CN111012924A - 一种基于牛奶外泌体的靶向载药体系 - Google Patents
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Abstract
本发明属于制药工程领域,具体涉及一种基于牛奶外泌体的靶向载药体系。本发明公开了一种基于牛奶外泌体的靶向载药体系,构建方法为,将一端为脂链的靶向配体修饰到载药牛奶外泌体表面,所述靶向配体另一端为可特异性结合肿瘤细胞表面过表达的受体的分子。本发明中被修饰的载药牛奶外泌体可以靶向结合癌细胞,从而增强被癌细胞的摄取,达到特异杀伤肿瘤细胞的效果。
Description
技术领域
本发明属于制药工程领域,具体涉及一种基于牛奶外泌体的靶向载药体系。
背景技术
外泌体是由细胞分泌的天然纳米级别囊泡,可以起到细胞间通讯的作用,其中空的结构以及良好的生物相容性使得外泌体被广泛用于纳米载药体系。由于细胞培养带来的复杂操作及高成本,限制了外泌体的大批量生产。研究表明,提取牛奶中的外泌体可以解决这一问题,甚至由于牛奶外泌体对胃肠环境的耐受性,使得其具备口服递药的潜力。
载药体系的靶向修饰通常选用可与肿瘤细胞表面过表达受体进行特异性结合的分子,将其锚定在载药体系表面,达到将药物靶向递送至肿瘤的效果。
因此,靶向修饰牛奶外泌体对建立纳米载药体系具有重要意义。
发明内容
基于上述问题,本发明提供了一种基于牛奶外泌体的靶向载药体系,所述靶向载药系包括疏水性药物,所述疏水性药物被牛奶外泌体装载,所述牛奶外泌体外部连接有靶向配体,所述靶向配体具有脂链端和靶向端。
在一种实施例中,所述靶向配体的脂链端包括二硬脂酰基磷脂酰乙醇胺(DSPE)、二硬脂酰磷脂酰胆碱(DSPC)、大豆磷脂酰胆碱(SPC);
所述靶向配体的靶向端包括叶酸(FA)、透明质酸(HA)、核酸适配体AS1411。
在一种实施例中,所述靶向配体脂链端和靶向端通过聚乙二醇(PEG2000)连接。
在一种实施例中,所述靶向配体通过酰胺缩合反应所制得。
在一种实施例中,所述疏水性药物包括化学药物、基因药物。
所述化学药物包括广谱抗癌药;基因药物包括肿瘤生长抑制基因。
在一种实施例中,所述化学药物包括阿霉素;所述基因药物包括miRNA-204。
本发明的另一个目的在于,提供一种于牛奶外泌体的靶向载药体系的制备方法,制备方法包括:
S1,靶向配体的制备;
S2,牛奶外泌体装载疏水性药物,得到载药牛奶外泌体;
S3,将S2中的载药牛奶外泌体装载靶向配体,得到所述基于牛奶外泌体的靶向载药体系。
在一种实施例中,S2、S3中所述装载的方法为孵育。
在一种实施例中,所述牛奶外泌体的制备方法:通过2~4M的盐酸调节牛奶的pH值为4.55~4.64,用离心力为0.7×104~1.0×104g进行离心去除杂蛋白后,用离心力为11.0×104~13.5×104g进行离心富集牛奶外泌体。
在一种实施例中,所述S1制备的靶向配体是通过酰胺缩合反应得到,并且通过LH20柱分离纯化。
有益效果
与现有研究相比,本发明通过具有脂链端和靶向端的靶向配体修饰到载药牛奶外泌体表面,提供了一种基于牛奶外泌体的靶向载药体系及其制备方法,可以增强对肿瘤细胞的结合亲和力,实现药物在肿瘤细胞中的有效积累。本发明所构建的牛奶外泌体靶向载药体系,与普通载药体系相比具有更高的生物相容性,有望增强牛奶外泌体口服给药的药效,可以促进牛奶外泌体这一天然易得的纳米载体在肿瘤靶向治疗中的应用。
附图说明
图1是牛奶外泌体的电镜图;
图2是动态光散射法测量的牛奶外泌体的粒径分布及表面电位;
图3是靶向修饰后的载药牛奶外泌体的粒径分布及表面电位,A图是透明质酸修饰后的载药牛奶外泌体的粒径分布及表面电位,B图是叶酸修饰后的载药牛奶外泌体的粒径分布及表面电位;
图4是细胞摄取荧光,定性分析透明质酸牛奶外泌体靶向载药体系对肿瘤细胞和普通细胞的结合能力,A图是MDA-MB-231细胞,B图是MCF-7细胞,C图是A549细胞,D图是HEK293细胞。
图5是细胞摄取荧光,定性分析叶酸牛奶外泌体靶向载药体系对肿瘤细胞和普通细胞的结合能力,A图是HT-29细胞,B图是MCF-7细胞,C图是HEK293细胞。
图6是流式细胞术法定量分析透明质酸牛奶外泌体靶向载药体系对肿瘤细胞的结合能力。
图7是流式细胞术法定量分析叶酸牛奶外泌体靶向载药体系对肿瘤细胞的结合能力。
图8是CCK8法测定透明质酸牛奶外泌体靶向载药体系对细胞的生长抑制能力,A图是MDA-MB-231细胞,B图是MCF-7细胞,C图是A549细胞,D图是HEK293细胞。
图9是CCK8法测定叶酸牛奶外泌体靶向载药体系对肿瘤细胞HT-29的生长抑制能力。
具体实施方式
下面结合实施例对本发明做进一步说明,但应当理解这些实施例并非限制本发明的范围。
实施例1透明质酸修饰的牛奶外泌体载药体系
在此实施例中,具有肿瘤靶向功能的分子为透明质酸(HA),装载药物为阿霉素。
靶向配体的制备
取45mg的HA(MW 30kDa)与0.5mgEDCI(1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐)、0.2mgNHS(N-羟基琥珀酰亚胺)于4℃反应4h,形成HA的活化酯形式,再与4.1mg的DSPE-PEG2000-NH2(二棕榈酰磷脂酰乙醇胺-聚乙二醇-氨基,华腾制药公司购买。)于室温反应12h,溶剂为DMF,反应后的溶液用氮气吹干,重新溶解于PBS中,并用LH20自装柱纯化,纯化产物经核磁验证后分装保存至-20℃,得到靶向配体DSPE-PEG2000-HA。
牛奶外泌体的制备
牛奶分装,13,000g,4℃,离心30分钟,离心后,将上层脂肪、底部酪蛋白残渣和细胞碎片除去,收集中间部分的乳液得到脱脂奶;将脱脂奶与蒸馏水按体积比1:1进行稀释;使用2M盐酸,将溶液pH值调整到4.6,使溶液中的酪蛋白析出;将其均量分装,10,000g,离心40分钟(离心力为0.7×104g),弃除底部蛋白质沉淀,收集上层乳清并将其过0.22μm的滤膜,减少可能夹杂的杂蛋白;得到的乳清在超高速冷冻离心机中,135,000g,离心1小时(离心力为13.5×104g),收获沉淀。PBS预冷,并洗涤沉淀3次,再用适量的PBS将其重悬,即牛奶外泌体。
用透射电镜观察及动态光散射测量粒径和电位,结果分别如图1和图2所示,牛奶外泌体显示“杯托状”,水合粒径为123.9nm,电位-4.24mV,生物学性质良好。
牛奶外泌体装载阿霉素及表征
牛奶外泌体与阿霉素以质量比5:1进行装载,37℃避光孵育30min。用10000Da的超滤管7500rpm超滤10min进行纯化,得到的样品用NanoDrop仪器于480nm处检测吸光值,依据阿霉素标准曲线计算浓度和包封率,包封率计算公式如下。
包封率=[(纯化样品中阿霉素浓度*样品体积)/阿霉素总质量]*100%,包封率为22%。
基于牛奶外泌体的靶向载药体系HA-mExo-DOX制备及表征
上述制备好的装载阿霉素的牛奶外泌体与DSPE-PEG2000-HA溶液(0.2mg/mL)于37℃孵育48h,期间拿出震荡1-2次,得到的基于牛奶外泌体的靶向载药体系HA-mExo-DOX。
将HA-mExo-DOX体系用纳米粒度仪检测粒径分布及表面电位。结果如图3(A)所示,粒径和电位的变化表示HA的成功修饰。
基于牛奶外泌体的靶向载药体系HA-mExo-DOX的靶向性检测
HA-mExo的制备:将牛奶外泌体与DSPE-PEG2000-HA溶液(0.2mg/mL)于37℃孵育48h,期间拿出震荡1-2次,得到透明质酸修饰的牛奶外泌体HA-mExo。
DOX溶液的制备:将阿霉素固体试剂溶解至pH7.4的PBS溶液中配成浓度为1μg/μL的DOX溶液。
HA-mExo-DOX溶液的制备:用上述方法制备的HA-mExo-DOX溶液浓度为10ng/μL,以DOX定量。
本研究采用透明质酸HA作为牛奶外泌体的靶向修饰工具,为验证靶向修饰的效果,分别将HA-mExo-DOX与人乳腺癌细胞MCF-7、MDA-MB-231、人肺癌细胞A549和人肾上皮细胞HEK293进行共培养,通过阿霉素本身的红色荧光判定该体系与几种细胞的靶向结合情况。
细胞摄取荧光实验
细胞消化并计数,以每孔4万个细胞种至24孔板中,在细胞培养箱中过夜培养。待细胞全部贴壁后,将培养基换成新鲜的基础培养基,分别加入等体积PBS、HA-mExo溶液、HA-mExo-DOX溶液和DOX溶液,在含有DOX组的溶液中DOX终浓度均为5ng/μL,于培养箱中孵育6h后,吸出每孔培养液弃掉。用4%多聚甲醛避光条件下固定15min,灭菌PBS洗3次。用4',6-二脒基-2-苯基吲哚(DAPI)对细胞核进行染色,最后用灭菌PBS洗三次。放在倒置荧光显微镜下进行观察并用相机拍照。结果如图4所示,HA受体高表达的癌细胞系MCF-7、MDA-MB-231、A549中HA-mExo-DOX组荧光摄取显著增强;HA受体非高表达的普通细胞系HEK293中HA-mExo-DOX组荧光摄取微弱,定性证明了HA-mExo-DOX体系的靶向性。
流式细胞术
细胞消化并计数,将细胞液浓度调整至20万个/mL,分装于1.5mL灭菌离心管中,每管500μL。离心机1500rpm,离心5min,弃去培养基上清,用200μL灭菌PBS重悬后再次离心。用100μL流式液重悬细胞,再分别加入100μL的PBS、HA-mExo溶液、HA-mExo-DOX溶液和DOX溶液,在含有DOX组的溶液中DOX终浓度均为5ng/μL,于37℃,孵育2h。孵育后于1500rpm,离心5min,弃上清,用200μL预冷的流式液重悬细胞后用流式细胞仪进行检测。结果如图6所示,HA受体高表达的癌细胞系MCF-7、MDA-MB-231、A549中HA-mExo-DOX组荧光摄取显著(P<0.05)高于DOX组;HA受体非高表达的普通细胞系HEK293中HA-mExo-DOX组荧光摄取低于DOX组,定量证明了HA-mExo-DOX体系的靶向性。
HA-mExo-DOX体系的细胞毒性检测
以每孔2000个将细胞种在96孔板中,过夜培养。待细胞全部贴壁后,换成新鲜培养基,分别加入10μL等体积HA-mExo溶液和不同浓度的HA-mExo-DOX溶液(0、2、4、8、10ng/μL),每组重复三个复孔。培养24h后,吸去培养液,每孔加入100μL含10%CCK-8的完全培养基,另设一个空白细胞孔作为背景,避光操作。37℃孵育2h后,用酶标仪检测450nm处吸光值,各组数值减去空白值,绘制细胞活性曲线。结果如图8所示,细胞活性呈现出药物浓度依赖性,随着药物浓度增高,细胞活性逐渐降低,并且当药物浓度达到较高水平,HA-mExo-DOX体系比DOX体系显示出更强的肿瘤细胞杀伤性。
实施例2叶酸修饰的牛奶外泌体载药体系
在此实施例中,具有肿瘤靶向功能的分子为叶酸(FA),装载药物为阿霉素。
靶向配体的制备
DSPE-PEG2000-FA购于华腾制药公司
牛奶外泌体的制备
牛奶分装,13,000g,4℃,离心30分钟,离心后,将上层脂肪、底部酪蛋白残渣和细胞碎片除去,收集中间部分的乳液得到脱脂奶;将脱脂奶与蒸馏水按体积比1:1进行稀释;使用2M盐酸,将溶液pH值调整到4.6,使溶液中的酪蛋白析出;将其均量分装,10,000g,离心40分钟(离心力为0.7×104g),弃除底部蛋白质沉淀,收集上层乳清并将其过0.22μm的滤膜,减少可能夹杂的杂蛋白;得到的乳清在超高速冷冻离心机中,135,000g,离心1小时(离心力为13.5×104g),收获沉淀,即外泌体。PBS预冷,并洗涤沉淀3次,再用适量的PBS将其重悬。用透射电镜观察及动态光散射测量粒径和电位,结果分别如图1和图2所示,牛奶外泌体显示“杯托状”,水合粒径为123.9nm,电位-4.24mV,生物学性质良好。
牛奶外泌体装载阿霉素及表征
牛奶外泌体与阿霉素以质量比5:1进行装载,37℃避光孵育30min。用10000Da的超滤管7500rpm超滤10min进行纯化,得到的样品用NanoDrop仪器于480nm处检测吸光值,依据阿霉素标准曲线计算浓度和包封率,包封率计算公式如下。
包封率=[(纯化样品中阿霉素浓度*样品体积)/阿霉素总质量]*100%,包封率为21%。
基于牛奶外泌体的靶向载药体系FA-mExo-DOX制备及表征
上述制备好的装载阿霉素的牛奶外泌体与DSPE-PEG2000-FA溶液(0.2mg/mL)于37℃孵育48h,期间拿出震荡1-2次,得到的基于牛奶外泌体的靶向载药体系FA-mExo-DOX。
得到的FA-mExo-DOX体系用纳米粒度仪检测粒径分布及表面电位。结果如图3(B)所示,粒径和电位的变化表示FA的成功修饰。
FA-mExo-DOX体系的靶向性检测
FA-mExo的制备:将牛奶外泌体与DSPE-PEG2000-FA溶液(0.2mg/mL)于37℃孵育48h,期间拿出震荡1-2次,得到叶酸修饰的牛奶外泌体FA-mExo。
DOX溶液的制备:将阿霉素固体试剂溶解至PBS溶液(pH7.4)中,配成浓度为1mg/mL的DOX溶液。。
FA-mExo-DOX溶液的制备:用上述方法制备的FA-mExo-DOX溶液浓度为10ng/μL,以DOX定量。
本研究采用叶酸FA作为牛奶外泌体的靶向修饰工具,为验证靶向修饰的效果,分别将FA-mExo-DOX与人乳腺癌细胞MCF-7、人结直肠癌细胞HT-29和人肾上皮细胞HEK293进行共培养,通过阿霉素本身的红色荧光判定该体系与几种细胞的靶向结合情况。
细胞摄取荧光实验
细胞消化并计数,以每孔4万个细胞种至24孔板中,在细胞培养箱中过夜培养。待细胞全部贴壁后,将培养基换成新鲜的基础培养基,分别加入等体积PBS、FA-mExo溶液、FA-mExo-DOX溶液和DOX溶液,在含有DOX组的溶液中DOX终浓度均为5ng/μL,于培养箱中孵育6h后,吸出每孔培养液弃掉。用4%多聚甲醛避光条件下固定15min,灭菌PBS洗3次。用4',6-二脒基-2-苯基吲哚(DAPI)对细胞核进行染色,最后用灭菌PBS洗三次。放在倒置荧光显微镜下进行观察并用相机拍照。结果如图5所示,FA受体高表达的癌细胞系HT-29和FA受体中表达的癌细胞系MCF-7中FA-mExo-DOX组荧光摄取显著增强;FA受体低表达的普通细胞系HEK293中FA-mExo-DOX组荧光摄取微弱,定性证明了FA-mExo-DOX体系的靶向性。
流式细胞术
细胞消化并计数,将细胞液浓度调整至20万个/mL,分装于1.5mL灭菌离心管中,每管500μL。离心机1500rpm,离心5min,弃去培养基上清,用200μL灭菌PBS重悬后再次离心。用100μL流式液重悬细胞,再分别加入100μL的PBS、FA-mExo溶液、FA-mExo-DOX溶液和DOX溶液,在含有DOX组的溶液中DOX终浓度均为5ng/μL,于37℃,孵育2h。孵育后于1500rpm,离心5min,弃上清,用200μL预冷的流式液重悬细胞后用流式细胞仪进行检测。结果如图7所示,FA受体高表达的癌细胞系HT-29中FA-mExo-DOX组荧光摄取高于DOX组,FA受体中表达的癌细胞系MCF-7中FA-mExo-DOX组荧光摄取与DOX接近,FA受体低表达的普通细胞系HEK293中FA-mExo-DOX组荧光摄取低于DOX组,定量证明了FA-mExo-DOX体系的靶向性。
FA-mExo-DOX体系的细胞毒性检测
以每孔2000个将细胞种在96孔板中,过夜培养。待细胞全部贴壁后,换成新鲜培养基,分别加入10μL的mExo溶液(蛋白浓度1.5μg/μL)、FA-mExo溶液和不同浓度的FA-mExo-DOX溶液(5、15、30ng/μL),每组重复三个复孔。培养24h后,吸去培养液,每孔加入100μL含10%CCK-8的完全培养基,另设一个空白细胞孔作为背景,避光操作。37℃孵育2h后,用酶标仪检测450nm处吸光值,各组数值减去空白值,绘制细胞活性柱状图。结果如图9所示,牛奶外泌体本身(mExo)以及修饰了FA的牛奶外泌体(FA-mExo)都对细胞无杀伤作用,随着FA-mExo-DOX浓度增高,HT-29细胞存活率逐渐降低,呈现浓度梯度依赖型,证明了FA-mExo-DOX对癌细胞有效的杀伤作用。
Claims (10)
1.一种基于牛奶外泌体的靶向载药体系,其特征在于,所述靶向载药体系包括疏水性药物,所述疏水性药物被牛奶外泌体装载,所述牛奶外泌体外部连接有靶向配体,所述靶向配体具有脂链端和靶向端。
2.根据权利要求1所述的一种基于牛奶外泌体的靶向载药体系,其特征在于,所述靶向配体的脂链端包括二硬脂酰基磷脂酰乙醇胺、二硬脂酰磷脂酰胆碱、大豆磷脂酰胆碱;所述靶向配体的靶向端为特异性结合肿瘤表面过表达受体功能的分子,包括叶酸、透明质酸、核酸适配体AS1411。
3.根据权利要求1或2所述的一种基于牛奶外泌体的靶向载药体系,其特征在于,所述靶向配体脂链端和靶向端通过聚乙二醇连接。
4.根据权利要求1或2所述的一种基于牛奶外泌体的靶向载药体系,其特征在于,所述靶向配体通过酰胺缩合反应所制得。
5.根据权利要求1所述的一种基于牛奶外泌体的靶向载药体系,其特征在于,所述疏水性药物包括化学药物、基因药物。
6.根据权利要求5所述的一种基于牛奶外泌体的靶向载药体系,其特征在于,所述化学药物包括阿霉素;所述基因药物包括miRNA-204。
7.根据权利要求1、2、5或6所述任一一种基于牛奶外泌体的靶向载药体系的制备方法,其特征在于,制备方法包括:
S1,靶向配体的制备;
S2,牛奶外泌体装载疏水性药物,得到载药牛奶外泌体;
S3,将S2中的载药牛奶外泌体装载靶向配体,得到所述基于牛奶外泌体的靶向载药体系。
8.根据权利要求7所述的一种基于牛奶外泌体的靶向载药体系的制备方法,其特征在于,S2、S3中所述装载的方法为孵育。
9.根据权利要求7所述的一种基于牛奶外泌体的靶向载药体系的制备方法,其特征在于,所述牛奶外泌体的制备方法:通过2~4M的盐酸调节牛奶的pH值为4.55~4.64,用离心力为0.7×104~1.0×104g进行离心去除杂蛋白后,用离心力为11.0×104~13.5×104g进行离心富集牛奶外泌体。
10.根据权利要求7所述的一种基于牛奶外泌体的靶向载药体系的制备方法,其特征在于,所述S1制备的靶向配体是通过酰胺缩合反应得到,并且通过LH20柱分离纯化。
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CN114982958B (zh) * | 2022-06-13 | 2024-03-19 | 大连工业大学 | 靶向修饰负载虾青素牛乳外泌体纳米制剂及其制备方法 |
CN116036310A (zh) * | 2023-01-13 | 2023-05-02 | 广东医科大学附属医院 | 琥珀酰化壳聚糖修饰外泌体及其制备方法和应用 |
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