CN117045811A - 一种骨靶向纳米载药体系及其制备方法和应用 - Google Patents
一种骨靶向纳米载药体系及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种骨靶向纳米载药体系及其制备方法和应用,将天然药物单体槲皮素与六氯环三磷腈自交联形成纳米微球,并在表面修饰氨基聚乙二醇以及骨靶向配体阿仑膦酸,构建具有良好水分散性和骨靶向功能的复合纳米载药体系。本发明利用六氯环三磷腈的固有化学特性,与槲皮素自交联形成纳米微球;通过对纳米微球表面修饰聚乙二醇,能克服槲皮素本身水溶性差的特性,还能避免纳米药物被体内单核‑巨噬细胞的吞噬,从而延长其循环时间;进一步在表面修饰骨靶向配体阿仑膦酸,能赋予其骨靶向性,将纳米药物靶向输送至骨组织中。最终,本发明制备的纳米载药体系具有良好的骨靶向性,在抗骨质疏松领域具有良好的前景以及很高的潜在应用价值。
Description
技术领域
本发明涉及生物医药领域,尤其涉及一种骨靶向纳米载药体系及其制备方法和应用。
背景技术
目前,与年龄相关的健康问题日益增多,骨质疏松症便是其中之一。骨质疏松症是一种全身性代谢性骨病,其特征为骨量降低、骨微结构破坏,导致骨脆性增加,易发生脆性骨折等。目前,世界卫生组织已将骨质疏松症列为仅次于心血管疾病的第二大危害人类健康的慢性疾病。因此,深入开展骨质疏松症的病理学研究,积极探索新的防治措施具有重大的社会意义和经济价值。
人体内的骨组织并非静止不变的,成骨细胞不断产生新的骨质,破骨细胞对旧的以及毁损的骨质进行吸收,从而维持着骨代谢的动态平衡。在骨质疏松症的发病过程中,破骨细胞扮演着重要角色,破骨细胞过度活化或其功能的异常增强是造成骨量丢失的关键原因。除此之外,作为骨代谢的另一方,成骨细胞数目减少或活性降低亦将导致骨再生能力减弱,使得新骨形成减少,骨小梁厚度变薄、间隙增加,最终导致骨显微结构破坏,脆性增加,诱发骨质疏松性骨折。考虑到骨质疏松病理状态下骨吸收和骨形成的失衡,抑制过度活跃的骨吸收,同时促进新骨形成,恢复骨代谢平衡是治疗骨质疏松症的理想办法。目前,已有多种抗骨质疏松药物相继问世,包括:双膦酸盐类,选择性雌激素受体调节剂,RANKL单克隆抗体,重组人甲状旁腺激素等。然而,尽管抗骨质疏松药物众多,但其治疗结果却并不理想:双膦酸盐类药物长期使用后下颌骨坏死、股骨非特异性骨折等并发症时有发生;选择性雌激素受体调节剂在使用中可造成下肢的疼痛性痉挛,血管舒缩样不稳,甚至出现严重的脑卒中;RANKL单克隆抗体抗骨吸收作用确切,但临床试验中发现有诱发恶性肿瘤的潜在风险,药物安全性还有待进一步论证;而作为唯一具有成骨效应的甲状旁腺激素,在临床使用中发现常出现高钙血症、短暂性低血压甚至过敏性休克等不良反应,加上其价格昂贵,仍未广泛应用。有鉴于此,研发新一代更加安全且兼具抑制骨吸收和促进骨形成双重功效的抗骨质疏松药物,是当下的迫切需求。
因此,现有技术需要改进。
发明内容
鉴于上述现有技术的不足,本发明的目的在于提供一种骨靶向纳米载药体系及其制备方法和应用,旨在解决现有技术中缺乏安全、兼具抑制骨吸收和促进骨形成双重功效的抗骨质疏松药物的问题。
本发明的技术方案如下:
一种骨靶向纳米载药体系,其中,包括药物单体、六氯环三磷腈、氨基聚乙二醇以及骨靶向配体;其中,所述药物单体与六氯环三磷腈自交联形成纳米微球,所述纳米微球表面修饰所述氨基聚乙二醇以及骨靶向配体。
所述的骨靶向纳米载药体系,其中,所述药物单体为槲皮素,所述骨靶向配体为阿仑膦酸。
所述的骨靶向纳米载药体系,其中,所述载药体系包括槲皮素、六氯环三磷腈、氨基聚乙二醇以及阿仑膦酸;所述槲皮素与六氯环三磷腈自交联形成纳米微球,所述纳米微球表面修饰所述氨基聚乙二醇以及阿仑膦酸。
一种骨靶向纳米载药体系的制备方法,其中,包括步骤:
使六氯环三磷腈与药物单体发生自交联,形成纳米微球;
将氨基聚乙二醇溶于水中,得到氨基聚乙二醇溶液;
将所述纳米微球分散在溶剂中,之后加入所述氨基聚乙二醇溶液、骨靶向配体以及催化剂,超声反应预定时间一;
反应完成后,分离固体产物并提纯、干燥,得到所述骨靶向纳米载药体系。
所述的骨靶向纳米载药体系的制备方法,其中,所述药物单体为槲皮素。
所述的骨靶向纳米载药体系的制备方法,其中,使六氯环三磷腈与槲皮素发生自交联,形成纳米微球的步骤具体包括:
提供六氯环三磷腈和槲皮素;
将所述六氯环三磷腈和槲皮素分别溶于二甲基甲酰胺中;
将得到的六氯环三磷腈溶液和槲皮素溶液混合,并加入三乙胺,超声反应预定时间二;
反应完成后,分离固体产物并洗涤、冻干,得到所述纳米微球。
所述的骨靶向纳米载药体系的制备方法,其中,所述六氯环三磷腈和槲皮素的摩尔比为1:1。
所述的骨靶向纳米载药体系的制备方法,其中,所述氨基聚乙二醇的制备方法包括步骤:
将2000分子量的聚乙二醇溶于二氯甲烷中,之后加入三乙胺冰浴30分钟,得到聚乙二醇溶液;
将对甲苯磺酰氯溶于二氯甲烷中,得到对甲苯磺酰氯溶液,将所述对甲苯磺酰氯溶液加入到所述聚乙二醇溶液中发生反应;
反应结束后,将产物旋蒸浓缩并经石油醚萃取,得到聚乙二醇-OTS;
将所述聚乙二醇-OTS与氨水混合,置于反应釜中,在80℃下反应24h;
反应结束后,将产物加入乙醇旋干后再烘干过夜,并经石油醚萃取,得到所述氨基聚乙二醇。
一种骨靶向纳米载药体系的应用,其中,将如上任一所述的骨靶向纳米载药体系或者如上任一所述的制备方法得到的骨靶向纳米载药体系用于抗骨质疏松药物的制备。
一种骨靶向纳米载药体系的应用,其中,将如上任一所述的骨靶向纳米载药体系或者如上任一所述的制备方法得到的骨靶向纳米载药体系用于骨代谢平衡的分子机制以及骨质疏松中骨代谢调控机理的研究。
有益效果:本发明提供的骨靶向纳米载药体系,将筛选的天然药物单体与六氯环三磷腈(HCCP)自交联形成纳米微球,并在表面修饰氨基聚乙二醇(NH2-PEG)以及骨靶向配体阿仑膦酸(ALN),构建具有良好水分散性和骨靶向功能的复合纳米载药体系。本发明利用HCCP的固有化学特性,能与天然药物单体——槲皮素自交联形成纳米微球;通过对纳米微球表面修饰氨基聚乙二醇,能克服槲皮素本身水溶性差的特性,还能避免纳米药物被体内单核-巨噬细胞的吞噬,从而延长其循环时间;进一步在表面修饰骨靶向配体阿仑膦酸,能赋予其骨靶向性,将纳米药物靶向输送至骨组织中。不仅如此,阿仑膦酸也是一种抗骨吸收药物,能协同槲皮素共同发挥抗骨质疏松的作用。最终,本发明制备的纳米载药体系具有良好的骨靶向性,在体外能有效的抑制破骨细胞的分化及功能,同时能明显促进成骨细胞的分化;在体内能明显减少骨质疏松模型小鼠的骨丢失;而且,所述纳米载药体系安全无毒,对生物友好,因此其在抗骨质疏松领域具有良好的前景以及很高的潜在应用价值。
附图说明
图1为本发明所述负载槲皮素的骨靶向纳米载药体系的制备流程示意图以及作用机理示意图。
图2为本发明实施例制备的HQPA纳米微球的表征结果示意图,其中,核磁共振氢谱(A)、紫外-可见吸收光谱(B)、傅里叶变换红外光谱(C)、热重分析(D)。
图3为本发明实施例制备的HQPA纳米微球的粒径、投射电镜图(TEM)和释放曲线图,其中,DLS(A)、TEM(B)、槲皮素标准曲线(C)及不同pH下槲皮素释放曲线(D)。
图4为本发明实施例制备的HQPA纳米微球的体外骨靶向实验结果示意图,其中,左侧为未接枝阿仑膦酸的纳米微球,右侧为为阴性对照。
图5为本发明实施例制备的HQPA纳米微球在小鼠骨髓原代细胞(BMMs)内的分布示意图。
图6为本发明实施例制备的HQPA纳米微球影响破骨细胞分化的结果示意图。
图7为本发明实施例制备的HQPA纳米微球影响破骨细胞肌动蛋白环形成的结果示意图。
图8为本发明实施例制备的HQPA纳米微球影响成骨分化的结果示意图。
图9为本发明实施例制备的HQPA纳米微球对卵巢切除小鼠骨丢失的影响结果示意图。
图10为本发明实施例不同组胫骨通过脱钙处理后进行石蜡包埋H&E染色和破骨细胞特异性的TRAP染色结果示意图。
图11为本发明实施例不同组小鼠重要器官切片的H&E染色(心、肝、脾、肺、肾)结果示意图。
具体实施方式
本发明提供一种骨靶向纳米载药体系及其制备方法和应用,为使本发明的目的、技术方案及效果更加清楚、明确,以下对本发明进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
本发明实施例提供一种骨靶向纳米载药体系,包括药物单体、六氯环三磷腈、氨基聚乙二醇以及骨靶向配体。
在一些实施方式中,所述药物单体为槲皮素。
天然药物单体因具有毒性小、获取方便、药理作用广泛等特性引起研究者的关注,尤其是多酚类及黄酮类药物,能够通过多种机制调控骨代谢,从而发挥抗骨质疏松作用。近几年,研究发现槲皮素(quercetin,Que)不仅具有抑制破骨细胞生成,还能通过调节炎症反应、抑制脂肪分化,减少氧化应激等多种途径促进成骨分化,其显示出良好的双向调控骨代谢作用,是一个理想的抗骨质疏松药物。但槲皮素水溶性差,缺乏靶向性且发挥疗效的有效浓度较大等缺点极大限制了其应用。因此,本发明构建了具有骨靶向的载药体系,改善了天然药物单体的水溶性,将其靶向输送至骨组织,特异性发挥双向调控作用,有效的规避了上述问题。
在一些实施方式中,所述药物单体与六氯环三磷腈自交联形成纳米微球,所述纳米微球表面修饰所述氨基聚乙二醇以及骨靶向配体。
具体的,所述药物单体为槲皮素。
具体的,所述骨靶向配体为阿仑膦酸。
在一些实施方式中,所述槲皮素与六氯环三磷腈自交联形成纳米微球,所述纳米微球表面修饰所述氨基聚乙二醇以及阿仑膦酸。
纳米材料具有降低药物毒副作用、提高药物稳定性和缓释控释药物等优点,是较为理想的药物载体。六氯环三磷腈(Hexachlorocyclotriphosphonitrile,HCCP)是以N、P原子以交替的单双键排列而成,其无机骨架在生物体系中可以降解,产物无毒、无腐蚀性、无炎症反应,同时因P-Cl键的活性大,反应位点多,所有的Cl都能够和槲皮素上的酚羟基(-OH)反应并自交联形成纳米微球。另外,纳米微球表面剩余的P-C键可以进一步和氨基聚乙二醇(NH2-PEG)以及骨靶向配体阿仑膦酸(Alendronic Acid,ALN)上的氨基(-NH2)反应,从而构建了一个具有良好水分散性和骨靶向功能的复合纳米载药体系。
本发明实施例还提供一种骨靶向纳米载药体系的制备方法,包括步骤:
S10、使六氯环三磷腈与药物单体发生自交联,形成纳米微球;
S20、将氨基聚乙二醇溶于水中,得到氨基聚乙二醇溶液;
S30、将所述纳米微球分散在溶剂中,之后加入所述氨基聚乙二醇溶液、骨靶向配体以及催化剂,超声反应预定时间一;
S40、反应完成后,分离固体产物并提纯、干燥,得到所述骨靶向纳米载药体系。
本发明将筛选的天然药物单体与HCCP自交联形成纳米微球,并在表面修饰PEG及ALN,构建了具有良好水分散性和骨靶向功能的纳米复合载药体系。
在一些实施方式中,制备氨基聚乙二醇的步骤包括:
S201、将2000分子量的聚乙二醇溶于二氯甲烷中,之后加入三乙胺冰浴30分钟,得到聚乙二醇溶液;
S202、将对甲苯磺酰氯溶于二氯甲烷中,得到对甲苯磺酰氯溶液,将所述对甲苯磺酰氯溶液加入到所述聚乙二醇溶液中发生反应;
S203、反应结束后,将产物旋蒸浓缩并经石油醚萃取,得到聚乙二醇-OTS;
S204、将所述聚乙二醇-OTS与氨水混合,置于反应釜中,在80℃下反应24h;
S205、反应结束后,将产物加入乙醇旋干后再烘干过夜,并经石油醚萃取,得到所述氨基聚乙二醇。
在一个具体的实施例中,氨基聚乙二醇(NH2-PEG)的合成步骤为:
a.将2000分子量的聚乙二醇(PEG)溶于一定量的二氯甲烷中,并加入2-3ml三乙胺冰浴30分钟;
b.将3-4g对甲苯磺酰氯(TsCl)溶于二氯甲烷中,并缓慢滴入步骤a含有PEG的溶液中,过夜反应;
c.反应结束后,将产物旋蒸浓缩,然后滴加入冷冻的石油醚中,反复操作几次,得到淡黄色产物PEG-OTS;
d.随后将得到的产物PEG-OTS与40ml的氨水混合,加入150ml的反应釜中,在80℃下反应24h后冷却至室温;
e.反应结束后,将产物加入乙醇旋干,再烘干过夜,最后加入少量的二氯甲烷溶剂,并用冷冻石油醚反复沉淀数次,最终得到纯净的产物,即氨基聚乙二醇。
在一些实施方式中,预定时间一为1~3h,优选为3h。
在一些实施方式中,步骤S30中,超声反应所用的超声条件为功率为100W。
在一些实施方式中,所述催化剂为三乙胺。三乙胺(TEA)可作为反应的催化剂及缚酸剂。
在一个具体的实施例中,所述骨靶向配体为阿仑膦酸,所述药物单体为槲皮素,所述六氯环三磷腈-药物单体纳米微球为六氯环三磷腈-槲皮素纳米微球。
具体的,负载槲皮素的骨靶向纳米载药体系的制备方法包括步骤:
S100、使六氯环三磷腈与槲皮素发生自交联,形成六氯环三磷腈-槲皮素纳米微球;
S200、制备氨基聚乙二醇,并将所述氨基聚乙二醇溶于水中,得到氨基聚乙二醇溶液;
S300、将所述六氯环三磷腈-槲皮素纳米微球分散在溶剂中,之后加入所述氨基聚乙二醇溶液、预定浓度的阿仑膦酸溶液以及催化剂,超声反应预定时间一;
S400、反应完成后,分离固体产物并提纯、冻干,得到所述负载槲皮素的骨靶向纳米载药体系。
图1所示为本发明实施例中所述负载槲皮素的骨靶向纳米载药体系的制备流程示意图以及作用机理。为解决天然药物槲皮素水溶性差、缺乏靶向性等缺点,拓宽其在骨质疏松领域的应用,本发明通过自组装的方式设计了一个pH响应型骨靶向纳米载药体系,将槲皮素定向输送至骨组织内,发挥其双向调控骨代谢抗骨质疏松作用。而且,所述纳米载药体系具有良好的pH响应性,在pH=5.4时能释放60%槲皮素,且根据槲皮素标准曲线计算出槲皮素载药率达30%以上。
在一些实施方式中,使六氯环三磷腈与槲皮素发生自交联,形成六氯环三磷腈-槲皮素纳米微球的步骤包括:
S101、提供六氯环三磷腈和槲皮素;
S102、将所述六氯环三磷腈和槲皮素分别溶于二甲基甲酰胺中;
S103、将得到的六氯环三磷腈溶液和槲皮素溶液混合,并加入三乙胺,超声反应预定时间二;
S104、反应完成后,分离固体产物并洗涤、冻干,得到所述六氯环三磷腈-槲皮素纳米微球。
在一些实施方式中,所述六氯环三磷腈和槲皮素的摩尔比为1:1。
在一些实施方式中,步骤S102中,原料溶解后需要在水浴超声下超声10分钟,以活化官能团。
在一些实施方式中,预定时间二为1~3h,优选为3h。
在一些实施方式中,步骤S103中,超声反应所用的超声条件为功率为100W。
本发明将筛选的天然药物单体与HCCP自交联形成纳米微球,并在表面修饰PEG及ALN,构建具有良好水分散性和骨靶向功能的复合纳米载药体系。与现有技术相比,本发明利用HCCP的固有化学特性,能与槲皮素自交联形成纳米微球,通过对微球表面修饰聚乙二醇,能克服槲皮素本身水溶性差的特性,还能避免纳米药物被体内单核-巨噬细胞的吞噬,从而延长其循环时间,进一步在表面修饰阿仑膦酸,能赋予其骨靶向性,将纳米药物靶向输送至骨组织中。不仅如此,阿仑膦酸也是一种抗骨吸收药物,能协同槲皮素共同发挥抗骨质疏松的作用。在此基础上,本发明对纳米载药体系进行形貌特征、药物装载量、pH响应性及骨靶向功能检测分析,并验证该载药体系能在体内、体外双重调控骨代谢治疗骨质疏松的作用。最终试验证明,本发明制备的纳米载药体系具有良好的骨靶向性,并能在体外能有效的抑制破骨细胞的分化及功能,同时能明显促进成骨细胞的分化;在体内能明显减少骨质疏松模型小鼠的骨丢失;而且,所述纳米载药体系安全无毒,对生物友好,因此其在抗骨质疏松领域具有良好的前景以及很高的潜在应用价值。
本发明还提供了一种骨靶向纳米载药体系的应用,将上述的骨靶向纳米载药体系或者上述的制备方法得到的骨靶向纳米载药体系用于抗骨质疏松药物的制备。
在一些实施方式中,所述的骨靶向纳米载药体系用于核因子kB受体活化因子配体(RANKL)诱导的骨髓源性巨噬细胞(BMMs)融合生成破骨细胞。
本发明还提供了一种骨靶向纳米载药体系的应用,将上述的骨靶向纳米载药体系或者上述的制备方法得到的骨靶向纳米载药体系用于骨代谢平衡的分子机制以及骨质疏松中骨代谢调控机理的研究。
在一些实施方式中,所述的骨靶向纳米载药体系用于成骨细胞的成骨分化,或者骨质疏松症模型的研究。
下面通过具体实施例对本发明一种骨靶向纳米载药体系及其制备方法和应用做进一步的解释说明:
实施例1骨靶向纳米载药体系的制备
1、HCCP-Que纳米微球的制备:
(1)称取34mg HCCP及30mg Que,将其分别溶解于20ml二甲基甲酰胺(DMF)溶液中,在水浴中超声10分钟使其充分溶解;
(2)将两种溶液置入反应瓶中混合,并加入2ml三乙胺(TEA)作为催化剂及缚酸剂,在探头超声下超声3小时;
(3)反应完成后将产物倒入50ml离心管中,在10000转/分下离心5分钟沉淀,用超纯水及乙醇反复冲洗产物,直至上清液清澈;
(4)最后用冻干机将产物冻干,干燥保存。
2、氨基聚乙二醇(NH2-PEG)的合成:
(1)将2000分子量的聚乙二醇(PEG)溶于一定量的二氯甲烷中,并加入2-3ml三乙胺冰浴30分钟;
(2)将3-4g对甲苯磺酰氯(TsCl)溶于二氯甲烷中,并缓慢滴入含有PEG的溶液中,过夜反应;
(3)反应结束后,将产物旋蒸浓缩,然后滴加入冷冻的石油醚中,反复操作几次,得到淡黄色产物PEG-OTS;
(4)随后将得到的产物PEG-OTS与40ml的氨水混合,加入150ml的反应釜中,在80℃下反应24h后冷却至室温;
(5)反应结束后,将产物加入乙醇旋干,再烘干过夜,最后加入少量的二氯甲烷溶剂,并用冷冻石油醚反复沉淀数次,最终得到纯净的产物。
3、骨靶向纳米微球HQPA的制备:
(1)(NH2-PEG)称取32mg阿仑膦酸(ALN)及30mg氨基聚乙二醇分别溶解于10ml超纯水中,水浴超声10分钟使其充分溶解;
(2)将预先制备的HCCP-Que微球分散于20ml DMF中,随后与NH2-PEG及ALN水溶液混合,同样加入2ml TEA作为催化剂及缚酸剂,探头超声3小时,通过离心法对产物进行分离、提纯、冻干,最终得到淡黄色粉末状产物HQPA。
同时,制备作为对照的纳米微球HQP,其与HQPA的区别在于,步骤(2)中仅加入NH2-PEG,用同样的步骤合成未接枝阿仑膦酸的纳米微球HQP。
实施例2HQPA纳米微球的表征
对上述实施例1制备的HQPA纳米微球进行性能测试:
(1)将产物分别进行核磁共振氢谱(H-NMR)、傅里叶变换红外光谱(FTIR)、热重分析(TGA)、紫外-可见吸收光谱(UV-Vis)、X射线光电子能谱分析(XPS)等分析,验证HCCP、药物、PEG及ALN成功交联成一起构成一个完整的纳米结构;
(2)将产物进行扫描电镜(SEM)、透射电镜(TEM)等表征,对其进行结构、形貌特征表征。
图2以及图3中A-B为本实施所得的HQPA纳米微球的表征数据,其中图2核磁共振氢谱(A)、紫外-可见吸收光谱(B)、傅里叶变换红外光谱(C)、热重分析(D)证明该纳米微球成功合成,且具有良好的水分散性,粒径<200nm(图3中A);TEM显示其形貌特征为一致的类球型,大小均匀(图3中B)。
实施例3pH响应性检测
(1)槲皮素标准曲线的测定:首先配制不同浓度梯度的槲皮素溶液,利用UV-Vis分光光度计测试上述不同浓度的天然药物单体溶液在其紫外最大吸收波长处的吸光度数值。然后将测得的数值与对应的药物浓度线性拟合,从而获得天然药物单体的标准曲线。
(2)测定不同pH下药物释放曲线:将两份5mg骨靶向纳米微球分别分散到5mL pH分别为7.4、5.4的磷酸盐缓冲溶液(PBS)中,然后各加入到1kDa的透析袋中,透析袋两头夹住后分别放入100ml的pH=7.4和5.4的PBS溶液中,常温下磁力搅拌,然后在固定的时间(1h,2h,3h,4h,5h,6h,7h,8h,9h,10h,11h,12h,24h,36h,48,60h)各收集透析液三份,检测其在紫外最大吸光波长处的吸光度数值,测试完毕后将透析液倒回原来溶液中。然后根据之前测定的标准曲线计算不同时间点透析液中药物的浓度,从而获得骨靶向纳米微球的实时释放曲线,通过对比不同pH下的释放曲线明确其释放是否具有pH响应性。
图3中C-D结果显示实施例1所得的HQPA纳米微球具有良好的pH响应性,在pH=5.4时能释放60%槲皮素,且根据槲皮素标准曲线计算出槲皮素载药率达30%以上。
实施例4骨靶向性能检测
称取5mg的骨靶向纳米微球溶解于装有30ml pH=7.4的PBS缓冲液的离心管中,检测其最大紫外吸收波长处的光度值并记录,随后称取一份质量为100mg临床骨组织标本研磨成的粉末,加入骨靶向纳米微球的水溶液中,在常温下搅拌4小时,搅拌完毕后放入离心机中,在1000r/分的转速下离心5分钟,取出上清液,再次测量其最大紫外吸收波长处的光度值并记录,用公式骨结合率(%)=(搅拌前吸光度-搅拌后吸光度)/搅拌前吸光度*100%,计算样品的骨靶向性能。同时在相同情况下,用未接枝阿仑膦酸的纳米微球作为对照组。
图4为本发明实施例1制备的HQPA纳米微球的体外骨靶向实验图,其中左侧为未接枝阿仑膦酸的纳米微球,右侧为为阴性对照。结果显示所得的HQPA纳米微球与羟基磷灰石混合后紫外吸收光度值明显下降,颜色也明显变淡,通过计算羟基磷灰石结合率达66.4%,明显高于对照组HQP纳米微球溶液的结合率20.5%,说明合成的HQPA具有明显的骨靶向性。
实施例5BMMs对HQPA纳米微球的摄取与胞内分布研究
(1)体外分离培养小鼠骨髓来源的BMMs/BMSCs细胞,并将细胞接种至6孔板,加入培养基,过夜培养直至细胞贴壁生长;
(2)将HQPA纳米微球加入细胞中,共培养12小时,随后用PBS缓冲液冲洗两次,清除未进入细胞的药物;
(3)在48℃的温度下,用2%的戊二醛溶液和1%的锇酸对细胞进行固定2小时,固定完成后用PBS缓冲液清洗多余的固定液;
(4)随后将固定好的细胞用不同浓度梯度的乙醇进行脱水(30%,50%,70%,80%,95%,100%),每个梯度持续10分钟;
(5)在脱水后的培养基中加入环氧树脂,在60℃下持续48小时,然后切片,并用柠檬酸铅进行染色,随后在透射电镜下观察HQPA纳米微球在细胞内的分布情况。
图5为本发明实施例1制备的HQPA纳米微球在小鼠骨髓原代细胞(BMMs)内的分布,其中左侧图为细胞核,中间图为药物荧光,右侧图为二者叠加;结果显示所得的HQPA纳米微球能通过内吞作用进入细胞内,并缓慢释放槲皮素进入核内发挥作用。
实施例6HQPA纳米微球对破骨细胞分化及骨吸收功能的影响
(1)从细胞层面研究HQPA纳米微球对破骨细胞形成的影响
a.选取4-6周龄的C57/BL6小鼠,分离培养骨髓原代细胞(BMMs),分别接种于96孔板和6孔板内,待细胞完全贴壁后给予不同的浓度的HQPA纳米微球刺激48小时,然后通过CCK-8检测观察不同药物浓度对BMMs增殖、凋亡的影响以明确药物毒性范围。
b.体外分离培养小鼠骨髓来源的BMMs,M-CSF刺激下使其定向分化单核/巨噬细胞。
c.将上一步骤得到的细胞接种于96孔板内,每孔细胞数为7×103,待细胞贴壁生长24小时后,根据步骤a中得出的细胞毒性结果,给予不同浓度的HQPA纳米微球刺激,同时加入M-CSF(30ng/ml)和RANKL(50ng/ml)诱导破骨细胞分化,隔日诱导一次。
d.一方面,在诱导分化的第5-7天,待成熟的破骨样细胞形成时,4%多聚甲醛固定;另一方面,分别在诱导分化的第1、3、5天固定,而后进行破骨细胞特异性的TRAP染色,导致显微镜下统计三个或三个以上核的破骨细胞数目(OC.N)及破骨细胞铺展面积(SA)。
图6为本发明实施例1制备的HQPA纳米微球影响破骨细胞分化的结果图,其中对BMMS细胞的毒性结果(A)、能明显抑制破骨细胞分化及其定量分析图(B)的结果显示所得的HQPA纳米微球对破骨细胞分化的影响,A显示HQPA纳米微球在300μg/ml浓度以下对BMMs无明显毒性,B显示其能明显抑制破骨细胞的分化,且呈浓度依赖性。
(2)研究HQPA纳米微球对破骨细胞骨吸收功能的影响
a.体外分离培养小鼠骨髓来源BMMs细胞,在M-CSF的刺激下定向分化为单核-巨噬细胞;
b.BMMs来源的单核细胞分别接种于含有玻璃片和牛骨片(已经过无菌处理)的48孔板内,在含有M-CSF和RANKL的培养基中诱导破骨细胞分化,隔日换液;
c.在诱导分化第5天后,加入不同浓度的HQPA纳米微球,药物处理48小时后,4%的多聚甲醛固定细胞;
d.接种于玻璃片上的细胞进行DAPI和鬼笔环肽,利用激光共聚焦显微镜观察破骨细胞褶皱边缘(肌动蛋白环)的形成及破骨细胞胞浆内PH的情况;
e.取出牛骨片,洗去表面粘附的细胞,通过扫描电镜观察破骨细胞骨吸收陷窝的形成情况并利用3D Optical Profilometer技术(用于分析骨吸收后骨片表面形貌特征),统计分析破骨细胞骨吸收的表面积和体积,以此明确HQPA纳米微球对破骨细胞骨吸收功能的影响。
图7为本发明实施例1制备的HQPA纳米微球影响破骨细胞肌动蛋白环形成的结果图。结果显示所得的HQPA纳米微球能明显抑制破骨细胞骨吸收功能,A显示其能明显抑制破骨细胞的骨吸收功能,且呈现浓度依赖性;B显示其能明显抑制破骨细胞肌动蛋白环的形成,且呈浓度依赖性。
实施例7HQPA纳米微球对成骨细胞活性的影响规律及作用机制
(1)探究药物对MC3T3细胞的细胞毒性:培养MC3T3细胞,分别接种于96孔板和6孔板内,待细胞完全贴壁后给予不同的浓度的HQPA纳米微球刺激48小时,然后通过CCK-8检测观察不同药物浓度对BMMs增殖、凋亡的影响以明确药物毒性范围。
(2)药物成骨活性的研究:培养MC3T3细胞,接种于48孔板内,每孔细胞数1×104,24小时待细胞完全贴壁后开始成骨诱导并根据上一步骤得出的结果,在药物安全浓度范围内给予不同浓度的HQPA纳米微球刺激,隔日更换诱导液,分别于诱导分化的第0、1、5、7、14天进行ALP染色,在诱导分化的第21天进行Alizarin Red染色。
图8为本发明实施例1制备的HQPA纳米微球影响成骨分化的结果图。对MC3T3细胞的细胞毒性(A)、碱性磷酸酶染色(ALP)及茜素红染色(ARS)的结果图(B)显示所得的HQPA纳米微球能明显促进成骨细胞活性与功能。A显示HQPA纳米微球在100μg/ml浓度以下对MC3T3细胞无明显毒性,B、C显示HQPA纳米微球能明显促进碱性磷酸酶的生成及钙沉积,从而明显增强成骨细胞的成骨作用。
实施例8HQPA纳米微球在体内防止骨丢失的治疗效果及作用机理
本实施例拟构建去卵巢小鼠的骨质疏松模型,通过在体实验,获取实验动物标本,利用Micro-CT等技术手段系统研究HQPA纳米微球在小鼠体内的防治骨丢失的治疗效果。
a.骨质疏松小鼠模型的建立:30只8周龄的C57BL/6小鼠随机分为五组,每组6只,其中四组(共24只小鼠)10%水合氯醛(0.4ml/100g)麻醉后,常规消毒、铺巾。于大鼠两侧肋脊角区(第十二肋骨下1cm与脊柱外侧缘1cm交角处)逐层切开皮肤、皮下组织、深筋膜、腹部肌肉、腹膜,进入腹腔,找到并将卵巢动脉结扎,顺势将全部的卵巢切除,逐层缝合伤口。其中,假手术组只切开腹膜,不做卵巢切除。
b.动物饲养与药物干预:手术后次日起,连续三日腹腔注射青霉素(10万单位/500g)预防感染。然后开始给予药物干预,假手术组(Sham)和单纯卵巢切除组(Vehicle)腹腔注射生理盐水作为对照,其余三组分别给予腹腔注射槲皮素(10mg/kg)作为阳性对照组(Que),低剂量HQPA NPs(10mg/kg)组(Low)和高剂量HQPA NPs(20mg/kg)组(High),隔日一次。
c.动物处死与标本获取:于给药后第4周,完全麻醉后,随后将动物处死。动物处死后,完整获取大鼠双侧股骨、胫骨、腰椎各椎体以及心、肺、肝、肾、脾、等内脏组织。
d.标本检测分析:每组右侧的股骨,4%的多聚甲醛固定后先行Micro-CT扫描,然后将标本随机分为两部分,一部分脱钙处理后进行石蜡包埋H&E染色和破骨细胞特异性的TRAP染色,利用BioQuant软件进行骨形态计量学分析破骨细胞数目分析。
图9结果显示实施例1所得的HQPA纳米微球能防治卵巢切除后骨丢失,其中A显示不同组Micro-CT扫描结果,B是相关指标的定量分析,结果显示HQPA纳米微球能明显抑制卵巢切除后小鼠的骨丢失。
图10显示不同组胫骨通过脱钙处理后进行石蜡包埋H&E染色和破骨细胞特异性的TRAP染色,结果显示HQPA纳米微球能明显抑制破骨细胞的生成。
图11显示不同组小鼠重要器官切片的H&E染色(心、肝、脾、肺、肾),结果显示HQPANPs对其他重要器官无明显毒性。
综上,本实施例所设计的HQPA NPs在体内能够靶向地到达骨组织,并释放槲皮素有效发挥其抗骨质疏松作用。
综上所述,本发明提供了一种骨靶向纳米载药体系及其制备方法和应用。本发明提供的骨靶向纳米载药体系,将筛选的天然药物单体与六氯环三磷腈(HCCP)自交联形成纳米微球,并在表面修饰氨基聚乙二醇(NH2-PEG)以及骨靶向配体阿仑膦酸(ALN),构建具有良好水分散性和骨靶向功能的复合纳米载药体系。本发明利用HCCP的固有化学特性,能与天然药物单体——槲皮素自交联形成纳米微球;通过对纳米微球表面修饰氨基聚乙二醇,能克服槲皮素本身水溶性差的特性,还能避免纳米药物被体内单核-巨噬细胞的吞噬,从而延长其循环时间;进一步在表面修饰骨靶向配体阿仑膦酸,能赋予其骨靶向性,将纳米药物靶向输送至骨组织中。不仅如此,阿仑膦酸也是一种抗骨吸收药物,能协同槲皮素共同发挥抗骨质疏松的作用。最终,本发明制备的纳米载药体系具有良好的骨靶向性,在体外能有效的抑制破骨细胞的分化及功能,同时能明显促进成骨细胞的分化;在体内能明显减少骨质疏松模型小鼠的骨丢失;而且,所述纳米载药体系安全无毒,对生物友好,因此其在抗骨质疏松领域具有良好的前景以及很高的潜在应用价值。
应当理解的是,本发明的应用不限于上述的举例,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (10)
1.一种骨靶向纳米载药体系,其特征在于,包括药物单体、六氯环三磷腈、氨基聚乙二醇以及骨靶向配体;其中,所述药物单体与六氯环三磷腈自交联形成纳米微球,所述纳米微球表面修饰所述氨基聚乙二醇以及骨靶向配体。
2.根据权利要求1所述的骨靶向纳米载药体系,其特征在于,所述药物单体为槲皮素,所述骨靶向配体为阿仑膦酸。
3.根据权利要求1所述的骨靶向纳米载药体系,其特征在于,所述载药体系包括槲皮素、六氯环三磷腈、氨基聚乙二醇以及阿仑膦酸;所述槲皮素与六氯环三磷腈自交联形成纳米微球,所述纳米微球表面修饰所述氨基聚乙二醇以及阿仑膦酸。
4.一种骨靶向纳米载药体系的制备方法,其特征在于,包括步骤:
使六氯环三磷腈与药物单体发生自交联,形成纳米微球;
将氨基聚乙二醇溶于水中,得到氨基聚乙二醇溶液;
将所述纳米微球分散在溶剂中,之后加入所述氨基聚乙二醇溶液、骨靶向配体以及催化剂,超声反应预定时间一;
反应完成后,分离固体产物并提纯、干燥,得到所述骨靶向纳米载药体系。
5.根据权利要求4所述的骨靶向纳米载药体系的制备方法,其特征在于,所述药物单体为槲皮素。
6.根据权利要求5所述的骨靶向纳米载药体系的制备方法,其特征在于,使六氯环三磷腈与槲皮素发生自交联,形成纳米微球的步骤具体包括:
提供六氯环三磷腈和槲皮素;
将所述六氯环三磷腈和槲皮素分别溶于二甲基甲酰胺中;
将得到的六氯环三磷腈溶液和槲皮素溶液混合,并加入三乙胺,超声反应预定时间二;
反应完成后,分离固体产物并洗涤、冻干,得到所述纳米微球。
7.根据权利要求6所述的骨靶向纳米载药体系的制备方法,其特征在于,所述六氯环三磷腈和槲皮素的摩尔比为1:1。
8.根据权利要求4所述的骨靶向纳米载药体系的制备方法,其特征在于,所述氨基聚乙二醇的制备方法包括步骤:
将2000分子量的聚乙二醇溶于二氯甲烷中,之后加入三乙胺冰浴30分钟,得到聚乙二醇溶液;
将对甲苯磺酰氯溶于二氯甲烷中,得到对甲苯磺酰氯溶液,将所述对甲苯磺酰氯溶液加入到所述聚乙二醇溶液中发生反应;
反应结束后,将产物旋蒸浓缩并经石油醚萃取,得到聚乙二醇-OTS;
将所述聚乙二醇-OTS与氨水混合,置于反应釜中,在80℃下反应24h;
反应结束后,将产物加入乙醇旋干后再烘干过夜,并经石油醚萃取,得到所述氨基聚乙二醇。
9.一种骨靶向纳米载药体系的应用,其特征在于,将如权利要求1-3任一所述的骨靶向纳米载药体系或者如权利要求4-8任一所述的制备方法得到的骨靶向纳米载药体系用于抗骨质疏松药物的制备。
10.一种骨靶向纳米载药体系的应用,其特征在于,将如权利要求1-3任一所述的骨靶向纳米载药体系或者如权利要求4-8任一所述的制备方法得到的骨靶向纳米载药体系用于骨代谢平衡的分子机制以及骨质疏松中骨代谢调控机理的研究。
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