CN118078762A - 一种治疗白癜风的双层载药微针及其制备方法和应用 - Google Patents
一种治疗白癜风的双层载药微针及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及医用生物材料技术领域,特别是涉及一种治疗白癜风的双层载药微针及其制备方法和应用。所述微针具有双层结构针尖,第一层为较高机械强度的水凝胶外壳,可以将治疗白癜风的药物递送至更深处位置,促进黑色素的形成;第二层水凝胶内核可把纳米粒子递送至浅表层,加速表皮着色,双重、梯度和长效释放药物,不仅降低了给药频次,更极大增强白癜风的治疗效果。
Description
技术领域
本发明涉及医用生物材料技术领域,特别是涉及一种治疗白癜风的双层载药微针及其制备方法和应用。
背景技术
白癜风作为一种获得性自身免疫性色素脱失性疾病,主要表现为表皮黑素细胞缺失引起的皮肤或(和)毛发的斑片状色素脱失,流行病学显示其患病率为0.5%~2%。这种慢性皮肤病的治疗困难且易复发,不仅给患者带来了严重的生理及心理影响,还造成了极大的经济负担。目前,临床多种方法虽被证明对白癜风治疗有效,但刺激性强,易出现毛细血管扩张、光毒性等不良反应。近年来,针刺疗法在白癜风的治疗应用逐渐增多,其中微针治疗因其操作简单、安全有效等特点,已成为皮肤科研究热点。
微针是一种微凸起阵列组成的微创装置,能够穿透角质层到达表皮及真皮层,具有安全、无痛、微创、自我给药及便捷等优点。作为一种新型微针,水凝胶微针因其优良的性能在医学领域备受关注。水凝胶微针具有良好的生物相容性及力学性能,在皮肤作用之后可以被完整取下而不会在体内残留聚合物;其特有的溶胀性可以实现人体检测物微创提取及药物缓释。
微针疗法在皮肤病治疗中是一种较新颖的方法,已被用于色素异常性疾病、痤疮瘢痕和面部年轻化等的治疗。既往研究表明,微针疗法是白癜风的有效治疗方式之一。然而,由于白癜风具有易反复、易扩散、难根治的特点,载药单一的微针治疗周期长、复色率有限。如何最大程度地利用微针疗法的优势来治疗白癜风值得探索。因此,开发具有双层载药针尖结构的微针以应用于白癜风的治疗具有重要意义,而目前相关研究报道较少。
发明内容
针对上述问题,本发明提供一种用于治疗白癜风的水凝胶载药微针,该微针具有双层结构针尖,第一层为较高机械强度的水凝胶外壳,可以将治疗白癜风的药物递送至更深处位置,促进黑色素的形成;第二层水凝胶内核可把纳米粒子递送至浅表层,加速表皮着色,双重、梯度和长效释放药物,不仅降低了给药频次,更极大增强白癜风的治疗效果。
本发明的一方面提供了一种治疗白癜风的双层载药微针,包括针尖及位于所述针尖底部的基底;所述针尖为双层结构针尖,包括外壳和内核;所述外壳包括水凝胶成分,光引发剂和药物活性成分,所述水凝胶成分选自以下原料中的至少1种:甲基丙烯酰化明胶(GelMA)或甲基丙烯酸透明质酸(HAMA),所述药物活性成分选自以下原料中的至少1种:碱性成纤维细胞生长因子(bFGF)或酪氨酸酶(TYR);所述内核原料包括第一水溶性材料和多巴胺纳米粒子(PDA)。
该微针采用双层针尖结构,第一层外壳由具有良好力学性能和生物相容性的水凝胶形成,能够穿透皮肤角质层将bFGF或TYR药物递送至更深处位置,作用于黑素细胞,促进黑色素的形成;第二层内核把PDA递送至浅表层,加速表皮着色,双重长效给药,有效增强白癜风的治疗效果。
优选地,所述水凝胶成分由GelMA和HAMA组成,GelMA与HAMA的质量比为5-15:2-10。进一步优选地为10:2-10,机械强度好,利于穿刺。
优选地,所述第一水溶性材料为透明质酸(HA)或葡聚糖;进一步优选地,为HA。
优选地,所述多巴胺纳米粒子(PDA)为包裹细胞膜的多巴胺纳米粒子(M@PDA),所述细胞膜为人永生化角质形成细胞(HaCaT)膜,进一步提高治疗效果。
优选地,所述基底包括第二水溶性材料,所述第二水溶性材料选自以下原料中的至少1种:葡聚糖、HA或聚乙烯醇(PVA)。基底采用水溶性材料制成,微针刺破皮肤后吸水,使针尖和基底自动分离,针尖留存在皮肤内使药物成分释放。
光引发剂可以选用常用的引发剂,如苯基-2,4,6-三甲基苯甲酰基膦酸锂或Irgacure 2959等。
本发明的另一方面,还提供了上述所述微针的制备方法,包括以下步骤:
S1制备针尖:
S11将水凝胶成分,光引发剂和药物活性成分在水中溶解形成外壳溶液,加入到模具中,真空除泡,光交联固化形成水凝胶外壳,
S12将第一水溶性材料和多巴胺纳米粒子在水中溶解形成内核溶液,加入到S11形成的水凝胶外壳中,真空除泡,干燥,形成针尖内核,得到双层结构针尖;
S2制备微针:
将含有第二水溶性材料的基底溶液,加入步骤S1得到的具有双层结构针尖的微针模具中,干燥,形成基底,脱模,得到微针。
外壳溶液中,水凝胶的成分,影响成型后的微针强度和药物释放效果;光引发剂用量基于引发剂类型调整,bFGF或TYR的浓度影响给药效果,需要配合水凝胶成分进行调整;优选地,所述外壳溶液中,包括GelMA5-15wt%,HAMA 2-10wt%,光引发剂0.05-0.25wt%,bFGF浓度为100~300ng/ml,TYR浓度为100~300U/ml。强度好,且药物持续释放效果较好。
优选地,所述内核溶液中,包括2-4wt%第一水溶性材料,多巴胺纳米粒子浓度为50-150μg/ml,浅表层递送效果好,表皮着色快。
优选地,所述基底溶液中,包括2-4wt%第二水溶性材料,所述第二水溶性材料选自以下原料中的至少1种:葡聚糖、HA或PVA。
优选地,所述GelMA通过以下方法制备得到:明胶水溶液中加入甲基丙烯酸酐,50℃搅拌反应,透析,冻干,得到GelMA;优选地,所述明胶水溶液的浓度为10g/ml,所述明胶合甲基丙烯酸酐的质量比为5:3。
优选地,所述HAMA通过以下方法制备得到:透明质酸钠水溶液中加入甲基丙烯酸酐,pH在8~9之间,4℃搅拌反应,透析,冻干,得到HAMA;优选地,所述透明质酸钠水溶液的浓度为0.01g/ml,所述透明质酸钠和甲基丙烯酸酐的质量比为5:4。
优选地,所述多巴胺纳米粒子通过以下方法制备得到:将多巴胺盐酸盐和三羟甲基氨基甲烷缓冲液混合,搅拌,透析,冻干,得到多巴胺纳米粒子。得到的例子粒径均一,递送效果好。三羟甲基氨基甲烷缓冲液的PH值通常为7-9之间,优选地为8.5;所述多巴胺盐酸盐在缓冲液中的浓度为1×10-4~1×10-3g/ml;优选地,为5×10-4g/ml。
本发明的再一方面,还提供了一种透皮吸收药物制剂,包括所述的微针。
与现有技术相比,本发明具有以下有益效果:
本发明的微针具有优异的机械强度,适当的体内溶胀率和降解率以及良好的生物相容性。该微针采用双层针尖结构,第一层水凝胶外壳采用机械强度优异和良好生物相容性的GelMA/HAMA水凝胶形成,能够刺破角质层,将bFGF或酪氨酸酶等药物递送至更深处位置,bFGF可以抑制炎症反应,与酪氨酸酶双重作用于黑素细胞,促进黑色素的形成,增加色素沉着,有效改善皮肤脱色情况;第二层生物相容性良好的水凝胶内核把PDA递送至浅表层,进一步加速表皮着色;内外层载药结构双重长效给药,可以有效增强白癜风的治疗效果。
附图说明
图1为效果验证例1GelMA、Gelatin核磁测试结果;
图2为效果验证例1HAMA、HA核磁测试结果;
图3为效果验证例2PDA TEM图;
图4为效果验证例2M@PDA TEM图;
图5为实施例5制备的微针整体外观图;
图6为效果验证例3微针荧光图;
图7为效果验证例3微针SEM图;
图8为效果验证例4水凝胶的时间扫描曲线;
图9为效果验证例4水凝胶的频率扫描曲线;
图10为效果验证例5水凝胶溶胀曲线;
图11为效果验证例6水凝胶降解曲线;
图12为效果验证例7水凝胶的应力-应变曲线;
图13为效果验证例8微针的单针抗压强度曲线;
图14为效果验证例9不同浓度的PDA和M@PDA的细胞毒性检测结果;
图15为效果验证例10不同浓度的bFGF的细胞毒性检测结果;
图16为效果验证例10不同浓度的酪氨酸酶的细胞毒性检测结果;
图17为效果验证例11不同组分的GelMA/HAMA水凝胶的细胞毒性检测结果;
图18为效果验证例12不同组分的GelMA/HAMA水凝胶处理的HaCaT细胞体外划痕实验结果;
图19为效果验证例12不同组分的GelMA/HAMA水凝胶处理的HaCaT细胞划痕面积变化趋势图;
图20为效果验证例13不同组分的GelMA/HAMA水凝胶处理的ROS损伤HaCaT细胞的活死荧光染色结果;
图21为效果验证例13不同组分的GelMA/HAMA水凝胶处理的ROS损伤HaCaT细胞的CCK8活力定量检测结果;
图22为效果验证例13不同组分的GelMA/HAMA水凝胶处理的ROS损伤HaCaT细胞的ROS强度检测结果。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
本发明的描述中,除非另有明确的限定,加热、清洗、称取、冷冻等词语应做广义理解,所属技术领域技术人员可以结合技术方案的具体内容合理确定上述词语在本发明中的具体含义。
本发明的描述中,参考术语“一些实施例”、“示例”等的描述意指结合该实施例或示例描述的具体方法、材料包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体方法、材料可以在任何的一个或多个实施例或示例中以合适的方式结合。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
本发明实施例所用试剂、材料、设备如无特殊说明,均为市售来源;试验方法如无特殊说明,均为本领域的常规试验方法。
实施例
实施例1甲基丙烯酰化明胶GelMA的制备
将10.0g明胶(Gelatin)溶解在100mL的去离子水中,在50℃搅拌溶解。缓慢滴加6mL甲基丙烯酸酐(MA),在50℃搅拌5小时。最终产物GelMA用去离子水透析(MW:12000-14000Da)3天,8000rpm离心10分钟去除沉淀,收集上清液,冻干并储存在-20℃直至使用。
实施例2甲基丙烯酸透明质酸HAMA的制备
将1.0g透明质酸钠(HA)溶解于100ml蒸馏水中,然后加入0.8mL甲基丙烯酸酐(MA)。加入适量NaOH溶液(5M),保持pH在8~9之间,在4℃搅拌24h。最终产物(HAMA)用去离子水透析(MW:12000-14000Da)2天,8000rpm离心10分钟去除沉淀,收集上清液,冻干并储存在-20℃直至使用。
实施例3、聚多巴胺纳米粒子PDA的制备
将0.02g多巴胺盐酸盐加入到40mL三羟甲基氨基甲烷(Tris,0.05M,pH 8.5)缓冲液中,在室温、空气中搅拌6小时。最终产物PDA用去离子水透析(MW:1200Da)3天,冻干并储存在-20℃直至使用。
实施例4、M@PDA的制备
根据膜蛋白提取试剂盒使用说明提取角质细胞(HaCaT)膜。当HaCaT细胞培养至约为2000-5000万时,用细胞刮子收集细胞在冷PBS(10mM,pH 7.4)溶液中,在1500rpm下洗涤5min两次。将1mL添加了10ul PMSF的膜蛋白抽提试剂A加入细胞中,充分悬浮细胞,在冰浴中放置10-15min。将样品在液氮和室温反复冻融五次。4℃,2000rpm离心10min,收集上清液。4℃,12500rpm离心30min沉淀细胞膜碎片,吸走上清液。将细胞膜碎片冻干并储存于-80℃。
将1.0mg PDA纳米粒子溶于1.0mL PBS溶液中,超声波细胞粉碎机超声,得到1.0mL纳米材料溶液。将1.0mg细胞膜碎片溶于1.0mL纳米材料溶液中,超声并吹打5min,得到细胞膜/纳米材料混合液。将细胞膜/纳米材料混合液依次通过孔径为800nm和400nm的聚碳酸酯多孔膜,分别挤出10~13次来制备细胞膜包裹纳米粒子。将挤出的混合物溶液离心(4℃,13000rpm,10min),去除游离的细胞膜或纳米粒子,沉淀为制备的角质细胞膜包裹聚多巴胺纳米粒子(M@PDA)。
实施例5、GelMA/HAMA bFGF/TYR//HA/PDA凝胶微针的制备
一种治疗白癜风的双层载药微针,其采用如下方法制备,包括:
5.1制备针尖:
5.1.1将实施例1和2制备得到的GelMA和HAMA溶解在含有0.1wt%LAP的去离子水溶液中,并添加bFGF(200ng/ml)、酪氨酸酶(TYR,200U/ml),分别得到10% GelMA/2%HAMA、10% GelMA/5% HAMA、10% GelMA/10% HAMA的外壳凝胶溶液,加入到PDMS微针模具中,并将模具转移到亚克力真空脱泡箱中;将真空脱泡箱内的真空度调至-100kPa,除泡,使模具充满混合水凝胶溶液;在微针尖端完全填充后,去除多余的溶液并将微针尖端经405nm,30W/cm2紫外光照射120s交联以形成水凝胶外壳;
5.1.2将HA溶解在去离子水中,并添加实施例3的PDA(100μg/ml),得到3wt%HA和PDA内核溶液,加入到5.1.1形成的水凝胶外壳中,真空、除泡,在微针尖端完全填充后,去除多余的溶液,晾干形成水凝胶内核,得到双层结构针尖;
5.2制备微针:
把含有3wt%HA的基底水溶液添加到模具的基底中,干燥72小时,分别得到10%GelMA/2%HAMA//3%HA、10%GelMA/5% HAMA//3%HA、10% GelMA/10%HAMA//3%HA双层针尖水凝胶微针样品。
实施例6、GelMA/HAMA bFGF/TYR//HA/M@PDA凝胶微针的制备
一种治疗白癜风的双层载药微针,其采用如下方法制备,包括:
6.1制备针尖:
6.1.1将实施例1和2制备得到的GelMA和HAMA溶解在含有0.1wt%LAP的去离子水溶液中,并添加bFGF(200ng/ml)、酪氨酸酶(TYR,200U/ml),得到10% GelMA/10% HAMA的外壳凝胶溶液,加入到PDMS微针模具中,并将模具转移到亚克力真空脱泡箱中;将真空脱泡箱内的真空度调至-100kPa,除泡,使模具充满混合水凝胶溶液;在微针尖端完全填充后,去除多余的溶液并将微针尖端经405nm,30W/cm2紫外光照射120s交联以形成水凝胶外壳;
6.1.2将HA溶解在去离子水中,并添加实施例4的M@PDA(100μg/ml),得到3wt%HA和M@PDA内核溶液,继续加入到6.1.1形成的水凝胶外壳中,真空、除泡,在微针尖端完全填充后,去除多余的溶液,晾干形成水凝胶内核,得到双层结构针尖;
6.2制备微针:
把含有3wt%HA的基底水溶液添加到模具的基底中,干燥72小时,得到微针。
效果验证例
效果验证例1、核磁共振氢谱(1H NMR)
(1)样品:实施例1-2中的Gelatin、GelMA、HA、HAMA。
(2)测试方法:分别称取3-5mg样品溶解于适量的氘代试剂中(重水D2O),然后装入干净的核磁管中,利用核磁共振光谱仪在室温条件下进行核磁结构测定,并采用MestReNova软件进行图谱分析。
如图1、图2为Gelatin、GelMA、HA、HAMA核磁测试结果,从图中可以看到,与Gelatin相比,GelMA在δ=5.65ppm及5.33ppm左右出现明显的两个信号峰,表明实施例中的Gelatin改性成功;与HA相比,HAMA在δ=6.10ppm及5.71ppm左右处出现两个新的信号峰,而在δ=1.86ppm左右出现一个分叉的峰型,分别是甲基丙烯酸酯基上双氢键的核磁峰与甲基氢的核磁峰;δ=1.96ppm左右处的核磁峰则是HA侧链上甲基氢的峰,这证明实施例中的HA已被成功甲基丙烯酸化。
效果验证例2、透射电镜(TEM)
(1)样品:实施例3、4制备得到的PDA、M@PDA样品。
(2)测试方法:将样品均匀分散于水溶液中后进行TEM检测分析。
如图3TEM图显示PDA纳米颗粒的形状为球形,平均直径为100nm左右。用细胞膜修饰PDA后,TEM图像4也显示出M@PDA具有核壳结构,表明PDA、M@PDA制备成功。
效果验证例3、微针外观
(1)样品:实施例5、实施例6的微针样品。
(2)测试方法:使用荧光显微镜或扫描电子显微镜(SEM)观察、拍摄其外观。
如图5为实施例5所制备微针外观图,从图中可以看到,整片微针结构完整,无针体断裂、弯曲。
图6-7为实施例6的微针样品图,如图6微针荧光图所示,微针双层结构明显,并且荧光在微针中分布均匀,说明了微针具备载药能力。图7微针SEM图可以更清晰观察到锐利针尖的金字塔形结构,具有金字塔形状和更锐利针尖的水凝胶微针可以表现出更良好的皮肤插入效果。
效果验证例4、水凝胶流变性能测试
(1)样品:实施例5中的10% GelMA/2% HAMA、10% GelMA/5% HAMA、10%GelMA/10%HAMA外壳水凝胶溶液
(2)测试方法:使用24孔板,每孔加400μl的水凝胶溶液,紫外光交联3min成胶。用直径为25mm的不锈钢平行板转头进行流变学测量。G'表征样品的弹性模量,G"表征样品的粘性模量。于常温下从0.1到10rad/s的进行动态应变扫描,确定水凝胶的线性粘弹性范围,记录储能模量(G')和损耗模量(G”)变化曲线。
如图8、9水凝胶的时间、频率扫描曲线所示,三组水凝胶在时间及频率变化下均表现为G’>G”的状态,说明水凝胶能够在适宜的条件下以凝胶的形式稳定存在。当HAMA质量浓度从2%增加到10%时,水凝胶的硬度、线性粘弹区均整体明显增加。
效果验证例5、水凝胶体外溶胀测试
(1)样品:实施例5中5.1.1得到的10% GelMA/2% HAMA、10% GelMA/5% HAMA、10% GelMA/10% HAMA的水凝胶外壳
(2)测试方法:水凝胶外壳的初始重量记为M0。然后将其完全浸入PBS缓冲液中。取出水凝胶,用称量纸擦去多余水分,记录不同时间溶胀后的重量为Mt,溶胀比按下式计算:
溶胀率=Mt/M0×100%
水凝胶适当的溶胀能力可以使其储存的药物释放到周围环境中,如图10所示,三组不同比例的水凝胶外壳在PH=7.4的PBS溶液中2小时后就基本上溶胀饱和,10% GelMA/2%HAMA组水凝胶溶胀率为69.7%左右,在增加HAMA的浓度后,水凝胶溶胀率逐渐升高,其中HAMA浓度为5%时水凝胶溶胀率明显升高,为185.9%,浓度为10%时水凝胶溶胀率虽达到220.8%,但溶胀率升高速度减缓。
效果验证例6、水凝胶体外降解测试
(1)样品:实施例5中5.1.1得到的交联后的10% GelMA/2% HAMA、10% GelMA/5%HAMA、10% GelMA/10% HAMA的水凝胶外壳
(2)测试方法:冻干后水凝胶外壳样品的初始重量记为W0。然后将水凝胶完全浸入PBS缓冲液中,置于37℃恒温摇床。每3天更换一次PBS缓冲液。取出水凝胶,弃去多余的降解液,冻干,称重,并记录水凝胶的重量,记为Wt。降解率按下式计算:
如图11水凝胶降解曲线所示,三组水凝胶在PBS溶液中均可以降解,虽然由于只存在水解,导致降解速率较慢,但均可表明GelMA/HAMA水凝胶具有良好的生物降解性。
效果验证例7、水凝胶压缩测试
(1)样品:实施例5中10% GelMA/2% HAMA、10% GelMA/5% HAMA、10% GelMA/10%HAMA外壳水凝胶溶液,在405nm,30W/cm2紫外光照射下交联,制备成圆柱形样品( 高度=6mm)。
(2)测试方法:以恒定速率(0.05mm/s)压缩样品直至断裂。
如图12水凝胶的应力-应变曲线所示,随着HAMA的浓度增大,水凝胶的杨氏模量增加速度也有所增加,当HAMA浓度为10%时,水凝胶压缩到63%左右时便破裂了,此时的杨氏模量为412kPa左右,含5%HAMA组水凝胶在应变为65%左右时出现破裂,此时的杨氏模量为552kPa左右,抗压强度最高,而含2%HAMA组水凝胶在应变为70%左右时才出现破裂,相较于其他两组表现出更好的韧性,此时的杨氏模量为388kPa左右。
效果验证例8、微针针尖压缩强度测试
(1)样品:实施例1得到的10%GelMA/2%HAMA//3%HA、10%GelMA/5% HAMA//3%HA、10% GelMA/10% HAMA//3%HA双层针尖水凝胶微针
(2)测试方法:将水凝胶微针放置在样品台上,尖端向上。然后通过万能试验机以0.05mm/s的恒定速率按压尖端。
如图13微针的单针抗压强度曲线所示,单针在400μm处的压力随HAMA浓度的增加而增大,水凝胶微针的单针强度越高,则更容易刺穿角质层。
效果验证例9、多巴胺细胞毒性实验
(1)样品:PDA和M@PDA溶液(浓度梯度:20、40、60、80、100、200、400μg/mL)
(2)测试方法:
人HaCaT细胞消化重悬后,稀释细胞,并滴加细胞悬浮液于细胞计数板上计算细胞数量。根据10000个/孔的细胞量,加入至96孔板中,5%CO2,37℃培养过夜。24h后,将PDA和M@PDA溶液按100μl/孔加入到96孔板中,每个材料做6个平行孔。5%CO2,37℃培养。培养24h±2h后,取出细胞培养板,将细胞培养板中培养基吸弃,用PBS洗涤,拍照。加入已按1:10比例与全培稀释的CCK8溶液,100μl/孔。同时在无细胞孔内加入100ul混合液,作为阴性对照孔,与实验组别同数量。5%CO2,37℃培养0.5-1h。取出细胞培养板,于酶标仪中,450nm波长下测定OD值。
如图14PDA和M@PDA的细胞毒性检测结果所示,不同浓度的PDA和M@PDA对HaCaT细胞增殖影响较小,细胞毒性较低,适用于制备微针。
效果验证例10、生长因子和酪氨酸酶细胞毒性实验
(1)样品:bFGF溶液(浓度梯度:1/10/20/40/100/200ng/ml)和TYR溶液(浓度梯度:5/20/40/60/80/100/200U/ml)
(2)测试方法:
人HaCaT消化重悬后,稀释细胞,并滴加细胞悬浮液于细胞计数板上计算细胞数量。根据10000个/孔的细胞量,加入至96孔板中,5%CO2,37℃培养过夜。24h后,将bFGF溶液和TYR溶液按100μl/孔加入到96孔板中,每个材料做6个平行孔。5%CO2,37℃培养。培养24h±2h后,取出细胞培养板,将细胞培养板中培养基吸弃,用PBS洗涤,拍照。加入已按1:10比例与全培稀释的CCK8溶液,100μl/孔。同时在无细胞孔内加入100ul混合液,作为阴性对照孔,与实验组别同数量。5%CO2,37℃培养0.5-1h。取出细胞培养板,于酶标仪中,450nm波长下测定OD值。
如图15、16不同浓度的bFGF和酪氨酸酶的细胞毒性检测结果所示,不同浓度的bFGF和酪氨酸酶均可维持HaCaT细胞更好活力,对细胞增殖有一定的改善作用,均可用于制备微针。
效果验证例11、微针水凝胶细胞毒性实验
测试样品:参照实施例5步骤,分别称重、配制GelMA/HAMA//HA水凝胶、bFGF/TYR/GelMA/HAMA//HA水凝胶、GelMA/HAMA//HA/M@PDA水凝胶和bFGF/TYR/GelMA/HAMA//HA/M@PDA水凝胶
测试方法:人HaCaT细胞消化重悬后,稀释细胞,并滴加细胞悬浮液于细胞计数板上计算细胞数量,根据10000个/孔的细胞量,加入至96孔板中,5%CO2,37℃培养过夜。将水凝胶样品以0.1g/ml比例用DMEM全培浸提,培养箱中浸提24±2h。24h后,将浸提液吸出,加入到96孔板中,每个材料做6个平行孔。分别在培养1、3天后,取出细胞培养板,将细胞培养板中培养基吸弃,用PBS洗涤,拍照。加入已按1:10比例与全培稀释的CCK8溶液,100μl/孔。同时在无细胞孔内加入100ul混合液,作为阴性对照孔,与实验组别同数量。5%CO2,37℃培养0.5-1h。取出细胞培养板,于酶标仪中,450nm波长下测定OD值。
如图17不同组分的GelMA/HAMA水凝胶的细胞毒性检测结果所示,GelMA/HAMA水凝胶细胞毒性低,均可维持细胞较好的活力,特别是添加bFGF和TYR可以有效促进HaCaT细胞的增殖,表明GelMA/HAMA水凝胶无细胞毒性,生物相容性良好。
效果验证例12、微针水凝胶HaCaT细胞体外划痕实验
(1)样品:同效果验证例11
(2)测试方法:将HaCaT细胞接种于48孔板上,培养24h至形成融合层。用移液管尖端形成划痕。然后加入GelMA/HAMA//HA水凝胶、bFGF/TYR/GelMA/HAMA//HA水凝胶、GelMA/HAMA//HA/M@PDA水凝胶和bFGF/TYR/GelMA/HAMA//HA/M@PDA水凝胶浸提液。以DMEM高糖培养基培养的HaCaT细胞为对照组。采集不同时间的划痕图像,拍照的时间点为:0h、6h、24h、48h。利用ImageJ软件计算划痕面积。
如图18、19HaCaT细胞体外划痕实验测试结果所示,bFGF/TYR/GelMA/HAMA//HA/M@PDA水凝胶培养的细胞向划痕中心迁移的速度明显快于对照组(图18),说明bFGF/TYR有一定的促进细胞迁移的作用,这可能与其可以促进细胞增殖有关。
效果验证例13、微针水凝胶体外功能评价实验
(1)样品:同效果验证例11
(2)测试方法:使用1600μmol/L H2O2(过氧化氢)溶液诱导ROS环境对HaCaT细胞进行损伤,损伤后细胞活力为50%,建立ROS损伤模型。用GelMA/HAMA//HA水凝胶、bFGF/TYR/GelMA/HAMA//HA水凝胶、GelMA/HAMA//HA/M@PDA水凝胶和
bFGF/TYR/GelMA/HAMA//HA/M@PDA水凝胶浸提液进一步培养损伤细胞24h后:
(a)采用活死荧光染色,以及CCK-8试剂盒检测细胞活力,评价其对ROS损伤后HaCaT细胞的保护作用;
(b)用DCFH-DA活性氧ROS荧光探针检测ROS荧光强度,评价ROS清除效果。
bFGF可以抑制炎症反应和刺激色素细胞,而TYR促进黑色素的形成和M@PDA增强角质形成细胞的摄取以增加色素沉着,具有减轻炎症和保护细胞的作用。
经水凝胶浸提液培养的HaCaT细胞的活死荧光染色结果如图20所示,与GelMA/HAMA//HA水凝胶浸提液共培养24h后,相比对照组有更多细胞死亡。而经过bFGF、TYR及M@PDA处理后,死细胞数量明显减少,细胞整体表现出更好的活力。
如图21微针水凝胶浸提液处理的ROS损伤HaCaT细胞的活力定量统计结果所示,H2O2处理后的细胞用水凝胶浸提液培养后,含有bFGF、TYR及M@PDA组的细胞存活率明显高于其它组。
在白癜风的各种致病机制中,一个重要的机制是高ROS环境引起的细胞损伤,进而影响表皮色素沉着。如图22所示,经ROS环境损伤的细胞,与水凝胶浸提液共培养后,含有bFGF、TYR及M@PDA组的细胞中ROS生成明显减少,证实bFGF、TYR及M@PDA可以共同起到较好的ROS保护能力,所制备水凝胶微针具有良好的白癜风治疗效果。
以上实施例和效果验证例,说明了本发明方案的微针水凝胶体系具有优异的机械性能,适当的体内溶胀率和降解率以及良好的生物相容性。本发明的微针采用双层针尖结构,第一层水凝胶外壳采用机械强度优异能够刺破角质层,将bFGF或酪氨酸酶药物递送至更深处位置,bFGF可以抑制炎症反应,与酪氨酸酶双重作用于黑素细胞,促进黑色素的形成,增加色素沉着,有效改善皮肤脱色情况;第二层的水凝胶内核把PDA递送至浅表层,进一步加速表皮着色;内外层载药结构双重长效给药,可以有效增强白癜风的治疗效果。
本发明的微针可用于白癜风的透皮吸收药物制剂。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种治疗白癜风的双层载药微针,其特征在于,包括针尖及位于所述针尖底部的基底;所述针尖为双层结构针尖,包括外壳和内核;
所述外壳包括水凝胶成分,光引发剂和药物活性成分,
所述水凝胶成分选自以下原料中的至少1种:GelMA或HAMA,
所述药物活性成分选自以下原料中的至少1种:bFGF或TYR;
所述内核包括第一水溶性材料和多巴胺纳米粒子。
2.根据权利要求1所述的微针,其特征在于,所述水凝胶成分由GelMA和HAMA组成,GelMA与HAMA的质量比为5-15:2-10。
3.根据权利要求1所述的微针,其特征在于,所述第一水溶性材料为HA或葡聚糖。
4.根据权利要求1所述的微针,其特征在于,所述多巴胺纳米粒子为包裹细胞膜的多巴胺纳米粒子,所述细胞膜为人永生化角质形成细胞膜。
5.根据权利要求1所述的微针,其特征在于,所述基底包括第二水溶性材料,所述第二水溶性材料选自以下原料中的至少1种:葡聚糖、HA或PVA。
6.如权利要求1所述微针的制备方法,其特征在于,包括以下步骤:
S1制备针尖:
S11将水凝胶成分,光引发剂和药物活性成分在水中溶解形成外壳溶液,加入到模具中,真空除泡,光交联固化形成水凝胶外壳,
S12将第一水溶性材料和多巴胺纳米粒子在水中溶解形成内核溶液,加入到S11形成的水凝胶外壳中,真空除泡,干燥,形成水凝胶内核,得到双层结构针尖;
S2制备微针:
将基底溶液,加入步骤S1得到的具有双层结构针尖的微针模具中,干燥,形成基底,脱模,得到微针。
7.根据权利要求6所述的制备方法,其特征在于,
所述外壳溶液中,包括GelMA5-15wt%,HAMA2-10wt%,光引发剂0.05-0.25wt%,bFGF浓度为100~300ng/ml,TYR浓度为100~300U/ml;
所述内核溶液中,包括2-4wt%第一水溶性材料,多巴胺纳米粒子浓度为50-150μg/ml;
所述基底溶液中,包括2-4wt%第二水溶性材料,所述第二水溶性材料选自以下原料中的至少1种:葡聚糖、HA或PVA。
8.根据权利要求7所述的制备方法,其特征在于,
所述GelMA通过以下方法制备得到:明胶水溶液中加入甲基丙烯酸酐,50℃搅拌反应,透析,冻干,得到GelMA;
所述HAMA通过以下方法制备得到:透明质酸钠水溶液中加入甲基丙烯酸酐,pH在8~9之间,4℃搅拌反应,透析,冻干,得到HAMA。
9.根据权利要求7所述的制备方法,其特征在于,所述多巴胺纳米粒子通过以下方法制备得到:将多巴胺盐酸盐和三羟甲基氨基甲烷缓冲液混合,搅拌,透析,冻干,得到多巴胺纳米粒子。
10.一种透皮吸收药物制剂,其特征在于,包括权利要求1-5中任一项所述的微针,及权利要求6-9中任一项所述的制备方法制备得到的微针。
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