产生耐药,全身毒副作用明显。宫颈癌是放射敏感性肿瘤,放疗对于中晚期患者和复发患者的效果是得到肯定的,但其严重的并发症给治疗带来极大的困难。因此探索宫颈癌的发病机制,寻找宫颈癌治疗的新靶点,对宫颈癌的早期诊断、治疗及预后评估具有重要的临床意义。
发明内容
针对上述技术问题,本发明提供一种超声联合靶向CD133载sPD1微泡,并将其应用于制备抑制宫颈癌的药物。进一步优选方案中,应用于制备抑制U14宫颈癌细胞的药物。
所述的超声联合靶向CD133载sPD1微泡是通过薄膜水化法及机械震荡法制备的生物素化脂质微泡,具体步骤包括如下:
(1)将二硬脂酰磷脂酰胆碱(DSPC)、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-生物素(DSPE-PEG2000-Biotin)、硬脂酸—聚乙酰亚胺(PEI-600)混于玻璃试管中,60℃水浴锅预热后,涡旋状态下充入氮气使混合液成膜于试管壁;(二硬脂酰磷脂酰胆碱(DSPC)、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-生物素 (DSPE-PEG2000-Biotin)、硬脂酸—聚乙酰亚胺(PEI-600),均为美国Avanti公司购买)。二硬脂酰磷脂酰胆碱、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-生物素、硬脂酸—聚乙酰亚胺摩尔比75-90:3-6:3-6:2-6。
优选方案中二硬脂酰磷脂酰胆碱、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-生物素、硬脂酸—聚乙酰亚胺摩尔比85:5:5:5。
(2)将步骤(1)中成膜后的玻璃管抽真空(抽真空3h以上,使液体充分挥发)后,加入 Tris缓冲液,水浴超声(使玻璃管壁上的薄膜充分溶解于Tris缓冲液中,液体澄清后停止水浴超声)后置于西林瓶中密封,冲入C3F8气体,冷藏保存;
(3)将步骤(2)中冷藏后的西林瓶经振荡后得到白色乳状液体(一般震荡25-30s);将该白色乳状液体移经离心(在1000rpm的离心速度下离心3min)后弃去下层液体即为生物素化脂质微泡(下层液体为游离的脂质和杂质);
(4)向步骤(3)中生物素化脂质微泡滴加链酶亲和素(美国Avanti公司购买),在2-4℃、避光下恒温振荡孵育,得到微泡混悬液;
(5)将步骤(4)中的微泡混悬液离心(在1000rpm的离心速度下离心3min)后,弃去下清液(以除去游离的链酶亲和素)后,采用PBS缓冲液漂洗、离心、弃下清(优选方案为重复PBS缓冲液漂洗、离心、弃下清的步骤3次),得到微泡;
(6)将生物素化的CD133抗体(美国ebioscience公司购买)加入步骤(5)的微泡中,在 2-4℃、避光的恒温振荡孵育过夜后离心(在1000rpm的离心速度下离心3min),弃下清液(以除去游离的生物素化的CD133抗体),加入PBS缓冲液漂洗、离心、弃下清(优选方案为重复PBS缓冲液漂洗、离心、弃下清的步骤3次),即可得到靶向CD133微泡。
(7)将所得的靶向CD133微泡和sPD1质粒(pcDNA3.1(-)-sPD1)室温下孵育,再用PBS 冲洗,离心(在1000rpm的离心速度下离心3min),除去未结合在微泡表面的sPD1质粒,得到靶向CD133载sPD1脂质微泡,即为靶向CD133载sPD1微泡。
所得靶向微泡通过血球计数板计数,粒度仪测量粒径,显微镜下观察微泡形态;通过激光扫描共聚焦荧光显微镜观察。
所述的PE标记链酶亲和素的加入量相对于生物素化脂质微泡过量;生物素化的CD133 抗体的加入量相对于微泡过量;sPD1质粒的加入量相对于靶向CD133微泡的加入量过量。
本发明将所制备得到的靶向CD133微泡或靶向CD133载sPD1微泡在制备抑制宫颈癌的药物上的应用。
所述的靶向CD133微泡或靶向CD133载sPD1微泡在制备抑制U14宫颈癌细胞的药物上的应用。
所述的靶向CD133微泡或靶向CD133载sPD1微泡的浓度为1×107-1.5×109个/ml。
将所得靶向CD133载sPD1微泡与CD133高表达的Hela宫颈癌细胞共同孵育,经PBS反复洗涤后,光学显微镜下观察靶向CD133微泡在细胞水平上的靶向聚集性。将制备的靶向CD133载sPD1微泡和空微泡(未载入CD133抗体及sPD1质粒)尾静脉注射到荷瘤小鼠体内,通过超声诊断仪观察微泡在小鼠移植瘤内的超声成像效果。
实施本申请的技术方案所采用的小鼠和细胞系的情况如下:
SPF级BALB/c小鼠,雌性,18g左右,由三峡大学动物实验中心提供,饲养于三峡大学动物实验中心无特定病原体屏障环境中,严格SPF级实验动物操作规程。U14宫颈癌细胞瘤株,由三峡大学肿瘤微环境与免疫治疗湖北省重点实验室提供。
小鼠宫颈癌移植瘤的建立及分组处理
从荷U14宫颈癌细胞的小鼠腹腔内抽取腹水,调整U14宫颈癌细胞浓度约2×107/ml于小鼠右前腋皮下接种0.2ml,一周后形成约0.5cm皮下肿瘤结节。将成功建立的小鼠U14宫颈癌皮下移植瘤模型,随机分为5组,每组10只:对照组、空微泡组、靶向CD133微泡、靶向CD133载sPD1微泡。根据分组给予不同种类微泡制剂的注射,每两天经尾静脉注射 (0.2ml/只)及超声辐照瘤体一次,辐照条件:频率1MHZ,功率1W/cm2,占空比50%,辐照时间90s/只,其中对照组给予相同剂量的生理盐水处理,并于治疗前测量瘤体长径(a) 短径(b),按公式V=1/2ab2计算肿瘤体积,绘制肿瘤生长曲线。治疗5次后处死部分小鼠,剥离瘤体,计算肿瘤体积及质量抑瘤率。剩余小鼠继续治疗,隔天每组取一只小鼠处死,取脾脏,分离脾细胞。
本发明的试验数据采用SPSS18.0软件进行统计学分析,结果用(x±s)表示,P<0.05 为差别具有统计学意义。
附图说明
图1靶向CD133载sPD1微泡激光共聚焦图像,其中A为可见光下微泡形态,B为PE标记 (链霉亲和素)发射的红色荧光图像,C为FITC标记(抗鼠IgG)的图像,反映微泡上有CD33抗体,D为B和C图叠加。
图2为PI染色的sPD-1质粒在激光共聚焦图像反映微泡载DNA的作用,其中,A为可见光下的微泡形态,B为PI染色的微泡的图像。
图3靶向CD133载sPD1微泡稳定性试验。
图4为靶向CD133载sPD1微泡载sPD-1质粒结合能力的电泳图。
图5为靶向CD133载sPD1微泡中sPD-1质粒降解试验电泳图。
图6为靶向CD133载sPD1微泡体外靶向作用。
图7为不同组处理抑制小鼠肿瘤作用,其中,A.肿瘤体积增长曲线;B.各组小鼠体重变化。
图8为不同组小鼠的肿瘤实物图。
图9为Tunel试验反映靶向CD133载sPD1微泡联合低频超声诱导宫颈癌细胞凋亡的作用,其中A.对照,B.空微泡,C.靶向CD133微泡,D.载sPD1脂质微泡,E.靶向CD133载sPD1微泡。
具体实施方式
实施例1
靶向CD133载sPD1微泡的制备及其特征
1)将二硬脂酰磷脂酰胆碱(DSPC)、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000 (DSPE-PEG2000)、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-生物素(DSPE-PEG2000-Biotin)、硬脂酸—聚乙酰亚胺(PEI-600)按摩尔比85:5:5:5混于玻璃试管中,轻轻摇晃,充分混匀, 60℃水浴锅预热后,将玻璃试管放在涡旋振荡器上,用N2吹玻璃试管中的液体,边吹边涡旋,在玻璃管壁底部形成一层均质的薄膜。
2)将成膜后的玻璃管连接真空抽气机,抽真空3h,使液体充分挥发。
3)取出玻璃试管后,向其中加入5ml Tris缓冲液,放入超声清洗机中水浴5min,使玻璃管壁上的薄膜充分溶解于Tris缓冲液中,液体澄清后停止水浴超声。使用移液枪将其分装到5个西林瓶中并加盖密封。
4)分别向每个西林瓶中冲入C3F8气体,放入4℃冰箱保存。
5)将西林瓶固定在高速振荡仪上,调节振荡时间为30s,振荡后液体呈白色乳状液体;将液体移至离心管中,使用台式低速离心机1000rpm离心3min,离心后弃去下层液体即为普通生物素化脂质微泡,下层液体为游离的脂质和杂质。
6)取生物素化脂质微泡1ml于试管中,向其中缓慢滴加过量的PE标记链酶亲和素,在4℃、避光的恒温振荡器里孵育30min。
7)使用台式低速离心机将上述微泡混悬液以1000rpm离心3min,弃下清液以除去游离的链酶亲和素;加入PBS缓冲液漂洗、离心、弃下清,如此重复3次。
8)将生物素化的CD133单克隆抗体加入上述微泡中,在4℃、避光的恒温振荡器里孵育过夜;使用台式低速离心机将孵育后的微泡混悬液以1000rpm离心3min,弃下清液以除去游离的生物素化的CD133单克隆抗体;加入PBS缓冲液漂洗、离心、弃下清,如此重复 3次,制备出靶向CD133微泡。
9)将所得的靶向CD133微泡和sPD1质粒(pcDNA3.1(-)-sPD1)室温下孵育,再用PBS冲洗,离心(在1000rpm的离心速度下离心3min),除去未结合在微泡表面的sPD1质粒,得到靶向CD133载sPD1脂质微泡,即为靶向CD133载sPD1微泡。
靶向微泡的物理性质
本发明制备的目标阳离子微球具有空心球形,形态稳定,粒径均匀。靶向CD133载sPD1微泡(TCMB)浓度为(2.1±0.4)×108/ml。TCMB的zeta电位为(32.4±2.8)mV。平均直径为(929.7±23.9)nm。在激光共聚焦下观察目标阳离子超声微泡。A中在可见光激发下,表现出无荧光现象,B在绿色激发波下,微泡表面呈红色荧光其中,红色荧光表明 PE标记的链霉亲和素与微泡表面生物素结合。C为在蓝光激发下,微泡表面呈现绿色荧光,绿色荧光表明FITC标记的第二抗体与微泡表面的CD133抗体结合,反映微泡上有CD33 抗体。D为绿色和红色光完全重叠的情况下,形成黄色荧光(图1)。
靶向阳离子微泡的基因载量
在共聚焦显微镜下,观察到质粒可以结合TCMB表面,红色荧光表示被PI染色sPD-1质粒(图2),从图中可以看出PI染色的sPD-1质粒在激光共聚焦显微镜下显示红色荧光并且结合在TCMB周围。
图3为携带基因的TCMB的浓度随时间而变化,从第7天开始显着下降,具有统计学意义,P<0.05。
sPD-1质粒不与正常微泡(NMB)结合,但可与靶向阳离子微泡结合,1ug sPD-1质粒可与接近饱和状态的25μl TCMB孵育,表明基因携带能力约为20μg/108微泡,即25μl TCMB可以完全阻止sPD-1质粒向正极移动,而NMB则不能(图4)。众所周知,质粒直接进入血液并被体内的酶破坏,模拟机体内环境,琼脂糖凝胶电泳结果显示结合TCMB表面的sPD-1质粒在4分钟内保持完整,表明TCMB对基因有一定的保护效果(图5)。将 sPD-1质粒与微泡表面结合,然后置于4℃环境中5天,浓度未发现明显变化,表明稳定性良好。
微泡的靶向性分析
将相同浓度的NMB和TCMB(1×106/mL)与U14细胞一起孵育半小时,然后在倒置显微镜下观察微泡和细胞是否结合。结果显示:NMB基本上不与细胞结合,而TCMB可以与细胞表面紧密结合,这表明自制的靶向CD133阳离子微泡可以特异性结合U14细胞(见图6所示)。
实施例2
靶向CD133载sPD1微泡在制备抑制宫颈癌的药物上的应用
小鼠U14宫颈癌皮下移植瘤模型的建立
U14细胞接种于小鼠右前腋下10天后,可见肿瘤形成,多数呈圆形,直径7cm左右,少数不规则。50只小鼠用于实验,其中45只小鼠成功建模,成瘤率90%。小鼠生长状态良好,未见异常死亡。将模型小鼠随机分为五组,每组9只。
超声联合靶向载基因微泡对小鼠宫颈癌的抑制作用
各组小鼠的肿瘤抑瘤率
小鼠的右腋下出现了膨出的肿块。从小鼠的腋下切除的肿瘤组织是结节状的,质地稍硬,表面是假包膜的。它易于从周围组织剥离,并且可以在肿瘤组织中看到局部液化坏死(图8)。在治疗期间,小鼠存活良好,在将微泡注入尾静脉期间注射少量空气后,两只小鼠意外死亡。记录小鼠体重观察,第1天,第6天和第11天之间体重没有显着差异(图7B)。随着病程的延长,各组肿瘤继续增加,呈指数增长趋势,治疗组肿瘤生长速度不同程度减慢(图7A)。各组小鼠的肿瘤体积和重量抑制率如表1所示。可以看到每个治疗组小鼠的肿瘤体积和体重均有不同程度的降低,与对照组有统计学差异(*P<0.05)。CD133/sPD1组的体积抑制率和体重抑制率分别为78.01%和72.25%,与其他组有统计学差异(#P<0.05)(表1)。
表1不同组之间肿瘤抑制率的比较
注:与对照组相比,*P<0.05;各组间相比,#P<0.05
Tunel实验检测肿瘤组织凋亡
用Tunel试验评估负载sPD-1的TCMB联合低频超声对宫颈癌的抑制作用。红染的细胞核表明细胞处于凋亡状态,可以清楚地看到,通过不同的处理,各组小鼠的肿瘤组织中均出现了大量的凋亡细胞(图9)。与对照组相比,空微泡组细胞凋亡率无统计学差异(P>0.05)。 CD133组,sPD1组和CD133/sPD1组凋亡率增加(*P<0.05),CD133/sPD1组凋亡率增加最明显(#P<0.05)。
Claims (9)
1.靶向CD133微泡的制备方法,其特征在于,包括如下步骤:
(1)二硬脂酰磷脂酰胆碱、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-生物素、硬脂酸—聚乙酰亚胺混于玻璃试管中,50-60℃水浴锅预热后,涡旋状态下充入氮气使混合液成膜于试管壁;
(2)将步骤(1)中成膜后的玻璃管抽真空后,加入Tris缓冲液,水浴超声后置于西林瓶中密封,冲入C3F8气体,冷藏保存;
(3)将步骤(2)中冷藏后的西林瓶经振荡后得到白色乳状液体;将该白色乳状液体移经离心后弃去下层液体即为生物素化脂质微泡;
(4)向步骤(3)中生物素化脂质微泡滴加链酶亲和素,在2-4 °C、避光下恒温振荡孵育,得到微泡混悬液;
(5)将步骤(4)中的微泡混悬液离心后,弃去下清液后,采用PBS缓冲液漂洗、离心、弃下清,得到微泡;
(6)将生物素化的CD133抗体加入步骤(5)的微泡中,在2-4 °C、避光的恒温振荡孵育过夜后离心,弃下清液,加入PBS缓冲液漂洗、离心、弃下清,即可得到靶向CD133微泡。
2.靶向CD133载sPD1微泡的制备方法,其特征在于,采用权利要求1制备得到的靶向CD133微泡和sPD1质粒室温下孵育后,再用PBS冲洗,离心,即可得到靶向CD133载sPD1微泡。
3.根据权利要求1所述的靶向CD133微泡或靶向CD133载sPD1微泡的制备方法,其特征在于,二硬脂酰磷脂酰胆碱、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-生物素、硬脂酸-聚乙酰亚胺摩尔比75-90:3-6:3-6:2-6。
4.根据权利要求3所述的靶向CD133微泡或靶向CD133载sPD1微泡的制备方法,其特征在于,二硬脂酰磷脂酰胆碱、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-生物素、硬脂酸-聚乙酰亚胺摩尔比85:5:5:5。
5.根据权利要求1所述的靶向CD133微泡的制备方法,其特征在于,链酶亲和素的加入量相对于生物素化脂质微泡的加入量过量;生物素化的CD133抗体的加入量相对于微泡的加入量过量。
6.根据权利要求2所述的靶向CD133载sPD1微泡的制备方法,其特征在于,sPD1质粒的加入量相对于靶向CD133微泡的加入量过量。
7.根据权利要求1-6任一项所制备得到的靶向CD133微泡或靶向CD133载sPD1微泡在制备抑制宫颈癌的药物上的应用。
8.根据权利要求7所述的应用,其特征在于,所述的靶向CD133微泡或靶向CD133载sPD1微泡在制备抑制U14宫颈癌细胞的药物上的应用。
9.根据权利要求7所述的应用,其特征在于,所述的靶向CD133微泡或靶向CD133载sPD1微泡的浓度为1×107-1.5×109个/ml。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111249471A (zh) * | 2020-01-18 | 2020-06-09 | 广州医科大学附属第三医院(广州重症孕产妇救治中心、广州柔济医院) | 一种基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105106977A (zh) * | 2015-07-27 | 2015-12-02 | 深圳市人民医院 | 一种携带iRGD穿膜肽双靶向阳离子超声微泡制备方法 |
CN108079322A (zh) * | 2016-11-23 | 2018-05-29 | 韩会义 | 一种rgd靶向微泡造影剂 |
-
2019
- 2019-07-15 CN CN201910636105.9A patent/CN110339371B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105106977A (zh) * | 2015-07-27 | 2015-12-02 | 深圳市人民医院 | 一种携带iRGD穿膜肽双靶向阳离子超声微泡制备方法 |
CN108079322A (zh) * | 2016-11-23 | 2018-05-29 | 韩会义 | 一种rgd靶向微泡造影剂 |
Non-Patent Citations (6)
Title |
---|
XIAONAN GUO ET AL.: "The sustained and targeted treatment of hemangiomas by propranolol loaded CD133 aptamers conjugated liposomes-in-microspheres", 《BIOMEDICINE&PHARMACOTHERAPY》 * |
YAN-MIN LIU ET AL.: "Ultrasound-targeted microbubble destruction-mediated downregulation of CD133 inhibits epithelial-mesenchymal transition,stemness and migratory ability of liver cancer stem cells", 《ONCOLOGY REPORTS》 * |
刘芸等: "前列腺癌超声微泡介导药物及基因治疗研究进展", 《天津医药》 * |
秦琪等: "PD-1/PD-L1信号通路及相关抗体在宫颈癌免疫治疗中的应用", 《生命的化学》 * |
赵苗等: "超声微泡靶向介导miR-206抑制宫颈癌生长", 《中国老年学杂志》 * |
金征宇主编: "《基因与纳米探针-医学分子成像理论与实践中》", 30 November 2017, 天津科学技术出版社 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111249471A (zh) * | 2020-01-18 | 2020-06-09 | 广州医科大学附属第三医院(广州重症孕产妇救治中心、广州柔济医院) | 一种基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法 |
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