CN117398482A - 一种卵清蛋白负载硝基自由基氮氧化物纳米复合材料及其制备方法和应用 - Google Patents
一种卵清蛋白负载硝基自由基氮氧化物纳米复合材料及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种卵清蛋白负载硝基自由基氮氧化物纳米复合材料及其制备方法和应用。属于材料制备技术领域。硝基自由基氮氧化物纳米复合材料形状规则,平均粒径为100nm‑150nm,具有良好的分散性。制备方法如下:将壳聚糖和四甲基哌啶‑氮氧化物分别溶于水和二氯甲烷或二甲基亚砜,搅拌完全溶解之后将其二者混合,待反应结束后透析,透析结束后用超纯水洗涤,离心,分散在超纯水中。此外,将卵清蛋白负载在硝基自由基氮氧化物纳米复合材料上,可赋予该纳米材料免疫原性。该方法制备的材料具有核磁共振成像增强效果和作为免疫佐剂的潜力。
Description
技术领域
本发明涉及一种纳米复合材料的制备方法,具体涉及一种卵清蛋白负载硝基自由基氮氧化物纳米复合材料的制备方法,该方法简单稳定,可广泛应用于生物、医学领域。
背景技术
肿瘤早已成为地球上引起人类死亡最多的疾病之一,在20世纪导致了数百万人的死亡。为了应对其对人类的威胁,近些年来科学家们进行了无数次的尝试,开创了许多种治疗方法。但是由于肿瘤的异质性和一部分并发症,导致患者的存活率并不高。因此,发展更精准的肿瘤诊断技术和有效的肿瘤治疗方法对挽救肿瘤患者生命仍具有重大现实意义。
近年来,将纳米药物与癌症疗法相结合成为了肿瘤诊疗发展的一大趋势,而且已经有一部分纳米药物通过批准进入市场。纳米级诊疗体系成为一种替代性诊疗肿瘤的模型,它有良好的靶向性和较小的副作用,克服了传统疗法的局限性。新型癌症治疗方法中,癌症免疫疗法已经成为继手术疗法、放疗、化疗和靶向治疗后的第五大主流疗法,已经取得了令人瞩目的成果。癌症免疫疗法是通过训练和激活人体自身的免疫系统,增强自身的免疫能力,进而检测并杀死肿瘤细胞的治疗方法。而基于纳米药物的癌症免疫疗法有三个特有的优势:一是唤醒免疫系统,二是触发抗原的特异性免疫,三是提供长期的免疫记忆作用。纳米材料可以调节宿主的抗癌免疫应答,为开发新型癌症治疗方法提供了新的思路。
随着临床诊断技术和生物医学研究领域的发展,开发更加安全有效的疾病诊断和治疗方案成为研究的重中之重。从W.C.伦琴发现X射线算起,医学成像技术已经发展了100多年,包括核磁共振成像(MRI)、计算机断层扫描(CT)、伽马射线成像和用于精确诊断的超声成像技术。其中,核磁共振成像技术(Magnetic Resonance Imaging,即MRI)是一种通过原子核自旋运动,在外加磁场作用下被射频脉冲激发后会产生信号,再经电脑的处理转化为图像的断层成像技术,是临床上常用的诊断技术之一。同时,通过核磁共振成像技术还可以实现对生物组织的高空间分辨率和3D动态监测。然而,在临床应用中发现某些正常组织和病变组织的弛豫时间会相互重叠,进而导致诊断困难,为解决此问题,研究者引入核磁共振造影剂。目前市售核磁共振造影剂多以钆(Gd)基造影剂为主,但由于其较高的肾毒性和脑部沉积,使其安全性有待考量。因此开发对人体无损伤、生物相容性优异的核磁共振造影剂成为大势所趋和研究热点。
合成基于有机氮氧化物(TEMPO)的新型有机MRI造影剂并负载模型抗原OVA(卵清蛋白),提高其生物相容性和组织靶向性,增强其抗肿瘤效果和激活机体免疫功能的能力,增强核磁共振成像对比度。
发明内容
本发明的目的在于克服现有技术的不足,提供一种具有核磁共振增强效果和增强免疫应答的卵清蛋白负载硝基自由基氮氧化物纳米复合材料及其制备方法。
为了解决技术问题,本发明首先提供了一种卵清蛋白负载硝基自由基氮氧化物纳米复合材料的制备方法,包括如下步骤:
1)将壳聚糖溶解在超纯水中,同时加入冰醋酸,混合搅拌后得到溶液A;
2)将4-羧基-2,2,6,6-四甲基哌啶-氮氧化物溶解在二氯甲烷或二甲基亚砜中,之后加入1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和N-羟基琥珀酰亚胺(NHS),在0℃~5℃下混合搅拌后得到溶液B;
3)在步骤1)所得的A溶液中,加入吐温80溶液;再加入步骤2)所得的B溶液,混合得到混合液后进行搅拌,待反应结束进行透析,离心洗涤后得到CS-TEMPO纳米粒子,将洗涤得到的CS-TEMPO纳米粒子分散在超纯水中,得到CS-TEMPO纳米粒子分散液;
4)将卵清蛋白,1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和N-羟基琥珀酰亚胺(NHS)在0℃~5℃下混合搅拌,再加入步骤3)得到的CS-TEMPO纳米粒子分散液,混合搅拌,反应结束后进行离心洗涤,得到CS-TEMPO-OVA纳米粒子。
作为本发明的优选方案,所述步骤1)的A液中所添加的壳聚糖的脱乙酰度大于85%,壳聚糖和超纯水的摩尔比为650:1~700:1,加入的冰醋酸和超纯水的体积比为1:100~1:200。
作为本发明的优选方案,所述步骤2)的B液中所添加的4-羧基-2,2,6,6-四甲基哌啶-氮氧化物、1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和N-羟基琥珀酰亚胺(NHS)的摩尔比为1:(0.6~0.7):(0.5~0.7),4-羧基-2,2,6,6-四甲基哌啶-氮氧化物和二氯甲烷或二甲基亚砜的摩尔比为4:1~6:1。
作为本发明的优选方案,其特征在于,所述步骤3)中,A溶液和B溶液的体积比为4:3~1:1;所添加的吐温80溶液体积浓度为1%~3%,添加的吐温80溶液和混合液体积比为1:35~2:35。
作为本发明的优选方案,所述步骤3)中,反应温度为5℃~25℃,反应条件为避光,搅拌速度为500-700rpm,所述步骤3)中,混合搅拌时间为36h-50h。
作为本发明的优选方案,所述步骤3)中,透析时间为2~4天,透析袋分子量为250~300Da。
作为本发明的优选方案,所述步骤4)所添加的卵清蛋白、1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)、N-羟基琥珀酰亚胺(NHS)和CS-TEMPO的摩尔比为(100~200):200:(300~400):1。
作为本发明的优选方案,所述步骤4)中得到的CS-TEMPO-OVA纳米粒子分散到PBS中,并在4℃进行保存。
本发明还提供了上述的卵清蛋白负载硝基自由基氮氧化物纳米复合材料在制备核磁共振成像造影剂或肿瘤免疫治疗药物中的应用。
与现有技术相比,本发明具有的有益效果包括:
(1)本发明制备的卵清蛋白负载硝基自由基氮氧化物纳米复合材料(CS-TEMPO-OVA纳米粒子),r1弛豫率为3.1mM-1s-1,在核磁共振成像造影剂方面有潜在的应用;
(2)本发明制备的负载了模型抗原OVA的CS-TEMPO纳米粒子还在体外表现出有效的提高树突状细胞成熟的作用,能够间接激活T细胞免疫应答,从而达到免疫治疗的效果;
(3)本发明的制备方法对实验仪器的要求低,方法简单易于操作,得到的纳米颗粒形状尺寸均一,分散性良好。
附图说明
图1为本发明实施例1所得CS-TEMPO纳米粒子的透射电镜(TEM)图。
图2为本发明实施例1所得CS-TEMPO-OVA纳米粒子的透射电镜(TEM)图。
图3为本发明实施例1所得CS-TEMPO-OVA纳米粒子的红外光谱(FT-IR)图。
图4为本发明实施例1所得CS-TEMPO-OVA纳米粒子的核磁共振成像图片和T1弛豫率比例图。
图5为本发明实施例1所得产物与(b)BMDC细胞、(a)4T1细胞共培养的细胞毒性结果图。
图6为本发明实施例1所得CS-TEMPO-OVA纳米粒子与BMDC细胞共培养24h之后的流式分析结果图。
图7为本发明实施例1所得CS-TEMPO-OVA纳米粒子与BMDC细胞共培养24h之后的流式分析结果图。
图8为本发明实施例1所得CS-TEMPO-OVA纳米粒子与BMDC、T细胞共培养24h之后的ELISA酶联免疫吸附试验结果图。
图9为本发明对比例所得产物的透射电镜(TEM)图。
具体实施方式
下面结合具体实施方式对本发明做进一步阐述和说明。所述实施例仅是本公开内容的示范且不圈定限制范围。本发明中各个实施方式的技术特征在没有相互冲突的前提下,均可进行相应组合。
实施例一
将500mg脱乙酰度大于85%的壳聚糖溶于20mL超纯水,为使壳聚糖溶解完全,加入200μL的冰醋酸,搅拌2h,直至完全溶解且无气泡,以制备溶液A。将250mg 4-羧基-2,2,6,6-四甲基哌啶-氮氧化物溶于15mL二氯甲烷或二甲基亚砜,加入180mg的1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和100mg N-羟基琥珀酰亚胺(NHS),在0℃下避光混合搅拌0.5h,以制备溶液B。在A溶液中加入添加1mL浓度为2%的吐温80溶液,搅拌完全溶解之后将A、B溶液混合,在室温下以500rpm速度搅拌48h。待反应结束后透析,用分子量为300Da的透析袋透析2天,结束后用超纯水洗涤,离心,得到呈现橙色的中间产物,分散在10mL超纯水中,即为CS-TEMPO纳米粒子分散液。将4mg卵清蛋白(OVA)溶解在1mL超纯水中,加入1mg的1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和1mg N-羟基琥珀酰亚胺(NHS),加入1mL所得CS-TEMPO纳米粒子分散液,在0℃~5℃下混合搅拌8h,离心得到OVA负载的CS-TEMPO纳米粒子。
实施例1所得产物的透射电镜图片如图1和2所示,图1为CS-TEMPO纳米粒子,图2为CS-TEMPO-OVA纳米粒子。所得产物的红外光谱(FTIR)图片如图3所示,所得产物的核磁共振成像图片和T1弛豫率比例如图4所示。从图1和2中可见,所得的卵清蛋白负载硝基自由基氮氧化物纳米复合材料(CS-TEMPO-OVA纳米粒子)具有良好的分散性,且粒径均一稳定;从图3中可见,所得的卵清蛋白负载硝基自由基氮氧化物纳米复合材料(CS-TEMPO-OVA纳米粒子)纳米复合材料确实含CS和等TEMPO基团;从图4中可见,卵清蛋白负载硝基自由基氮氧化物纳米复合材料(CS-TEMPO-OVA纳米粒子)具有优异的核磁共振增强效果。
如图5所示,将不同浓度的卵清蛋白负载硝基自由基氮氧化物纳米复合材料(CS-TEMPO-OVA纳米粒子)与4T1(小鼠乳腺癌细胞)细胞、BMDC(小鼠骨髓源树突状细胞)细胞共培养24h,通过CCK-8法检测细胞生存率,卵清蛋白负载硝基自由基氮氧化物纳米复合材料(CS-TEMPO-OVA纳米粒子)有良好的生物相容性。
如图6和图7所示,将一定浓度的卵清蛋白负载硝基自由基氮氧化物纳米复合材料(CS-TEMPO-OVA纳米粒子)和BMDC(小鼠骨髓源树突状细胞)细胞共培养24h,用流式抗体CD11c、CD80和CD86染色之后,通过流式细胞仪检测,发现CS-TEMPO-OVA纳米粒子可高效促进树突状细胞成熟。
如图8所示,将一定浓度的卵清蛋白负载硝基自由基氮氧化物纳米复合材料(CS-TEMPO-OVA纳米粒子)和BMDC(小鼠骨髓源树突状细胞)细胞、T(小鼠脾脏T细胞)细胞共培养24h,再通过ELISA酶联免疫吸附试剂盒检测培养上清中细胞因子浓度,发现CS-TEMPO-OVA纳米粒子可以激活T细胞免疫应答,促进T细胞产生杀伤癌细胞的细胞因子,从而证明此纳米复合材料具有良好的抗肿瘤效果。
实施例二
将500mg脱乙酰度大于85%的壳聚糖溶于20mL超纯水,为使壳聚糖溶解完全,加入200μL的冰醋酸,搅拌2h,直至完全溶解且无气泡,以制备溶液A。将250mg 4-羧基-2,2,6,6-四甲基哌啶-氮氧化物溶于20mL二甲基亚砜,加入180mg的1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和100mg N-羟基琥珀酰亚胺(NHS),在0℃下避光混合搅拌0.5h,以制备溶液B。在A溶液中加入添加3mL浓度为2%的吐温80溶液,之后将A、B溶液混合,在室温下以500rpm速度搅拌48h。之后透析,用分子量为300Da的透析袋透析2天,结束后用超纯水洗涤,离心,得到呈现橙色的产物,进一步分散在10mL超纯水中,即为CS-TEMPO纳米粒子分散液。将5mg卵清蛋白(OVA)溶解在1mL超纯水中,加入1mg的1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和1mg N-羟基琥珀酰亚胺(NHS),加入1mL所得CS-TEMPO纳米粒子分散液,在0℃~5℃下混合搅拌8h,离心得到CS-TEMPO-OVA纳米粒子。
对比例
将500mg脱乙酰度大于85%的壳聚糖溶于20mL超纯水,为使壳聚糖溶解完全,加入200μL的冰醋酸,搅拌2h,直至完全溶解且无气泡,以制备溶液A。将250mg 4-羧基-2,2,6,6-四甲基哌啶-氮氧化物溶于20mL二氯甲烷或二甲基亚砜,加入200mg的1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和100mg N-羟基琥珀酰亚胺(NHS),室温下避光混合搅拌1h,以制备溶液B,搅拌完全溶解之后将A、B溶液混合,在室温下以500rpm速度搅拌48h。待反应结束后透析,用分子量为300Da的透析袋透析2天,结束后用超纯水洗涤,离心,分散在10mL超纯水中,即为CS-TEMPO纳米粒子分散液。将5mg卵清蛋白(OVA)溶解在1mL超纯水中,加入1mg的1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和1mg N-羟基琥珀酰亚胺(NHS),加入1mL所得CS-TEMPO纳米粒子分散液,室温下混合搅拌8h,离心得到CS-TEMPO-OVA纳米粒子。
如图9所示,为本发明对比例所得产物的透射电镜(TEM)图;从图8中可见,通过此对比例得的卵清蛋白负载硝基自由基氮氧化物纳米复合材料(CS-TEMPO-OVA纳米粒子)粒径不均一,且分散效果不好。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (10)
1.一种卵清蛋白负载硝基自由基氮氧化物纳米复合材料的制备方法,其特征在于,包括如下步骤:
1)将壳聚糖溶解在超纯水中,同时加入冰醋酸,混合搅拌后得到溶液A;
2)将4-羧基-2,2,6,6-四甲基哌啶-氮氧化物溶解在二氯甲烷或二甲基亚砜中,之后加入1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和N-羟基琥珀酰亚胺(NHS),在0℃~5℃下混合搅拌后得到溶液B;
3)在步骤1)所得的A溶液中,加入吐温80溶液;再加入步骤2)所得的B溶液,混合得到混合液后进行搅拌,待反应结束进行透析,离心洗涤后得到CS-TEMPO纳米粒子,将洗涤得到的CS-TEMPO纳米粒子分散在超纯水中,得到CS-TEMPO纳米粒子分散液;
4)将卵清蛋白、1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和N-羟基琥珀酰亚胺(NHS)在0℃~5℃下混合搅拌,再加入步骤3)得到的CS-TEMPO纳米粒子分散液,混合搅拌,反应结束后进行离心洗涤,得到CS-TEMPO-OVA纳米粒子。
2.根据权利要求1所述的卵清蛋白负载硝基自由基氮氧化物纳米复合材料的制备方法,其特征在于,所述步骤1)的A液中所添加的壳聚糖的脱乙酰度大于85%,壳聚糖和超纯水的摩尔比为650:1~700:1,加入的冰醋酸和超纯水的体积比为1:100~1:200。
3.根据权利要求1所述的卵清蛋白负载硝基自由基氮氧化物纳米复合材料的制备方法,其特征在于,所述步骤2)的B液中所添加的4-羧基-2,2,6,6-四甲基哌啶-氮氧化物、1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和N-羟基琥珀酰亚胺(NHS)的摩尔比为1:(0.6~0.7):(0.5~0.7),4-羧基-2,2,6,6-四甲基哌啶-氮氧化物和二氯甲烷或二甲基亚砜的摩尔比为4:1~6:1。
4.根据权利要求1所述的卵清蛋白负载硝基自由基氮氧化物纳米复合材料的制备方法,其特征在于,所述步骤3)中,A溶液和B溶液的体积比为4:3~1:1;所添加的吐温80溶液体积浓度为1%~3%,添加的吐温80溶液和混合液体积比为1:35~2:35。
5.根据权利要求1所述的卵清蛋白负载硝基自由基氮氧化物纳米复合材料,其特征在于,所述步骤3)中,反应温度为5℃~25℃,反应条件为避光,搅拌速度为500-700rpm,所述步骤3)中,混合搅拌时间为36h-50h。
6.根据权利要求1所述的卵清蛋白负载硝基自由基氮氧化物纳米复合材料的制备方法,其特征在于,所述步骤3)中,透析时间为2~4天,透析袋分子量为250~300Da。
7.根据权利要求1所述的卵清蛋白负载硝基自由基氮氧化物纳米复合材料的制备方法,其特征在于,所述步骤4)所添加的卵清蛋白、1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)、N-羟基琥珀酰亚胺(NHS)和CS-TEMPO的摩尔比为(100~200):200:(300~400):1。
8.根据权利要求1所述的卵清蛋白负载硝基自由基氮氧化物纳米复合材料的制备方法,其特征在于,所述步骤4)中得到的CS-TEMPO-OVA纳米粒子分散到PBS中,并在4℃进行保存。
9.一种根据权利要求1-8任一项所述的制备方法制备的卵清蛋白负载硝基自由基氮氧化物纳米复合材料。
10.一种权利要求9所述的卵清蛋白负载硝基自由基氮氧化物纳米复合材料在制备核磁共振成像造影剂或肿瘤免疫治疗药物中的应用。
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