CN116650665A - 一种纳米靶向载药复合物及其制备方法和在防治血管钙化中的应用 - Google Patents
一种纳米靶向载药复合物及其制备方法和在防治血管钙化中的应用 Download PDFInfo
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Abstract
本发明公开了一种纳米靶向载药复合物及其制备方法和在防治血管钙化中的应用。本发明以Ti3C2MXenes纳米材料作为防治血管钙化的载体,并且进行了双抗体修饰,负载黄酮类化合物‑矢车菊素,使其具有靶向血管钙化的能力,从而达到靶向治疗目的;经过实验研究证明,对于高磷诱导的钙化雄性小鼠和HASMC诱导钙化模型,本发明提供了黄酮类矢车菊素来源的纳米化合物,有效缓解血管钙化,并且在毒性实验HE染色中也未对小鼠心脏、肝脏、脾脏、肺脏、肾脏造成损伤,具有较高的安全性,有较高的安全性,其有望应用到血管钙化疾病的防治当中;本发明为治疗血管钙化提供了一种新的思路和防治方向。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种纳米靶向载药及其制备方法和在防治血管钙化中的应用。
背景技术
血管钙化(vascular calcification,VC)是慢性肾脏病(chronic kidneydisease,CKD)最常见的合并症之一,并且是心血管疾病的独立危险因素和死亡原因。VC是一种类似骨形成的生物学过程,涉及从血管平滑肌细胞(vascular smooth muscle cell,VSMC)转向成骨样细胞的表型转换。其致病因素除了传统上建立的成骨信号外,功能失调的钙磷代谢异常、微细胞器功能障碍导致防御机制的丧失,包括线粒体碎片化、氧化应激、炎症反应、内质网应激等都可以导致血管钙化的发展。目前临床研究已证明,VC与CKD患者的发病率和死亡率相关,并且CKD患者的钙化要比正常人群早几年发生。目前血管钙化是普遍难治疾病之一,虽然有研究证明葡萄柚汁可以缓解血管钙化,但无靶向性,化合物体内利用度低等特点,目前尚未实际应用。因此,采用主动靶向可以更加有效的将纳米复合物运送到血管钙化部位。
MXene是一种新兴的二维(2D)过渡金属碳化物/氮化物家族,近年来因其具有大比表面积、高导电性、低毒性和可生物降解性等突出特性而得到了广泛的探索。制备主动靶向MXene纳米材料已有公开报道。中国专利文件CN202211049693A和CN113144206A均形成具有肿瘤靶向载药体系,目前尚无应用于血管钙化方向。
发明内容
本发明第一方面的目的,在于提供一种纳米靶向载药复合物。
本发明第二方面的目的,在于提供上述纳米靶向载药复合物的应用。
本发明第三方面的目的,在于提供一种包含上述纳米靶向载药复合物的药物。
本发明第四方面的目的,在于提供制备上述纳米靶向载药复合物的制备方法。
本发明所采取的技术方案是:
本发明的第一方面,提供一种纳米靶向载药复合物,包括Ti3C2纳米材料载体、黄酮类化合物、靶向性抗体。
优选地,所述黄酮类化合物包括花青素。
优选地,所述花青素包括矢车菊素。
优选地,所述靶向性抗体为血管钙化特异性蛋白抗体。
优选地,所述血管钙化特异性蛋白包括人骨钙素(osteocalcin,OCN)、核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANK)、骨形态发生蛋白2(bone morphogenetic protein2,BMP2)、平滑肌肌动蛋白22a(smooth muscle22alpha,SM22a)、核相关转录因子2(runt-related transcription factor-2,Runx2)、NAD+依赖的组蛋白去乙酰化酶Sirt6(NAD-dependent deacetylase sirtuin-6,SIRT6)中的至少一种。
优选地,所述血管钙化特异性蛋白抗体包括人骨钙素抗体(osteocalcinantibody,OCN Ab)、核因子κB受体活化因子配体抗体(receptor activator of nuclearfactor-κB ligand antibody,RANKL Ab)、骨形态发生蛋白2(bone morphogeneticprotein2,BMP2)抗体、平滑肌肌动蛋白22a(smooth muscle 22alpha,SM22a)抗体、核相关转录因子2(runt-related transcription factor-2,Runx2)抗体、NAD+依赖的组蛋白去乙酰化酶Sirt6(NAD-dependent deacetylase sirtuin-6,SIRT6)抗体中的至少一种。
优选地,所述Ti3C2纳米材料的水合粒径为80nm~150nm。
优选地,所述纳米材料载体与靶向性抗体采用化学键连接方式进行,所述化学键为共价键连接。
优选地,所述共价键连接方式为碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺化学法,但并不局限于此。
优选地,所述矢车菊素为氯化矢车菊素,其分子式为:C15H11ClO6,结构式如下所示:
本发明的第二方面,提供本发明第一方面所述的纳米靶向载药复合物在制备预防、缓解和/或治疗血管和/或血管细胞钙化的药物中的应用。
优选地,所述血管细胞包括血管平滑肌细胞。
优选地,用于血管细胞时,纳米靶向复合物浓度为2~32μg/ml。
对于不同的对象,根据换算规则对药物剂量进行换算。
本发明的第三方面,提供一种药物,所述产品包含本发明第一方面所述的纳米靶向载药复合物。
优选地,所述药物还包括药学上可接受的辅料。
优选地,所述药学上可接受的辅料包括:稀释剂、黏合剂、润湿剂、润滑剂、崩解剂、溶剂、乳化剂、助溶剂、防腐剂、pH调节剂、渗透压调节剂、表面活性剂、包衣材料、抗氧剂、抑菌剂或缓冲剂中的至少一种。
优选地,所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、乳剂、溶液剂、滴丸剂、注射剂、栓剂、灌肠剂、气雾剂、贴剂或滴剂中的至少一种。
优选地,所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射中的至少一种。
优选地,所述药物还包括至少一种其他可用于预防、缓解和/或治疗血管和/或血管细胞钙化的成分。
本发明的第四方面,提供一种纳米靶向载药复合物的制备方法,包括以下步骤:
S1:将黄酮类化合物负载于Ti3C2纳米材料载体表面,制备复合物1;
S2:将靶向性抗体连接至复合物1表面,制备得到纳米靶向载药复合物。
优选地,所述Ti3C2纳米材料的制备方法包括:称取MXene-多层Ti3C2粉体加入四丙基氢氧化铵溶液中;混合均匀后超声;离心、取沉淀、洗涤、重复2~4次;得Ti3C2纳米材料。
优选地,所述MXene-多层Ti3C2粉体和四丙基氢氧化铵溶液的质量体积比为1:0.5~1.5。
优选地,所述四丙基氢氧化铵溶液的浓度为20~30%。
优选的,所述混合均匀的方式包括搅拌。
优选地,所述搅拌的条件为磁力搅拌速率800~1200rpm,1.5~2.5天。
优选地,所述超声的条件为:超声功率250~350W,超声时间1.5~2.5天。
优选地,所述离心的条件为:12000~14000rpm,8~12min。
优选地,所述洗涤为有机溶剂离心洗涤后再用超纯水离心洗涤。
优选地,所述有机溶剂包括无水乙醇、乙二醇、丙二醇中的至少一种。
优选地,黄酮类化合物与Ti3C2纳米材料载体通过氢键作用连接。
优选地,所述黄酮类化合物与Ti3C2纳米材料载体的质量比为1:1~3。
优选地,所述步骤S1的具体操作为:将Ti3C2纳米材料溶液与黄酮类化合物溶液混合均匀,离心,洗涤,即得复合物1。
优选地,所述Ti3C2纳米材料溶液的溶剂为水。
优选地,所述黄酮类化合物溶液的溶剂为DMSO。
优选地,所述步骤S1中混合均匀的方式包括搅拌。
优选地,所述步骤S1中的搅拌条件为:24~26℃,避光、搅拌20~28h。
优选地,所述步骤S1离心的条件为:11000~13000rpm离心4~6min。
优选地,所述步骤S2中的连接的方式为化学键连接,所述化学键连接为共价键间接。
优选地,所述共价键连接方式为碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺化学法。
优选地,所述步骤S2的具体操作为:将包含碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺的溶液与复合物1溶液混合均匀后加入靶向性抗体混合后离心。
优选地,所述包含碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺的溶液的溶剂为水。
优选地,所述包含碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺的溶液中碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺的质量比为1:0.5~1.5。
优选地,所述复合物1溶液的溶剂为PBS。
优选地,所述包含碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺的溶液与复合物1溶液混合条件为搅拌。
优选地,所述搅拌的条件为磁力搅拌速率800~1200rpm,3~5h,24~26℃。
优选地,加入靶向性抗体后的混合条件为搅拌。
优选地,所述搅拌的条件为磁力搅拌速率800~1200rpm,22~26h,24~26℃。
优选地,所述步骤S2中的离心的条件为12000~14000rpm,4~6min。
优选地,所述靶向性抗体与复合物1的质量比为1:15~17。
优选地,所述靶向性抗体为血管钙化特异性蛋白抗体。
优选地,所述血管钙化特异性蛋白抗体包括人骨钙素抗体(osteocalcinantibody,OCN Ab)和/或核因子κB受体活化因子配体抗体(receptor activator ofnuclear factor-κB ligand antibody,RANKL Ab)。
优选地,所述人骨钙素抗体与因子κB受体活化因子配体抗体的质量比为1:0.5~1.5。
本发明的有益效果是:
本发明以Ti3C2 MXenes纳米材料作为防治血管钙化的载体,并且进行了双抗体修饰,负载黄酮类化合物,使其具有靶向血管钙化的能力,从而达到靶向治疗目的;经过实验研究证明,对于高磷诱导的钙化雄性小鼠和HASMC诱导钙化模型,本发明提供了黄酮类矢车菊素来源纳米化合物,可以有效缓解血管钙化,并且在毒性实验HE染色中也未对小鼠心脏、肝脏、脾脏、肺脏、肾脏造成损伤,具有较高的安全性,有较高的安全性,其有望应用到血管钙化疾病的防治当中;本发明为治疗血管钙化提供了一种新的思路和防治方向。
附图说明
图1为本发明中实施例2中纳米表征,1a为Ti3C2;1b为纳米靶向复合物(TROC)。1c为利用动态光散射测得Ti3C2以及TROC的粒径分析图,1d为Ti3C2以及TROC的Zeta电位图。
图2为本发明中实施例3中抗体负载率结果。
图3为本发明中实施例4中不同浓度矢车菊素为代表的黄酮类化合物纳米靶向化合物对动物溶血实验结果。
图4为本发明中实施例5中同等浓度矢车菊素为代表的黄酮类化合物纳米靶向化合物对小鼠器官的HE染色安全性测定结果。
图5为本发明实施例6中不同浓度的矢车菊素为代表的黄酮类化合物纳米靶向化合物对小鼠主动脉环体外诱导钙化Von Kossa染色结果。
图6为本发明实施例7中不同浓度的矢车菊素为代表的黄酮类化合物纳米靶向化合物对小鼠钙化组织主动脉大体茜素红染色结果。
图7为本发明实施例8中不同浓度的矢车菊素为代表的黄酮类化合物纳米靶向化合物对高磷诱导的人血管平滑肌细胞(HVSMCs)的缓解钙化作用结果。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
实施例1矢车菊素为代表黄酮类化合物纳米靶向载药复合物的制备方法
称取10mg MXene-多层Ti3C2粉体,分散于10mL的25%的四丙基氢氧化铵溶液中,置于25℃下用转速为1000rpm的磁力搅拌器搅拌2天,随后用300W的水浴超声仪超声2天,继续用离心机以13000rpm速度离心10min,沉淀用无水乙醇13000rpm速度离心10min洗涤后再用超纯水离心洗涤,共三次,最后制得Ti3C2纳米片。
进一步称5mg制备成功的Ti3C2纳米片,分散到5mL的超纯水中,用超声仪超声使其均匀分散。美国西格玛(sigma)购买纯度≥98%矢车菊素(Cyanidin),取10mg的矢车菊素溶解在DMSO溶液中,加入1ml均匀分散在超纯水中的Ti3C2,混合后,25℃下避光磁力搅拌24h,用离心机12000rpm速度离心5min,最后用超纯水洗涤3次,沉淀即为Ti3C2-矢车菊素纳米复合物。
因黄酮类化合物其靶向特异性差等问题,需要大量化合物才能发挥作用,提出设计具有靶向性抗体联合抑制血管钙化。
在Ti3C2表面通过化学键修饰连接RANKL和OCN抗体:
称取Ti3C2-矢车菊素纳米复合物1mg分散于1mL PBS(pH=7.4)中,超声30min中使其分散均匀。分别称取8mg碳化二亚胺盐酸盐和2mgN-羟基琥珀酰亚胺超声溶于0.2mL超纯水中,将其加入Ti3C2-Cyanidin混合液中,25℃下磁力搅拌4h,平均分成3份,每份0.4mL。最后向每份分别加入10μL RANKL(1mg/mL)抗体、10μL OCN抗体(1mg/mL),继续搅拌24h。最后,将上述混合液用离心机离心13000rpm速度离心5min;即为矢车菊素纳米靶向复合物(TROC)。
实施例2矢车菊素为代表黄酮类化合物纳米靶向载药复合物表征测定
对实施例1得到的Ti3C2纳米片以及矢车菊素纳米靶向复合物进行电镜检测。电镜上样方法:将滤纸放入10cm皿内,先将电镜网轻轻用电镜镊子夹出,小心轻柔地放到滤纸上,一般默认有黑色油花的面为正面,如果样品多,则先将铜网周围浸湿(如果样品不多则无需这步),轻轻加入10μl样品到铜网上,最后样品浓度足够则静置48h,干透后既制样成功。
图1a和图1b为透射电镜检测Ti3C2纳米片以及矢车菊素纳米靶向复合物形貌。图1c为用粒径分析仪检测制备的Ti3C2纳米片以及矢车菊素纳米靶向复合物的水合粒径,图1d为Ti3C2纳米片以及矢车菊素纳米靶向复合物的Zeta电位图;利用动态光散射测得制备好的Ti3C2纳米片平均水合粒径为83.7nm,电位为-26mV,结果证明Ti3C2纳米片状态保持稳定以及TROC制备成功。
实施例3矢车菊素为代表黄酮类化合物纳米靶向载药复合物抗体负载率结果
配制0μg/mL、2.5μg/mL、5μg/mL、10μg/mL、20μg/mL、30μg/mL、40μg/mL浓度梯度的蛋白标准品溶液。通过BCA蛋白浓度检测试剂盒进行测定,根据试剂盒说明书,将试剂A和试剂B以50:1的体积比充分混合。将20μL的标准品加入至96孔板中,每个标准品浓度设置3个复孔;向各个孔内加入200μL上述配好的工作液,37℃下孵育30min后,通过酶标仪检测其在562nm处的吸光度值,通过吸光度对浓度绘制蛋白标准曲线。
TROC的制备过程同实施例1,并以同样的方法制备TRC(Ti3C2-Cyanidin/RANKL)和TOC(Ti3C2-Cyanidin/OCN),在最后一步离心后将上清液稀释适宜倍数后测定其吸光度值,对其中的RANKL和OCN进行定量,计算负载上的抗体含量。
通过标准蛋白的紫外吸光度与浓度标准曲线,得到Ti3C2-RANKL/OCN的蛋白负载率为1.33%(图2)。
实施例4纳米靶向化合物溶血实验安全性测定
取雄性6~8周龄C57BL/6小鼠眼球血制备2%的红细胞,首选取500μl的眼球血,均匀震荡摇匀10分钟,加入0.9%的医用生理盐水,均匀摇匀后用1000rpm离心15分钟,去除上清后,继续加入0.9%的医用生理盐水洗涤3次,即制备成功。分别制备Ti3C2-Cyanidin/RANKL/OCN溶于0.9%生理盐水中配置为6.25μg/ml、12.5μg/ml、25μg/ml、50μg/ml、100μg/ml,给予蒸馏水为阳性对照组、2%的红细胞悬液为阴性对照组,按比例分别加入EP管内,观察其颜色变化。
图3可以清楚观察到不会引起任何明显的溶血反应(<5%)。
实施例5纳米靶向化合物对脏器安全性测定
给予小鼠尾静脉注射TROC 5mg/kg,每周一次共注射2周,解剖后取心脏、肝脏、脾脏、肺脏、肾脏进行解剖出,放置于20倍样本体积的4%多聚甲醛/通用型组织固定液24h以上,进行切片。将切片放入二甲苯中,共2个缸浸泡,每个20分钟。无水乙醇液设置浓度梯度,100%、95%、75%的无水乙醇,分别各泡5分钟。自来水洗,蒸馏水洗三遍。用苏木素染液染色,再用伊红染色,切片以此放入75%的无水乙醇、95%的无水乙醇、100%的无水乙醇各5分钟处理后,放入二甲苯中,共2个缸浸泡,各5分钟。最后中性树胶封片。再显微镜镜下观察组织形态。
图4为解剖后观察其主要器官脏器心脏、肝脏、脾脏、肺脏、肾脏等结构变化,HE染色后发现,各脏器组织无结构破坏或者改变,纳米靶向化合物安全性高。
实施例6不同浓度的矢车菊素纳米靶向化合物对体外诱导小鼠主动脉环钙化的作用
购买8-10周C57BL/6J雄性小鼠,动物来源于(北京维通利华实验动物技术有限公司)。用颈椎脱臼法,处死第8周C57BL/6J雄性小鼠后。用手术剪剪开小鼠的胸腔和膈肌,暴露主动脉。在髂动脉分叉处进行剪断,镊子夹住离断的主动脉,小心沿着主动脉向上,一直分离主动脉根部,连同心脏一起取出,放置皿中。用显微镊轻轻仔细的分离周围的脂肪组织并把心脏离断,保留主动脉,并用PBS进行冲洗2-3次。主动脉,进行横断面切,形成血管环。
在6孔板中放置血管环,取高糖培养基DMEM 44.5ml加入5ml血清,再加入500μl双抗,配置成为10%的完全培养基。用无菌水将Na2HPO4溶液稀释为200mM,按比例加入6孔板后终浓度为2.8mM高磷诱导的完全培养基诱导钙化。每孔加入2ml的钙化诱导培养基,2天更换一次完全培养基。分为完全培养基(NC)组、高磷刺激诱导钙化(M)组、高磷刺激诱导钙化+材料(M+Ti3C2)组、高磷刺激诱导钙化+纳米靶向组(M+TRO)、高磷刺激诱导钙化+纳米靶向药物组(M+TROC)、高磷刺激诱导钙化+矢车菊素组(M+Cyanidin),Ti3C2、TRO、TROC、Cyanidin的浓度均为均8μg/ml,进行体外钙化诱导培养和干预。7天后放置4%的组织固定液中固定24小时,进行切片。将切片放入二甲苯中,共2个缸浸泡,每个20分钟。无水乙醇液设置浓度梯度,100%、95%、75%的无水乙醇,分别各泡5分钟。自来水洗,蒸馏水洗三遍。切片滴加VonKossa染液,用紫外灯连续照射4小时,用蒸馏水进行冲洗。苏木素染液染5分钟,用流水冲洗至无色。切片以此放入75%的无水乙醇、95%的无水乙醇、100%的无水乙醇各5分钟处理后,放入二甲苯中,共2个缸浸泡,各5分钟。最后中性树胶封片。
结果参考图5,主动脉血管环在高磷诱导刺激7天后出现钙化,在等浓度作用下,纳米靶向药物组比药物治疗组血管钙化程度减轻,深色代表钙化颜色变浅。
实施例7不同浓度的纳米靶向化合物对高磷饮食诱导钙化小鼠主动脉大体的影响
给予C57BL/6J雄性小鼠高腺嘌呤高磷饲料(high adenine and high phosphate,AP)使用0.2%的腺嘌呤和2%的磷的饲料共12周。所用饲料为广州省医学实验动物定制。所有的试验小鼠均生活每日光照12小时和黑暗12小时的具有合格资质的SPF级动物房。打耳标对小鼠进行标记,笼牌标记小鼠的分类。每天给予充足的食物和水,每周给小鼠进行体重称量。在建模第3周开始给予小鼠尾静脉注射,共注射10次药物,每次5mg/kg注射剂量。共饲养12周后对小鼠进行主动脉大体解剖,将经过深度麻醉后的小鼠,以腹壁做正中纵切口暴露腹腔,使主动脉清楚暴露。在主动脉弓部结扎主动脉,使用一次性采血针用生理盐水进行灌注轻柔冲洗,清除残存的血液后,取出整条主动脉。小鼠主动脉组织用95%乙醇固定24小时,并用0.003%茜素红(溶于1%氢氧化钠制成)染色30小时。然后用2%氢氧化钠清洗2次主动脉,染色结果见图6。
结果参考图6,与钙化组相比,治疗组小鼠主动脉血管钙化程度明显减轻,并且纳米靶向化合物治疗组比矢车菊素治疗组效果更好,颜色变浅更多,证实纳米靶向化合物可减轻高磷高腺嘌呤诱导的C57B/L6小鼠血管钙化。
实施例8纳米靶向化合物对HVSMCs钙化的缓解钙化作用
将HVSMCs细胞按照1×105个/孔接种在6孔板中,用无菌水将Na2HPO4溶液稀释为200mM,分装储存,使用前摇匀,按比例加入6孔板后终浓度为2.8mM。在6孔板给予2.8mM的Na2HPO4刺激,同时各种均加入等浓度8μg/ml溶液,分为完全培养基(NC)组、高磷刺激诱导钙化(M)组、高磷刺激诱导钙化+材料(M+Ti3C2)组、高磷刺激诱导钙化+纳米靶向组(M+TRO)、高磷刺激诱导钙化+纳米靶向药物组(M+TROC)、高磷刺激诱导钙化+矢车菊素组(M+Cyanidin),2天更换一次培养基。
图7所见,HSAMC在高磷诱导刺激7天后出现钙化,表现为茜素红染色阳性(颜色为红),加入纳米靶向药物组颜色比高磷诱导钙化颜色浅,在细胞层面也同样证明,纳米靶向化合物组可缓解血管钙化,略优于单纯药物组。
综合实施1-8可见,纳米靶向化合物可缓解血管钙化,并优于单纯药物,有望应用到血管钙化疾病的防治当中。
上述具体实施方式对本发明作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
Claims (10)
1.一种纳米靶向载药复合物,包括Ti3C2纳米材料载体、黄酮类化合物、靶向性抗体。
2.根据权利要求1所述的纳米靶向载药复合物,其特征在于,所述黄酮类化合物包括花青素,优选地,所述花青素包括矢车菊素。
3.根据权利要求1所述的纳米靶向载药复合物,其特征在于,所述靶向性抗体为血管钙化特异性蛋白抗体;优选地,所述血管钙化特异性蛋白包括人骨钙素、核因子κB受体活化因子配体、骨形态发生蛋白2、平滑肌肌动蛋白22a、核相关转录因子2、NAD+依赖的组蛋白去乙酰化酶Sirt6中的至少一种。
4.根据权利要求1所述的纳米靶向载药复合物,其特征在于,所述Ti3C2纳米材料的水合粒径为80nm~150nm。
5.权利要求1~4任一项所述的纳米靶向载药复合物在制备预防、缓解和/或治疗血管和/或血管细胞钙化的药物中的应用。
6.根据权利要求5所述的应用,其特征在于,所述血管细胞包括血管平滑肌细胞。
7.一种药物,所述药物包含权利要求1~4任一项所述的纳米靶向载药复合物;优选地,所述药物还包括至少一种其他可用于预防、缓解和/或治疗血管和/或血管细胞钙化的成分。
8.权利要求1~4任一项所述纳米靶向载药复合物的制备方法,包括以下步骤:
S1:将黄酮类化合物负载于Ti3C2纳米材料载体表面,制备复合物1;
S2:将靶向性抗体连接至复合物1表面,制备得到纳米靶向载药复合物。
9.根据权利要求8所述的制备方法,其特征在于,所述黄酮类化合物与Ti3C2纳米材料载体的质量比为1:1~3。
10.根据权利要求8所述的制备方法,其特征在于,所述靶向性抗体与复合物1的质量比为1:15~17;
优选地,所述靶向性抗体包括人骨钙素抗体和核因子κB受体活化因子配体抗体;优选地,所述靶向性抗体包括人骨钙素抗体和核因子κB受体活化因子配体抗体的质量比为1:0.5~1.5。
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