CN109394730A - 一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒及其制备方法和应用 - Google Patents
一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒及其制备方法和应用,由载药纳米核心和包裹在该载药纳米核心之外的红细胞膜构成。本发明具有良好的生物相容性和分散性,在激光的照射下产生热量,可以杀死肿瘤细胞,还具有很强的荧光性能,可以在荧光成像模式下指导光热治疗与化疗的联合治疗,通过红细胞在纳米粒表面的修饰,实现在生物体内的长循环,以提高载药纳米粒的疗效。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒及其制备方法和应用。
背景技术
近几十年来,癌症已成为危害人类健康的最严重疾病之一。全球人口特征预测未来几十年癌症发病率将持续增长,到2025年,每年癌症新增病例将大于2000万。而传统的放化疗无法改善癌症死亡率高和治疗费用昂贵的现状,新的治疗方案亟待研发以满足患者的需要。
光热疗法作为热疗的一种,其原理是利用激光照射光热剂,将光能迅速转化为热能,使肿瘤所在部位温度升高并能维持一段时间,从而达到杀死肿瘤细胞的目的。由于皮肤、组织、血液和水在近红外光区域的吸收和散射低,波长为700-950nm的近红外光诱发的光热疗法成为光热疗法的重点。
吲哚菁绿(简称ICG)具有减少光散射、良好的组织渗透性和对自体荧光生物样品干扰小等优点,在生物医学的诸多领域备受青睐。多年来,ICG被应用于临床眼科造影术、心输出量测量和肝功能的研究。目前为止,ICG是唯一被FDA批准应用于临床研究的唯一一种近红外光热剂。ICG具有良好的光热转换效率,单位质量的光热转换效率可达SWNTs的7倍,甚至可达普通纳米金棒的8500倍。但是ICG在生理介质中容易发生结构转变,如发生自聚集和不可逆降解等,导致其光学性质的改变,甚至失去近红外吸收能力。
发明内容
本发明的目的在于克服现有技术缺陷,提供一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒。
本发明的另一目的在于提供上述红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒的制备方法。
本发明的再一目的在于提供上述红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒的应用。
本发明的技术方案如下:
一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒,由载药纳米核心和包裹在该载药纳米核心之外的红细胞膜构成,其中,
载药纳米核心包括由牛血清白蛋白交联而成,且该牛血清白蛋白上具有通过疏水作用力吸附其上的藤黄酸和吲哚菁绿。
在本发明的一个优选实施方案中,其粒径为100-200nm。
在本发明的一个优选实施方案中,所述藤黄酸和吲哚菁绿的摩尔比为0.8-1.2∶0.8-1.2。
在本发明的一个优选实施方案中,所述藤黄酸和吲哚菁绿的含量为其质量的22-26%。
上述红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒的制备方法,包括如下步骤:
(1)将吲哚菁绿溶液和藤黄酸溶液加入到牛血清白蛋白溶液中,搅拌并加入戊二醛固定,然后固液分离,获得载药纳米核心;
(2)将步骤(1)所得的载药纳米核心加入到红细胞膜溶液中,超声处理,即得;
上述吲哚菁绿溶液和藤黄酸溶液的溶剂为甲醇,牛血清白蛋白溶液的溶剂为磷酸盐缓冲液,红细胞膜溶液的溶剂为无菌超纯水。
上述红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒在制备抗肿瘤药物中的应用。
本发明的有益效果是:
1、本发明中的红细胞膜(Red blood cell membranes,RBCm)包裹纳米粒药物载体由作为内核的纳米粒子和外围的红细胞膜构成,这种联合方式能同时发挥这两类药物载体的各自优势。红细胞膜载体来源于红细胞,具有许多良好的特性,降解不会产生有毒或有害物质,无免疫原性、半衰期长具有较大的比表面积等。
2、本发明中的藤黄酸为藤黄科植物藤黄树分泌的干燥树脂中提取纯化获得的有效成分。近年研究发现,藤黄酸可显著抑制人肝癌及胃腺癌细胞的增殖,用于肿瘤治疗,具有活性成分性质稳定、疗效显著且不良反应少的特点。
3、本发明具有良好的生物相容性和分散性,在激光的照射下产生热量,可以杀死肿瘤细胞,还具有很强的荧光性能,可以在荧光成像模式下指导光热治疗与化疗的联合治疗,通过红细胞在纳米粒表面的修饰,实现在生物体内的长循环,以提高载药纳米粒的疗效。
附图说明
图1为本发明实施例4中GI NPs和RBCm-GI NPs的透射电镜图;其中a为GI NPs的透射电镜图,b为RBCm-GI NPs的透射电镜图。
图2为本发明实施例4中不同藤黄酸和吲哚菁绿比列下的紫外可见近红外吸收光谱图;其中从下往上的比例依次为20∶0,15∶5,12∶8,10∶10,8∶12和5∶15。
图3为本发明实施例5中RBCm-GI NPs不同浓度ICG在808nm的激光下的升温曲线。
图4为本发明实施例6中GI NPs和RBCm-GI NPs对MCF-7和HeLa细胞的细胞存活图;其中a为24h,b为48h后的细胞存活率。
图5为本发明实施例7中RBCm-GINPs在细胞水平的联合治疗效果图。
具体实施方式
以下通过具体实施方式对本发明的技术方案进行进一步的说明和描述。
实施例1:白蛋白藤黄酸吲哚菁绿纳米粒的制备
以牛血清白蛋白(BSA)为基底,通过疏水作用力吸附藤黄酸和吲哚菁绿,利用戊二醛化学交联剂的作用交联形成形成白蛋白藤黄酸吲哚菁绿纳米粒(GI NPs,即所述载药纳米核心)。具体方法如下:
将藤黄酸和吲哚菁绿溶于1mL的甲醇中,配制成不同浓度的比例;称取牛血清白蛋白(BSA)溶于10mL的pH 7.4磷酸盐缓冲液中,配制成不同浓度的牛血清白蛋白溶液。将溶有藤黄酸(GA)和吲哚菁绿(ICG)的甲醇溶液滴入到搅拌的牛血清白蛋白溶液中,加入20μL戊二醛交联,继续避光搅拌24h。最后将得到的混合物离心,除去没有上清液,即得到白蛋白藤黄酸吲哚菁绿纳米粒。
实施例2:红细胞膜的提取
取老鼠全血放入离心机中离心,在1000rpm转速、4℃条件下离心10min,移除血浆和白细胞层;得到的沉淀即红细胞用pH 7.4PBS缓冲液清洗后,再加入灭菌后的超纯水,冰浴条件下放置30-60min,使红细胞破裂并释放血红素,在5000rpm转速下离心10min;最后用灭菌后的超纯水清洗多次后,即得到红细胞膜。
实施例3:红细胞膜包裹白蛋白藤黄酸吲哚菁绿纳米粒的制备
取适量的红细胞膜分散在无菌超纯水中,加入对应比例的白蛋白藤黄酸吲哚菁绿纳米粒,100W超声3min后,1000rpm离心5min,即得到藤黄酸和吲哚菁绿的含量为其质量的22-26%的红细胞膜包裹白蛋白藤黄酸吲哚菁绿纳米粒(RBCm-GINPs,即本发明的红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒)。
实施例4:红细胞膜包裹白蛋白藤黄酸吲哚菁绿纳米粒的表征
分别采用透射电镜和紫外可见近红外光扫描观察实施例1和3制得的纳米粒。结果见图1和2。白蛋白藤黄酸吲哚菁绿纳米粒(GINPs)和红细胞膜包裹白蛋白藤黄酸吲哚菁绿纳米粒(RBCm-GI NPs)滴到铜网上后,磷钨酸进行复染后,透射电镜进行表征。白蛋白藤黄酸吲哚菁绿纳米粒(GI NPs)分散在水用紫外可见近红外光扫描来进行表征。
图1为白蛋白藤黄酸吲哚菁绿纳米粒(GINPs)和红细胞膜包裹白蛋白藤黄酸吲哚菁绿纳米粒(RBCm-GI NPs)的透射电镜图。由图1可以看出GI NPs的粒径在100nm左右,RBCm-GI NPs的表面包裹有一层红细胞膜,表明红细胞膜成功的包裹上GI NPs了。
图2是白蛋白藤黄酸吲哚菁绿纳米粒(GINPs)分散在水用紫外可见近红外光扫描图。由图中可知在不同的藤黄酸(GA)和吲哚菁绿(ICG)的比例下,ICG的吸收峰不同,因此综合GA和ICG的摩尔比,选择了GA∶ICG的摩尔比为8∶12。
实施例5:RBCm-GI NPs在激光照射下的升温曲线
将不同浓度下的RBCm-GI NPs分散在水中,用808nm的激光(功率为1.5W/cm2)直接照射在样品上,利用红外热成像仪测定温度的变化,结果如图3所示。由图3结果表明,在5min内,随着RBCm-GI NPs中ICG的浓度升高温度也越高,说明吸附ICG后的纳米粒具有很高的光热转换率。RBCm-GI NPs具有较强的光学吸收性质,可以作为光热治疗的材料。
实施例6:G1NPs和RBCm-GI NPs在细胞水平的毒性研究
取不同GA浓度的GI NPs和RBCm-GI NPs分别与MCF-7和HeLa细胞孵育24h和48h,采用MTT试剂进行细胞活性检测,结果如图4所示。由图4中可知,当GA的浓度很低时,GI NPs和RBCm-GI NPs均有非常明显的化疗作用。
实施例7:RBCm-GI NPs在细胞水平的联合治疗
按照不同的步骤将GI NPs、ICG和RBCm-GI NPs分别加入到含有100μL的HeLa细胞中,用808nm、1.5W/cm2的激光照射细胞3min,洗去多余的材料。继续培养24h后,采用MTT试剂进行细胞活性检测,结果如图5所示。由图5可以看出,在激光照射下和没有激光照射的情况下,对细胞活性有一定的影响。在激光照射下,RBCm-GI NPs的光热性质产生的升温可以增加细胞膜的通透性,从而促进RBCm-GI NPs进入细胞的能力,进而杀死细胞,表明RBCm-GINPs在细胞水平的联合治疗效果显著。
以上所述,仅为本发明的较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
Claims (6)
1.一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒,其特征在于:由载药纳米核心和包裹在该载药纳米核心之外的红细胞膜构成,其中,
载药纳米核心包括由牛血清白蛋白交联而成,且该牛血清白蛋白上具有通过疏水作用力吸附其上的藤黄酸和吲哚菁绿。
2.如权利要求1所述的一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒,其特征在于:其粒径为100-200nm。
3.如权利要求1所述的一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒,其特征在于:所述藤黄酸和吲哚菁绿的质量比为0.8-1.2∶0.8-1.2。
4.如权利要求3所述的一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒,其特征在于:所述藤黄酸和吲哚菁绿的含量为其质量的22-26%。
5.权利要求1至4中任一权利要求所述的一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒的制备方法,其特征在于:包括如下步骤:
(1)将吲哚菁绿溶液和藤黄酸溶液加入到牛血清白蛋白溶液中,搅拌并加入戊二醛固定,然后固液分离,获得载药纳米核心;
(2)将步骤(1)所得的载药纳米核心加入到红细胞膜溶液中,超声处理,即得;
上述吲哚菁绿溶液和藤黄酸溶液的溶剂为甲醇,牛血清白蛋白溶液的溶剂为磷酸盐缓冲液,红细胞膜溶液的溶剂为无菌超纯水。
6.权利要求1至4中任一权利要求所述的一种红细胞膜包裹共载藤黄酸和吲哚菁绿白蛋白纳米粒在制备抗肿瘤药物中的应用。
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