CN118021965A - 一种基于温和光热联合天然药物的抗肥胖透皮递药系统及其制法和应用 - Google Patents
一种基于温和光热联合天然药物的抗肥胖透皮递药系统及其制法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于温和光热联合天然药物的抗肥胖透皮递药系统及其制法和应用,所述递药系统以可溶性微针作为载体,载体上负载光热剂黑磷纳米片,载体内装载双药纳米粒而构成;所述双药纳米粒为姜黄素和甘草次酸无载体自组装构建而得,通过双药纳米粒联合黑磷纳米片,在808nm激光照射下,提供了脂肪组织局部温和热量,实现温和光热治疗,通过减少脂肪细胞分化、促进白色脂肪棕色化、缓解脂肪组织慢性炎症,从而发挥较好的体内抗肥胖效果;同时微针给药改善注射给药可能带来的脂肪组织感染、皮下结节等,实现局部无痛微创给药,提高患者依从性,在治疗肥胖方面具有良好的应用前景。
Description
技术领域
本发明涉及一种抗肥胖透皮递药系统,尤其涉及一种基于温和光热联合天然药物的抗肥胖透皮递药系统,还涉及上述递药系统的制法和应用。
背景技术
肥胖是一种慢性代谢疾病,定义为对健康产生不利影响的身体脂肪过度或异常积累。近年来,肥胖症的患病率在全球范围内有所增加,现已达到大流行水平。到2030年,五分之一的女性和七分之一的男性将患有肥胖症,全球将有超过10亿人与肥胖症作斗争。
治疗肥胖的策略分为两大类,外科手术和非手术方法。对于严重肥胖症或患有多种并发症的患者采取胃绕道等外科手术,但具有手术风险,例如感染、疤痕、血肿等。非手术方法中,限制饮食和运动是一种较为安全的减肥方法。患者须遵循科学合理的饮食习惯,在减少能量摄入的同时增加有效运动强度,以消耗体内多余的脂肪和能量。然而,大多数人很难长期坚持下去。非手术的化学药物治疗,例如氯卡色林、芬特明和托吡酯,可以通过改变自身的代谢率来控制体重增加。然而,这些药物具有严重的多重副作用,包括中风、抑郁等。随着纳米技术的迅速发展,纳米递药系统不仅能够提高疏水性药物的溶解性和稳定性,还能同时负载多种药物,实现药物的协同治疗,从而进一步加强肥胖的治疗作用。2020年,美国食品药品监督管理局(FDA)批准的天然新分子实体中约有25%来自植物。相较于流行的合成化学药物,天然药物价格合理、可用性增加、天然安全、副作用低或无副作用。而随着纳米技术的迅速发展,纳米载体负载药物能够提高疏水性药物的溶解度和稳定性。因此,寻求合适的负载天然药物的抗肥胖纳米递药系统应用于低风险、无痛苦减脂具有广阔的发展前景。
人体内有两种主要的脂肪组织:白色脂肪组织(White adipose tissue,WAT)和棕色脂肪组织(Brown adipose tissue,BAT)。与含有大脂滴和少量线粒体的白色脂肪细胞不同,棕色脂肪细胞含有多室小脂滴,且有丰富的线粒体。因此,棕色脂肪细胞在燃烧葡萄糖和脂质以维持BAT的产热方面发挥着重要作用。研究表明,WAT中出现的棕色样脂肪(即所谓的米色脂肪),其静息时表现出白色脂肪的特质,而在寒冷或β肾上腺素能受体激动剂等激活的情况下,具有棕色化潜力,促进产热和能量消耗,改善机体糖脂代谢。
正常脂肪组织中10-15%为抗炎的M2型巨噬细胞,表达白细胞介素-10(Interleukin-10,IL-10)和精氨酸酶-1(Arginase-1,Arg-1),这有助于维持局部免疫和代谢稳态。然而,在肥胖的人类和啮齿动物,脂肪组织释放趋化剂如单核细胞趋化蛋白-1(Monocyte chemoattractant protein-1,MCP-1),此时脂肪组织巨噬细胞约占比为40-50%,并将表型转变为促炎的M1型巨噬细胞,同时分泌促炎细胞因子,如肿瘤坏死因子(Tumor necrosis factor,TNF-α)和白细胞介素-6(Interleukin-6,IL-6)。这些炎症因子进而引起一系列病理效应,导致葡萄糖耐受不良、胰岛素抵抗和血管功能障碍。
目前,常见的含天然药物的纳米制剂有纳米胶束、纳米金颗粒等,如2022年四川大学公开了用于抗肥胖的载雷公藤红素的纳米胶束(CN115154422A),其载体为两亲性的糖胺聚糖衍生物载体,用于提高药物溶解度和稳定性。而纳米载体具有一定的结构限制性,载体装载药物的载药量、包封率通常较低,且部分载体有一定的生物毒性。当过量的纳米载体进入体内后,易被单核巨噬细胞和网状内皮系统所清除,从而累积和滞留在肝脏中,造成身体的过重负担。由于缺乏脂肪细胞特异性表面标志物,静脉注射给药方式药物递送到脂肪组织效率极低,常规皮下注射随之而来的脂肪组织感染、皮下结节等导致患者依从性不高。
发明内容
发明目的:本发明的目的是提供一种安全无毒的基于温和光热联合天然药物的抗肥胖透皮递药系统,还提供上述透皮递药系统的制法和应用;该递药系统促进白色脂肪棕化增加能量消耗和产热,同时恢复肥胖个体的M2型巨噬细胞,减少M1型巨噬细胞浸润,以实现天然药物治疗联合温和光热治疗的无痛苦减肥目的。
技术方案:本发明的基于温和光热联合天然药物的抗肥胖透皮递药系统,以可溶性微针作为载体,载体上负载光热剂黑磷纳米片,载体内装载双药纳米粒而构成;所述双药纳米粒为姜黄素和甘草次酸无载体自组装构建而得。
其中,所述双药纳米粒,是取姜黄素和甘草次酸分散于溶剂中制得混合溶液,将混合溶液滴入水中进行超声,避光透析而得。
所述双药纳米粒CG NPs外壳光滑圆润,平均粒径为109.50±1.30nm,PDI为0.121±0.012,电位为-19.2±0.46mV。
其中,所述混合溶液中,姜黄素和甘草次酸的质量比为4:1~1:4,混合溶液与水的体积比为1:5~1:50。
其中,所述黑磷纳米片的粒径为100~200nm,PDI为0.205±0.029,电位为-18.2±0.37mV。
其中,所述可溶性微针中黑磷纳米片和双药纳米粒的质量比为10:1~1:5。
上述抗肥胖透皮递药系统的制备方法,包括以下步骤:
(1)取姜黄素(Curcumin,Cur)和甘草次酸(Glycyrrhetinic acid,GA)分散于溶剂中制得混合溶液,将混合溶液滴入水中进行超声,避光透析即得双药纳米粒CG NPs;
(2)取黑磷晶体,研磨,分散于丙酮溶液,超声剥离,离心沉淀,混悬后即得黑磷纳米片BP(Black phosphorus,BP);
(3)取双药纳米粒与黑磷纳米片溶于聚乙烯吡咯烷酮溶液中,注入微针模具真空干燥,加入PVA-1788溶液作为基底层,干燥脱模取出即得抗肥胖透皮递药系统CG@BP MN。
其中,步骤(1)中,所述溶剂为无水甲醇、无水乙醇、二甲基亚砜、二氯甲烷、乙酸乙酯、乙腈的任意一种或多种,所述超声功率为50~300W,时间为1~10min。
其中,步骤(2)中,所述超声剥离,超声功率为80~200W,时间为2~24h。
其中,步骤(3)中,所述干燥,时间为8~24h。
制得的CG@BP MN贴片呈正方形,CG@BP MN针体完整,呈现子弹头型。显微镜观察微针高度约为890.21±5.52μm,间隔约为792.64±3.94μm。各针体外层针尖显示出均匀金属光泽,为光热剂黑磷纳米片均匀附着在针体上,同时针体内部呈现橙黄色,为CG NPs负载。
发明原理:本发明针对肥胖疾病中脂肪组织的生理环境,以脂肪组织中的白色脂肪细胞和巨噬细胞为目标,通过分子间非共价作用力构建双纳米粒,以可溶性微针作为载体,构建抗肥胖透皮递药系统,实现天然药物治疗联合温和热疗无痛安全地治疗肥胖,为肥胖治疗提供新的方法与思路。
姜黄素作为天然来源的抗氧化剂,能够抑制和调节促炎细胞因子的产生和分泌,如白细胞介素、TNF-α等,可以减少肥胖引起的脂肪组织炎症。同时研究表明,姜黄素通过抑制增强子结合蛋白α(CCAAT/enhancer binding proteins alpha,C/EBPα)和过氧化物酶体增殖物激活受体γ(Peroxisome proliferator-activatedreceptor,PPARγ)来抑制前脂肪细胞的分化,减少脂滴堆积,降低脂质积累水平;同时上调脂肪细胞中棕色脂肪标志物线粒体棕色脂肪解偶联蛋白1(Uncouplingprotein 1,UCP1)的表达,诱导白色脂肪棕色化,促进产热从而消耗能量,减少体重。
甘草次酸是甘草根的主要活性成分,在多种中药配方中用于配伍,起到减毒增效的作用。甘草次酸能够降低炎症介质的分泌,如IL-6,白介素1β(Interleukin1β,IL-1β)等,发挥抗炎作用,同时在脂肪细胞水平上抑制I型11β-羟基类固醇脱氢酶,通过抑制PPARγ实现抑制前脂肪细胞分化,减少脂滴堆积,降低脂质积累水平。
黑磷在808nm处具有较大的消光系数和高光热转换效率,可快速有效地将近红外激发光转换为热能。与传统碳纳米材料和金属纳米材料相比,黑磷在生理条件下会氧化进而降解成磷酸根离子和亚磷酸根离子等安全的小分子;磷元素亦是人体不可或缺的元素,降解的磷酸类产物可以参与蛋白磷酸化、碱基合成等过程,用于维持正常机体功能。黑磷具有可降解性和生物相容性的优势,是一种性能优良的光热治疗剂。研究表明,通过红外光照射,利用41±0.5℃的温度实现局部温和热疗,诱导白色脂肪褐变,增加局部产热,在不影响中枢交感神经系统和免疫系统的情况下,减轻肥胖并改善代谢紊乱,并且无不良反应。
姜黄素的水溶性较差、极易被水解,只有极少的姜黄素进入血液循环,甘草次酸水溶性差,经皮吸收效率较低。因此构建一款提高难溶性药物溶解度、生物利用度高、且适用于局部减脂的纳米递送系统,具有合理的开发意义。
随着纳米技术的迅速发展,纳米载体负载药物能够提高疏水性药物的溶解度和稳定性。然而,纳米载体由于自身结构的限制,载体装载药物的载药量、包封率通常较低,且部分载体生物有一定的毒性。当过量的纳米载体进入体内后,纳米载体易被单核巨噬细胞和网状内皮系统所清除,从而累积和滞留在肝脏中,造成身体的过重负担。发明人选择采用无载体纳米递药系统,在使用一种或者多种药物在几乎不使用载体材料的情况下,通过非共价作用力组装得到纳米递药系统,该递药系统易制备、载药量高,不仅有传统纳米递送系统的优势,可以借助纳米制剂的小尺寸(20~200nm)特点通过EPR效应(脂肪组织的高通透性和滞留效应)高效聚集在脂肪组织,实现被动靶向。还能避免载体带来的毒性作用,从而提高药物递送效率、减轻载体毒副作用。微针作为一种新型透皮给药方式,不仅能够刺破角质层,将纳米递药系统直接递送至真皮层,进而在脂肪组织聚集,同时避免注射给药带来的脂肪组织感染、皮下结节等,从而提高生物利用度,改善患者依从性。
本发明的抗肥胖透皮递药系统,双药纳米粒联合黑磷纳米片进行温和光热治疗,降低成熟脂肪细胞中的脂肪含量,发挥良好的体外抗肥胖效果,促进M2型巨噬细胞转变,抑制M1型极化,起到较好的体内抗炎的作用,通过减少脂肪细胞分化、促进白色脂肪棕色化、缓解脂肪组织慢性炎症,从而发挥较好的体内抗肥胖效果。
具体为,利用两种药物分子间的相互作用如疏水作用力、氢键自组装形成的无载体纳米粒,将疏水性药物姜黄素和甘草次酸通过纳米技术实现联合用药,提高了药物包封率和载药量,有效避免传统载体材料带来的潜在毒性,且提高药物的生物利用度和对脂肪细胞脂肪生成的抑制作用,同时缓解肥胖带来的慢性炎症环境,为纳米技术应用于肥胖治疗领域提供了新思路。引入光热剂黑磷片,实现了近红外光热诱导条件下的温和光热治疗,与传统杀伤破坏脂肪细胞方式相比,进一步降低了光热效应对正常细胞的副作用,为白色脂肪棕色化提供了新方法。
有益效果:本发明和现有技术相比,具有如下优点:(1)提高了药物包封率和载药量,有效避免传统载体材料带来的潜在毒性,且提高药物的生物利用度和对脂肪细胞脂肪生成的抑制作用,同时缓解肥胖带来的慢性炎症环境;(2)本发明制备方法简单,反应条件温和,能够降低生产和使用成本,无载体纳米载药系统在肥胖治疗方面具有良好的应用前景。
附图说明
图1为实施例1中CG NPs纳米粒的粒径、PDI及电位表征图;
图2为实施例1中CG NPs的紫外光谱图;
图3为实施例1中CG NPs的傅里叶变换红外吸收光谱图;
图4为实施例1中CG NPs的透射电子显微镜图;
图5为实施例1中CG NPs的体外稳定性考察结果,其中A图为CG NPs置于4℃环境下的粒径、PDI变化图,B图为CG NPs置于室温环境下的粒径、PDI变化图;
图6为实施例1中的体外毒性研究结果,其中A图为CG NPs对小鼠成纤维细胞3T3-L1的细胞毒性,图B为CG NPs对小鼠巨噬细胞RAW264.7的细胞毒性;
图7为实施例4中BP的粒径、PDI及电位表征图;
图8为实施例4中BP的透射电子显微镜图;
图9为实施例5中CG@BP MN的立体显微镜拍摄图;
图10为实施例5中CG@BP MN的溶解性能研究结果;
图11为实施例6中不同组别对3T3-L1细胞油红O染色图;
图12为实施例8中不同载药微针对肥胖小鼠UCP1表达的免疫组化图;
图13为实施例8中不同载药微针对肥胖小鼠各脂肪因子表达水平图,A、B图分别为TC、TG表达水平图;
图14为实施例8中不同载药微针对肥胖小鼠M1、M2型巨噬细胞比例的流式分析图;
图15为实施例8中不同载药微针对肥胖小鼠各炎症因子表达水平图,A、B图分别为TNF-α、MCP-1表达水平图;
图16实施例9中CG@BP MN的体内安全性评价,小鼠心、肝、脾、肺、肾组织切片的H&E染色结果图。
具体实施方式
下面结合实施例对本发明的技术方案作进一步说明,实施例中所用的试验材料均可通过常规途径购得。
实施例1
双药无载体自组装纳米递药系统(CG NPs)的制备及表征:
(1)CG NPs的制备
称量姜黄素和甘草次酸分散于溶剂中制成质量比为2:1的混合溶液,将溶解后的混合溶液滴加入超纯水中进行超声,混合溶液与超纯水体积比为1:15,超声功率为300W,超声时间为60s,得到CG NPs,将其转移到相对分子质量为3500Da的透析袋中,室温避光条件下,在超纯水中透析过夜,透析后透析袋中的液体为同时负载姜黄素和甘草次酸的双药无载体自组装纳米递药系统(CG NPs);
(2)CG NPs纳米粒的表征
采用动态光散射法(Dynamic light scattering method,DLS)测量CG NPs水合粒径,结果如图1所示,该纳米粒平均粒径为109.50±1.30nm,PDI为0.121±0.012,电位为-19.2±0.46mV。
利用紫外可见分光光度计(Ultraviolet visible spectrophotometer,UV-Vis)测定紫外光谱,结果如图2所示,证实CG NPs由两种小分子药物组装而成。
利用傅里叶红外光谱仪(Fourier Transform Infrared Spectroscopy,FITR)测定姜黄素原料药,甘草次酸原料药,二者原料药混合物,CG NPs冻干粉的红外吸收图谱。结果如图3所示,游离姜黄素在3450-3090cm-1范围内检测到一个较宽的吸收峰,3502cm-1出现特征吸收峰,对应Cur中的酚羟基(-OH),1602cm-1为苯环骨架振动,1627cm-1为羰基C=O和C=C的混合振动(共轭酮的羰基拉伸振动),1510cm-1归属于C-C和C-O的伸缩振动,1427cm-1吸收峰为烯烃结构(C-H)的弯曲振动,1232cm-1为芳香环的C-O伸缩振动,1026cm-1是醚(C-O-C)的拉伸振动。游离甘草次酸的FTIR光谱在3437cm-1(羟基-OH)、2945cm-1(亚甲基-CH2)、1705cm-1(羧基-COOH)、1660cm-1(羰基-C=O)、1384cm-1(甲基-CH3)、1178cm-1(亚甲氧基O-CH2)处均可见明显特征峰,1386cm-1(甲基-CH3)和1367cm-1齐墩果烷型五环三萜骨架的特征峰。物理混合后姜黄素的多处特征峰(3502、1627、856cm-1),甘草次酸的多出特征峰(3437,1705,1664,1384,1367cm-1)有所减弱但是并没有消失,这可能是因为甘草次酸具有较高的峰形会掩盖部分姜黄素的指纹区使其特征峰强度降低,这表明物理混合并不会使甘草次酸与姜黄素发生相互作用。而CG NPs中,姜黄素和甘草次酸在3502cm-1和3437cm-1处的-OH拉伸振动峰合并同时迁移至3417cm-1,说明姜黄素和甘草次酸之间存在强氢键相互作用。而CG NPs在姜黄素指纹图谱区域(500-1300cm-1)的吸收峰的强度和数量明显降低,同时姜黄素分子在1627、1510、1153和1026cm-1特征峰的变化主要与芳香环和环间链的振动有关,再加入甘草次酸后,姜黄素中的吸收峰迁移至1656、1514、1161、1124cm-1,表明姜黄素的疏水基团可能通过疏水相互作用与甘草次酸相互作用。姜黄素的苯环骨架吸收峰由1602cm-1迁移至1587cm-1,甘草次酸的亚甲氧基1178cm-1迁移至1172cm-1,表明二者之间存在静电作用。综上所述,说明CG NPs制备成功。
通过透射电子显微镜(Transmission electron microscope,TEM)观察CG NPs形貌,结果如图4所示,外壳圆润光滑,实际大小与水合粒径相近,说明CG NPs制备成功。
实施例2
CG NPs的体外稳定性考察:
将实施例1制得的CG NPs分别分散在超纯水、生理盐水(0.9%NaCl)、PBS、DMEM培养基、含10%血清的DMEM培养基中,于4℃或室温下保存,每天取出等体积的纳米溶液,用DLS分别于1、2、3、4、5、6、7天时测定其粒径与PDI,并记录数据。
结果如图5所示,在水、生理盐水(0.9%NaCl)、PBS、DMEM培养基、含10%血清的DMEM培养基中,CG NPs的粒径与PDI均无明显变化,表明其具有良好的稳定性,可为后续细胞实验的开展提供实验基础。
实施例3
CG NPs纳米粒对RAW264.7和3T3-L1的体外毒性研究:
通过MTT法测定实例1制备的CG NPs纳米粒对RAW 264.7和3T3-L1的增殖抑制活性。具体步骤为:取处于对数生长期的RAW264.7细胞,经过胰酶消化后,以5×104个/孔的细胞密度在96孔板中培养,置于37℃、5%CO2培养箱中培养24h后。设置游离姜黄素、复合游离药、CG NPs组别,将0.78125,1.5625,3.125,6.25,12.5,25,50μM(以姜黄素浓度计算)的样品溶液加入到培养基中培养24h。然后除去培养基,加入10μL MTT(5mg/mL)并孵育4h。用150μL DMSO溶解甲臜晶体,振荡10分钟后,使用酶标仪在490nm处测试吸光度,按下述公式计算细胞存活率。
其中,ODSample为各组药物处理后的细胞吸光度,ODControl为未经各组药物处理的细胞吸光度,ODBlank为PBS溶液的吸光度。
结果如图6中的A所示,3T3-L1细胞的存活率随各组给药浓度增加,姜黄素游离药、复合游离药组无明显下降趋势,CG NPs组略降低,CG NPs对3T3-L1的最低细胞毒性浓度IC20为44.021μM(以姜黄素浓度)。如图6中的B所示,RAW 264.7细胞的存活率呈现浓度依赖性,高浓度给药时RAW 264.7呈现一定的细胞毒性,CG NPs对RAW264.7的最低细胞毒性浓度IC20为17.732μM(以姜黄素浓度)。在相同药物的浓度下,CG NPs能更多地被细胞摄取,更高浓度地姜黄素从而蓄积在细胞内部,具有更强的细胞毒性。
实施例4
黑磷纳米片BP的制备及性质研究:
(1)黑磷纳米片BP的制备
精密称取25mg黑磷晶体置于研钵中研磨,待大块晶体研磨为黑磷粉末后均匀分散于丙酮溶液中,冰水浴中超声剥离24h;待超声结束后,离心去除黑磷大块沉淀,收集上层分散液再次离心,底部沉淀用超纯水混悬,即得到水分散的黑磷纳米片BP;
(2)黑磷纳米片BP的性质研究
通过DLS测量BP的粒径,结果图7所示,粒径为189.10±2.50nm,PDI为0.205±0.029,电位为-18.2±0.37mV;通过TEM观察结果如图8(scale bar=200nm)所示,BP粒径约为100~200nm。
实施例5
CG@BP MN递送系统的制备、表征与性质研究:
(1)CG@BP MN递送系统的制备
称取3mg的CG NPs纳米粒与3mg BP溶于1mL的20%聚乙烯吡咯烷酮溶液中,充分溶解后注入微针模具;放入真空干燥器真空干燥2h,加入20%PVA-1788溶液作为基底层,常温干燥过夜,脱模取出即得CG@BP MN。
(2)CG@BP MN递送系统的表征
采用体视显微镜,观察针尖长度。将微针放置于体视显微镜下,通过明场光源拍摄微针的形状,并测量微针针尖的实际长。结果如图9所示,微针贴片呈正方形,CG@BP MN针体完整,呈现子弹头型。显微镜观察微针高度约为890.21±5.52μm,间隔约为792.64±3.94μm,scale bar=200μm,500μm;各针体外层针尖显示出均匀金属光泽,为光热剂黑磷纳米片均匀附着在针体上,同时针体内部呈现橙黄色,为CG NPs负载,说明CG@BP MN制备成功。
(3)CG@BP MN递送系统的性质研究
将CG@BP MN的针尖浸没于生理盐水中,分别于0、1、2、3、5min取出并置于体视显微镜明场光源下拍摄,结果如图10所示,scale bar=200μm,500μm。结果可见,CG@BP MN在3min时,针尖溶解将近100%,在5min完全溶解,说明该微针是快速溶解型微针。
实施例6
CG@BP递送系统的体外抗肥胖效果研究
将处于对数生长期的3T3-L1细胞悬液(1×105/mL)接种在24孔板中,每孔1mL,然后按照传统鸡尾酒诱导方法诱导3T3-L1前脂肪细胞分化为成熟的脂肪细胞,待细胞融合度达100%并接触抑制2天后开始给药,用分化诱导培养基I(Differentiation medium I,DMI)配制含游离姜黄素、游离姜黄素和甘草次酸混合物、CG NPs、CG@BP(+)组(姜黄素浓度为6.25μM),每孔中加入1mL药液并置于细胞培养箱中培养4天,接着用分化诱导培养基II(Differentiationmedium I,DMII)继续培养4天,最后再用基础培养基培养2天结束分化过程。基础培养期间进行温和光热治疗,光照条件为光照时间3min,黑磷浓度30μg/mL,光照功率1W/cm2。前脂肪细胞诱导分化为成熟脂肪细胞的过程中会导致脂质积聚,油红O染色可直观显示出脂肪细胞的脂质积累程度,对诱导结束的3T3-L1细胞进行油红O染色,显微镜下观察拍照,通过Image J图像处理软件统计红色脂滴面积。
结果如图11所示(scale bar=500μm),诱导分化结束后,可见诱导分化组细胞大都分化为典型的成熟脂肪细胞,由梭状的成纤维细胞变成圆形肥大的“戒状”成熟脂肪细胞,胞浆内脂滴明显增多,部分融合成较大脂滴,脂滴环绕四周。与诱导分化组相比,CG NPs组胞内脂滴减少,有较好的减脂效果;而CG@BP(+)组引入温和光热,进一步促使脂肪细胞棕化,胞内脂滴含量进一步下降,从而发挥抗肥胖效果。
实施例7
CG@BP MN递送系统的体内抗肥胖作用研究
(1)CG@BP MN递送系统的体内促棕化作用研究
选取健康的6周龄雄性C57BL/6J小鼠,适应性饲养后随机分组为正常饲料组和高脂饲料组(High fat diet,HFD),自由饮水,喂养约8周后,小鼠肥胖模型构建完成。将小鼠随机分为六组,分别为正常组、肥胖模型组、Cur MN、Cur+GA MN、CG MN和CG@BP MN。各组以姜黄素每只小鼠5mg/kg的剂量经皮给药,光照组在微针完全溶解后,然后用808nm近红外激光按照1.5W/cm2的功率下照射小鼠一侧腹股沟白色脂肪组织(Inguinal white adiposetissue,iWAT),以41±0.5℃实现小鼠局部温和热疗5min,每三天给药一次。给药5周后,所有小鼠禁食12h称重,记录小鼠体重。
取各组小鼠iWAT,用生理盐水润洗干净,清洗后用滤纸吸干脏器表面的水分,置于4%多聚甲醛中固定24h,经石蜡包埋切片后,进行UCP-1免疫染色,镜下观察UCP-1表达并拍照。如图12所示(scale bar=100μm),免疫组化结果显示,相较于对照组小鼠,肥胖组小鼠iWAT中的UCP1表达下降,CG MN组的UCP1表达显著恢复,提示肥胖小鼠的皮下白色脂肪被成功棕色化。而CG@BP MN(+)干预后小鼠iWAT中的UCP1表达更为显著,说明温和光热治疗进一步促进白色脂肪组织棕色化,从而增加脂肪组织的能量消耗和产热,改善机体代谢。
(2)CG@BP MN递送系统的体内减脂作用研究
采用异氟烷麻醉小鼠,随后用镊子摘去小鼠眼球取血,全血静置1h,3000rpm离心15min,取血清以备后续检测使用,按照试剂盒说明书检测总胆固醇(Total Cholesterol,TC)、甘油三酯(Triglyceride,TG)含量。
如图13所示,与正常组相比,肥胖模型组TC、TG含量显著升高,说明肥胖小鼠模型造模成功,同时长期的高脂饮食致使小鼠体内脂质代谢水平紊乱;与肥胖模型组相比,CurMN组、Cur+GA MN组、CG@BP MN组、CG@BP MN(+)组TC、TG水平均显著降低,各组均改善小鼠的脂质代谢情况,其中CG@BPMN(+)可恢复肥胖小鼠血脂接近至正常水平,改善肥胖可能引起的高脂血症的脂代谢异常,具有最佳的体内抗肥胖及血脂恢复效果。
实施例8
CG@BP MN递送系统的体内抗炎作用研究
将新鲜iWAT用预冷的PBS润洗后,用剪刀将脂肪组织切成小块,置于200目筛网一边滴加PBS一边研磨,制备细胞悬液。将细胞悬液于1000rpm、4℃条件下离心10min,吸去顶部的白色脂肪层,底层白色或红色的沉淀为巨噬细胞所在。向每份样本中加入4mL1X红细胞裂解液,轻弹试管将沉淀重悬,室温下孵育5min,偶尔轻轻摇晃,使样本中的红细胞充分裂解。裂解完成后,调整细胞浓度为106个细胞/管,在避光条件下加入APC标记的抗CD86抗体,PE标记的抗CD206抗体,FITC标记的抗F4/80抗体,并以染色缓冲液(含5%FBS的PBS)稀释样本至总体积为100μL,将每管细胞过300目筛网后转移到流式管中,使用流式细胞仪测定平均荧光强度。
收集各组小鼠血液,静置1h,随后4℃,5000rpm离心取上清液,将其置于2mL离心管中,使用试剂盒测定血清中TNF-α、MCP-1炎症因子含量。
由图14可知,与对照组相比,肥胖组M1型数量显著增加,M2型数量显著降低,M1/M2比值显著增加。与肥胖组相比,CG MN治疗组F4/80+CD206+细胞群(M2型巨噬细胞)显著恢复,F4/80+CD86+细胞群(M1型巨噬细胞)比例显著减少,M1/M2比值趋于正常小鼠比值,说明CGMN相比于游离药(Cur)或混合游离药(Cur+GA)具有更好的抗炎效果,从而减轻肥胖小鼠的脂肪组织炎症。CG@BP MN(+)相较于CG MN的M1/M2比值变化较小,且各细胞群比例均与正常小鼠无显著性差异,说明温和光热治疗能够联合CG MN而不诱发局部组织炎症,采用温和热疗的方式能够将对正常组织的副作用减少至较低水平。
由图15可知,与对照组相比,肥胖模型组TNF-α、MCP-1含量均极显著升高,同时MCP-1具有招募巨噬细胞的能力,进一步增加炎症因子和趋化因子的释放,形成恶性循环,而最终导致肥胖小鼠体内呈现全身性慢性低度炎症;与肥胖模型组相比,CG@BP MN(+)组TNF-α、MCP-1含量均极显著降低,因此表明CG@BP MN(+)可调节肥胖小鼠的慢性炎症水平,恢复肥胖小鼠炎症因子至正常水平,具有最佳的体内抗炎恢复效果。
实施例9
CG@BP MN递送系统的体内安全性评价
选取各治疗组小鼠心、肝、脾、肺、肾,用4%多聚甲醛充分固定,石蜡包埋后进行H&E染色。结果如图16(scale bar=100μm)所示,与正常组相比,CG@BP MN(+)给药组的各组织未发现病变、坏死、形态异常等病理变化,其组织学形态正常,说明CG@BP MN(+)递药系统具有良好的体内安全性。
Claims (10)
1.一种基于温和光热联合天然药物的抗肥胖透皮递药系统,其特征在于,所述透皮递药系统以可溶性微针作为载体,载体上负载光热剂黑磷纳米片,载体内装载双药纳米粒而构成;所述双药纳米粒为姜黄素和甘草次酸无载体自组装构建而得。
2.根据权利要求1所述的透皮递药系统,其特征在于,所述双药纳米粒,是取姜黄素和甘草次酸分散于溶剂中制得混合溶液,将混合溶液滴入水中进行超声,避光透析而得。
3.根据权利要求2所述的透皮递药系统,其特征在于,所述混合溶液中,姜黄素和甘草次酸的质量比为4:1~1:4,混合溶液与水的体积比为1:5~1:50。
4.根据权利要求1所述的透皮递药系统,其特征在于,所述黑磷纳米片的粒径为100~200nm。
5.根据权利要求1所述的透皮递药系统,其特征在于,所述可溶性微针中黑磷纳米片和双药纳米粒的质量比为10:1~1:5。
6.一种权利要求1所述抗肥胖透皮递药系统的制备方法,其特征在于,包括以下步骤:
(1)取姜黄素和甘草次酸分散于溶剂中制得混合溶液,将混合溶液滴入水中进行超声,避光透析即得双药纳米粒CG NPs;
(2)取黑磷晶体,研磨,分散于丙酮溶液,超声剥离,离心沉淀,混悬后即得黑磷纳米片BP;
(3)取双药纳米粒与黑磷纳米片溶于聚乙烯吡咯烷酮溶液中,注入微针模具真空干燥,加入聚乙烯醇溶液作为基底层,干燥脱模取出即得抗肥胖透皮递药系统CG@BP MN。
7.根据权利要求6所述的制备方法,其特征在于,步骤(1)中,所述溶剂为无水甲醇、无水乙醇、二甲基亚砜、二氯甲烷、乙酸乙酯、乙腈的任意一种或多种,所述超声功率为50~300W,时间为1~10min。
8.根据权利要求6所述的制备方法,其特征在于,步骤(2)中,所述超声剥离,超声功率为80~200W,时间为2~24h;步骤(3)中,所述干燥,时间为8~24h。
9.一种权利要求1所述的抗肥胖透皮递药系统在制备肥胖治疗药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述药物降低脂肪组织含量和/或改善脂肪组织炎症。
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