CN114983972B - 水凝胶包被miRNA-200s纳米微粒缓释系统在制备视神经炎疾病类药物中的应用 - Google Patents
水凝胶包被miRNA-200s纳米微粒缓释系统在制备视神经炎疾病类药物中的应用 Download PDFInfo
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Abstract
本发明公开了水凝胶包被miRNA‑200s纳米微粒缓释系统在制备视神经炎疾病类药物中的应用和药物及模型构建方法，涉及生物医药领域。视神经炎疾病类药物通过水凝胶包被miRNA‑200s纳米微粒缓释系统(NSDDS)制备获得；验证视神经炎疾病保护剂模型构建方法包括：对视神经炎疾病造模后小鼠玻璃体腔内注射视神经炎疾病类药物。本发明研究发现水凝胶包被miRNA‑200s纳米微粒缓释系统可制备视神经炎疾病类药物，从而改善视神经炎类疾病损伤，预防及保护炎症及其他原因所致的视力下降甚至失明。本方法治疗的灵敏度高，改善效果显著。本发明具有很好的应用价值和推广前景。
Description
技术领域
本发明涉及寄生虫及神经损伤保护领域，更具体地，涉及一种水凝胶包被miRNA-200s纳米微粒缓释系统在制备预防及保护视神经炎疾病及其它原因所致视力下降甚至失明类药物中的应用和药物及模型构建方法。
背景技术
视神经炎(optic neuritis，ON)是一种累及视神经的常见疾病，涉及炎症脱髓鞘以及轴索损伤。疾病过程可以导致视网膜节细胞(retinal ganglion cell，RGC)死亡、黄斑体积减小以及视觉损伤，甚至永久性的视觉丧失。视神经炎常常伴随眼球疼痛和视力减退。本病病因广杂,无论是毗邻视神经的感染病灶或全身各种感染因子,还是中枢神经系统脱髓鞘疾病或系统性自身免疫性疾病均可能促发或并发视神经炎。
根据病因不同，视神经炎被分为特发性脱髓鞘性视神经炎(亦称经典多发性硬化相关性视神经炎(MS-ON))、视神经脊髓炎相关性视神经炎和其他中枢神经系统脱髓鞘疾病相关性视神经炎。视神经炎的常见临床表现为单眼或双眼视力下降、视神经乳头水肿、视网膜静脉迂曲、扩张及视乳头周围渗出。患者还会出现多种形式的神经纤维束型视野缺损；视觉动作电位(VEP)检查表现表现为潜伏期延长和(或)波幅降低；部分患者视力恢复较差，会遗留严重视力障碍。目前临床上对视神经炎的常规治疗方案有糖皮质激素、多发性硬化症(MS)修正药物(如β-干扰素、醋酸格拉默、米托蒽醌和那他珠单抗等)、免疫抑制剂等。但这些治疗方法难以做到根治，容易形成激素依赖，且病情易反复发作。视神经炎与多发性硬化密切相关，无论是单独治疗视神经炎或是治疗多发性硬化等与视神经炎相关的疾病，目前均未发现何种治疗方法能够改善患者的最终预后。
miRNA是一类广泛存在于真核生物中的单链小分子非编码RNA，通过与靶基因特异性结合，抑制靶基因的翻译甚至降解，参与基因转录后的调控。大量研究已经鉴定出多种miRNA及其靶基因参与CNS修复过程，促进神经保护、轴突再生、神经元可塑性、血管生成等。与其他神经系统疾病一样，选择合适的miRNA提供显着的RGC神经保护作用是首要的关键步骤之一。对视网膜来讲，miRNA及其模拟物或抑制剂可以通过包括病毒和非病毒载体在内的多种方法进入RGC，可作为视觉疾病一种潜在的治疗手段。
有研究发现，miR-200s(141、200a)可调控视网膜节细胞(RGC)的存活。已有文献报道在人类视网膜色素上皮细胞(RPEs)和(RGCs)中过表达miR-141可以下调Keap1(Nrf2的抑制剂)，进而激活Nrf2，从而减少紫外线辐射造成的RPEs和视网膜节细胞凋亡。antagomiR-141和Nrf2 shRNA敲除实验进一步验证了上述结果。在青光眼小鼠模型中，有相关研究结果表明miR-141靶向对接蛋白5(DOK5)，激活MAPK信号通路，最终抑制视网膜血管上皮细胞的增殖和血管生成，促进RGCs的凋亡。也有研究发现在小鼠青光眼模型中，miR-200a的表达下调，而成纤维细胞生长因子7(FGF7)显著上调。他们进一步研究发现，将miR-200a模拟物静脉注射到青光眼小鼠模型中，可以显著的保护视网膜节细胞和保存视神经纤维层的厚度，其机制可能是通过抑制FGF7激活MAPK通路来实现的。但miR-200s对视神经炎的作用目前尚无相关研究。
研究miRNA对靶细胞作用的方法通常有两种，一种为过表达或传递miRNA到靶细胞；另一种为在靶细胞内下调/沉默待研究的miRNA。在对视网膜节细胞的研究中，为减少实验过程中miRNA的降解并提高其作用效果，miRNA及其模拟物或抑制剂可以通过包括病毒和非病毒载体在内的多种方法(如脂质体/外泌体转染、AntagomiRs和miRNA海绵等)被递送到RGC中作为潜在的治疗剂。这些通常首先在体外进行测试，例如视网膜培养系统，然后在视网膜疾病的体内动物模型中进行测试。眼内直接注射是最常用的给药途径。但由于玻璃体腔内频繁注射易引起眼内出血、视网膜脱离、眼内炎等缺陷，近些年，一些眼内缓释给药系统的开发等在不同程度上克服了上述缺点。包括脂质体、纳米粒、微粒、眼植入物等方法获得较满意的效果成为治疗玻璃体视网膜疾病的发展趋势之一。
本申请为一种水凝胶包被miRNA-200s纳米微粒缓释系统在制备视神经炎疾病类药物中的应用和药物及模型构建方法，从而改善视神经炎等疾病。
发明内容
本发明的目的是为了提供一种水凝胶包被miRNA-200s纳米微粒缓释系统在制备预防及保护视神经炎疾病及其它原因所致视力下降甚至失明类药物中的应用和药物及模型构建方法，本方法治疗的灵敏度高，改善效果显著。
本发明的第一个目的是水凝胶包被miRNA-200s纳米微粒缓释系统在制备视神经炎疾病类药物中的应用。
本发明的第二个目的是水凝胶包被miRNA-200s纳米微粒缓释系统可制备神视神经炎疾病类药物，从而改善视神经炎损伤等疾病。
本发明的第三个目的是水凝胶包被miRNA-200s纳米微粒缓释系统在制备视神经炎疾病类药物中的应用，所述视神经炎疾病包括以广州管圆线虫感染引起的各种视神经炎疾病类损伤。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
水凝胶包被miRNA-200s纳米微粒缓释系统，该系统包含一种与视神经损伤保护相关的miRNA-200s标记物，该miRNA标记物负载到STH水凝胶制成miRNA-200s纳米颗粒即水凝胶包被miRNA-200s纳米微粒缓释系统。
一种水凝胶包被miRNA-200s纳米微粒缓释系统的制备方法，包括如下步骤：
一、STH水凝胶制备：
1)向预冷的超纯水(4℃)中加入硅酸盐纳米片粉末Laponite，然后使用台式高速离心机离心三次混合均匀，制备浓度为9％(w/w)的Laponite水凝胶；
2)磁力搅拌下将猪皮明胶溶于超纯水中，制备浓度为18％(w/w)的明胶原液；
3)分别制备6NC40、6NC50、6NC75水凝胶(即为STH水凝胶)；所述6NC40、6NC50、6NC75中：“6”指的是固体总质量为凝胶质量的6％；“75”，“50”和“40”指的是硅酸盐水凝胶与明胶的质量比分别为75:25,50:50和40:60。
二、将miR-200s负载到STH制成miR-200s纳米颗粒：
1)用不同比例(6NC75、6NC50、6NC40)的Laponite/明胶比的STH负载miR-200s(携带荧光)；
2)在制备过程中，将miR-200s按设计浓度1nmol/10g加入PBS缓冲液中，然后向含有miR-200s的溶液中分别加入明胶原液和Laponite凝胶；然后3500rpm下离心5min，重复三次；
3)得到负载miR-200s的STH，置于4℃储存。
一种水凝胶包被miRNA-200s纳米微粒缓释系统在制备视神经炎疾病类药物中的应用。
上述应用中，所述视神经炎疾病表现为视网膜神经节细胞的损伤及炎症细胞浸润。
上述应用中，所述视神经炎被分为特发性脱髓鞘性视神经炎(亦称经典多发性硬化相关性视神经炎(MS-ON))、视神经脊髓炎相关性视神经炎和其他中枢神经系统脱髓鞘疾病相关性视神经炎，可导致视力明显下降甚至失明。
上述应用中，所述水凝胶包被miRNA-200s纳米微粒缓释系统用药剂量为2nmol/20g体重，用药对象为Balb/c小鼠及其它视神经损伤动物，用药途径为玻璃体腔内注射，治疗方法快速、简洁、合理、有效。
上述应用中，所述视神经炎疾病包括以广州管圆线虫感染引起的各种视神经炎类疾病，可引起视力下降甚至失明。
一种视神经炎疾病类药物验证模型的构建方法，所述模型构建方法为：对视神经炎疾病造模后的小鼠注射所述视神经炎疾病类药物，且对模型动物安全有效，无明显副作用及并发症。
上述疾病类药物验证模型的构建方法，具体包括以下步骤：
1)选取小鼠进行视神经炎疾病造模；
2)造模后，对小鼠通过玻璃体腔内注射所述视神经炎疾病类药物；
3)随后检测小鼠的视网膜神经节细胞的损伤情况及视力损伤程度。
上述视神经炎疾病类药物验证模型的构建方法，所述检测方法为免疫荧光、磁共振成像技术、透射电镜技术和/或HE、LFB染色。
本发明通过建立广州管圆线虫感染Balb/c小鼠所致视神经炎模型，microRNA芯片技术及RT-qPCR定量分析发现miRNA-200s感染早期明显上调，后期下降。选择外源性过表达miRNA-200s，采用western blot、RT-qPCR、H&E染色、透射电镜、免疫荧光等技术进行鉴定分析，判定所述miRNA-200s为该损伤模型的治疗靶点；通过水凝胶包被miRNA-200s纳米微粒缓释系统制备视神经炎疾病类药物来预防、保护及改善感染小鼠的视神经损伤及视力下降等症状，从而发挥对感染及其他原因所致视神经炎的保护作用，提出了一种新型治疗方法。
因此本发明要求保护一种水凝胶包被miRNA-200s纳米微粒缓释系统在制备预防及保护视神经炎疾病及其它原因所致视力下降甚至失明类药物中的应用。
所述的使用水凝胶包被miRNA-200s纳米微粒缓释系统制备视神经炎疾病类药物在改善广州管圆线虫病中的应用也属于本发明的保护范围。
一种水凝胶包被miRNA-200s纳米微粒缓释系统可制备视神经炎疾病类药物，从而改善视神经炎等损伤。且对模型动物安全有效，无明显副作用及并发症。
视神经炎疾病类药物验证模型的构建方法，采用如权利要求1-5任一所述的视神经炎疾病类药物，所述模型构建方法为：对视神经炎疾病造模后的小鼠玻璃体腔内注射所述视神经炎疾病类药物。
最优选地，水凝胶包被miRNA-200s纳米微粒缓释系统在制备视神经炎疾病类药物中的应用，所述视神经炎疾病为特发性脱髓鞘性视神经炎(亦称经典多发性硬化相关性视神经炎(MS-ON))、视神经脊髓炎相关性视神经炎和其他中枢神经系统脱髓鞘疾病相关性视神经炎中的一种。
其治疗方法包括以下步骤：
1、建立Balb/c小鼠的视神经损伤模型。
2、外源性过表达水凝胶包被miRNA-200s纳米微粒缓释系统所制备的药物。
水凝胶包被miRNA-200s纳米微粒缓释系统所制备的药物通过玻璃体腔内注射入视神经损伤动物玻璃体。
3、各指标分析：
MRI检测大脑皮质、胼胝体、海马和视皮质损伤情况；视觉电生理等检测损伤动物的视觉改变情况；视网膜HE染色、RGC免疫荧光计数、视神经LFB染色及透射电镜等检测视神经脱髓鞘及轴突损伤程度；全脑荧光观察视觉通路神经元及髓鞘损伤程度。过表达水凝胶包被miRNA-200s纳米微粒缓释系统后可部分逆转上述现象，改善视神经损伤。
与现有技术相比，本发明具有如下有益效果：
本发明公开了水凝胶包被miRNA-200s纳米微粒缓释系统在制备视神经炎疾病类药物中的应用和药物及模型构建方法，涉及生物医药领域。视神经炎疾病类药物通过水凝胶包被miRNA-200s纳米微粒缓释系统(NSDDS)制备获得；验证视神经炎疾病保护剂模型构建方法包括：对视神经炎疾病造模后小鼠玻璃体腔内注射视神经炎疾病类药物。本发明研究发现水凝胶包被miRNA-200s纳米微粒缓释系统可制备视神经炎疾病类药物，从而改善视神经炎类疾病损伤，预防及保护炎症及其它原因所致的视力下降甚至失明。本方法治疗的灵敏度高，改善效果显著。本发明具有很好的应用价值和推广前景。
附图说明
图1a为广州管圆线虫感染引起Balb/c小鼠明显神经行为学评分降低及眼部炎症症状；
图1b为感染小鼠的眼底镜检查变化；
图1c为感染小鼠的免疫组化病理切片；
图1d为感染小鼠的视觉动作电位(VEP)变化；
图2为过表达miR-200s可显著改善广州管圆线虫所致视神经炎的脱髓鞘现象及神经元损伤，并显著提高感染小鼠的视觉及降低致盲率。
图3为负载miRNA剪切变稀水凝胶(STHs)纳米颗粒缓释系统的建立。
图4为神经系统受损小鼠miRNA-200s治疗前后视网膜的病理学及透射电镜分析以及相应的视觉诱发电位分析和视网膜神经节细胞的蛋白定量分析。
图5为水凝胶包被miRNA-200s纳米微粒缓释系统在制备视神经炎疾病类药物中的应用和药物及所涉及的神经系统疾病模型构建方法。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1广州管圆线虫感染引起Balb/c小鼠明显视神经炎，可作为研究视神经炎的理想动物模型
一、测试方法
从感染第0天开始，观察小鼠神经行为学及视觉功能变化；使用眼底镜观察小鼠视乳头及眼底静脉变化；HE染色及电镜观察视网膜及节细胞损伤情况；视觉动作电位(VEP)观察是否有视力下降及炎症损伤。
二、测试结果
神经行为学评分显示A.cantonensis感染21d旷场和平衡能力下降明显(图1a中第一幅图所示)，而且视觉功能明显下降，部分小鼠眼睑完全闭合(箭头所致)(图1a中第二幅图)。眼底镜检查显示感染21d感染小鼠视神经乳头水肿，眼底静脉充血扩张(黑色箭头所指)(图1b所示)。HE染色显示感染21d小鼠感染视网膜四层结构出现细胞松散，且节细胞层可见明显炎症细胞浸润；节细胞电镜显示节细胞结构完全破坏(图1c所示)。视觉动作电位(VEP)显示21d感染小鼠潜伏期与正常组相比明显延长(图1c所示)。
实施例2过表达miR-200s可显著改善广州管圆线虫所致视神经炎的脱髓鞘现象及神经元损伤，并显著提高感染小鼠的视力及降低致盲率
一、测试方法
采用统计学方法估计小鼠死亡率、致盲率、偏瘫率及视觉动作电位(VEP)的潜伏期变化；同时采用HE染色及透射电镜技术分析视网膜及节细胞损伤修复情况。
二、测试结果
广州管圆线虫感染21d后，小鼠死亡率、致盲率、偏瘫率明显升高，而过表达miRNA-200s后可逆转上述变化(图2中第一行第一幅图所示)。在过表达miR-200s后，A.cantonensis感染小鼠与无处理组相比，无一例出现失明症状，且视觉动作电位(VEP)的潜伏期较无处理组明显缩短(图2中第一行第二幅图所示)。病理检测显示视神经脱髓鞘现象明显减轻，视网膜节细胞的数目及形态均接近正常组(图2中第二行三组图所示)。而全脑的免疫荧光扫描结果也显示，过表达miR-200s后，视皮质及上丘区神经元的数目较感染组明显上升(图2中第三行第一幅图所示)。以上结果提示miR-200s对A.cantonensis所致视神经炎的脱髓鞘现象及神经元损伤等退行性变具有明显的保护作用。
实施例3负载miRNA剪切变稀水凝胶(STHs)纳米颗粒缓释系统的建立，显著提高miR-200s作用于视网膜的血药浓度
一、测试方法
用硅酸盐纳米片粉末(Laponite)负载携带荧光的miR-200s模拟物，制备出负载miR-200s剪切变稀(STH)水凝胶纳米颗粒。
一、STH水凝胶制备：
1)向预冷的超纯水(4℃)中加入硅酸盐纳米片粉末(Laponite)，然后使用台式高速离心机离心三次混合均匀，制备浓度为9％(w/w)的Laponite水凝胶；
2)磁力搅拌下将猪皮明胶溶于超纯水中，制备浓度为18％(w/w)的明胶原液；
3)分别制备6NC40、6NC50、6NC75水凝胶(即为STH水凝胶)。注：6NC75和6NC50：“6”指的是固体总质量为凝胶质量的6％；“75”，“50”和“40”指的是硅酸盐水凝胶与明胶的质量比分别为75:25,50:50和40:60。
二、将miR-200s负载到STH制成miR-200s纳米颗粒：
1)用不同比例(6NC75、6NC50、6NC40)的Laponite/明胶比的STH负载miR-200s(携带荧光)；
2)在制备过程中，将miR-200s按设计浓度(1nmol/10g)加入PBS缓冲液中，然后向含有miR-200s的溶液中分别加入明胶原液和Laponite凝胶；然后3500rpm下离心5min，重复三次；
3)得到负载miR-200s的STH，置于4℃储存。
二、测试结果
因Laponite纳米片表面带负电，边缘带正电，可以通过静电吸附带负电的miRNA，从而达到延缓了释放miRNA的目的。因视网膜紧邻玻璃体，而玻璃体为无色透明的水分占99％的胶状体。如若将miR-200s制成水凝胶纳米颗粒并注射入玻璃体，则操作较颅内注射简单方便且较为安全；并显著提高miR-200s作用于视网膜的血药浓度；更重要的是，同为水凝胶的玻璃体将和miR-200s传递的剪切变稀水凝胶纳米颗粒融合成缓释系统，明显提高miR-200s对视网膜内细胞的作用时间。我们前期结果初步显示，miR-200s能成功负载到STH水凝胶中；而且随着释放时间的增加，释放的miR-200s的量也逐渐增加，说明miR-200s纳米颗粒缓释系统体外初步建立成功。
实施例4神经系统受损小鼠miRNA-200s治疗前后视网膜的病理学及透射电镜分析以及相应的视觉诱发电位分析和视网膜神经节细胞的蛋白定量分析
一、测试方法
Western blot检测蛋白量变，通过制胶、上样、电泳、电转、封闭、一抗孵育、二抗孵育、显影等一系列常规步骤，最后在化学发光显影系统显影成像。透射电镜观察脑组织结构，用ImageJ软件测量髓鞘厚度和轴突直径，以轴突直径除以神经束直径计算gratio。同时采用视觉动作电位(VEP)观察是否有视力下降及炎症损伤。
二、测试结果
实验结果也表明miR-200s在广州管圆线虫感染两天时大量表达于视网膜。在miR-200s过表达后，视网膜处损伤较感染21d时有明显改善(图4中第一行图所示)。视觉诱发电位(VEP)检测发现潜伏期延长及波幅降低，提示可能有视神经炎所致的视觉损伤及视力下降(图4中第二行及第三行第一、二幅图所示)。Western Blot对RGCs计数也表明视网膜神经节细胞丢失(图4中第三行第三、四幅图所示)，过表达miRNA-200s后发现上述视神经损伤现象有所改善。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步的详细说明，应当理解，以上所述仅为本发明的具体实施例而已，并不用于限定本发明的保护范围。特别指出，对于本领域技术人员来说，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.水凝胶包被miRNA-200s纳米微粒缓释系统，其特征在于，该系统包含一种与视神经损伤保护相关的miRNA-200s 标记物，该miRNA标记物负载到STH水凝胶制成miRNA-200s纳米颗粒即水凝胶包被miRNA-200s纳米微粒缓释系统；
包括如下步骤：
一、STH水凝胶制备：
1）向预冷的超纯水中加入硅酸盐纳米片粉末Laponite，然后使用台式高速离心机离心三次混合均匀，制备浓度为9% (w/w)的Laponite水凝胶；
2）磁力搅拌下将猪皮明胶溶于超纯水中，制备浓度为18%（w/w）的明胶原液；
3）分别制备6NC40、 6NC50、6NC75水凝胶（即为STH水凝胶）；所述6NC40、 6NC50、6NC75中：“6”指的是固体总质量为凝胶质量的6%；“75”，“50”和“40”指的是硅酸盐水凝胶与明胶的质量比分别为75:25,50:50和40:60；
二、将miR-200s负载到STH制成miR-200s纳米颗粒：
1）用不同比例的Laponite/明胶比的STH负载miR-200s；
2）在制备过程中，将miR-200s按设计浓度1 nmol/10 g加入PBS缓冲液中，然后向含有miR-200s的溶液中分别加入明胶原液和Laponite凝胶；然后3500 rpm下离心5 min，重复三次；
3）得到负载miR-200s的STH，置于4 ℃储存。
2.权利要求1所述水凝胶包被miRNA-200s纳米微粒缓释系统在制备视神经炎疾病药物中的应用。
3.根据权利要求2所述应用，其特征在于，所述视神经炎疾病表现为视网膜神经节细胞的损伤及炎症细胞浸润。
4.根据权利要求2所述应用，其特征在于，所述视神经炎被分为特发性脱髓鞘性视神经炎、视神经脊髓炎相关性视神经炎和其他中枢神经系统脱髓鞘疾病相关性视神经炎。
5.根据权利要求2所述应用，其特征在于，所述水凝胶包被miRNA-200s纳米微粒缓释系统用药剂量为2nmol/20g体重，用药对象为Balb/c小鼠及其它视神经损伤动物，用药途径为玻璃体腔内注射。
6.根据权利要求2所述应用，其特征在于，所述视神经炎疾病包括以广州管圆线虫感染引起的各种视神经炎疾病。
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