CN116253891A - Chta-dsb-da高分子载体及其载药纳米材料的制备和在眼科药物中的应用 - Google Patents
Chta-dsb-da高分子载体及其载药纳米材料的制备和在眼科药物中的应用 Download PDFInfo
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Abstract
本发明属于纳米材料领域,具体涉及一种CHTA‑DSB‑DA高分子载体,本发明还提供了所述的高分子载体的制备和负载N‑GSDMD抑制剂的纳米材料。本发明所述的CHTA‑DSB‑DA高分子载体具有良好的ROS识别和降解能力,将其和N‑GSDMD抑制剂以及Cu2+联合,能够实现协同,能够有效改善青光眼的治疗效果。
Description
技术领域
本发明属于药物领域,具体涉及眼科药物及其载体材料领域。
背景技术
急性青光眼是一种进行性视神经退化的眼科疾病,可造成不可逆性失明,其典型特征是眼压(IOP)突然大幅增加、严重眼痛和不可逆的视力丧失。视网膜神经节细胞(RGC)将视觉信息从视网膜传递到大脑的神经元,它的损伤和死亡是急性青光眼的病理生理标志。氧化应激产生的ROS已被认为是RGC损伤和死亡的重要机制。ROS的累积不仅可诱导线粒体双层膜孔开放,释放钙离子、细胞色素C、凋亡诱导因子AIF,引起胱冬蛋白酶caspase 9激活caspase 3/6/7;还可使线粒体电子传递链解偶联,下调ATP产生水平,上调促凋亡蛋白Bax的表达水平,最终导致线粒体外膜破裂、细胞凋亡。最近的研究表明,ROS还能导致细胞焦亡。
焦亡是一种新发现的由炎症小体依赖性Casp1活化介导的细胞死亡机制。焦亡已经被证实在心血管疾病、肝脏疾病和癌症等多种疾病中起重要作用,是生物学界研究的热点问题。最新研究表明,焦亡是急性青光眼RGCs死亡和视网膜损伤的新机制。GSDMD是一种由242个氨基酸组成的蛋白,具有两个保守的结构域:N-末端效应结构域和C-末端抑制结构域,N端为主要的功能结构域,是导致RGCs焦亡的关键因素。研究表明,一方面,在急性青光眼模型中,GSDMD的N末端裂解,形成了更多的RGCs膜孔,引起了乳酸脱氢酶(LDH)和IL-1β释放的增加,最终导致RGC细胞发生焦亡;另一方面,敲除RGCs中的N-GSDMD,RGCs的生存率显著提升,视网膜组织损伤降低。因此,抑制N-GSDMD介导的焦亡,可能为治疗急性青光眼视网膜损伤提供新思路。
现阶段,急性青光眼的治疗策略主要分为两大类:一方面,是利用抗氧化剂清除RGC细胞内ROS。然而,抗氧化剂进入RGCs效率低,作用时间短且不可控,有可能产生一定的副作用。此外,人们还开发了多种消耗ROS的材料(含硒/碲化物的聚合物、含二硒化物/碲化物的聚合物、多草酸盐等)。但此类材料存在着合成困难、可控性差、降解性差以及潜在的毒性,作用机制单一,最终效力不足等问题。
另外一方面,开发了小分子药物、蛋白类药物和基因治疗来抗焦亡。小分子药物或蛋白类药物存在着可溶性差,易降解以及作用持续时间短的问题;而基因治疗因为常采用病毒载体,存在着生物安全性的问题。因此,至今对于如何通过抑制RGCs的焦亡来治疗急性青光眼仍然知之甚少。
双硫仑(DSF)是一种用于治疗酒精成瘾的药物,具有较高的安全性,正在被应用于多种疾病的治疗和预防中。新近的研究表明,DSF主要通过共价修饰N-GSDMD中的Cys191,使反应性Cys191残基失活,抑制N-GSDMD成孔,从而抑制焦亡。此外,DSF还可在细胞内迅速代谢为二乙基二硫代碳酸盐(DTC),后者通过与葡萄糖酸铜(Cu(II))络合形成CuET,极大地增强DTC在体内活性,提高它抑制细胞焦亡的能力,展现出协同效应。然而,DSF仅可在细胞内发挥作用,而且它是小分子化合物,水溶性差。因此,通过一种有效的递送系统将DSF导入RGCs,同时清除ROS,并抑制RGCs焦亡的新方法将为急性青光眼的治疗带来革命性进展。
然而,DSF需要进入RGCs,才可高效发挥功效。因此通过递送系统递送DSF进入眼部RGCs,显得十分必要,但这一策略始终存在着一系列问题。一方面,眼部递送药物存在角结膜渗透性差以及血眼屏障,导致到达眼部视网膜药物的量非常有限;另一方面,经眼内注射的药物,其药物滞留时间短、细胞吸收率不理想且作用时间维持短,导致人们需要增加注射次数,但这样又给眼睛带来更大的创伤和更多的感染风险。此外,过量的药物还可引起较大的毒副作用。
发明内容
针对眼科药物活性成分如N-GSDMD抑制剂药效不理想的问题,本发明第一目的在于,提供一种全新的CHTA-DSB-DA高分子载体(本发明也称为高分子载体),旨在改善眼科药物活性成分的药效。
本发明第二目的在于,提供所述的CHTA-DSB-DA高分子载体的制备方法。
本发明第三目的在于,提供所述的CHTA-DSB-DA高分子载体在制备眼科药物方面的应用。
本发明第四目的在于,提供基于所述的载体负载有N-GSDMD抑制剂复合纳米材料;旨在改善复合材料的药效。
本发明第五目的在于,提供所述的负载有N-GSDMD抑制剂复合纳米材料的制备和在制药方面的应用。
本发明第六目的在于,提供包含所述的负载有N-GSDMD抑制剂复合纳米材料的治疗青光眼的药物。
一种CHTA-DSB-DA高分子载体,具有式1结构式:
所述的m为100~150的整数;
所述的x为5~30的整数。
本发明提供了全新结构的高分子载体。研究发现,所述的全新的高分子载体,基于链段以及结构的联合协同,可以达到ROS响应的同时双重消耗ROS的效果。
本发明中,所述的m优选为110~130;x优选为10~15。
本发明还提供了一种所述的CHTA-DSB-DA高分子载体的制备方法,将式2、式3进行聚合反应,随后用式4进行封端处理,再采用式5进行接枝修饰,制得所述的CHTA-DSB-DA高分子载体:
本发明中,式2和式3的摩尔比为1:1~1.2;
优选地,聚合反应的溶剂为有机溶剂,优选为DMF、DMSO中的至少一种;
优选地,聚合反应温度为30~60℃。
优选地,式4和式2的重量比为1:1.5~3.5。
优选地,封端反应后,经透析处理,获得封端产物。
优选地,对封端产物的羧基进行活化后再和式5进行接枝反应。
本发明中,可基于现有的手段对封端产物的羧基进行活化。
本发明可基于现有的手段进行羧基活化处理。
本发明中,将活化的封端产物中的羧基和式5的摩尔比为1:1~1.2。
优选地,接枝反应在缚酸剂下进行,所述的缚酸剂没有特别要求例如为TEA。
本发明还提供了一种所述的CHTA-DSB-DA高分子载体的应用,将其和活性药物复合,制得眼科药物。
本发明优选的应用,所述的活性药物为N-GSDMD抑制剂。优选地,所述的N-GSDMD抑制剂为双硫仑。
优选的应用,将其和活性药物、铜离子源联合,用于制备眼科药物。本发明研究发现,在所述的高分子载体创新下,进一步配合所述的铜离子源和N-GSDMD抑制剂,能够实现载体和双重药效成分的联合协同,能够获得更优的药效以及长效效果。
优选地,所述的铜离子源为水溶性铜盐;例如,硝酸铜、硫酸铜、有机酸铜、氯化铜中的至少一种。
优选地,所述的眼科药物为用于治疗RGCs渐进性死亡及其轴突的丢失的药物;
优选地,所述的眼科药物为治疗青光眼的药物。
本发明还提供了一种负载有N-GSDMD抑制剂复合纳米材料,包括载体及其负载的N-GSDMD抑制剂;所述的载体为本发明所述的CHTA-DSB-DA高分子载体。
本发明优选的负载有N-GSDMD抑制剂复合纳米材料,所述的N-GSDMD抑制剂为双硫仑;
优选地,载体和N-GSDMD抑制剂的重量比为5~20:1,优选为10~15:1;
优选地,所述的复合纳米材料中,还包含铜离子源;所述的铜离子源优选为水溶性铜盐;
优选地,所述的复合纳米材料的尺寸为100~300nm。
本发明还提供了一种所述的负载有N-GSDMD抑制剂复合纳米材料的制备方法,将N-GSDMD抑制剂和载体分散在有机溶剂中,随后加入水,混合后进行透析处理,即得。
本发明还提供了一种所述的负载有N-GSDMD抑制剂复合纳米材料的应用,将其用于制备治疗RGCs渐进性死亡及其轴突的丢失的眼科药物;
优选地,将其制备治疗青光眼的眼科药物;
优选地,将其制备用于治疗青光眼的注射药物制剂。
本发明还提供了一种治疗青光眼的药物,包括药学有效量的所述的负载有N-GSDMD抑制剂复合纳米材料。优选地,还包含药学上可接受的辅料。优选地,为局部注射制剂。
有益效果
1、本发明提供了一种全新式1结构的高分子载体,其基于所述的聚合物链以及结构的联合,能够表现出良好的ROS响应和消耗的性能;
2、将所述的高分子载体和N-GSDMD抑制剂如双硫仑的联合,能够实现协同,能够进一步清除RGCs细胞内的有害ROS,提高其保护RGCs免于焦亡的性能;
3、在所述的式1高分子载体上负载N-GSDMD抑制剂和铜离子源的联合,能够实现载体以及活性成分的进一步协同,有助于进一步改善药效以及长效效果。
附图说明
图1为实施例1制得的P1聚合物的H-NMR图;
图2为实施例1制得的P2聚合物的H-NMR图;
图3为实施例2制得的DSF-NPs纳米颗粒TEM以及粒径分布图;
图4为实施例3不同药物和浓度的效果图;
图5为实施例4材料对H2O2和ABTS+清除效果的数据。
图6为实施例5的不同纳米颗粒对细胞内ROS的清除情况
图7为实施例6的激光共聚焦检测各种干预条件下对ROS的清除情况。
图8为实施例7的各种干预条件对视网膜ROS的清除情况;
图9为实施例8的HE染色验证不同条件对青光眼模型小鼠视网膜RGCs的保护作用
图10为实施例9的视网膜铺片验证纳米粒子与铜离子联用能够有效保护视网膜RGCs免于急性高眼压的损伤。
图11为实施例10不同浓度的双硫仑以及双硫仑与铜离子两用对视网膜RGCs的保护作用
图12为实施例11的视网膜铺片发现纳米粒子在第七天时仍然大量存在于视网膜RGCs中
图13为实施例12的不同干预组模型小鼠瞳孔对光反射的情况;
图14为实施例13的不同干预组模型小鼠pERG波幅的情况;
图15为实施例14的不同干预组模型小鼠fVEP波幅的情况。
具体实施方案
注:以下案例中,纳米颗粒的用量以其中的药物DSF用量计。
实施例1
合成线路如下:
将DSB(式2;2mmol,392.6mg)和CHTA(式3;2.1mmol,470.8mg)溶解在5ml无水DMF中。在50℃下磁力搅拌后,将mPEG5000(式4;863mg)加入反应系统并再反应24小时。随后,将混合物放入透析袋(MWCO:8000Da)中并透析48小时。48小时后,溶液在冻干后得到P1,并通过1H NMR进行分析。P1(ca.25mmol羧基,210mg),DMAP(0.05mmol,6.1mg),EDC(0.5mmol,96mg)和NHS(0.5mmol,58mg)溶解在4ml的DMF中并搅拌15分钟。将盐酸多巴胺(式5;0.275mmol,70mg)与52ul的TEA在小型EP管中混合,然后将混合物加入上述DMF溶液中并搅拌过夜。随后,将混合物放入透析袋(MWCO:8000Da)中并透析48小时。透析完成后,溶液通过冻干后得到P2。
实施例2-纳米颗粒制备
A:在搅拌条件下将双硫仑(DSF,2.5mg)和P2(30mg)的DMSO溶液逐渐滴加入去离子水(30mL)中。将混合物放入透析袋(MWCO:8000Da)中透析12h。制得DSF-NPs。
B:在搅拌条件下将P2(30mg)的DMSO溶液逐渐滴加入去离子水(30mL)中。将混合物放入透析袋(MWCO:8000Da)中透析12h。制得NPs。
C:在搅拌条件下将双硫仑(2.5mg)和P1(30mg)的DMSO溶液逐渐滴加入去离子水(30mL)中。将混合物放入透析袋(MWCO:8000Da)中透析12h。制得ND-NPs。
实施例3
实验方法:将R28细胞以5×103细胞/孔的密度接种在96孔板中,并在37℃下与含有10%FBS的DMEM低葡萄糖孵育过夜。然后以0.01μM至100μM的浓度用DSF、DSF+Cu(II)、DSF-NPs和DSF-NPs+Cu(II)处理细胞。药物干预7小时后,相继建立OGD模型(R28OGD)。再灌注12小时后,向每个孔中加入10μL CCK8试剂,然后在37℃下在黑暗中孵育1小时。随后,通过Bio-Rad微板读取器(SpetraMax M3)读取450nm处的OD值。
实验见图4:DSF+Cu(II),DSF-NPs和DSF-NPs+Cu(II)对R28OGD的保护作用随着剂量浓度的升高逐渐增强,而当DSF的浓度为50mM时,DSF表现出明显的细胞毒性。我们还发现,DSF-NPs与Cu(II)联合能够明显保护R28OGD细胞免于损伤,保护作用明显强于DSF+Cu(II)组和DSF-NPs组。这表明了对于急性青光眼的RGC细胞,DSF-NPs联合Cu(II)可能是最为有效的治疗手段。
实施例4-NPs体外ROS测定
ROS测定
实验方法:采用过氧化氢检测试剂盒(Beyotime,Shanghai,China)检测DSF-NPs对H2O2的清除能力。首先,DSF-NPs(10μM)是孵化2毫升含有50mM的PBS过氧化氢在室温下5分钟,1h,2h,4h,8h,或不同浓度的DSF-NPs(从1μM,5μM,10μM)被孵化2毫升含有50mM的PBS过氧化氢在室温下2小时。然后添加50μL的样品或标准溶液(1、2、5、10、20、50、100μM)96孔板。每孔加入100μL过氧化氢检测试剂。室温孵育30min后,Bio-Rad多板读取仪(SpetraMax M3)测量560nm处的吸收,测定剩余H2O2的浓度,并计算H2O2-清除能力。·ABTS+实验中,DSF-NPs(100μL,2μM)与·ABTS+溶液(100μL,7mM)在黑暗中混合1min、30min、1h、2h和4h。随后,用Bio-Rad多平板阅读器(SpetraMax M3)测量混合物在734nm处的吸光度。DSF-NPs清除·ABTS+活性的计算公式如下
·ABTS+清除活性(%)=(A0-Ai)/A0×100%
式中A0和Ai分别表示·ABTS+溶液加入DSF-NPs样品前后的吸光度。
实验见图5:我们选择了H2O2和2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt free radicals(·ABTS+)来进一步评价DSF-NPs清除ROS的能力。结果表明,DSF-NPs对H2O2的清除具有时间依赖性。DSF-NPs与H2O2共孵育8h后,清除了近65%的H2O2(图5),该清除效果具有高分子剂量的依赖性。同样地,我们检测了DSF-NPs清除ABTS+的能力。结果显示,DSF-NPs s与·ABTS+共孵育4小时后,·ABTS+的清除率达到80%左右,且该清除过程具有时间依赖性。由此可见,DSF-NPs s具有明显清除ROS的能力。
实施例5
采用DSF-NPs和ND-NPs进行细胞ROS研究,步骤为:
实验方法:R28细胞以1×106细胞/孔密度接种于六孔平板过夜。然后用DSF-NPs和ND-NPs分别以DSF浓度10μM处理细胞7h,OGD建模12h后处理成单细胞悬液,按照说明书用ROS探针进行染色。上述样品均用PBS冲洗2次,进行荧光定量检测。数据用CytExpert软件进行分析。结果见图6;
实验见图6:为探究DSF-NPs是否具有消耗ROS的能力,我们用活性氧荧光探针DCFH-DA检测了DSF-NPs作用R28OGD细胞后,细胞内的ROS水平。DCFH-DA是一种ROS探针,它在细胞内可被ROS氧化,生成DCF,发出强绿色荧光产物。绿色荧光越强,标志着细胞内ROS水平越高。研究结果显示,R28OGD细胞内ROS的水平最高,无处理的R28细胞内的ROS水平最低。而DSF-NPs处理的R28OGD细胞内ROS水平明显低于ND-NPs组和R28OGD组,ND-NPs处理的R28OGD细胞内的ROS水平明显低于R28OGD组(图6),DSF-NPs处理后的R28OGD细胞ROS水平约为R28OGD组的60%。以上结果表明,ND-NPs的缩硫酮结构在一定程度上消耗ROS,而DSF-NPs消耗R28OGD细胞内的ROS的能力更强。
实施例6-载体-双硫仑+铜离子的案例
R28细胞以1×106细胞/孔密度接种于六孔平板过夜。然后用:(A):10μM DSF;(B):10μM DSF+0.02μM Cu(II);(C):1μM DSF-NPs;(D):1μM DSF-NPs+0.002μM Cu(II)分别处理细胞7h,OGD建模12h后处理成单细胞悬液,按照说明书用AnnexinV/PI或DCFH-DA染色。上述样品干燥后均用PBS冲洗2次,进行荧光定量检测。数据用CytExpert软件进行分析。
实验见图7:DSF-NPs处理后R28OGD的ROS水平约为R28OGD的60%。随后,通过CLSM研究不同处理下R28OGD中ROS水平的变化。CLSM结果显示,DSF-NPs处理的R28OGD的ROS水平明显低于DSF+Cu(II)处理的R28OGD和R28OGD(图7)。值得注意的是,DSF-NPs处理的R28OGD的绿色荧光强度约为R28OGD的60%,这与流式细胞术结果一致(图7)。
实施例7-动物模型方面的数据;
实验方法:动物组织中的ROS:各组视网膜在无血清培养基中收集。4℃组织匀浆后,将DCFH-DA探针加入组织样本中,37℃黑暗孵育30分钟。然后,样品洗三次,然后用PBS重悬。使用Bio-Rad酶标仪(激发波长:488nm,发射波长:525nm)读取吸光度。
注:ND-NPs、DSF-NPs的用量(以DSF计)均为1μM。
实验见图8:检测不同处理下miceI/R视网膜ROS水平(图8)。结果表明,与ND-NPs相比,DSF-NPs能显著降低miceI/R视网膜ROS。经DSF-NPs处理的小鼠视网膜ROS水平约为未处理小鼠的60%。
实施例8
实验方法:I/R建模48h后,经视盘平行于眼球最大周长在垂直经络上切取石蜡包埋的视网膜组织切片(6μm)。切片置于显微镜载玻片上,脱蜡,用苏木精和伊红染色。使用倒置荧光显微镜(Olympus IX 83,Japan)生成染色视网膜的显微照片。
注:图9中,DSF(4mM)+Cu(II)(8μM);DSF-NPs(400μM);DSF-NPs(400μM)+Cu(0.8μM)。
实验结果见图9,miceI/R的视网膜在单位扫描面积内未死亡的RGC(7个左右)细胞数量最少。DSF+Cu(II)治疗的miceI/R(15个左右)和DSF-NPs治疗的miceI/R(17个左右)视网膜内存活的RGCs细胞数量明显多于miceI/R。值得一提的是,DSF-NPs+Cu(II)联合治疗后活RGC细胞数量(32个左右)明显多于其它实验组。上述结果充分说明了DSF-NPs和Cu(II)联用能够协同抑制急性高眼压对视网膜RGCs细胞的损伤,其保护效果明显强于DSF+Cu(II)和DSF-NPs。
实施例9
实验方法:视网膜铺片免疫荧光,立即剜除小鼠眼球,置于冰冷的PBS溶液中,仔细解剖清洗视网膜。视网膜在4%多聚甲醛(pH 7.4)中浸泡30分钟,然后转移到新鲜PBS中2次,5分钟。随后在室温下,5%BSA+0.5%Triton-X-100中孵育2小时。与Brn-3a抗体(1:500)4℃孵育过夜。PBS洗涤3次后,用山羊抗兔AlexaPlus 555(1:500)孵育视网膜。从这一步开始,所有的操作都是在黑暗中进行的。用一滴抗褪色安装介质(Beyotime,上海,中国)小心地将视网膜放置在载玻片上,并在其上放置一个盖子。RGCs在每个视网膜象限分别以中心、中、边缘等距的三个帧样本拍摄。在CLSM(蔡司LSM 880,德国)下观察和拍照视网膜细胞。采用ImageJ 1.52i软件统计和测量RGCs。
实验结果见图10:注,DSF-NPs组:(400μM);DSF-NPs+Cu(II)组:DSF-NPs(400μM)+Cu(0.8μM)。
miceI/R视网膜在单位成像面积内的Brn3a阳性的细胞数量(23个左右)最少,DSF-NPs治疗后的miceI/RBrn3a阳性的细胞数量(84个左右)明显多于未处理的miceI/R。DSF-NPs+Cu(II)治疗后miceI/R视网膜的Brn3a阳性细胞数量(103个左右)与DSF+Cu(II)和DSF-NPs治疗后的miceI/R相比数量最多。以上结果同样说明,DSF-NPs和Cu(II)联用能够更有力地保护视网膜RGCs免于急性高眼压的损伤。
实施例10
实验方法:在C57小鼠IR模型构建前,玻璃体腔分别注射2mM、4mM、6mM的DSF以及4mM DSF+Cu(II)。在构建IR模型后3天进行视网膜铺片免疫荧光,立即剜除小鼠眼球,置于冰冷的PBS溶液中,仔细解剖清洗视网膜。视网膜在4%多聚甲醛(pH 7.4)中浸泡30分钟,然后转移到新鲜PBS中2次,5分钟。随后在室温下,5%BSA+0.5%Triton-X-100中孵育2小时。与Brn-3a抗体(1:500)4℃孵育过夜。PBS洗涤3次后,用山羊抗兔AlexaPlus 555(1:500)孵育视网膜。从这一步开始,所有的操作都是在黑暗中进行的。用一滴抗褪色安装介质(Beyotime,上海,中国)小心地将视网膜放置在载玻片上,并在其上放置一个盖子。rgc在每个视网膜象限分别以中心、中、边缘等距的三个帧样本拍摄。在CLSM(蔡司LSM 880,德国)下观察和拍照视网膜细胞。采用ImageJ 1.52i软件统计和测量RGC。
实验结果见图11:4mM的DSF对miceI/R的保护作用明显强于2mM和6mM。并且以4mM的浓度与Cu(II)联用后效果明显强于单纯使用4mM的DSF。
实施例11
实验方法:C57小鼠玻璃体腔内注射包裹有Cy5.5的DSF-NPs,分别于1天,2天和7天取眼球进行视网膜铺片,观察DSF-NPs是否进入RGCs以及存留时间的情况。所用视网膜铺片方法同前。
实验结果见图12:第一天是DSF-NPs已经明显进入视网膜RGCs。在第二天时,DSF-NPsCy5.5在视网膜RGCs中的内吞明显强于第一天。在第七天时,视网膜RGCs内仍然存在大量的DSF-NPsCy5.5。
实施例12
实验方法:C57小鼠I/R模型构建前两天,小鼠眼内分别注射DSF+Cu(II)、DSF-NPs和DSF-NPs+Cu(II)。在构建I/R模型后的第七天对小鼠的瞳孔对光反射情况进行检测。
实验结果见图13:实验结果显示,miceI/R对光反射的瞳孔面积约为76%左右,明显大于DSF+Cu(II)(32%)和DSF-NPs(36%)治疗的miceI/R。然而,经DSF-NPs+Cu(II)治疗miceI/R,其对光反射瞳孔面积约为12%左右,明显小于DSF+Cu(II)和DSF-NPs治疗的miceI /R。以上结果表明,DSF-NPs联合Cu(II)协同作用能够更有效的保护视网膜RGC细胞传递视网膜信息的功能。
实施例13
实验方法:对于视网膜电图(fERG),首先将小鼠在暗室中暗适应2小时以上,然后将两个接触测量电极放置在双眼角膜上,并将接地电极连接到尾部。接下来,使用ERG模式获得数据。实验完成后,将小鼠置于温暖的笼子中进行恢复。fERG使用Roland眼科电生理诊断系统(德国)进行。
实验结果见图14:miceI/R的f-ERG的波幅为2.86μv左右,明显低于DSF+Cu(II)(6.03μv)和DSF-NPs(6.98μv)治疗后的miceI/R。在DSF-NPs+Cu(II)治疗后,miceI/R的f-ERG的波幅为12.88μv,明显高于其他实验组。
实施例14
实验方法:将小鼠用1%戊巴比妥钠麻醉后,将它们放在桌子上,将三个记录电极分别插入前囟(阴极)、枕骨(阳极)和耳朵(接地电极)的皮下。在暗室中从右眼和左眼获得单侧闪光VEP数据。fVEP使用Roland眼科电生理诊断系统(德国)进行。
实验结果见图15:miceI/RVEP的P波波幅约为227.57μV,明显低于DSF+Cu(II)(354.78μV)和DSF-NPs(343.65μV)治疗的miceI/R。然而,在DSF-NPs+Cu(II)治疗后,miceI/R的VEP的P波波幅为420.51μV,明显高于其他实验组。VEP检查结果表明,DSF-NPs和Cu(II)联用能够更加有效的保护RGC细胞至视觉中枢的传导功能。
本发明设计合成了一种可降解高分子材料P1,P1的合成过程是CHTA和DSB通过缩聚反应聚合,然后用亲水性聚合物mPEG5K-OH封端,最终得到ROS响应性可生物降解的聚合物。将盐酸多巴胺(DA)分子键连到高分子P1的侧链,形成了高分子P2。随后,本发明利用P2包裹DSF,形成了DSF-NPs。在体外,本发明利用对R28细胞系构建氧糖剥夺模型(OGD)证明了NPs1(DSF-NPs)可高效地进入并蓄积在R28内。随后在OGD病理模型中高水平的ROS作用下,P2分子中的缩硫酮键发生断裂,从而导致DSF释放,DSF的代谢产物DTC与Cu(II)形成CuET,CuET与DSF联合作用抑制焦亡关键分子N-GSDMD,抑制RGCs焦亡。在动物水平,本发明还通过眼前房注射生理盐水,建立了小鼠急性青光眼模型(miceIR)。本发明验证了,一方面,DSF-NPs可消耗miceIR的RGCs内过多的ROS,从而减轻氧化应激损伤;另一方面,NPs1可抑制miceIR中RGCs的焦亡,起到保护RGCs,恢复青光眼小鼠视功能的作用。本研究为临床治疗青光眼药物的研发提供了新思路。
与现有技术的区别主要在于以下三点:第一,首次将FDA认证的具有抑制焦亡功效的Disulfiram构建成纳米药物应用于青光眼治疗;第二,该纳米药物释放的DSF的代谢产物DTC与Cu(II)形成CuET,CuET与DSF双重抑制RGCs焦亡;第三,该纳米药物兼具ROS响应释放和ROS消耗,即可响应释放药物的同时,缩硫酮键与酚羟基双重清除病理性生成过多的ROS。
Claims (10)
3.如权利要求2所述的CHTA-DSB-DA高分子载体的制备方法,其特征在于,式2和式3的摩尔比为1:1~1.2;
优选地,聚合反应温度为30~60℃;
优选地,式4和式2的重量比为1:1.5~3.5;
优选地,对封端产物的羧基进行活化后再和式5进行接枝反应;
优选地,将活化的封端产物中的羧基和式5的摩尔比为1:1~1.2。
4.一种权利要求1所述的CHTA-DSB-DA高分子载体或权利要求2~3任一项所述制备方法制得的CHTA-DSB-DA高分子载体的应用,其特征在于,将其和活性药物复合,制得眼科药物。
5.如权利要求4所述的应用,其特征在于,所述的活性药物为N-GSDMD抑制剂;
优选地,所述的N-GSDMD抑制剂为双硫仑;
优选地,将其和活性药物、铜离子源联合,用于制备眼科药物;
优选地,所述的铜离子源为水溶性铜盐;
优选地,所述的眼科药物为用于治疗RGCs渐进性死亡及其轴突的丢失的药物;
优选地,所述的眼科药物为治疗青光眼的药物。
6.一种负载有N-GSDMD抑制剂复合纳米材料,其特征在于,包括载体及其负载的N-GSDMD抑制剂;
所述的载体为权利要求1所述的CHTA-DSB-DA高分子载体或权利要求2~3任一项所述制备方法制得的CHTA-DSB-DA高分子载体。
7.如权利要求6所述的负载有N-GSDMD抑制剂复合纳米材料,其特征在于,所述的N-GSDMD抑制剂为双硫仑;
优选地,载体和N-GSDMD抑制剂的重量比为5~20:1;
优选地,所述的复合纳米材料中,还包含铜离子源;所述的铜离子源优选为水溶性铜盐;
优选地,所述的复合纳米材料的尺寸为100~300nm。
8.一种权利要求6或7所述的负载有N-GSDMD抑制剂复合纳米材料的制备方法,其特征在于,将N-GSDMD抑制剂和载体分散在有机溶剂中,随后加入水,混合后进行透析处理,即得。
9.一种权利要求6或7所述的负载有N-GSDMD抑制剂复合纳米材料的应用,其特征在于,将其用于制备治疗RGCs渐进性死亡及其轴突的丢失的眼科药物;
优选地,将其制备治疗青光眼的眼科药物;
优选地,将其制备用于治疗青光眼的注射药物制剂。
10.一种治疗青光眼的药物,其特征在于,包括药学有效量的权利要求6或7所述的负载有N-GSDMD抑制剂复合纳米材料;
优选地,还包含药学上可接受的辅料;
优选地,为局部注射制剂。
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