CN114681429B - 一种细菌外膜囊泡包被的具有光热-化疗-免疫功能的纳米粒子的制备方法 - Google Patents
一种细菌外膜囊泡包被的具有光热-化疗-免疫功能的纳米粒子的制备方法 Download PDFInfo
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Abstract
本发明涉及生物医药领域,公开了一种细菌外膜囊泡包被的具有光热‑化疗‑免疫功能的纳米粒子的制备方法。本发明首先通过液相机械剥离法和差速离心得到锑烯纳米颗粒,再利用二硬脂酰基磷脂酰乙醇胺‑聚乙二醇和氟尿嘧啶包覆在锑烯纳米颗粒表面,最后用细菌外膜囊泡包覆锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺‑聚乙二醇‑氟尿嘧啶,得到细菌外膜囊泡包覆的锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺‑聚乙二醇‑氟尿嘧啶。该方法得到的纳米粒子具有多功能性,可以实现光声成像,光热/光动力/化疗/免疫反应四协同治疗,可增加以锑烯为基体的纳米药物的稳定性,增强抗肿瘤的效果,有望实际运用于肿瘤治疗。
Description
技术领域
本发明涉及生物医药领域,尤其涉及一种细菌外膜囊泡包被的具有光热-化疗-免疫功能的纳米粒子的制备方法。
背景技术
超薄二维(2D)纳米材料是一种新型纳米材料。由于2D纳米材料的大表面体积、高药物和基因负载、高光热转换效率和光动力特性,使它们在药物和基因传递、光热治疗(PTT)、光动力治疗(PDT)、磁共振成像(MRI)、光声断层成像(PAT)、计算机断层成像(CT)等个性化癌症纳米医学领域具有巨大的应用潜力。锑烯(AM)作为一种新型二维材料,自2015年首次报道以来,因其优异的理化性质引起了研究人员广泛的关注。经研究发现锑烯在近红外区具有优异的光热转换效率,高达45.5%,在抗肿瘤治疗具有良好的应用前景。
虽然锑烯纳米材料是最近出现的一种新的光热治疗剂,但它在生理介质中的快速降解极大地限制了它的直接利用,在肿瘤治疗方面鲜有报道;此外,锑烯表面活性基团少,化学改性难度大,而且通过范德华力、氢键疏水作用结合得到的产物稳定性差。
发明内容
为了解决上述技术问题,本发明提供了一种细菌外膜囊泡包被的具有光热-化疗-免疫功能的纳米粒子的制备方法。本发明首先通过液相机械剥离法和差速离心得到锑烯纳米颗粒,再利用二硬脂酰基磷脂酰乙醇胺-聚乙二醇和氟尿嘧啶包覆在锑烯纳米颗粒表面,最后用细菌外膜囊泡包覆锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶,得到细菌外膜囊泡包覆的锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶。该方法得到的纳米粒子具有多功能性,可以实现光声成像,光热/光动力/化疗/免疫反应四协同治疗,可增加以锑烯为基体的纳米药物的稳定性,增强抗肿瘤的效果,有望实际运用于肿瘤治疗。
本发明的具体技术方案为:
一种细菌外膜囊泡包被的具有光热-化疗-免疫功能的纳米粒子的制备方法,包括如下步骤:
(1)二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液的配制:将二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶于去离子水,配制成1-2mg/mL的二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液,冷藏备用。
(2)细菌外膜囊泡的制备:采用超滤离心法制备细菌外膜囊泡。
(3)氟尿嘧啶溶液的配制:将氟尿嘧啶溶于去离子水,配制成1-2mg/mL的氟尿嘧啶溶液,锡纸包封后冷藏备用。
(4)锑烯纳米颗粒分散液的制备:取锑粉2-4g分散于40-80mL N-甲基-2-吡咯烷酮中,对容器封口后,用超声波探头冰浴超声处理,接着在超声清洗器中冰浴超声处理,最后用超声波细胞粉碎仪超声处理;8-10℃下将所得分散液离心,弃去沉积的块状锑,再离心,收集沉淀,加入10-15mL去离子水得到锑烯纳米颗粒分散液,冷藏备用。
(5)将步骤(4)所得锑烯纳米颗粒分散液进行稀释后配制成浓度为1-2mg/mL的锑烯纳米颗粒稀释分散液。
(6)取0.5-1mL二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液和0.5-1mL 氟尿嘧啶溶液加入到0.5-1ml 锑烯纳米颗粒分散液中,搅拌,离心,收集沉淀,加入1-2ml去离子水得到锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶分散液。
二硬脂酰基磷脂酰乙醇胺-聚乙二醇可以和细菌外膜囊泡表面的脂质结合,聚乙二醇化修饰可以降低细菌外膜囊泡的免疫原性,提高其胶体稳定性。
(7)包覆细菌外膜囊泡:用mini-extruder通过聚碳酸酯膜挤压细菌外膜囊泡与二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶分散液的混合液,在1-5℃下进行离心,去除上清液后重悬,制备得到细菌外膜囊泡包覆的锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶。
本发明首先通过液相机械剥离法和差速离心得到锑烯纳米颗粒,再利用二硬脂酰基磷脂酰乙醇胺-聚乙二醇和氟尿嘧啶包覆在锑烯纳米颗粒表面,最后用细菌外膜囊泡包覆锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶,得到细菌外膜囊泡包覆的锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶。该方法得到的纳米粒子具有多功能性,可以实现光声成像,光热/光动力/化疗/免疫反应四协同治疗,可增加以锑烯为基体的纳米药物的稳定性,增强抗肿瘤的效果。
锑与磷同族,形态功能相似。锑烯具有与黑磷相似的蜂窝结构,可以用来装载药物和输送,具有优异的光热转换效率,其环境稳定性显著好于BP。除此之外,锑烯纳米颗粒具有优异的声光性能,在2D材料家族中表现出最强的光声信号,这一特点赋予了其光声成像性能;并且锑烯纳米颗粒在X射线照射下会转换为对癌细胞有特异性细胞毒性的Sb2O3和Sb2O5,Sb2O3通过诱导活性氧的过度产生和线粒体破坏而发挥强细胞毒性作用,这一特点赋予了锑烯纳米颗粒在光动力治疗肿瘤中的潜力;锑烯的自发氧化加速了光能的释放,通过加速非辐射跃迁速率进一步提高光热转换,同时还拓宽了近红外区的吸收范围,这一特点赋予了锑烯纳米颗粒在光热治疗肿瘤中的潜力。
基于锑烯的上述众多功能,本发明引入生物相容性和生物降解性良好的二硬脂酰基磷脂酰乙醇胺-聚乙二醇,提高其在生理环境下的生物相容性和纳米粒子的结构稳定性,使其成功负载氟尿嘧啶,进一步优化锑烯的生物学特性,最后用细菌外膜囊泡包覆此载药平台,最终得到的纳米药物同时具有光动力/光热/化疗/免疫四协同治疗效果以及多模成像特性(荧光/光声/光热成像),为抗肿瘤治疗的纳米药物研究提供新方向。
作为优选,步骤(4)中,首次离心转速为2000-4000rpm,离心时间为10-15min;第二次离心转速为8000-12000rpm,离心时间为30-35min;离心温度不超过10℃。
作为优选,步骤(4)中,所述超声波探头的规格为φ4-8,冰浴超声时间为8-10h,功率为800-1200w,每超声2-4s停1s;所述超声清洗器冰浴超声的时间为2-3h,功率为200-400w;所述超声波细胞粉碎仪冰浴超声时间为2-3h。
作为优选,步骤(1)、步骤(3)和步骤(4)中,所述冷藏的温度为1-5℃。
作为优选,步骤(6)中,所述搅拌的时间为8-12h;离心转速为10000-12000rpm,离心时间为3-5min。
作为优选,步骤(7)中,所述聚碳酸酯膜厚度为50-150nm,细菌外膜囊泡与锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶的质量比为1-2:1,挤出次数为10-20,制备温度为20-30℃,每次离心时间为3-7min,离心转速为12000-15000xg。
与现有技术对比,本发明的有益效果是:
(1)本发明通过液相剥离和差速离心法成功制备得到锑烯纳米颗粒。
(2)良好的生物相容性、生物安全性和生理环境稳定性是考察光热剂优异性的重要指标。本发明利用静电吸附作用在锑烯纳米颗粒外面包覆二硬脂酰基磷脂酰乙醇胺-聚乙二醇,不仅提高纳米颗粒的生物相容性和结构稳定性,还减缓了该材料在生理环境下的降解,即提高了其在生理环境稳定性。
(3)相较于石墨烯纳米片、黑磷、过渡金属硫化物纳米片,锑烯纳米颗粒不仅具有更高的光热转换效率,光热性能优异,锑烯的自发氧化性使其具备光动力性能,赋予材料光热和光动力协同治疗效果,同时,材料表面还负载了氟尿嘧啶,大大增强纳米药物的抗肿瘤效果。外表面用细菌外膜囊泡包覆,细菌外膜囊泡可以激活宿主免疫反应进行癌症免疫治疗,进一步优化锑烯的生物学特性,提高了血液相容性,使纳米材料具备肿瘤靶向和药物缓释功能。
(4)本发明使用的锑烯具备光声成像的功能,,因此,赋予了材料荧光热和光声成像的效果,大大提高了纳米药物局部治疗的精准性。
(5)本发明使用细菌外膜囊泡包覆载药平台,细菌外膜囊泡可以激活宿主免疫反应进行癌症免疫治疗。
具体实施方式
下面结合实施例对本发明作进一步的描述。
实施例1
(1)二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液配制:称取20ml二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液加入20ml去离子水,配制成1mg/mL的二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液,置于冰箱中4℃下保存。
(2)细菌外膜囊泡的制备:采用超滤离心法制备细菌外膜囊泡。500 mL细菌培养液在4℃下以5 000xg离心10 min后,收集上清液,再用0.45umn的滤膜过滤,滤液用截留相对分子质量为100 000的超滤离心管浓缩。浓缩液在4℃下超速离心(1500xg)4 h,沉淀物加50mmolL的HEPES (pH6.8)缓冲液重悬,于-20℃保存。采用BCA蛋白浓度测定试剂盒测定细菌外膜囊泡蛋白浓度,采用鲎试剂测定细菌外膜囊泡的内毒素含量,严格按照说明书进行操作。
(3)氟尿嘧啶溶液配制:称取10mg氟尿嘧啶溶于10ml去离子水中,配制成1mg/mL的氟尿嘧啶溶液,用锡纸包封置于冰箱中4℃下保存。
(4)AMNPs制备:室温下取2g锑粉分散于装有40ml NMP的干净烧杯中,保鲜膜封口,超声波细胞粉碎仪下用φ6超声波探头冰浴超声8h,功率为1000w,每超声3s停1s,每隔30min用玻璃棒搅拌分散液使其均匀分散,同时观察冰浴中冰块是否溶解,并及时补充冰块,接着在超声清洗器中冰浴超声2h,功率为300w,再用超声波细胞粉碎仪超声2h;最后进行离心:8℃下将得到的分散液以3000rpm转速离心10min,弃去沉积的块状锑,保留上清液,将上清液以8000rpm的转速离心45min后收集沉淀,加入10ml去离子水得到锑烯纳米颗粒(AMNPs)分散液,并将其放置于4℃下保存。
适当稀释后在紫外-可见光分析仪测定该AMNPs分散液在808nm波长下的吸光度,根据锑标准曲线计算得到该方法制备的AMNPs分散液中锑的浓度为7mg/mL,稀释7倍后得到1mg/mL的AMNPs分散液用于后续实验。
(5)二硬脂酰基磷脂酰乙醇胺-聚乙二醇功能化AMNPs:取0.5mL二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液和1mLAMNPs分散液,搅拌,离心,收集沉淀,加入2ml去离子水得到AMNPs@二硬脂酰基磷脂酰乙醇胺-聚乙二醇纳米颗粒分散液。
(6)负载氟尿嘧啶:取1ml的氟尿嘧啶溶液加入到上述得到的纳米颗粒分散液,磁力搅拌12h,陈化过夜后以10000rpm的转速离心5min后,加入2ml去离子水超声分散后以同样的离心参数再进行一次离心,收集沉淀,加入1ml去离子水,最终得1mg/mL的载药纳米粒子分散液,冷藏于4℃冰箱。
(7)包覆细菌外膜囊泡:用mini-extruder通过100 nm的聚碳酸酯膜挤压细菌外膜囊泡与AMNPs@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶的混合液(质量比为1.5:1),挤出次数为13次,制备温度为25℃。在4℃下,样品以14000xg离心5 min,去除上清液后重悬,制备得到细菌外膜囊泡包覆的AMNPs@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶。
实施例2
(1)二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液配制:称取20ml 二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液加入10ml去离子水,配制成2mg/mL的二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液,置于冰箱中4℃下保存。
(2)细菌外膜囊泡(细菌外膜囊泡)的制备:采用超滤离心法制备细菌外膜囊泡。500 m L细菌培养液在4℃下以5 000xg离心10 min后,收集上清液,再用0.45umn的滤膜过滤,滤液用截留相对分子质量为100 000的超滤离心管浓缩。浓缩液在4℃下超速离心(1500xg)4 h,沉淀物加50 mmolL的4-羟乙基哌嗪乙磺酸 (p H6.8)缓冲液重悬,于-20℃保存。采用BCA蛋白浓度测定试剂盒测定细菌外膜囊泡蛋白浓度,采用鲎试剂测定细菌外膜囊泡的内毒素含量,严格按照说明书进行操作。
(3)氟尿嘧啶溶液配制:称取20mg氟尿嘧啶溶于10ml去离子水中,配制成2mg/mL的氟尿嘧啶溶液,用锡纸包封置于冰箱中4℃下保存
(4)锑烯纳米颗粒制备:室温下取4g锑粉分散于装有80ml NMP的干净烧杯中,保鲜膜封口,超声波细胞粉碎仪下用φ6超声波探头冰浴超声10h,功率为1000w,每超声3s停1s,每隔30min用玻璃棒搅拌分散液使其均匀分散,同时观察冰浴中冰块是否溶解,并及时补充冰块,接着在超声清洗器中冰浴超声3h,功率为300w,再用超声波细胞粉碎仪超声3h;最后进行离心:10℃下将得到的分散液以3000rpm转速离心15min,弃去沉积的块状锑,保留上清液,将上清液以12000rpm的转速离心30min后收集沉淀,加入15ml去离子水得到锑烯纳米颗粒分散液,并将其放置于4℃下保存。
适当稀释后在紫外-可见光分析仪测定该锑烯纳米颗粒分散液在808nm波长下的吸光度,根据锑标准曲线计算得到该方法制备的锑烯纳米颗粒分散液中锑的浓度为8mg/mL,稀释4倍后得到2mg/mL的锑烯纳米颗粒分散液用于后续实验。
(5)二硬脂酰基磷脂酰乙醇胺-聚乙二醇功能化锑烯纳米颗粒:取0.5mL二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液和1mL锑烯纳米颗粒分散液,搅拌,离心,收集沉淀,加入1.5ml去离子水得到AMNPs@二硬脂酰基磷脂酰乙醇胺-聚乙二醇纳米颗粒分散液。
(6)负载氟尿嘧啶:取1ml的氟尿嘧啶溶液加入到上述得到的纳米颗粒分散液,磁力搅拌12h,陈化过夜后以10000rpm的转速离心5min后,加入2ml去离子水超声分散后以同样的离心参数再进行一次离心,收集沉淀,加入1ml去离子水,最终得1mg/mL的载药纳米粒子分散液,冷藏于4℃冰箱。
(7)包覆细菌外膜囊泡:用mini-extruder通过100 nm的聚碳酸酯膜挤压细菌外膜囊泡与AMNPs@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶的混合液(质量比为1.5:1),挤出次数为13次,制备温度为25℃。在4℃下,样品以14000xg离心5 min,去除上清液后重悬,制备得到细菌外膜囊泡包覆的AMNPs@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶。
本发明中所用原料、设备,若无特别说明,均为本领域的常用原料、设备;本发明中所用方法,若无特别说明,均为本领域的常规方法。
以上所述,仅是本发明的较佳实施例,并非对本发明作任何限制,凡是根据本发明技术实质对以上实施例所作的任何简单修改、变更以及等效变换,均仍属于本发明技术方案的保护范围。
Claims (7)
1.一种细菌外膜囊泡包被的具有光热-化疗-免疫功能的纳米粒子的制备方法,其特征在于包括如下步骤:
(1)二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液的配制:将二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶于去离子水,配制成1-2mg/mL的二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液,冷藏备用;
(2)细菌外膜囊泡的制备:采用超滤离心法制备细菌外膜囊泡;
(3)氟尿嘧啶溶液的配制:将氟尿嘧啶溶于去离子水,配制成1-2mg/mL的氟尿嘧啶溶液,锡纸包封后冷藏备用;
(4)锑烯纳米颗粒分散液的制备:取锑粉2-4g分散于40-80mL N-甲基-2-吡咯烷酮中,对容器封口后,用超声波探头冰浴超声处理,接着在超声清洗器中冰浴超声处理,最后用超声波细胞粉碎仪超声处理;8-10℃下将所得分散液离心,弃去沉积的块状锑,再离心,收集沉淀,加入10-15mL去离子水得到锑烯纳米颗粒分散液,冷藏备用;
(5)将步骤(4)所得锑烯纳米颗粒分散液进行稀释后配制成浓度为1-2mg/mL的锑烯纳米颗粒稀释分散液;
(6)取0.5-1mL二硬脂酰基磷脂酰乙醇胺-聚乙二醇溶液和0.5-1mL氟尿嘧啶溶液加入到0.5-1ml 锑烯纳米颗粒稀释分散液中,搅拌,离心,收集沉淀,加入1-2ml去离子水得到锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶分散液;
(7)包覆细菌外膜囊泡:用mini-extruder通过聚碳酸酯膜挤压细菌外膜囊泡与二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶分散液的混合液,在1-5℃下进行离心,去除上清液后重悬,制备得到细菌外膜囊泡包覆的锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶。
2.如权利要求1所述的制备方法,其特征在于,步骤(4)中,首次离心转速为2000-4000rpm,离心时间为10-15min;第二次离心转速为8000-12000rpm,离心时间为30-35min;离心温度不超过10℃。
3.如权利要求1所述的制备方法,其特征在于,步骤(4)中,所述超声波探头的规格为φ4-8,冰浴超声时间为8-10h,功率为800-1200w,每超声2-4s停1s;所述超声清洗器冰浴超声的时间为2-3h,功率为200-400w;所述超声波细胞粉碎仪冰浴超声时间为2-3h。
4.如权利要求1所述的制备方法,其特征在于,步骤(1)、步骤(3)和步骤(4)中,所述冷藏的温度为1-5℃。
5.如权利要求1所述的制备方法,其特征在于,步骤(6)中,所述搅拌的时间为8-12h;离心转速为10000-12000rpm,离心时间为3-5min。
6.如权利要求1所述的制备方法,其特征在于,步骤(7)中,细菌外膜囊泡与锑烯纳米颗粒@二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氟尿嘧啶的质量比为1-2:1。
7.如权利要求1所述的制备方法,其特征在于,步骤(7)中,所述聚碳酸酯膜厚度为50-150nm,挤出次数为10-20,制备温度为20-30℃,每次离心时间为3-7min,离心转速为12000-15000xg。
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