CN115006547B - 负载氯沙坦钾的响应性zif-8纳米颗粒及其制备方法和应用 - Google Patents
负载氯沙坦钾的响应性zif-8纳米颗粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种负载氯沙坦钾的响应性ZIF‑8纳米颗粒的制备方法,通过锌离子、二甲基咪唑及氯沙坦,原位封装得到LP@ZIF‑8,将其尾静脉注射至斑块小鼠的体内,该药物可以通过EPR效应聚集到斑块内,由于斑块内部的代谢方式是无氧糖酵解,所以其pH低于生理值,在这一pH下,ZIF‑8解离,Zn离子促进细胞自噬,实现胆固醇逆向转运,从而达到降脂的作用,而氯沙坦则发挥其在局部的强抗炎作用,降低白介素‑6、白介1‑β、TNF‑α等炎症因子,有望应用于动脉粥样硬化的治疗。
Description
技术领域
本发明涉及及生物医药技术领域,具体涉及ZIF-8纳米材料载药体系及其在预防、缓解、治疗动脉粥样硬化中的应用。
背景技术
近年来,心脑血管疾病引发的患病率和死亡率已经远远高于其他重大疾病,而动脉粥样硬化(Atherosclerosis,AS)是导致心脑血管疾病的病理基础。其具体的生理病理过程包括单核细胞招募,在局部分化增殖为巨噬细胞,巨噬细胞吞噬脂质形成泡沫细胞,泡沫细胞释放炎症因子,活性氧等进一步招募单核细胞形成恶性循环。其中,炎症和脂质是两个最重要的致病因素。
目前临床内科治疗的手段以生活方式干预和药物治疗为主,但尽管控制了传统的危险因素,在中国,心血管疾病的死亡率仍然逐年攀升,如何提高内科治疗的疗效是一个亟待解决的关键问题。究其原因一是因为临床药物无靶向性,生物利用度较差;二是因为临床药物治疗大多是针对全身脂代谢进行治疗,忽略了炎症的重要性,因而治标不治本。
自噬是一个关键的修复和细胞应激的过程,可以被营养物质缺乏、缺氧、活性氧(Reactive oxygen species,ROS)、DNA损伤、蛋白质聚集、细胞器受损或细胞内病原体等激活。自噬能够重新利用细胞内的能量资源以应对营养不良,同时能够在细胞面临不利条件时去除细胞毒性蛋白和受损的细胞器,因此它在维持细胞内稳态方面发挥着至关重要的作用。自噬过程有四个关键步骤:诱导、成核、延伸和闭合。简而言之,自噬过程始于内质网双层膜结构的自噬泡,吞噬细胞内大分子物质或细胞器成分形成自噬体。自噬体与溶酶体融合形成自噬溶酶体。自噬溶酶体富含水解酶,这些酶降解自噬的物质并将降解的成分释放到细胞质中以供再利用。
诸多研究表明,AS的进展会损害巨噬细胞的自噬作用。而自噬能够促进脂滴相关胆固醇的动员,从而在巨噬细胞衍生的泡沫细胞中促进胆固醇逆向转运。胆固醇进出失衡,是诱发动脉壁巨噬细胞泡沫化的关键因素。具体而言,在AS中,自噬能够介导细胞质内的脂质被包裹在自噬小体双层膜囊泡中,并将脂滴递送至溶酶体,在溶酶体酸性脂肪酶的作用下,水解生成游离胆固醇流出,并依赖ATP结合盒转运蛋白A1(ABCA-1)流出,减少脂滴在细胞中的集聚进而抑制细胞泡沫化进程。有研究证实,巨噬细胞ATG5特异性敲除的小鼠模型中,自噬水平显著降低,巨噬细胞胆固醇流出受阻,这加速了AS的形成。目前已有研究表明,诱导自噬的药物如:雷帕霉素,维拉帕米,辣椒素等。但这些诱导自噬的小分子药物特异性不强,在体内不具备靶向性,调控方式不可控等,容易引起免疫抑制,肝脏毒性等一系列严重的毒副作用。
然而,仅对脂质代谢做出调节还远远不够。免疫微环境紊乱应同时予以纠正。血管紧张素II(AngII)是RAS的关键因子,它不仅作为外周激素,而且作为局部炎症调节剂,具有生理和病理生理功能。氯沙坦钾(Losartan potassium,LP)是一种典型的RAS抑制剂,已被确定为ROS和炎症免疫微环境的调节因子。它可以降低IL-6、IL-1β和TNF-α的水平,降低肝脏中NF-κB的核浓度,已用于神经炎症、神经鞘瘤、骨关节炎和川崎病。此外,它可以通过改善一氧化氮(NO)和超氧化物(O-2)之间的不平衡来改善内皮功能,这使其十分适于递送至斑块。然而,只有高剂量口服(50-100mg/kg)才能改善心血管预后。
纳米药物递送系统(nano-DSS)已被广泛运用于生物医学领域,用于延长血液循环和在动脉粥样硬化部位更准确的药物递送。目前许多研究均使用同一种纳米载体靶向递送两种药物(一种抗炎药物加一种降脂药物)至AS斑块中。然而,共传递系统一直因其药物共载效率低、剂量比调整不便、系统稳定性差和潜在的药物泄漏而备受诟病。因此,迫切需要开发安全、高效、全面的AS治疗的新策略。
大量研究证明,纳米颗粒可以作为一种新型的自噬诱导剂,在疾病的预防和治疗中发挥着重要和关键的作用。例如,铜钯合金纳米颗粒、氧化铈纳米颗粒、氧化锌纳米颗粒和碳量子点等都已被详细的证明能在不同地系统中诱导自噬。因此,研究新型纳米载体的自噬诱导能力,及其在泡沫细胞调控中的作用,将有助于优化胆固醇逆向转运。
ZIF-8纳米金属有机框架材料孔径较大,可以通过主客体相互作用,荷载具有较大分子结构的药物和生物分子,且药物分子一旦封装于ZIF-8空腔中,很难通过自由扩散的方式向外界释放,只能通过其本身的降解释放药物。ZIF-8在中性的生理环境中结构稳定,在酸性环境下(pH=4.5-6.4),ZIF-8中的2-MI发生质子化,破坏锌离子与咪唑环之间的配位作用,从而引起ZIF-8结构的降解,进而释放药物。因其固有的pH响应性降解行为,目前许多研究均用其构建pH响应性药物递送系统。
此外,随着近年来人们对ZIF-8的研究不断深入,其自身的生物效应不断被揭示。部分学者认为,ZIF-8颗粒能够诱导PI3K调控的肿瘤细胞自噬,导致肿瘤细胞死亡。这一特性可能是由于其金属核心Zn2+引起的,这一点在ZnO纳米粒子中也得到了证实。此外,还有研究表明,ZIF-8具有类过氧化氢酶活性,在过量H2O2等自由基所致氧化应激下能够保持抗氧化活性,这一特点主要是来自于其配体2-MI。
发明内容
发明目的:针对现有技术的不足,本发明提供一种负载氯沙坦钾的响应性ZIF-8纳米颗粒(LP@ZIF-8),所制备的纳米药物通过静脉注射,将LP@ZIF-8靶向递送至AS斑块部位,由于斑块部位的pH值较低,ZIF-8解离释放出的氯沙坦钾LP在局部发挥高效抗炎的作用,而ZIF-8框架则达到恢复泡沫细胞自噬水平,协助胆固醇逆向转运的功能,如此抗炎及调脂双管齐下,更加有效、安全的治疗动脉粥样。
为了解决上述技术问题,本发明提供一种负载氯沙坦钾的响应性ZIF-8纳米颗粒的制备方法,包括如下步骤:
(1)将2-甲基咪唑和Zn(NO3)2·6H2O分别溶于溶剂中得到2-甲基咪唑溶液和Zn(NO3)2·6H2O溶液;
(2)将氯沙坦钾溶液逐滴滴加至Zn(NO3)2·6H2O溶液中,室温下搅拌约5min,将上述溶液逐滴滴加至2-甲基咪唑溶液中,室温搅拌24h,得到白色混悬液,离心,将得到的固体用溶剂洗若干次,然后放置于真空烘箱烘干,即得负载氯沙坦钾的响应性ZIF-8纳米颗粒。
其中,步骤(1)中,所述的溶剂为甲醇、乙醇、水中的任意一种或几种的组合。
优选地,2-甲基咪唑、Zn(NO3)2·6H2O和氯沙坦钾的质量比为6:3:1到6:3:7。
优选地,2-甲基咪唑溶液的浓度为20-30mg/mL,Zn(NO3)2·6H2O溶液的浓度为30-35mg/mL。
步骤(2)中的滴加速率为1-50mL/min。
步骤(2)烘箱温度为60℃-180℃。
本发明进一步提出了上述负载氯沙坦钾的响应性ZIF-8纳米颗粒在制备用于预防、缓解或治疗炎症相关疾病和脂代谢疾病的药物上的应用。
其中,所述炎症相关疾病包括变态反应性炎症、非特异性炎症和感染性炎症;所述脂代谢疾病包括动脉粥样硬化、高脂血症、肝脏脂肪变性和心肌脂肪变性。
所述负载氯沙坦钾的响应性ZIF-8纳米颗粒在降脂的同时发挥抗炎作用。
有益效果:与现有技术相比,本申请具有如下优点:
(1)本发明制备的LP@ZIF-8同时具有调节脂质及抗炎的效果,具有良好的病变主动脉靶向效果;选择氯沙坦钾不仅是因为其对炎症免疫微环境具有强大的调节效应,还因为其结构可能通过配体替代法取代部分ZIF-8的配体,2-甲基咪唑,从而在更大程度上提高其载药能力;
(2)本发明所述纳米药物能够实现该类抗炎药物的有效负载;
(3)本发明所述线粒体靶向的抗炎多肽纳米药物具有良好的体内生物相容性,在体内可降解,且降解产物对机体无毒副作用;
(4)本发明所述纳米药物给药后,能够响应与炎症性病灶局部的低pH微环境,同时通过被动靶向作用在炎症病灶部位靶向富集,与健康对照组相比能够显著提高纳米药物的靶向效果;
(5)本发明所述纳米药物在给药后,能够通过靶向作用、清除局部活性氧和调节局部炎症反应,对炎症相关性疾病包括哮喘、心力衰竭、心肌缺血/再灌注损伤、缺血性脑卒中、炎症性肠炎,骨关节炎,牙周炎的治疗效果明显优于对照组、纯材料组,纯药物;
(6)本发明所述纳米药物在给药后,能够通过靶向作用、诱导局部细胞发生自噬,从而诱导胆固醇逆向转运,降低局部胆固醇,对脂代谢疾病包括动脉粥样硬化,高脂血症,肝脏脂肪变性,心肌脂肪变性等的治疗效果明显优于对照组、纯材料组,纯药物;
(7)本发明所述纳米药物制备方法相对简单且成本较低,易于实现所述纳米药物的产业化。
附图说明
图1为LP@ZIF-8的载药效率随氯沙坦钾加入量的变化图;
图2为不同载药率的LP@ZIF-8紫外可见光谱;
图3为紫外-可见分光光度法检测不同pH值缓冲液的药物释放量;
图4为本发明效果中活性氧染色的结果;
图5为本发明效果中炎症因子IL-1β分泌结果;
图6为本发明效果中炎症因子IL-6分泌结果;
图7为本发明效果中炎症因子TNF-α分泌结果;
图8为本发明效果中纳米药物诱导自噬及脂质的双免疫荧光染色结果;
图9为本发明效果中纳米药物诱导自噬的结果;
图10为本发明效果中脂质含量的油红染色结果;
图11斑块小鼠及野生型健康对照小鼠的体内分布示意图;
图12斑块小鼠及野生型健康对照小鼠的离体主动脉分布示意图;
图13为疗结束后对主动脉进行ORO和HE染色结果图。
具体实施方式
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。
除非另有指明,下述实施例中使用的试剂均为普通可以市购的试剂。
本发明拟基于多功能ZIF-8金属有机框架纳米平台,构建一种通过斑块破损内皮,实现被动靶向至斑块的纳米药物,本发明的目的是,通过静脉注射,将LP@ZIF-8靶向递送至AS斑块部位,由于斑块部位的pH值较低,ZIF-8解离释放出的氯沙坦钾LP在局部发挥高效抗炎的作用,而ZIF-8框架则达到恢复泡沫细胞自噬水平,协助胆固醇逆向转运的功能,如此抗炎及调脂双管齐下,更加有效、安全的治疗动脉粥样。
实施例1负载氯沙坦钾的纳米药物LP@ZIF-8的合成。
采用原位合成的方式,首先配置LP储备液(5-40mg/mL甲醇储备液),取320mg2-MI溶于10mL甲醇中,称取Zn(NO3)2·6H2O 150mg溶于10mL甲醇中,随后将药物储备液逐滴滴加至Zn(NO3)2溶液中,室温下搅拌约5min;再将上述溶液逐滴滴加至2-MI溶液中,室温搅拌24h,载药LP@ZIF-8纳米颗粒自发形成,得到白色混悬液,10000r离心甲醇溶液洗3次去除剩余反应物,真空烘箱烘干即得LP@ZIF-8纳米颗粒,所合成纳米药物在75-150nm之间。电镜图和动态光散射表征图空载的ZIF-8和最大负载量的LP@ZIF-8,结果如图1所示,其中,图1B为ZIF-8的DLS和TEM;图1C为LP@ZIF-8的DLS和TEM。空载的ZIF-8和最大负载量的LP@ZIF-8粒径范围的平均值分别是75nm和105.7nm。
本实施例进一步考察了氯沙坦钾投入量对LP@ZIF-8的载药效率的变化,其中,载药效率通过如下方法进行计算:体系中被包封的药物的质量(mg)/体系中的材料加药物总质量(mg)。图1A为随着所投入的氯沙坦钾含量的不同,LP@ZIF-8的载药效率变化,从结果中可以看出,随着氯沙坦钾含量的增加,载药率逐渐增加至平稳,最高载药率高达40%左右。图2为不同载药率的LP@ZIF-8紫外可见光谱。
下述实施例中,均在用载药量为40%的纳米药物。
实施例2纳米药物LP@ZIF-8pH的响应性。
将LP@ZIF-8(制备条件和方法如上,载药量为40%)分散在PBS(pH=5.5和7.4)中,并在37℃的恒温旋转摇床上摇匀。在每个时间点,取出样品,离心,用等体积的新鲜PBS替换。采用紫外-可见分光光度法检测不同pH值缓冲液的药物释放量。结果如图3所示。
实施例3纳米药物LP@ZIF-8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ROS的生成。将RAW 264.7细胞以每孔5×105的密度接种于6孔板中,每组设置3个平行样本。然后,用200ng/mL的脂多糖LPS(或不含LPS,作为阴性对照)(脂多糖)刺激细胞,然后用PBS、LP@ZIF-8(37μMLP)、LP或ZIF-8(等效浓度的LP@ZIF-8)处理24h。在荧光显微镜下对细胞进行观察并拍照。结果如图4所示。试验结果表明,LP@ZIF-8能够很好的清除巨噬细胞产生的活性氧。
实施例4纳米药物LP@ZIF-8对炎症因子的影响。
用200ng/mL的LPS(不含LPS组作为阴性对照)刺激RAW264.7细胞,然后用PBS、LP@ZIF-8(37μMLP)、LP或ZIF-8(同等浓度的LP@ZIF-8)处理细胞。处理24h后,收集培养基,用ELISA试剂盒(Abcam,USA)分析TNF-α、IL-6和IL-1β的浓度。结果如图5-7所示。结果表明,LP@ZIF-8对于炎症因子白介素-6,白介1-β,TNF-α等均具有较好的抑制作用。
实施例5纳米药物LP@ZIF-8诱导自噬及脂质调节的研究。
将细胞以2×105的密度接种在4个20mm共聚焦培养皿中,培养24小时。然后,用100μg/mL的ox-LDL(不含ox-LDL的作为阴性对照)激活细胞,同时用PBS、LP@ZIF-8(37μMLP)、LP或ZIF-8(等效浓度的LP@ZIF-8)处理24h。然后,用BODIPY493/503(Sigma)染色脂质,用4%多聚甲醛固定,用10%正常山羊血清封闭,用0.2%TritonX-100渗透。然后,用抗lc3(1:200,ab192890)的抗体在4℃孵育过夜,然后进一步用IgG H&L(ab150080)和2-(4-酰胺苯基)-6-盐酸盐,DAPI1μg/mL(Sigma)染色,在激光共聚焦显微镜下(德国徕卡)观察拍照。图8为本发明效果中诱导自噬及脂质的双免疫荧光染色结果,结果表明,随着ZIF-8的加入,泡沫细胞的自噬增强,而自噬的增强又同时伴随着泡沫细胞脂质含量的降低,证明了LP@ZIF-8能够通过诱导自噬,清除泡沫细胞中的脂质。
细胞处理和给药方法同上。孵育时间为24h。24h后将细胞裂解并收集裂解后的蛋白。用BCA测定法定量测定蛋白浓度。在4-12%的三甘氨酸凝胶上跑蛋白,随后将其转移到聚偏氟乙烯(PVDF)膜上,然后与抗lc3(1:1000)和β-actin(1:1000)孵育过夜。最后用辣根过氧化物酶连接的二抗再孵育这些样本。用AlphaEaseFC分析这些蛋白的相对数量。结果如图9所示,通过WB结果表明,体系中ZIF-8能够诱导泡沫细胞的自噬,进一步通过油红染色观察泡沫细胞。将RAW264.7细胞(2.0mL,5×105)接种于6孔板中。然后以Raw264.7细胞为阴性对照。用ox-LDL(100μg/mL)处理24h的细胞作为阳性对照。其他组分别用100μg/mL ox-LDL以及30μMLP、ZIF-8和LP@ZIF-8(含30μMLP)同样处理24h。PBS洗涤后,RAW264.7细胞用4%多聚甲醛固定15分钟,用60%异丙醇洗涤,用油红O染脂滴。用荧光显微镜观察泡沫细胞并拍照(奥林巴斯,日本)。结果如图10所示。结果表明,LP@ZIF-8能够使得细胞红染的面积减少,即清除泡沫细胞中的脂质。
实施例6纳米药物LP@ZIF-8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ApoE-/-小鼠构建动脉粥样硬化模型(n=4)。野生型小鼠正常饮食作为健康对照组。通过尾静脉注射IR820@ZIF-8,每组IR820(新吲哚菁绿,一种荧光染料,用于可视化材料在主动脉中的富集区域和富集多少)的剂量为2mg/kg。分别在3h、6h、12h、24h和48h拍摄荧光图像。48h后处死小鼠。收集主动脉进行离体的荧光图像拍摄。如11为IR820@ZIF-8在斑块小鼠及野生型健康对照小鼠的体内分布示意图,图12为斑块小鼠及野生型健康对照小鼠的离体主动脉分布示意图。结果表明,ZIF-8纳米颗粒能够通过斑块内不完整的内皮,被动靶向到病变的主动脉部位而不会在健康的主动脉中富集。
治疗结束后对主动脉进行ORO和HE染色,反应脂质含量和总斑块面积,具体地,将动脉粥样硬化小鼠随机分为4组,正常小鼠作为健康对照,分别给予实验鼠(a)PBS、(b)ZIF-8、(c)LP或(d)LP@ZIF-8,每周3次,共6周。游离LP的剂量与ZIF-8时LP的剂量相当(5mg/kg)。ZIF-8的剂量与LP@ZIF-8的载体剂量相当。在治疗期间维持高脂饮食。最后一次治疗后,处死小鼠取出主动脉,对全主动脉进行油红O染色和HE染色。结果如图13所示。从结果中可以看出,HE染色可见,LP@ZIF-8治疗组与其他各组相比,斑块面积减少;油红O染色可见,LP@ZIF-8治疗组与其他各组相比,能够减少动脉粥样硬化小鼠的脂质含量。
总上所述,本申请通过锌离子、二甲基咪唑及氯沙坦,原位封装LP@ZIF-8,尾静脉注射至斑块小鼠的体内,该药物可以通过EPR效应聚集到斑块内,由于斑块内部的代谢方式是无氧糖酵解,所以其pH低于生理值,在这一pH下,ZIF-8解离,Zn离子促进细胞自噬,实现胆固醇逆向转运,从而达到降脂的作用,而氯沙坦则发挥其在局部的强抗炎作用,降低白介素-6,白介1-β,TNF-α等炎症因子。
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 (4)
1.一种负载氯沙坦钾的响应性ZIF-8纳米颗粒在制备用于预防、缓解或治疗炎症相关疾病和脂代谢疾病的药物上的应用,所述炎症相关疾病为变态反应性炎症、非特异性炎症和感染性炎症;所述脂代谢疾病为动脉粥样硬化、高脂血症、肝脏脂肪变性和心肌脂肪变性,其中,所述负载氯沙坦钾的响应性ZIF-8纳米颗粒通过如下方法制备得到:
(1)将2-甲基咪唑和Zn(NO3)2•6H2O分别溶于溶剂中得到2-甲基咪唑溶液和Zn(NO3)2•6H2O溶液;
(2)将氯沙坦钾溶液逐滴滴加至Zn(NO3)2•6H2O溶液中,室温下搅拌5 min,将上述溶液逐滴滴加至2-甲基咪唑溶液中,室温搅拌24 h,得到白色混悬液,离心,将得到的固体用溶剂洗若干次,然后放置于真空烘箱烘干,即得负载氯沙坦钾的响应性ZIF-8纳米颗粒;
其中,2-甲基咪唑、Zn(NO3)2•6H2O和氯沙坦钾的质量比为6:3:1到6:3:7;2-甲基咪唑溶液的浓度为20-30 mg/mL,Zn(NO3)2•6H2O溶液的浓度为30-35 mg/mL。
2.根据权利要求1所述的应用,其特征在于,步骤(1)中,所述的溶剂为甲醇、乙醇、水中的任意一种或几种的组合。
3.根据权利要求1所述的应用,其特征在于,步骤(2)中的滴加速率为1-50 mL/ min。
4.根据权利要求1所述的应用,其特征在于,步骤(2)烘箱温度为60℃-180 ℃。
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