CN116531524B - 一种脑靶向纳米粒及其制备方法和应用 - Google Patents
一种脑靶向纳米粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及脑损伤药物技术领域,提供了一种脑靶向纳米粒及其制备方法和应用。本发明提供的脑靶向纳米粒包括载药纳米粒和连接在所述载药纳米粒表面的转铁蛋白;所述载药纳米粒包括载体和负载在所述载体中的药物;所述载体为两亲性嵌段共聚物;所述药物为莱菔硫烷。采用本发明的脑靶向纳米粒治疗急性一氧化碳中毒脑损伤,不仅具有高效性和安全性,而且由于修饰脑靶向蛋白的原因可以明显提高抗凋亡能力,增强抗氧化活性,降低神经系统损害。本发明提供的制备方法直接将药物包裹进纳米粒中再进行靶向修饰,制备步骤简单,效率高。
Description
技术领域
本发明涉及脑损伤药物技术领域,尤其涉及一种脑靶向纳米粒及其制备方法和应用。
背景技术
传统药物治疗一氧化碳中毒主要包括高压氧疗法,或采用脑保护剂、神经营养剂和皮质类固醇等进行治疗,这些治疗方法在一定程度上能够缓解症状和减轻损伤,但也存在一些引起副作用,如免疫系统损伤以及药物过量使用的问题。
莱菔硫烷主要来源于天然十字花科植物中的异硫氰酸酯,具有抗氧化应激、免疫炎症修复、抗肿瘤等多种药理作用。莱菔硫烷作为一种具有多种生物学活性的药物,主要用于癌症治疗,尤其是结肠癌和直肠癌。近年来,研究发现莱菔硫烷也可以用于治疗一氧化碳(CO)中毒引起的脑损伤。相比传统治疗急性一氧化碳中毒的药物,莱菔硫烷具有一定的优势:(1)抗氧化应激作用:研究表明,莱菔硫烷可以减轻氧化应激反应,对抗CO中毒引起的氧化应激,从而减轻脑损伤。(2)抗炎作用:莱菔硫烷具有抗炎作用,可以减轻CO中毒引起的炎症反应,保护神经细胞。(3)抗凋亡作用:莱菔硫烷可以减少神经细胞的凋亡,降低CO中毒后神经细胞损伤。(4)通过血脑屏障:传统莱菔硫烷可以通过血脑屏障,但效率较低,经纳米修饰包装后更易通过血脑屏障,更有效地作用于受损的神经细胞。
但是,莱菔硫烷不具有靶向性,治疗效果还有待进一步提高。
发明内容
有鉴于此,本发明提供了一种脑靶向纳米粒及其制备方法和应用。本发明提供的脑靶向纳米粒脑靶向效果好,能够促进药物在脑组织中的分布和作用,减少细胞凋亡,对一氧化碳中毒脑损伤的治疗效果好。
为了实现上述发明目的,本发明提供以下技术方案:
一种脑靶向纳米粒,包括载药纳米粒和连接在所述载药纳米粒表面的转铁蛋白;所述载药纳米粒包括载体和负载在所述载体中的药物;所述载体为两亲性嵌段共聚物;所述药物为莱菔硫烷;
所述两亲性嵌段共聚物为聚乙二醇-聚乳酸羟基乙酸共聚物。
优选的,所述聚乙二醇-聚乳酸羟基乙酸共聚物中聚乙二醇嵌段的重均分子量为2K~10K,聚乳酸羟基乙酸嵌段的重均分子量为5K~20K。
优选的,所述脑靶向纳米粒的粒径为97~151nm;所述脑靶向纳米粒中莱菔硫烷的载药量为12wt%~21wt%。
本发明还提供了上述方案所述脑靶向纳米粒的制备方法,包括以下步骤:
将带有羧基的两亲性嵌段共聚物、莱菔硫烷和有机溶剂混合,得到有机相;
将所述有机相加入水中,然后将有机溶剂挥干,得到载药纳米粒混悬液;
将所述载药纳米粒混悬液、EDC和NHS混合进行活化,然后加入转铁蛋白进行偶联反应,得到所述脑靶向纳米粒。
优选的,所述有机相和水的体积比为1:(2~20)。
优选的,所述带有羧基的两亲性嵌段共聚物和莱菔硫烷的质量比为(5~300):(1.25~75)。
优选的,所述转铁蛋白和所述带有羧基的两亲性嵌段共聚物的摩尔比为(50~750):1。
优选的,所述带有羧基的两亲性嵌段共聚物、EDC和NHS的摩尔比为1:(2~10):(2~5)。
优选的,所述活化的温度为20~26℃,时间为0.5~4h;所述偶联反应的温度为20~26℃,时间为8~16h。
本发明还提供了上述方案所述的脑靶向纳米粒或上述方案所述制备方法制备的脑靶向纳米粒在制备治疗急性一氧化碳中毒脑损伤的药物中的应用。
本发明提供了一种脑靶向纳米粒,包括载药纳米粒和连接在所述载药纳米粒表面的转铁蛋白;所述载药纳米粒包括载体和负载在所述载体中的药物;所述载体为两亲性嵌段共聚物;所述药物为莱菔硫烷;所述两亲性嵌段共聚物为聚乙二醇-聚乳酸羟基乙酸共聚物。本发明在载药纳米粒表面修饰转铁蛋白,可以有效提高药物的脑靶向性,改善药物脑靶向富集能力,增强脑内组织药物分布而减少其他组织药物分布,促进药物在脑内靶向系统的表达,还可以延长药物的半衰期,进而明显增加脑内药物的靶向性及蓄积浓度,促进神经系统疾病的治疗。并且,本发明采用的两亲性嵌段共聚物为可生物降解且被FDA批准可用于人体的聚合物,生物相容性好,毒副作用小,且能提高药物的水溶性、稳定性和体内循环时间,所得脑靶向纳米粒具有粒径大小可控性强、包封率高、稳定性好、可控性释放的特点。
综上所述,使用本发明的脑靶向纳米粒治疗急性一氧化碳中毒脑损伤,不仅具有高效性和安全性,而且由于修饰脑靶向蛋白的原因可以明显提高抗凋亡能力,增强抗氧化活性降低神经系统损害。这为治疗一氧化碳中毒脑损伤提供了新的思路和方法,应用前景广阔。
本发明还提供了上述方案所述脑靶向纳米粒的制备方法,本发明将莱菔硫烷包裹在两亲性嵌段共聚物的纳米乳中,之后通过EDC/NHS活化,直接将转铁蛋白连接在纳米粒表面,从而实现靶向修饰,最终形成本发明的脑靶向纳米粒。本发明提供的制备方法直接将药物包裹进纳米粒中再进行靶向修饰,避免了先制备载药纳米粒再涂覆靶向聚合物的过程,简化了制备步骤,提高了制备效率。
附图说明
图1为本发明实施例中制备脑靶向纳米粒的流程示意图;
图2为SFN-PEG-PLGA NPs和SFN-TF-PEG-PLGA NPs的FTIR图谱;
图3为SFN-PEG-PLGA NPs和SFN-TF-PEG-PLGA NPs的粒径分布图;
图4为不同浓度PEG-PLGA纳米粒对PC12神经细胞的细胞毒性作用;
图5为荧光共聚焦显微镜下观察到的PC12细胞对不同纳米粒的内吞情况;
图6为内吞情况的统计学分析结果;
图7为流式细胞术对TF-PEG-PLGA纳米粒和PEG-PLGA纳米粒的细胞内摄取情况的评估结果,其中A为流式细胞仪定量分析PC12细胞对包载FITC纳米粒的摄取结果,B为统计学分析结果;
图8为TF-PEG-PLGA纳米粒和PEG-PLGA纳米粒在动物脑组织中的靶向效果,其中A为荧光成像仪观察结果,B为统计学分析结果;
图9为采用不同纳米粒治疗后大鼠的脑组织切片的HE染色结果;
图10为采用不同纳米粒治疗后大鼠的脑组织切片的透射电镜观察结果;
图11为尼氏染色观察细胞凋亡情况,其中A为显微镜观察结果,B为统计学分析结果;
图12为Western blot实验中特异性蛋白的表达情况;其中A为光学成像结果,B为P-P38/GAPDH的统计学分析结果,C为TGF-β1/GAPDH的统计学分析结果,D为BCL2/GAPDH的统计学分析结果,E为P-P13K/P13K的统计学分析结果,F为BAX/GAPDH的统计学分析结果。
具体实施方式
本发明提供了一种脑靶向纳米粒,包括载药纳米粒和连接在所述载药纳米粒表面的转铁蛋白;所述载药纳米粒包括载体和负载在所述载体中的药物;所述载体为两亲性嵌段共聚物;所述药物为莱菔硫烷;所述两亲性嵌段共聚物为聚乙二醇-聚乳酸羟基乙酸共聚物(PEG-PLGA)。
在本发明中,所述聚乙二醇-聚乳酸羟基乙酸共聚物中聚乙二醇嵌段的重均分子量优选为2K~10K,更优选为5K,聚乳酸羟基乙酸嵌段的重均分子量优选为5K~20K,更优选为10K,聚乳酸羟基乙酸嵌段中乳酸(LA)和羟基乙酸(GA)的摩尔比优选为(50~80):(20~50),更优选为75:25;在本发明的具体实施例中,所述聚乙二醇-聚乳酸羟基乙酸共聚物优选为羧基聚乙二醇聚乳酸-羟基乙酸共聚物(COOH-PEG-PLGA)。本发明对上述两亲性嵌段共聚物的来源没有特殊要求,采用市售的产品即可。
在本发明中,所述脑靶向纳米粒的粒径优选为97~151nm;所述脑靶向纳米粒中莱菔硫烷的载药量优选为12wt%~21wt%。
本发明还提供了上述方案所述脑靶向纳米粒的制备方法,包括以下步骤:
将带有羧基的两亲性嵌段共聚物、莱菔硫烷和有机溶剂混合,得到有机相;
将所述有机相加入水中,然后将有机溶剂挥干,得到载药纳米粒混悬液;
将所述载药纳米粒混悬液、EDC和NHS混合进行活化,然后加入转铁蛋白进行偶联反应,得到所述脑靶向纳米粒。
本发明将带有羧基的两亲性嵌段共聚物、莱菔硫烷(SFN)和有机溶剂混合,得到有机相。在本发明中,所述有机溶剂优选为四氢呋喃(THF),所述带有羧基的两亲性嵌段共聚物和莱菔硫烷的质量比优选为(5~300):(1.25~75),更优选为(50~200):(10~60);所述带有羧基的两亲性嵌段共聚物和有机溶剂的用量比优选为300mg:600~1000μL,更优选为300mg:750μL;在本发明中,所述带有羧基的两亲性嵌段共聚物即为COOH-PEG-PLGA。
得到有机相后,本发明将所述有机相加入水中,然后将有机溶剂挥干,得到载药纳米粒混悬液。在本发明中,所述水优选为去离子水;所述有机相和水的体积比优选为1:(2~20),更优选为1:(2~10);本发明优选在搅拌条件下将有机相注入水中,所述搅拌的转速优选为400r/min,所述搅拌在常温下进行即可;在本发明中,优选待有机相注入完毕后,继续在常温下搅拌,至挥尽有机溶剂,即得到载药纳米粒混悬液。
得到载药纳米粒混悬液后,本发明将所述载药纳米粒混悬液、EDC和NHS混合进行活化,然后加入转铁蛋白进行偶联反应,得到所述脑靶向纳米粒。在本发明中,所述转铁蛋白(TF)和所述带有羧基的两亲性嵌段共聚物的摩尔比优选为(50~750):1,更优选为(100~600):1;所述带有羧基的两亲性嵌段共聚物、EDC和NHS的摩尔比优选为1:(2~10):(2~5),更优选为1:(3~8):(3~4);在本发明中,所述EDC优选以EDC水溶液的形式使用,所述NHS优选以NHS水溶液的形式使用,本发明对所述EDC水溶液和NHS水溶液的浓度没有要求;所述转铁蛋白优选以转铁蛋白水溶液的形式使用,所述转铁蛋白水溶液的浓度优选为1mg/mL。
在本发明中,所述活化的温度优选为20~26℃,更优选为室温,所述活化的时间优选为0.5~4h,更优选为3~4h;所述偶联反应的温度优选为20~26℃,更优选为室温,所述偶联反应的时间优选为8~16h,更优选为12~16h;所述活化和偶联反应均在搅拌条件下进行,所述搅拌的转速优选为250r/min;本发明先利用EDC和NHS将共聚物中的羧基进行活化,在EDC/NHS的催化作用下,COOH-PEG-PLGA结构中的羧基可与转铁蛋白结构中的氨基反应生成酰胺键,从而将转铁蛋白修饰在纳米粒表面。
偶联反应完成后,本发明优选将所得产物料液进行离心,去除游离的转铁蛋白以及EDC和NHS,得到所述脑靶向纳米粒;所述离心的转速优选为12000r/min,时间优选为20min。
本发明还提供了上述方案所述的脑靶向纳米粒或上述方案所述制备方法制备的脑靶向纳米粒在制备治疗急性一氧化碳中毒脑损伤的药物中的应用。本发明提供的脑靶向纳米粒脑靶向效果好,促进药物在脑组织中的分布和作用,减少细胞凋亡,还可以有效抑制炎性细胞浸润和细胞肿胀,从而减轻一氧化碳中毒脑损伤;同时,本发明提供的脑靶向纳米粒生物相容性好,毒副作用小,安全性好,在急性一氧化碳中毒脑损伤的治疗方面具有广阔的应用前景。
下面将结合本发明中的实施例,对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例中采用的COOH-PEG-PLGA为市售产品,其中PEG嵌段的重均分子量为5K,PLGA嵌段的重均分子量为10K,LA和GA的摩尔比为75:25。
图1为本发明实施例中制备脑靶向纳米粒的流程示意图。
实施例1
(1)称取COOH-PEG-PLGA 300mg和莱菔硫烷(SFN)75mg共溶于THF 750μL中,构成有机相;将去离子水作为水相;有机相和水相的体积比为1:2;在400r/min转速下将有机相注入水相中,常温搅拌至挥尽有机溶剂,即得SFN-PEG-PLGA NPs混悬液。
(2)向SFN-PEG-PLGA NPs混悬液中逐滴加入EDC水溶液活化,之后再加入NHS水溶液继续在室温下以250 r/min的搅拌速率搅拌4h;再逐滴滴入TF水溶液(1mg/mL)继续搅拌16h,得到SFN-TF-PEG-PLGA NPs混悬液,其中,TF和COOH-PEG-PLGA的摩尔比为750:1,COOH-PEG-PLGA、EDC和NHS的摩尔比为1:10:5;以12000 r/min离心20min去除游离TF和催化剂,即得SFN-TF-PEG-PLGA NPs。
图2为SFN-PEG-PLGA NPs和SFN-TF-PEG-PLGA NPs的FTIR图谱;根据图2可以看出,红外光谱显示酰胺I带和酰胺II带的峰值发生了明显的变化,表明Tf与COOH基团之间发生了酰胺化反应,转铁蛋白成功偶联到 PEG-PLGA纳米粒表面。
图3为SFN-PEG-PLGA NPs和SFN-TF-PEG-PLGA NPs的粒径分布图;表1为SFN-PEG-PLGA NPs和SFN-TF-PEG-PLGA NPs的粒径分布、Zeta电位、PDI以及包封率和载药量。
表1 纳米粒的粒径分布、Zeta电位、PDI以及包封率和载药量
纳米粒 | 粒径(d.nm) | Zeta电位(mv) | PDI | 包封率(%) | 载药量(%) |
SFN-PEG-PLGA NPs | 130.1±2.7 | 19.2±1.6 | 0.25±0.01 | 87.5±0.16 | 21.8±0.12 |
SFN-TF-PEG-PLGA NPs | 151.3±3.6 | 11.2±0.7 | 0.52±0.76 | 83.4±0.68 | 21.3±0.16 |
根据图3和表1中的数据可以看出,修饰TF后,纳米粒的粒径变大,Zeta电位和PDI变大。
实施例2
纳米粒的安全性评估和靶向效果评估:实验选用CCK8细胞增殖试剂盒来测定PEG-PLGA纳米粒子的细胞毒性。
按照实施例1步骤(1)的方法,省略SFN的添加,制备空白无载药的PEG-PLGA纳米粒悬浮液,并调节其浓度分别为1、2、4、8、10mg/mL,按照以下方法测试细胞毒性:
首先,将PC12神经细胞按照每孔5000个细胞数100μL培养液接种到96孔板中,分为5个不同浓度PEG-PLGA纳米粒悬浮液处理实验组,培养至细胞贴壁充分后,细胞贴壁后分别用1、2、4、8、10mg/mL的PEG-PLGA纳米粒悬浮液处理细胞,每个浓度设置6个重复孔。24 h后去除原培养液,用PBS清洗2遍,每孔含100µL完全培养液和10µL CCK-8溶液的细胞培养液,继续培养2 h,酶标仪(波长450nm处)检测吸光度测定细胞的活力。
实验结果如图4所示。图4表明,PEG-PLGA纳米粒子对细胞的毒性较低。随着纳米粒浓度的提升,神经细胞活力基本保持在80%以上,材料具有低毒性,表明良好的生物安全性。
实施例3
通过共聚焦显微镜观察TF-PEG-PLGA纳米粒相对于PEG-PLGA纳米粒的细胞内摄取情况。实验利用FITC标记的纳米粒,观察不同纳米粒的内吞效率,并对结果进行统计学分析。
按照实施例1中的方法,省略SFN的添加,分别制备得到PEG-PLGA纳米粒和TF-PEG-PLGA纳米粒(后续均按照此方法制备,不再赘述);按照以下步骤进行实验:
首先,将PC12神经细胞分为两组,分别用FITC标记的TF-PEG-PLGA纳米粒和PEG-PLGA纳米粒进行处理。每组细胞加入5μg FITC标记的纳米粒,进行4h的培养。之后用PBS清洗一遍,加入4%多聚甲醛进行细胞固定,时间为15min,之后用PBS清洗3遍,加入5μg/mL的DAPI溶液进行细胞核染色10min,然后用PBS清洗一遍并滴加50%的甘油进行保湿。最后,使用共聚焦荧光显微镜观察两组细胞内吞纳米粒的情况。利用FITC标记的纳米粒,可以通过荧光显微镜直接观察到在细胞内的分布情况和数量。最后,对两组细胞的内吞情况进行统计学分析,比较两组细胞的内吞效率差异。
实验结果如图5~6所示,图5为荧光共聚焦显微镜下观察到的PC12细胞对不同纳米粒的内吞情况;图6为内吞情况的统计学分析结果。根据图5~6可以看出,偶联转铁蛋白后的纳米粒相较于单纯纳米粒表现出更高的靶向内吞能力。
实施例4
通过流式细胞术评估TF-PEG-PLGA纳米粒相对于PEG-PLGA纳米粒的细胞内摄取情况,目的是研究细胞对接有靶向头的纳米粒的摄取效率。
实验过程中,将PC12神经细胞接种在6孔板中,每孔控制细胞数在20万~50万,分为两个实验组进行处理,一组用的纳米粒是TF-PEG-PLGA,另一组用的是PEG-PLGA。通过荧光标记两组纳米粒,可以在细胞内观察纳米粒的内吞情况。在两组细胞培养基中分别加入含有5μg FITC的TF-PEG-PLGA和PEG-PLGA纳米粒悬浮液在细胞培养箱中避光培养4h。4h后,弃掉培养液并用PBS清洗3次,之后使用胰酶消化收集细胞,1200rpm离心3min使细胞沉淀,弃掉上清并用1mL PBS重悬,最后上机使用流式细胞分析仪检测两组细胞的摄取情况。流式细胞分析仪可以对细胞进行高通量检测,并对每个细胞进行细胞计数和荧光强度分析。通过流式细胞分析仪可以得到每个细胞内吞的纳米粒数目和荧光信号强度,并且可以对数据进行统计学分析。
实验结果如图7所示,图7中A为流式细胞仪定量分析PC12细胞对包载FITC纳米粒的摄取结果,B为统计学分析结果。图7中的结果表明,连接了转铁蛋白TF的纳米粒可以更有效地被细胞摄取,这对于开发针对急性一氧化碳中毒脑损伤疾病的靶向治疗具有重要意义。
实施例5
通过小动物活体成像系统评估纳米粒在动物脑组织中的靶向效果。
首先准备两种纳米粒,一种是TF-PEG-PLGA纳米粒,另一种是PEG-PLGA纳米粒,两种纳米粒都用荧光染料标记以便于观察。将大鼠随机分成两组,每组3只,其中一组给予TF-PEG-PLGA纳米粒,另一组给予PEG-PLGA纳米粒,两组的荧光染料控制在每只大鼠10μg~20μg,在滴鼻给药1h后,用活体成像仪检测两组纳米粒在脑内荧光的分布情况。在成像前,对大鼠进行腹腔注射麻醉剂,以保证它们在成像期间不会移动,然后使用荧光成像仪对大鼠进行扫描观察。
图8为TF-PEG-PLGA纳米粒和PEG-PLGA纳米粒在动物脑组织中的靶向效果,其中A为荧光成像仪观察结果,B为统计学分析结果。图8中的结果表明,连接了转铁蛋白TF的载药纳米粒具有更好的脑靶向效果。
实施例6
通过HE染色观察经转铁蛋白修饰的负载莱菔硫烷的载药纳米粒在治疗急性一氧化碳中毒脑损伤大鼠的脑靶向性治疗效果。
随机将大鼠分为五个组,NC组为正常对照组,ACOP组为急性一氧化碳中毒模型组;SFN组为游离莱菔硫烷治疗组;SFN- PEG-PLGA组为未经转铁蛋白修饰的载药纳米粒治疗组;SFN-TF-PEG-PLGA组为经TF修饰的载药纳米粒治疗组。给药方式采用滴鼻给药,每天给药一次,连续七天。其中,SFN、SFN- PEG-PLGA和SFN-TF-PEG-PLGA组的莱菔硫烷剂量为5mg/kg;正常对照组和一氧化碳中毒模型组给予治疗组同等体积的生理盐水治疗。连续七天治疗后,将大鼠进行麻醉处理,取出大鼠脑组织,经过石蜡包埋后制作切片,将制作好的脑组织切片进行HE染色,在显微镜下观察脑组织切片中观察脑组织切片中细胞形态变化情况。
图9为脑组织切片的HE染色结果,标尺为50μm。HE染色结果显示,经转铁蛋白TF修饰的负载莱菔硫烷的纳米粒(SFN-TF-PEG-PLGA NPs)治疗组的效果最好,可以更好地抑制炎性细胞浸润和细胞肿胀,减轻一氧化碳中毒脑损伤。
实施例7
通过透射电镜观察细胞超微结构损伤情况,通过观察脑组织的超微结构变化探究经转铁蛋白修饰的负载莱菔硫烷的载药纳米粒在治疗急性一氧化碳中毒脑损伤大鼠的脑靶向性治疗效果。
本实验选择雄性SD大鼠作为实验对象,体重在200~220g之间,随机将大鼠分为五个组,NC组为正常对照组,ACOP组为急性一氧化碳中毒模型组;SFN组为游离莱菔硫烷治疗组;SFN-PEG-PLGA组为未修饰的载药纳米粒治疗组;SFN- TF-PEG-PLGA组为经TF修饰的载药纳米粒治疗组。给药方式采用滴鼻给药,每天给药1次,连续7天。其中,SFN、SFN-PEG-PLGA和SFN-TF-PEG-PLGA组的莱菔硫烷剂量为5mg/kg;正常对照组和一氧化碳中毒模型组给予治疗组同等剂量的生理盐水治疗。
在连续治疗七天后,取出大鼠脑组织,进行脑组织固定和包埋制作成超薄切片,对脑组织切片进行透射电镜观察。
图10为脑组织切片的透射电镜观察结果,其中A1-E1为5000x的放大倍数,对应的靶尺为2μm,A2-E2为40000x的放大倍数,对应的靶尺为500nm。通过观察结果可以看出,连接了转铁蛋白TF的纳米粒(SFN- TF-PEG-PLGA NPs)具有较好的脑靶向性治疗效果,可以有效改善急性一氧化碳中毒脑损伤引起的超微结构损伤。
实施例8
通过尼氏染色观察经转铁蛋白修饰的负载莱菔硫烷纳米粒对急性一氧化碳中毒脑损伤的治疗效果,评估其脑靶向性。
随机将大鼠分为五个组,NC组为正常对照组,ACOP组为急性一氧化碳中毒模型组;SFN组为游离莱菔硫烷治疗组;SFN-PEG-PLGA组为未修饰的载药纳米粒治疗组;SFN-TF-PEG-PLGA组为经TF修饰的载药纳米粒治疗组。给药方式采用滴鼻给药,每天给药一次,连续七天。其中,SFN、SFN-PEG-PLGA和SFN-TF-PEG-PLGA组的莱菔硫烷剂量为5mg/kg;正常对照组和一氧化碳中毒模型组给予治疗组同等体积的生理盐水治疗。连续七天治疗后,取出大鼠脑组织,经过石蜡包埋后制作切片,将制作好的脑组织切片进行尼氏染色,在显微镜下观察脑组织切片中的尼氏小体损伤情况。
实验结果如图11所示,图11中A为显微镜观察结果(标尺为50μm),B为统计学分析结果。实验结果说明,转铁蛋白修饰的载药纳米粒(SFN- TF-PEG-PLGA NPs)可以有效地提高药物的靶向性,促进药物在脑组织中的分布和作用,减少细胞凋亡从而达到更好的治疗效果。
实施例9
通过Western blot实验检测特异性蛋白的表达情况,评估经转铁蛋白修饰的负载莱菔硫烷纳米粒对急性一氧化碳中毒脑损伤的治疗效果。
按照实施例8中的方法进行实验分组和给药,根据实验分组设计,取出治疗后7天的大鼠脑组织样本,并用磷酸缓冲盐水(PBS)清洗1~3次,去除表面的血液和其他污染。根据每10mg组织加40μL裂解液的比例加入提前预冷的RIPA裂解液(含有1%PMSF)并置于提前预冷的冷冻研磨仪中研磨90s,使细胞膜破裂,释放细胞内蛋白。通过离心将细胞膜和其他杂质沉淀,取出上清液。使用BCA比色法检测上清液中蛋白的浓度。固定每组40mg蛋白上样量将蛋白样品加入SDS-PAGE凝胶,进行电泳分离。将电泳分离后的蛋白转移到PVDF膜上,之后将PVDF膜放入5%的脱脂奶粉溶液中,阻断非特异性结合位点。根据抗体说明书加入适当稀释比例的一抗(P13K/P-P13K P-P38/TGF-β1/BCL2/BAX/GAPDH抗体),并在4℃下过夜孵育,使主抗体与膜上的目标蛋白发生特异性结合。第二天,将膜用TBST缓冲液洗涤3次,每次10min,去除未结合的抗体。然后根据一抗的种属来源选择对应的二抗,在室温下孵育1h,使二抗与一抗结合形成免疫复合物。再次用TBST缓冲液洗涤3次,每次10min,去除未结合的二抗。最后,加入ECL发光液,放入化学发光成像仪拍照检测,记录成像结果。
实验结果如图12所示,图12中A为光学成像结果,B为P-P38/GAPDH的统计学分析结果,C为TGF-β1/GAPDH的统计学分析结果,D为BCL2/GAPDH的统计学分析结果,E为P-P13K/P13K的统计学分析结果,F为BAX/GAPDH的统计学分析结果。
实验结果说明,转铁蛋白修饰的载药纳米粒(SFN-TF-PEG-PLGA NPs)可以有效地提高药物的靶向性,促进抗凋亡蛋白的表达,抑制促凋亡蛋白的表达,从而达到更好的治疗效果。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种脑靶向纳米粒,其特征在于,包括载药纳米粒和连接在所述载药纳米粒表面的转铁蛋白;所述载药纳米粒包括载体和负载在所述载体中的药物;所述载体为两亲性嵌段共聚物;所述药物为莱菔硫烷;
所述两亲性嵌段共聚物为聚乙二醇-聚乳酸羟基乙酸共聚物;
所述脑靶向纳米粒的制备方法包括以下步骤:将带有羧基的聚乙二醇-聚乳酸羟基乙酸共聚物、莱菔硫烷和有机溶剂混合,得到有机相;将所述有机相加入水中,然后将有机溶剂挥干,得到载药纳米粒混悬液;将所述载药纳米粒混悬液、EDC和NHS混合进行活化,然后加入转铁蛋白进行偶联反应,得到所述脑靶向纳米粒;所述带有羧基的聚乙二醇-聚乳酸羟基乙酸共聚物和莱菔硫烷的质量比为(5~300):(1.25~75);所述转铁蛋白和所述带有羧基的聚乙二醇-聚乳酸羟基乙酸共聚物的摩尔比为(50~750):1;所述活化的温度为20~26℃,时间为0.5~4h;所述偶联反应的温度为20~26℃,时间为8~16h。
2.根据权利要求1所述的脑靶向纳米粒,其特征在于,所述聚乙二醇-聚乳酸羟基乙酸共聚物中聚乙二醇嵌段的重均分子量为2K~10K,聚乳酸羟基乙酸嵌段的重均分子量为5K~20K。
3.根据权利要求1所述的脑靶向纳米粒,其特征在于,所述脑靶向纳米粒的粒径为97~151nm;所述脑靶向纳米粒中莱菔硫烷的载药量为12wt%~21wt%。
4.权利要求1~3任意一项所述脑靶向纳米粒的制备方法,其特征在于,包括以下步骤:
将带有羧基的聚乙二醇-聚乳酸羟基乙酸共聚物、莱菔硫烷和有机溶剂混合,得到有机相;
将所述有机相加入水中,然后将有机溶剂挥干,得到载药纳米粒混悬液;
将所述载药纳米粒混悬液、EDC和NHS混合进行活化,然后加入转铁蛋白进行偶联反应,得到所述脑靶向纳米粒;
所述带有羧基的聚乙二醇-聚乳酸羟基乙酸共聚物和莱菔硫烷的质量比为(5~300):(1.25~75);所述转铁蛋白和所述带有羧基的聚乙二醇-聚乳酸羟基乙酸共聚物的摩尔比为(50~750):1;所述活化的温度为20~26℃,时间为0.5~4h;所述偶联反应的温度为20~26℃,时间为8~16h。
5.根据权利要求4所述的制备方法,其特征在于,所述有机相和水的体积比为1:(2~20)。
6.根据权利要求4所述的制备方法,其特征在于,所述带有羧基的两亲性嵌段共聚物、EDC和NHS的摩尔比为1:(2~10):(2~5)。
7.权利要求1~3任意一项所述的脑靶向纳米粒或权利要求4~6任意一项所述制备方法制备的脑靶向纳米粒在制备治疗急性一氧化碳中毒脑损伤的药物中的应用。
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"基于网络药理学探讨莱菔硫烷治疗急性一氧化碳中毒脑损伤的作用机制",岳傲春等,《中国应用生理学杂志》,第38卷第6期,第725-729页;岳傲春等;《中国应用生理学杂志》;第38卷(第6期);第725-729页 * |
Transferrin-appended PEGylated nanoparticles for temozolomide delivery to brain: in vitro characterisation;Aviral Jain等;《Journal of Microencapsulation》;第28卷(第1期);第21-28页 * |
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