CN116036302A - 一种修饰cRGD肽的超小粒径黑色素纳米颗粒的制备方法和应用 - Google Patents
一种修饰cRGD肽的超小粒径黑色素纳米颗粒的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种修饰cRGD肽的超小粒径黑色素纳米颗粒(RpMP)的制备方法,通过用cRGD‑PEG2000‑NH2对黑色素纳米颗粒(MNP)进行表面处理得到。本发明进一步提出了上述修饰cRGD肽的超小粒径黑色素纳米颗粒在制备用于抑制巨噬细胞焦亡、预防和/或缓解或治疗炎症/焦亡相关疾病的药物上的应用。其在细胞水平可以显著抑制LPS刺激的巨噬细胞焦亡。在动物水平,由于cRGD可以特异性靶向动脉粥样硬化新生血管的αvβ3整合素,由尾静脉注射的RpMP高效地富集到动脉粥样硬化斑块部位,在局部发挥抗焦亡,抑制斑块进展的治疗效果,实现更加精准、有效、安全的动脉粥样硬化治疗。
Description
技术领域
本发明涉及生物医药领域,具体涉及一种修饰cRGD肽的超小粒径黑色素纳米颗粒(RpMP)的制备方法和应用
背景技术
目前,动脉粥样硬化性心血管疾病的患病率和死亡率远远高于其他重大疾病,造成了极大的社会经济负担。而动脉粥样硬化是多种心脑血管事件的主要病理基础。目前,戒烟、运动,以及对饮食、血压和胆固醇水平尤其是低密度胆固醇水平的控制仍然是冠心病临床二级预防的主要干预措施。然而,即使控制了传统的危险因素,心血管不良事件的发生率依然很高。因此,探索抗动脉粥样硬化进展的新策略是目前临床亟待解决的关键问题。
随着现代血管生物学的发展,动脉粥样硬化的病理生理学观点正在从强调脂质转向血管免疫学,特别是巨噬细胞动力学。大量证据表明;炎症在冠心病的一级和二级预防中,都与心血管事件密切相关,被证实是独立危险因素。值得一提的是,CANTOS大型临床实验表明:IL-1β单抗的应用,有效降低了心梗后患者的炎症水平和心血管不良事件复发率,这无疑直接证实了炎症对动脉粥样硬化发生发展的促进作用。
焦亡是一种特殊形式的程序性细胞死亡,其特征是炎性小体的组装和激活,细胞膜低聚孔的形成,以及炎症细胞因子(IL-1β等)的快速分泌和释放,因此也被称为炎性细胞死亡。表达于各种免疫细胞的模式识别受体(pattern recognition receptors,PRRs)是炎症小体的核心,它们能够识别各种刺激,即病原体相关分子模式(pathogen-associatedmolecular patterns,PAMPs)和损伤/危险相关分子模式(damage/danger-associatedmolecular patterns DAMPs),PRRs被激活后形成多蛋白复合体-炎症小体。而促进泡沫细胞形成及动脉粥样硬化发生发展的关键因素,包括氧化低密度脂蛋白(ox-LDL)和胆固醇晶体,已被证明能够通过活性氧(ROS)等多种机制激活炎症小体并促进焦亡。在多种PRRs中,核苷酸结合寡聚化结构域样受体(NLR)家族蛋白中的NLRP3最为独特,它可以识别多种刺激,特别是DAMPs。此外,越来越多的证据表明NLRP3炎性小体在动脉粥样硬化的发病中起着关键作用。并且,NLRP3/IL-1β经典通路是最具代表性和最广为人知的一种焦亡形式。活化的NLRP3炎症小体会激活caspase-1,进而促进焦亡的刽子手——Gasdermin D(GSDMD)的裂解和激活。激活的GSDMD会与细胞膜结合,形成内径为10-20nm的寡聚孔,导致水的内流和具有生物活性的IL-1β及其他DAMPs的释放,最终导致细胞裂解,以及与之伴随的细胞内成分的泄漏和广泛的炎症反应。血管内巨噬细胞、内皮细胞和平滑肌细胞焦亡的过度激活会加速动脉粥样硬化的进展,引起斑块破裂,进而导致心血管不良事件的发生。因此,抑制动脉粥样硬化中的过度焦亡或许可以起到关键治疗作用。
鉴于炎症在动脉粥样硬化和其他心血管疾病中的重要作用,针对炎症的治疗手段是心血管疾病防治最大的研究热点。无论是前临床研究还是临床试验都已证实:抗炎治疗可以有效减轻动脉粥样硬化,显著减少心血管事件的发生率。此外,药物下调焦亡水平在改善动脉粥样硬化方面也显示出很大的价值。然而,由于缺乏病变靶向能力,这种全身抗炎治疗可能引起脱靶毒性,削弱人体对感染的局部和全身炎症反应进而导致免疫抑制,因此难以向临床转化。以CANTOS实验为例,IL-1β单抗虽然有效减少了心梗后患者心血管不良事件的发生,但显著增加了致死性感染和脓毒血症的风险。因此,如何提高动脉粥样硬化抗炎治疗的针对性和靶向性,增加药物在斑块部位的递送效率,从而维护生物体的免疫稳态,增强抗动脉粥样硬化疗效,是促进动脉粥样硬化抗炎治疗亟待解决的关键科学问题。此外,针对NLRP3介导的细胞焦亡的精准治疗方法仍然有限。
纳米医学的发展,使得我们能够基于一系列纳米平台,实现体内示踪和靶向递送,延长药物的血液循环事件,从而以较低的药物剂量和较长的半衰期,达到病变部位较高浓度的药物积累,降低全身的毒副作用。但大部分传统的纳米平台存在生物相容性差,工艺复杂耗时等缺点。近年来,天然多酚类化合物,特别是表没食子儿茶素-3-没食子酸酯(epigallocatechin-3-gallate,EGCG)已被证实可以通过抑制NLRP3炎性小体和随后IL-1β的释放发挥良好的抗炎价值。由各类多酚物质衍生合成的多功能纳米平台也引发了极大的关注。其中,黑色素作为广泛存在于各种生物体内的天然的多酚类生物聚合物,具备固有的生物相容性和生物可降解性。在过去的几年里,黑色素样纳米颗粒(melanin likenanoparticles,MNPs)在分子成像,光热治疗和药物递送领域引发了越来越多的关注。更有趣的是,许多研究表明,MNPs可以作为一种非凡的纳米抗氧化剂,在ROS相关的炎性疾病,如缺血性中风和急性肾损伤中表现出非常优异的治疗效果。然而,尚无研究表明MNPs是否可以抑制NLRP3介导的细胞焦亡,MNPs在动脉粥样硬化中的治疗潜力也未曾被评估。
发明内容
发明目的:针对现有技术的不足,本发明公开了一种修饰cRGD肽的超小粒径黑色素纳米颗粒(RpMP)。在细胞水平,RpMP可以显著抑制LPS刺激的巨噬细胞焦亡。在动物水平,由于cRGD可以特异性靶向动脉粥样硬化新生血管的αvβ3整合素,由尾静脉注射的RpMP高效地富集到动脉粥样硬化斑块部位,在局部发挥抗焦亡,抑制斑块进展的治疗效果,实现更加精准、有效、安全的动脉粥样硬化治疗。
具体地,本发明公开了一种修饰cRGD肽的超小粒径黑色素纳米颗粒的制备方法,通过用cRGD-PEG2000-NH2对黑色素纳米颗粒进行表面处理得到。
其中,cRGD-PEG2000-NH2与黑色素纳米颗粒的投入质量比为9~11:1。其中,黑色素纳米颗粒先以1mg/mL的浓度溶于水中,cRGD-PEG2000-NH2粉末直接投入即可。在合成的过程中,优选地在小体系条件下合成,单次反应投入5-6mg黑色素纳米颗粒为宜,避免cRGD-PEG2000-NH2装载量降低。
具体地,所述修饰cRGD靶向肽的黑色素纳米颗粒通过如下方法制备得到:
(1)将黑色素粉末(Melanin,CAS号M8631)溶于0.1mol/L NaOH水溶液中,然后转移到离心管中,超声粉碎的同时,用0.1mol/L HCl调节溶液pH至中性,得到黑色溶液,转移至超滤离心管中,用去离子水洗涤若干次后,冷冻干燥,得到黑色素纳米颗粒(MNP),进一步溶于水中得黑色素纳米颗粒水溶液备用;
(2)将cRGD-PEG2000-NH2粉末溶于步骤(1)得到的黑色素纳米颗粒水溶液中,用NaOH水溶液调节pH至9-9.5,室温搅拌24h,将所得黑色溶液转移至超滤离心管中,用去离子水洗涤若干次后,冷冻干燥,即得到修饰了cRGD的PEG化的超小粒径黑色素纳米颗粒cRGD-PEG-MNP,记为RpMP。
其中,步骤(1)中,超声条件为:120-200W的功率下工作7-8min,使黑色素被充分超声粉碎,否则会导致所得颗粒的粒径分布不均匀。其中,每工作6-7s,间歇3-4s,仪器保护温度设置为35-38℃。
在一个具体的实施方式中,按照如下步骤制备修饰cRGD靶向肽的超小粒径黑色素纳米颗粒:
(1)将20mg黑色素(melanin)粉末倒入0.1mol/L NaOH水溶液中,室温搅拌40min,使其充分溶解。然后置于超声粉碎机中,用0.1mol/L HCl调节溶液pH至中性,并在120-200W的功率下超声粉碎7-8min(工作时间:间歇时间:总工作时间=6-7s:3-4s:7-8min,仪器保护温度:35-38℃)。将所得黑色溶液转移至30kDa的超滤离心管中,用去离子水超滤洗涤若干次,然后放置于冷冻干燥机中,冻干完成后即得到纯化的超小粒径黑色素纳米颗粒(MNP)。
(2)将50mg cRGD-PEG2000-NH2粉末溶于(1)所得5ml MNP水溶液中(1mg/mL),用NaOH水溶液调节pH至9-9.5,室温搅拌24h。将所得黑色溶液转移至30kDa的超滤离心管中,用去离子水洗涤若干次后,放置于冷冻干燥机中冻干,即得到修饰了cRGD的PEG化的超小粒径黑色素纳米颗粒cRGD-PEG-MNP,即RpMP。
本发明进一步提出了上述修饰cRGD肽的超小粒径黑色素纳米颗粒在制备用于抑制巨噬细胞焦亡、预防和/或缓解或治疗炎症/焦亡相关疾病的药物上的应用。
其中,所述炎症/焦亡相关疾病为动脉粥样硬化。
具体地,所述修饰cRGD肽的超小粒径黑色素纳米颗粒在体外抑制LPS刺激的巨噬细胞焦亡,在体内富集到动脉粥样硬化斑块部位,局部发挥抗焦亡和抑制斑块进展的作用。
有益效果:与现有技术相比,本申请具有如下优点:
(1)本发明所用黑色素纳米平台含有丰富的活性基团和配位位点,能够通过简单的工艺进一步负载药物或与各种金属离子螯合,是构建分子诊疗探针的优异平台,因此也易于根据实际需求进行优化;
(2)本发明所述纳米药物,是基于广泛存在于生物体内的天然黑色素衍生合成的超小粒径纳米平台,具有良好的生物相容性和可降解性,易于从体内清除,并且对肝肾功能有显著的保护作用;
(3)本发明所述纳米药物在体外可以显著抑制LPS诱导的巨噬细胞焦亡,主要体现为:有效清除LPS刺激产生的细胞内活性氧;降低细胞内NLRP3炎症小体、Caspase1、GSDMD和IL-1β蛋白水平;进一步抑制IL-1β,IL-6,and TNF-α等炎性因子的释放;
(4)本发明所述纳米药物RpMP,能够同时通过EPR效应介导的被动靶向和所修饰cRGD肽介导的主动靶向作用,高效地富集到动脉粥样硬化斑块部位。与健康对照组,和注射未修饰靶向肽的NpMP纳米颗粒组相比,修饰cRGD肽显著提高了纳米颗粒在动脉粥样硬化小鼠主动脉斑块部位的靶向效果;
(5)本发明所述纳米药物在给药后,能够靶向病变部位,通过清除活性氧和抑制细胞焦亡来调节局部炎症免疫微环境,从而进一步减轻动脉粥样硬化的发展,其治疗效果明显优于生理盐水对照组、他汀治疗组和未修饰cRGD靶向肽的NpMP纳米颗粒组。
附图说明
图1为MNP、NpMP、RpMP的形貌表征;
图2为RpMP的紫外可见光谱和红外衍射光谱;
图3为本发明效果中纳米药物清除巨噬细胞内活性氧的结果;
图4为本发明效果中巨噬细胞内焦亡相关蛋白表达的结果;
图5为本发明效果中巨噬细胞上清炎症因子IL-1β分泌的结果;
图6为本发明效果中巨噬细胞上清炎症因子IL-6分泌的结果;
图7为本发明效果中巨噬细胞上清炎症因子TNF-α分泌的结果;
图8为本发明效果中纳米药物靶向主动脉斑块和对应主动脉大体油红O染色的结果;
图9为本发明效果中治疗后主动脉大体油红O染色的结果;
图10为本发明效果中治疗后主动脉HE染色的结果;
图11为本发明效果中治疗后主动脉活性氧免疫荧光染色的结果;
图12为本发明效果中治疗后主动脉NLRP3和GSDMD免疫组化染色的结果。
具体实施方式
本发明拟基于超小粒径的多功能黑色素纳米平台,通过修饰cRGD靶向肽,开发一种能够有效靶向至斑块,调节细胞焦亡的纳米药物RpMP。本发明的目的是:在细胞水平,RpMP通过清除细胞内活性氧、降低焦亡相关蛋白水平和促炎细胞因子水平,来抑制LPS刺激的巨噬细胞焦亡。在动物水平,通过静脉注射,将RpMP靶向递送至动脉粥样硬化斑块部位,在局部发挥抗焦亡,抑制斑块进展的治疗效果,实现更加精准、有效、安全的动脉粥样硬化治疗。
下面结合附图和具体实施方式对本发明做更进一步的具体说明,本发明的上述和/或其他方面的优点将会变得更加清楚。
实施例1靶向动脉粥样硬化斑块新生血管的纳米药物RpMP的合成。
首先将20mg黑色素粉末(Melanin,CAS号M8631))溶于0.1mol/L NaOH水溶液中,室温搅拌40min,使其充分溶解。然后置于超声粉碎机中,在160W的功率下超7min(工作时间:间歇时间:总工作时间=7s:3s:7min,仪器保护温度:37℃),并在超声粉碎过程中用0.1mol/L HCl调节溶液pH至中性。将所得黑色溶液转移至30kDa的超滤离心管中,用去离子水超滤洗涤若干次后,放置于冷冻干燥机中,冻干24h得到纯化的超小粒径黑色素纳米颗粒(MNP)。将所得MNP纳米颗粒溶于水中配置成1mg/mL的储备液。称取50mg cRGD-PEG2000-NH2粉末(购于湖南华腾制药有限公司)溶于5ml MNP储备液中,用NaOH水溶液调节pH至9-9.5,室温搅拌24h。将所得黑色溶液转移至30kDa的超滤离心管中,用去离子水洗涤若干次后,放置于冷冻干燥机中冻干24h,即可以得到修饰了cRGD的PEG化的cRGD-PEG-MNP纳米颗粒,即RpMP。所合成RpMP纳米药物在8.6-11.0nm之间。
为了后续对比验证RpMP纳米药物的靶向性,本实施例合成了未修饰cRGD的纳米颗粒NH2-PEG-MNP,即NpMP。合成步骤相似,称取50mg NH2-PEG2000-NH2粉末(购于上海芃硕生物科技有限公司)溶于5ml MNP储备液中,用NaOH水溶液调节pH至9-9.5,室温搅拌12h。将所得黑色溶液转移至30kDa的超滤离心管中,用去离子水洗涤若干次后,放置于冷冻干燥机中冻干24h,即可以得到修饰了NpMP。
电镜图和动态光散射表征了MNP、NpMP和RpMP的形貌,结果如图1所示,其中,图1A为MNP的DLS和TEM;图1B为NpMP的DLS和TEM;图1C为RpMP的DLS和TEM。MNP、NpMP和RpMP粒径范围的平均值分别是5.3nm,7.6nm和9.8nm。
本实施例进一步通过紫外可见光谱和红外衍射光谱表征了RpMP纳米药物中PEG链和cRGD靶向肽的修饰。图2A为MNP、cRGD-PEG2000-NH2和RpMP的紫外可见光谱,可以看出,RpMP在275nm处显示出cRGD-PEG2000-NH2的特征性吸收峰;并且由紫外图可以得出,所得RpMP中,负载的cRGD-PEG与MNP的质量比约为1:1。图2B为MNP、cRGD-PEG2000-NH2和RpMP的红外衍射光谱,结果显示:RpMP在1114cm-1和2885cm-1处表现出PEG分子的特征吸收峰(分别与C-O-C和烷基C-H的伸缩振动有关);2990cm-1-3690cm-1的宽代吸收峰源自于MNP中羟基的伸缩振动;而1673cm-1处的吸收峰与cRGD分子中C=O和C=N的伸缩振动有关。
实施例2纳米药物RpMP对细胞活性氧的影响。
用2,7-二氯荧光素二乙酸酯(DCFH-DA)检测试剂盒检测细胞内活性氧(ROS)水平。将RAW 264.7细胞以每孔2×105的密度接种于共聚焦小皿中培养过夜。然后,用200ng/mL的脂多糖(LPS)刺激细胞,同时给予PBS(LPS阳性对照组)、NpMP(200μg/mL)或RpMP(200μg/mL)处理24h。另设一组不给予LPS和药物作为阴性对照(blank组)。在共聚焦显微镜下对细胞进行观察并拍照。结果如图3所示。试验结果表明,RpMP能够很好的清除LPS刺激的巨噬细胞内活性氧的产生。
实施例3纳米药物RpMP对巨噬细胞焦亡相关蛋白的影响。
将RAW 264.7细胞以每孔5×105的密度接种于六孔板中培养过夜。空白对照组(blank组)用无血清DMEM培养基培养,阳性对照组仅以100ng/mL的LPS处理细胞,实验组用LPS(100ng/mL)刺激细胞的同时给予NpMP(200μg/mL)或RpMP(200μg/mL)。共孵育24小时后,用PBS洗涤细胞,并用RIPA裂解液(碧云天)裂解细胞。然后用BCA试剂盒(碧云天)量化细胞裂解液中的蛋白质浓度。用4-12%的三-甘氨酸凝胶(Invitrogen Novex)分离后,将蛋白样品转移到聚偏氟乙烯(PVDF)膜上,封闭1h。然后用抗NLRP3(ab263899,Abcam)、Caspase 1(ab179515,Abcam)、GSDMD(ab219800,Abcam)、IL-1β(af5103,Affinity)和Actin(GB15001,Servicebio)抗体孵育过夜。再用辣根过氧化物酶(GB25301,GB23303,Servicebio)标记的二抗探针孵育。用AlphaEase FC对免疫反应蛋白进行可视化。结果如图4所示,RpMP能够逆转LPS刺激导致的巨噬细胞NLRP3和IL-1β蛋白水平的升高,并且有效降低了细胞内Caspase1和GSDMD蛋白水平。以上结果证实,RpMP能够有效抑制NLRP3介导的巨噬细胞焦亡。
实施例4纳米药物RpMP对巨噬细胞分泌的炎症因子的影响。
将RAW 264.7细胞以每孔5×105的密度接种于六孔板中培养过夜。空白对照组(blank组)用无血清DMEM培养基培养,阳性对照组仅以200ng/mL的LPS处理细胞,实验组用LPS(200ng/mL)刺激细胞的同时给予NpMP(200μg/mL)或RpMP(200μg/mL)处理细胞,每组设置3个平行样本。处理24h后,收集细胞上清,采用IL-1βELISA试剂盒(ab197742,Abcam)、IL-6ELISA试剂盒(ab222503,Abcam)和TNF-αELISA试剂盒(ab208348,Abcam)检测细胞在上述不同干预条件下上清液中的炎症因子水平。结果如图5-7所示。结果表明,RpMP可以有效抑制LPS刺激的巨噬细胞促炎因子IL-1β、IL-6和TNF-α的分泌。
实施例5纳米药物RpMP对动脉粥样硬化小鼠主动脉斑块的靶向效果。
高脂饮食喂养6周龄的ApoE-/-小鼠12周构建动脉粥样硬化模型,喂养12周后,将ApoE-/-小鼠分为俩组(n=10),分别通过尾静脉注射罗丹明-B(一种红色荧光染料,用于可视化材料在主动脉斑块部位的富集情况)标记的NpMP或RpMP溶液(25mg/kg,溶剂为PBS或生理盐水)。另设一组正常饮食喂养的C57BL/6小鼠,尾静脉注射相同剂量的罗丹明-B标记的RpMP作为健康对照组(n=10)。分别在注射药物后6和24小时,从每组中任意选取5只小鼠采集主动脉,用体内成像系统(IVIS)进行荧光成像和拍摄,以确定纳米颗粒对主动脉斑块的靶向效率。拍摄结束后,收集所有主动脉进行大体油红O染色,用来可视化主动脉的动脉粥样硬化斑块。图8A为各组小鼠两个时间点的离体主动脉荧光成像的结果。结果表明,在注射后6h,各组主动脉荧光信号较弱,无明显差异;而在注射后24h,ApoE-/-小鼠的主动脉荧光信号明显增强,其中RpMP在主动脉处的富集最为显著。图8B为与图8A对应的同一根主动脉大体油红O的结果。结果表明,高脂饮食喂养的ApoE-/-小鼠主动脉有明显的动脉粥样硬化斑块,而C57BL/6小鼠主动脉无斑块形成。此外,在纳米药物处理的ApoE-/-小鼠中,可以观察到油红O染色位点与荧光信号区域之间的有明确对应关系。综上所述,这些结果表明纳米药物RpMP对动脉粥样硬化斑块具有良好的靶向性。
实施例6纳米药物RpMP对动脉粥样硬化小鼠脂质总量和斑块的影响。
高脂饮食喂养6周龄的ApoE-/-小鼠构建动脉粥样硬化模型。然后随机分为4组(n=6),给予不同的治疗:一组通过尾静脉注射200μL生理盐水(saline)作为阳性对照组;一组给予10mg/kg辛伐他汀(simvastatin)灌胃治疗作为常规药物对照组;其余2组分别通过尾静脉注射200μL NpMP或RpMP溶液(15mg/kg,PBS或生理盐水作为溶剂)作为实验组,治疗期间维持高脂饮食。另设一组正常饮食喂养的C57BL/6小鼠,尾静脉注射200μL生理盐水作为健康对照组。每三天进行一次治疗,7次治疗后,处死小鼠剥离主动脉,每组随机选取3根主动脉进行大体油红O染;其余三根取主动脉弓部位进行石蜡包埋和后续HE染色。图9为本发明效果中,治疗后各组主动脉大体油红O染色的结果。结果显示,与其他各组相比,RpMP治疗能够明显减少动脉粥样硬化小鼠主动脉斑块的脂质含量。图10为发明效果中各组主动脉横断面HE染色的结果。结果表明,与其他各组相比,RpMP治疗组的斑块面积明显减少。
实施例7纳米药物RpMP对动脉粥样硬化小鼠主动脉局部焦亡的影响。
小鼠治疗和分组方法同上,将各组小鼠上述其余三根主动脉的腹主动脉节段制成冰冻切片,进行二氢乙啶(DHE)免疫荧光染色,来检测各组主动脉局部ROS水平。取上述主动脉弓部剩下的石蜡切片分别与抗NLRP3和GSDMD抗体孵卵,进行免疫组化分析。图11为治疗后各组主动脉ROS染色的结果。结果显示,RpMP治疗能够有效清除ApoE-/-小鼠主动脉局部产生的过量活性氧。图12为治疗后各组主动脉NLRP3和GSDMD免疫组化的结果。可以看出,RpMP明显降低ApoE-/-小鼠主动脉NLRP3和GSDMD水平。这些结果表明,RpMP在动脉粥样硬化小鼠主动脉局部能够发挥抗焦亡作用,这是其有效抑制斑块进展的潜在治疗机制。
综上所述,本申请通过简单的合成方法,构建超小粒径的多功能黑色素纳米药物RpMP。在体外试验中,RpMP通过清除细胞内活性氧、降低焦亡相关蛋白水平和促炎细胞因子水平,有效抑制LPS刺激的巨噬细胞焦亡。在体内试验中,通过尾静脉注射,纳米药物RpMP能够通过cRGD肽介导的靶向效应,有效富集到动脉粥样硬化小鼠斑块部位,在局部发挥抗焦亡,抑制斑块进展的治疗效果,实现更加精准、有效、安全的动脉粥样硬化治疗。
本发明提供了一种修饰cRGD肽的超小粒径黑色素纳米颗粒(RpMP)的制备方法及应用,具体实现该技术方案的方法和途径很多,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。本实施例中未明确的各组成部分均可用现有技术加以实现。
Claims (7)
1.一种修饰cRGD肽的超小粒径黑色素纳米颗粒的制备方法,其特征在于,通过用cRGD-PEG2000-NH2对黑色素纳米颗粒进行表面处理得到。
2.根据权利要求1所述的制备方法,其特征在于, cRGD-PEG2000-NH2与黑色素纳米颗粒的投入质量比为9~11:1。
3.根据权利要求1所述的制备方法,其特征在于,所述修饰cRGD靶向肽的黑色素纳米颗粒通过如下方法制备得到:
(1)将黑色素粉末溶于0.1 mol/L NaOH水溶液中,然后转移到离心管中,超声粉碎的同时,用0.1 mol/L HCl 调节溶液pH至中性;
得到黑色溶液,转移至超滤离心管中,用去离子水洗涤若干次后,冷冻干燥,得到超小粒径的黑色素纳米颗粒,进一步溶于水中得黑色素纳米颗粒水溶液备用;
(2)将cRGD-PEG2000-NH2粉末溶于步骤(1)得到的黑色素纳米颗粒水溶液中,用NaOH水溶液调节pH至9-9.5,室温搅拌24 h,将所得黑色溶液转移至超滤离心管中,用去离子水洗涤若干次后,冷冻干燥,即得到修饰了cRGD肽的超小粒径黑色素纳米颗粒cRGD-PEG-MNP,记为RpMP。
4.根据权利要求3所述的制备方法,其特征在于,步骤(1)中,超声条件为: 120-200 W的功率下工作7-8 min,其中,每工作6-7s,间歇3-4s,仪器保护温度设置为35-38摄氏度。
5.权利要求1-4任一项所述的制备方法制备得到的修饰cRGD肽的超小粒径黑色素纳米颗粒在制备用于抑制巨噬细胞焦亡、预防和/或缓解或治疗炎症/焦亡相关疾病的药物上的应用。
6.根据权利要求5所述的应用,其特征在于,所述炎症/焦亡相关疾病为动脉粥样硬化。
7.根据权利要求6所述的应用,所述修饰cRGD肽的超小粒径黑色素纳米颗粒在体外抑制LPS刺激的巨噬细胞焦亡,在体内富集到动脉粥样硬化斑块部位,局部发挥抗焦亡和抑制斑块进展的作用。
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