CN114191376B - 一种用于治疗阿尔兹海默症的微针贴片及其制备方法 - Google Patents
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Abstract
本发明公开了一种用于治疗阿尔兹海默症的微针贴片及其制备方法，属于生物医药技术领域。所述微针贴片由微针阵列和背衬层组成，其中：所述微针阵列由活性药物和基质制成，活性药物由极化药物与抗炎药物组成；所述极化药物选自尼古丁、二氢硫辛酸、强啡肽、红景天苷或氧化铈纳米粒；所述抗炎药物选自白藜芦醇、MCC950、CY‑09、原花青素B2、紫菀酮或麝香酮。本发明将两种药物联合使用制成微针贴片，具有协同增效的效果；同时制备的微针贴片具备良好的外观、硬度及穿刺能力，释放出治疗水平的药物。该方法同时递送不同性质的药物简单方便，顺应性高，临床用于治疗阿尔兹海默症具有十分优良的前景。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种用于治疗阿尔兹海默症的微针贴片及其制备方法。
背景技术
阿尔兹海默症（Alzheimer‘s disease, AD）是一种起病隐匿、进行性发展的中枢神经系统疾病，属于痴呆症形式的一种。痴呆症多发于65岁以上人群，预计到2050年，全球患痴呆症的人数将增加至1.52亿。AD的发病机制非常复杂，目前针对AD的机制假说主要分为以下四个方面：（1）基于Aβ淀粉样斑块的假说。Aβ肽是正常组织中的淀粉样前体蛋白（APP）经过β分泌酶和γ分泌酶水解得到，Aβ分子能够自发聚合，从而在细胞外自发组成寡聚体、纤维体以及斑块。淀粉样蛋白级联假说表明，毒性形式的Aβ能够作用于脑内细胞，通过诱导细胞凋亡，释放炎症因子IL-1β、TNF-α和IFN-γ等物质进一步促进邻近细胞的炎性化。此外还能进一步促进tau蛋白的磷酸化，造成神经元纤维缠结（NFT）。（2）基于tau蛋白磷酸化假说。tau在大脑中促进微管的组装、维持神经元的结构稳定，是一种含量最高的微管蛋白。但在AD病理状态下，tau蛋白发生化学变化，导致脱离细胞骨架并相互粘附，造成NFT。（3）基于神经元突触功能失调和神经元保护假说。目前FDA批准用于治疗AD的药物主要为乙酰胆碱酯酶抑制剂和N-甲基-D-天冬氨酸（NMDA）受体拮抗剂。而这些药物只能在一定程度上缓解AD的症状，改善记忆，无法彻底治愈AD，也无法预防AD。甚至经典的药物盐酸美金刚，在患者服用期间会出现如幻觉、头晕头痛等较为严重的不良反应。随着进一步的研究，对AD的认识逐渐加深，2021年新批准的Aβ单抗药Aducanumab上市也颇有争议，仍然需要后续的临床数据来支撑。因此对于AD的有效治疗研究十分重要。（4）基于长期慢性炎症反应造成脑损伤的免疫炎性假说。早期研究指出炎症引发的炎症反应导致突触的缺失，而突触是形成记忆的关键，这似乎会增加患AD的风险。同时Ji-Yeun Hur等人首次直接证明了炎症因子通过干扰素诱导的跨膜蛋白3（interferon-induced transmembrane protein 3，IFITM3蛋白），一种γ分泌酶调节蛋白，与神经元和星形胶质细胞的γ分泌酶结合，并上调其活性，促进了β淀粉样蛋白的产生。临床试验的结果进一步表明一些老年人大脑中存在淀粉样斑块，但认知正常，经检测该人群血液中炎症因子水平也高于AD患者。综上所述，AD的发病机制十分复杂，似乎和炎症具有更明显的相关性。与种种因素息息相关的则是脑内的一群免疫细胞小胶质细胞，和炎症尤其相关。正常生理状态下小胶质细胞通过与神经元交流维持其稳态；但是在病理状态下，一些错误蛋白的出现，促使小胶质细胞激活，并处于炎性的M1状态，使脑内处于炎性环境，造成微环境的失调，进而导致神经元营养不足并发生损伤，同时毒性蛋白的刺激甚至加重了神经元的死亡。因此对神经炎症的缓解以及抑制小胶质细胞的过度激活将其逆转为M2型十分重要，在一定程度上将可能更好地利于AD的治疗。
考虑到AD病人的特殊性，因其记忆出现障碍，利用微针的形式给药简单方便，顺应性较好。与传统的经皮给药系统相比，微针能穿透角质层和表皮层到达真皮层，有效促进了机体对药物的吸收，达到药物的治疗水平。此外采取模具依赖型的制备方法，所需的设备简单，制备得到的微针则更依赖于所用材料的特性和制备的过程。因此本发明设计通过筛选微针的材料和制备方法制备得到外观整齐，稳定并具有较好穿刺性能的微针，用于治疗AD。以一种新型的疗法有效地调控了脑内小胶质细胞，抑制了神经炎症并使神经元细胞得到保护。
发明内容
针对现有口服用药的局限性，本发明的目的是提供一种用于治疗阿尔兹海默症的微针贴片，其系统能够调控小胶质细胞正常化从而期望有效治疗AD。
为了实现上述发明目的，本发明采用以下技术手段：
一种微针贴片，由微针阵列和背衬层组成，所述微针阵列由活性药物和基质制成，所述活性药物由极化药物与抗炎药物组成；
所述极化药物选自尼古丁、二氢硫辛酸、强啡肽、红景天苷或氧化铈纳米粒；
所述抗炎药物选自白藜芦醇、MCC950、CY-09、原花青素B2、紫菀酮或麝香酮。
进一步地，所述活性药物中极化药物与抗炎药物的质量比为1：0.5~50。
进一步地，所述基质由以下材料制成：PVP k30、PCL、VP单体、PLGA、透明质酸或PVA。
上述微针贴片的制备方法，包括以下步骤：
步骤1，将活性药物溶液和基质溶液混合后注入微针阵列模具中，制成微针阵列；
步骤2，将背衬层溶液注入微针阵列模具中填充，制成背衬，干燥后脱模，即可得到微针贴片。
进一步地，所述微针阵列的外观为圆锥形或三棱锥形，针尖长度为600-900μm，直径为300-500μm。
有益效果：本发明制备的微针贴片外观规整，具有优良的穿刺性能并对组织无损伤，同时能正确释放治疗水平的药物。对于载入的两种药物的联合使用，其中极化药物用于调控小胶质细胞表型使其逆转为保护表型，抗炎药物用于抑制小胶质细胞分泌促炎因子，改善细胞所处的炎性环境；既使得产生不利因素的细胞正常化，又改善了整个微环境，从而显著地增强对神经元细胞的保护，有效地改善阿尔兹海默症患者症状。该贴片制备工艺简单，病人使用顺应性好易于临床转化，具有巨大的临床应用价值。
附图说明
图1为实施例1微针贴片的形貌。
图2为实施例1微针贴片穿刺小鼠皮肤示意图。
图3为实施例1微针贴片释放药物的释放曲线。
图4为极化药物促进小胶质细胞极化为M2型趋势。
图5为两种药物联合使用抑制小胶质细胞促炎因子分泌结果。
图6为两种药物联合使用对小胶质细胞和神经元细胞保护作用。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
实施例1
治疗AD的微针的制备
将针尖材料PVP k30、极化药物1尼古丁、抗炎药物1白藜芦醇用无水乙醇配制成质量分数分别为10%、0.01%、0.01%的混合溶液，混合均匀后作为针尖注模液。移取200μL针尖注模液到三棱锥型PDMS模具中，于3500rpm转速下离心10min得到针尖层。自然干燥10min后，加入背衬层溶液，背衬层材料为PVP360：PVP40（w/w 1:1）并用无水乙醇配制成13%的混合溶液，移取250μL到上述模具中并进行真空干燥。2h后重复加入背衬材料溶液一次，继续干燥24h后脱模得到制备的微针贴片。
实施例2
治疗AD的微针的制备
将针尖材料PCL（分子量为80000）、极化药物1尼古丁、抗炎药物1白藜芦醇用无水乙醇配制成质量分数分别为10%、0.01%、0.01%的混合溶液，混合均匀后作为针尖注模液。移取200μL针尖注模液到圆锥型PDMS模具中，于3500rpm转速下离心10min得到针尖层。自然干燥10min后，加入背衬层溶液，背衬层材料为PVP360：PVP40（w/w 1:1）并用无水乙醇配制成13%的混合溶液，移取250μL到上述模具中并进行真空干燥。2h后重复加入背衬材料溶液一次，继续干燥24h后脱模得到制备的微针贴片。
实施例3
治疗AD的微针的制备
将针尖材料PVP k30、极化药物1尼古丁、抗炎药物2 MCC950用无水乙醇配制成质量分数分别为10%、0.01%、0.005%的混合溶液，混合均匀后作为针尖注模液。移取200μL针尖注模液到三棱锥型PDMS模具中，于3500rpm转速下离心10min得到针尖层。自然干燥10min后，加入背衬层溶液，背衬层材料为PVP360：PVP40（w/w 1:1）并用无水乙醇配制成13%的混合溶液，移取250μL到上述模具中并进行真空干燥。2h后重复加入背衬材料溶液一次，继续干燥24h后脱模得到制备的微针贴片。
实施例4
治疗AD的微针的制备
将交联材料N,N’-亚甲基双丙烯酰胺（MBA）、光引发剂材料2-羟基-4’-（2-羟乙氧基）-2- 甲基丙噻吩酮（Irgacure 2959）、极化药物1尼古丁、抗炎药物2 MCC950用形成针尖的材料溶液N-乙烯基吡咯烷酮（VP单体液体状）溶解完全，其中质量分数分别为1%、0.9%、0.03%、0.015%，作为针尖注模液。移取200μL针尖注模液到三棱锥型PDMS模具中，于3500rpm转速下离心10min，并于紫外光源下交联15min（功率300W，距离20cm）后得到针尖层。随后加入背衬层溶液，MBA、Irgacure 2959（w/w 1：0.9）用N-乙烯基吡咯烷酮配制成1.9%的混合液溶解完全，移取300μL注入到已形成针尖的PDMS模具中，紫外光源下交联15min（功率300W，距离20cm），形成背衬层，脱模得到制备的微针贴片。
下面对实施例1中制备的微针贴片进行考察，包括微针的外观形貌、对皮肤穿刺性能以及体外对治疗AD的药物的释放。并考察细胞水平上，实施例1中涉及的两种药物对小胶质细胞、神经元细胞的作用，包括对细胞极化的影响、抑制炎症作用并能保护小胶质细胞和神经元细胞做出可行性的验证，以保证在治疗AD时在动物层面上有更严谨的依据。
应用例1：微针贴片的可应用性考察，包括外观、可穿刺性及释放。
1. 外观形貌
制备的微针宏观形态利用手机相机拍摄，针尖更清晰的形态则在倒置荧光显微镜下拍摄如图1所示（A为手机相机拍摄图，B为4×10倒置荧光显微镜下拍摄图）。制备的微针外观规整，具有明显且规整的针尖，针尖长度为600-900μm，直径为300-500μm。
2. 皮肤穿刺性能考察
为了证明制备的微针能准确地插入到活体小鼠中，将C57BL/6小鼠先用脱毛膏脱去背部毛发，将微针贴片迅速插入到脱毛的皮肤处，按压30s。随后剥离微针，颈椎脱臼法处死小鼠，立即用剪刀和镊子把该处皮肤取下，平铺在铝箔纸上，放入4%的多聚甲醛溶液中固定。24h后取出固定的皮肤组织进行石蜡包埋，然后进行切片并用HE（苏木精-伊红）染色。光学显微镜下观察并拍摄微针插入的位置。如图2所示，皮肤横截面的切口表明微针可以有效地刺穿小鼠背部皮肤。
3. 微针贴片中药物释放
用经过特殊处理的猪皮（完整保留角质层）经室温解冻后使用。将猪皮固定在立式扩散池上，角质层朝上，真皮层朝向接收液。将实施例1中的微针从角质层刺入皮肤，按压1min，固定好，接收池中加入15mL PBS缓冲液（10mM，PH7.4）。在整个实验期间透皮仪装置的温度维持在37±0.2℃，转速为300rpm。分别在不同的时间点取样4mL进行紫外和HPLC的测定，同时补充空白介质溶液4mL。累计释放按照下面的公式计算：
Q(%)=
式中Ct为不同时间点测得的药物浓度，V0为释放介质的总体积，V为每次取样的体积，X为微针中药物的总含量。
如图3所示，药物能较好地从微针中释放出来，可溶性微针刺入皮肤后可以较快溶解，在皮肤层上形成凝胶态，药物的释放还需要穿过凝胶层，因此表现出缓释行为，能维持相应的持续治疗浓度，并能减少给药过程带来的副作用。
应用例2：极化药物对细胞调控的考察
选择BV2细胞系作为小胶质细胞模型，并用LPS诱导作为炎症模型，考察药物的作用效果。取对数生长期的BV2细胞经胰酶消化后，调整细胞浓度为106个/孔接种到6孔板中，12h后小心吸去上层培养基，加入实施例1中的模型药物（2μg/mL）到孔板中，3h后加入LPS（1μg/mL），均用完全培养基稀释得到相应浓度。共同培养24h后，弃去上层培养液，PBS洗涤一遍后，用胰酶消化收集细胞。分别用荧光标记的CD80和CD206抗体对收集的细胞进行标记。对于CD80染色，细胞收集后直接重悬在1%FBS的溶液中，加入工作浓度的荧光标记的CD80抗体。对于CD206，因为CD206对应的抗体是在胞内区域结合，细胞则需要经过4%的多聚甲醛固定，0.1%的Triton X-100破膜后重悬在1%FBS的溶液中与荧光标记的CD206抗体进行染色。30min后离心弃去上层染色液，PBS洗涤两次后用流式细胞仪检测。
BV2细胞存在不同表型，尤其是经LPS刺激后激活其TLR受体，经过NF-kB信号通路激活诱导炎症，细胞处于极化的M1型状态，此时其标志物CD80水平有所升高。如图4B、4C所示，而经过极化药物处理的组CD80水平明显降低5%，同未刺激的细胞组无显著性差异。同时作为M2型的标志水平蛋白CD206，如图4A所示，经过药物干预的细胞组其CD206水平显著高于LPS刺激组。可见对小胶质细胞得到明显的M2表型调控，这是一种有利于保护神经元细胞的表型。
应用例3：两种药物协同使用对小胶质细胞分泌促炎因子的影响
选择BV2细胞系作为小胶质细胞模型，并用LPS诱导作为炎症模型，考察两种药物的联用效果。取对数生长期的BV2细胞经胰酶消化后，调整细胞浓度为25×104个/孔接种到24孔板中，12h后小心吸去上层培养基，给药分组如下：
LPS刺激组：加入1μg/mL LPS刺激培养
极化药物组：加入2μg/mL极化药物1，3h后加入1μg/mL的LPS刺激
抗炎药物组：加入2μg/mL抗炎药物1，3h后加入1μg/mL的LPS刺激
联合给药组：分别加入2μg/mL极化药物1和抗炎药物1，3h后加入1μg/mL的LPS刺激
加入不同的药物用完全培养基稀释，共孵育24h后，取出上清，1000g，4℃离心10min后收集上清液分别用TNF-α和IL-1β的ELISA试剂盒进行检测。
经LPS刺激的BV2细胞由于激活了炎症信号通路，分泌的IL-1β和TNF-α含量均上升，但是随着极化药物和抗炎药物的干预，如图5所示，有效地抑制了促炎因子的分泌，减少20%-40%。极化药物的作用机制则是通过逆转BV2极化为M2型，处于该表型的BV2细胞更倾向于抗炎状态。抗炎药物则通过阻断激活炎症的NF-kB通路发挥抑制炎症的作用，因此二者联合使用从不同的途径发挥更强大的抗炎作用，有效抑制了两种炎症因子IL-1β和TNF-α的产生，从而缓解了严重的炎症，重塑了细胞的微环境，有利于细胞更稳定的维持生理功能。
应用例4：两种药物协同使用对小胶质细胞和神经元细胞的保护
Aβ蛋白在脑内会形成寡聚体，随后聚集形成Aβ的纤维状结构，进一步进行缠绕则形成β淀粉样斑块。其中Aβ寡聚体会对小胶质细胞和神经元细胞造成一定的损伤。因此考察这两种药物是否能更好地缓解这种损伤。
1、小胶质细胞
选择BV2细胞模型，取对数生长期的BV2细胞经胰酶消化后，以培养液调整细胞浓度8000个/孔接种到96孔板中，12h后小心吸去上层培养基，分别加入以下孵育好的Aβ寡聚体（20μM）+极化药物1（2μg/mL）、Aβ寡聚体（20μM）+抗炎药物1（2μg/mL）和Aβ寡聚体（20μM）+极化药物1（2μg/mL）+抗炎药物1（2μg/mL）。上述溶液分别与BV2细胞37℃共孵育24h后，每孔加入20μL MTT溶液（5mg/mL），4 h后弃掉上层培养液，每孔加入150 μL的DMSO，震摇10 min以充分溶解底部甲瓒。用酶标仪测定各孔在490nm波长处的吸光度OD，以空白培养基为对照，计算细胞存活率。
2、神经元细胞
选择PC12细胞为神经元细胞模型。以8000个/孔种在96孔板中，同应用例4中小胶质细胞的操作进行药物干预，但是在孵育的过程中各药物均采取不完全的DMEM培养基进行稀释，MTT法测得细胞存活率。
另一方面直接观察PC12神经元细胞的形态，将PC12细胞分别用药物干预36h后，弃去上层培养基，PBS洗涤一次后用4%多聚甲醛固定15min，再用0.1%TritonX-100孵育15min破膜，用工作浓度的鬼笔环肽标记细胞，染色30min，弃去上层染色液，PBS洗涤两次后在细胞成像仪下观察拍摄得到形态图片。
MTT实验结果如图6B和6C所示，Aβ寡聚体在20μM时会造成明显的细胞损伤，其中BV2细胞和PC12细胞均只有接近50%的存活率，并且高度分化的PC12在正常条件下呈细长状态，并形成胞质细丝，有许多的神经突起。但是经Aβ寡聚体的刺激，其形态明显发生变化，导致细胞皱缩，逐渐死亡。而经过极化药物和抗炎药物的干预后，细胞形态如图6A所示，细胞得到一定的保护，尤其是联合应用后细胞形态得到更好的维持，和正常组细胞形态接近。同时MTT法的结果也进一步证明了极化药物和抗炎药物对Aβ诱导的细胞毒性均得到一定程度的缓解，与Aβ刺激组有显著性差异（P<0.05）。这为治疗AD带来巨大的希望，能够有效地保护好脑内的小胶质细胞和神经元细胞。
本发明将两种药物联合使用，具有协同增效的效果。在小胶质细胞系模型中，两种药物协同给药后能够显著促进其趋向于M2型，并能够抑制神经炎症，重塑中枢系统的微环境，从而利于对小胶质细胞和神经元细胞的保护。同时制备的微针贴片具备良好的外观、硬度及穿刺能力，释放出治疗水平的药物。该方法同时递送不同性质的药物简单方便，顺应性高，临床用于治疗阿尔兹海默症具有十分优良的前景。
Claims (3)
1. 微针贴片在制备阿尔兹海默症治疗药物中的应用，其特征在于，所述微针贴片由微针阵列和背衬层组成，所述微针阵列由活性药物和基质制成，所述活性药物为极化药物尼古丁与抗炎药物白藜芦醇，所述基质为PVP k30，所述活性药物中极化药物与抗炎药物的质量比为1：0.5~50。
2.一种治疗阿尔兹海默症的微针贴片的制备方法，其特征在于，包括以下步骤：
步骤1，将活性药物溶液和基质溶液混合后注入微针阵列模具中，制成微针阵列；
步骤2，将背衬层溶液注入微针阵列模具中填充，制成背衬，干燥后脱模，即可得到微针贴片；
所述微针贴片由微针阵列和背衬层组成，所述微针阵列由活性药物和基质制成，所述活性药物为极化药物尼古丁与抗炎药物白藜芦醇，所述基质为PVP k30，所述活性药物中极化药物与抗炎药物的质量比为1：0.5~50。
3.根据权利要求2所述的制备方法，其特征在于，所述微针阵列的外观为圆锥形或三棱锥形，针尖长度为600-900μm，直径为300-500μm。
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| CN109431965A (zh) * | 2015-06-03 | 2019-03-08 | 南京三迭纪医药科技有限公司 | 药品剂型及其使用 |
| CN110891554A (zh) * | 2017-05-19 | 2020-03-17 | 保宁制药株式会社 | 含有多奈哌齐的微针经皮贴剂 |
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| CN109431965A (zh) * | 2015-06-03 | 2019-03-08 | 南京三迭纪医药科技有限公司 | 药品剂型及其使用 |
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