CN117899006A - 一种负载双硫仑纳米囊泡的复合水凝胶及其在制备糖尿病皮肤创面愈合药物中的应用 - Google Patents
一种负载双硫仑纳米囊泡的复合水凝胶及其在制备糖尿病皮肤创面愈合药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,公开了一种负载双硫仑纳米囊泡的复合水凝胶。本发明以双硫仑和PLGA共聚物形成纳米药物的核心,将间充质干细胞膜用于包被双硫仑和PLGA共聚物合成得到纳米囊泡,将纳米囊泡与水凝胶混合制备负载双硫仑纳米囊泡的复合水凝胶。本发明中的纳米颗粒能够调节免疫炎症,促进细胞增殖,发挥促进伤口愈合的作用;纳米药物外层的生物膜能够增强纳米颗粒的生物隐蔽性,防止其在体内被迅速清除,同时,间充质干细胞来源的细胞外囊泡可发挥与间充质干细胞类似的组织修复功能,协同发挥促进伤口愈合;本发明中的水凝胶,改变传统的注射给药方式,同时起到保湿透气、缓释药物的作用,促进伤口愈合,该复合水凝胶可用于治疗糖尿病皮肤伤口及其他外伤,成为一种促进伤口愈合的新策略。
Description
技术领域
本发明属于生物医药技术领域,更具体地,涉及一种负载双硫仑纳米囊泡的复合水凝胶及其在制备糖尿病皮肤创面愈合药物中的应用。
背景技术
糖尿病足,是指糖尿病患者踝关节以远的皮肤及其深层组织破坏,常合并感染和(或)下肢不同程度的动脉闭塞症,严重者则累及肌肉和骨组织,是长期糖尿病患者的并发症之一。由于糖尿病在我国已从少见病变成流行病,糖尿病足的患病率也明显增加,我国50岁以上的糖尿病患者,糖尿病足的发病率高达8.1%。据估计,全球每20秒钟就有一例糖尿病患者截肢,糖尿病足溃疡患者年死亡率高达11%,而截肢患者死亡率更高达22%。因此,糖尿病足是糖尿病患者致残、致死的主要原因之一,造成社会的沉重负担,是急需解决重大公共卫生问题。现代糖尿病足创面护理的内容包括清创、使用促进创面湿性愈合的敷料、减压、血管评估及管理、治疗感染和血糖控制等几个方面。尽管许多研究都集中在加快伤口愈合过程上,但目前尚无令人满意的治疗方法。
近些年研究指明,由于体内代谢和生化异常,糖尿病患者体内的促炎中性粒细胞途径增强,主要是中性粒细胞细胞外陷阱(NETs)形成,细胞外ROS生成和促炎细胞因子生成上调,这些物质过量积累使炎症持续,造成组织损害,伤口愈合延迟。新发现的双硫仑抑制GSDMD孔形成从而抑制NETs的作用可能为重新利用双硫仑提供新的治疗适应症,以治疗由过度炎症引起或加剧的许多疾病。
细胞外囊泡(EVs)是细胞衍生的膜结构,具有天然脂质双层膜结构,可作为理想的药物载体将药物递送到所需的靶标部位,同时间充质干细胞衍生的细胞外囊泡(MSC-EVs)本身具有免疫调节,炎症抑制,细胞增殖促进,血管生成等功能,在皮肤损伤中被广泛应用。
近年来,EVs与壳聚糖、海藻酸盐等水凝胶生物材料的结合已成为研究的热点,包封EVs的水凝胶可在伤口部位提高EVs释放的持久性和稳定性,同时不影响EVs的结构和功能完整性。生物相容性水凝胶将大量EVs无创、稳定输送到靶部位,同时具有良好的保湿性和透气性,从而成为创面敷料的理想选择。
发明内容
为解决上述现有技术中的问题,本发明以双硫仑和D,L-乳酸-co-乙醇酸(PLGA)共聚物形成纳米药物的核心,将间充质干细胞膜用于包被双硫仑和D,L-乳酸-co-乙醇酸(PLGA)共聚物设计合成得到纳米囊泡,将纳米囊泡与水凝胶混合制备负载双硫仑纳米囊泡的复合水凝胶。旨在解决以下问题:1)双硫仑和间充质干细胞细胞外囊泡联合所制备出的双硫仑纳米囊泡,参与调节伤口免疫炎症,促进细胞增殖,解决糖尿病伤口处慢性炎症持续加重造成的组织损伤问题;2)利用水凝胶保湿透气及提高药物释放的特性,解决药物静脉注射被快速清除的局限。从而使制备出的负载双硫仑纳米囊泡的复合水凝胶达到改善伤口免疫环境,加速伤口愈合,治疗糖尿病皮肤伤口及其他外伤的目的。
为实现上述目的,本发明提供如下技术方案:
一种复合水凝胶,含有核-壳结构的纳米颗粒和凝胶成分,所述核由双硫仑和D,L-乳酸-co-乙醇酸(PLGA)共聚物组成;所述壳为间充质干细胞膜;所述凝胶成分为海藻酸钠。
优选地,所述纳米颗粒的直径为120~200nm。
优选地,所述聚(D,L-乳酸-co-乙醇酸)和双硫仑的质量比为10:0.5~3。
优选地,所述间充质干细胞膜(壳)和所述PLGA载药纳米(核)的混合质量比为0.125~0.5:1。
优选地,所述的仿生间充质干细胞膜纳米载药颗粒与海藻酸钠水凝胶质量比为5:1。
本发明同时要求保护所述复合水凝胶的制备方法,包括以下步骤:
S1、提取间充质干细胞膜;
S2、PLGA载药纳米内核的合成:加入聚(D,L-乳酸-co-乙醇酸)和药物双硫仑混合,磁力搅拌即得;
S3、将步骤S1的间充质干细胞膜和步骤S2的PLGA载药纳米内核混合,超声后即得仿生间充质干细胞膜纳米载药颗粒;
S4、海藻酸钠水凝胶的合成:海藻酸钠混合于水中,4℃摇床混合溶解过夜,加入氯化钙后充分混匀,即得海藻酸钠水凝胶;
S5、将步骤S3的仿生间充质干细胞膜纳米载药颗粒与步骤S4的海藻酸钠水凝胶4℃下充分混合。
优选地,步骤S2磁力搅拌的条件为300~500rpm,温度为4~25℃,时间为4~48h。
优选地,步骤S3所述水浴超声的条件为功率50W~100W,温度为4~25℃,时间为3~30分钟。
本发明上述负载仿生间充质干细胞膜纳米载药颗粒的复合水凝胶能够调节免疫,促进细胞增殖和血管生成,发挥促进伤口愈合的作用;另外其仿生纳米颗粒内核包裹的双硫仑,能够针对糖尿病皮肤创面处中性粒细胞,减少NETs产生,促进伤口愈合。本发明上述仿负载仿生间充质干细胞膜纳米载药颗粒的复合水凝胶能够抑制高血糖环境下中性粒细胞被刺激活化产生NETs,同时伴随炎症因子表达变化,调节炎症反应,可用于制备降低糖尿病患者伤口处的炎症因子的含量的药物中。
与现有技术相比,本发明的有益效果是:
本发明提供了一种仿生间充质干细胞膜纳米载药颗粒,其生物膜的表面能够增强纳米颗粒的生物隐蔽性,防止其在体内被迅速清除;该载药颗粒能够抑制中性粒细胞刺激产生NETs,减少NETs积累造成的组织损害,在糖尿病皮肤创面愈合治疗中起到免疫调理作用;间充质干细胞来源的细胞外囊泡可发挥与间充质干细胞类似的组织修复功能,协同发挥促进伤口愈合。本发明制备的仿生间充质干细胞膜纳米载药颗粒合成简单,原料为生物性膜和可降解的PLGA,生物相容性高,适用于糖尿病皮肤创面治疗。
附图说明
图1为DSF-EP制备及在糖尿病皮肤伤口修复中的应用原理图;
图2为DSF-EP的表征,其中图2A外泌体表面标志蛋白;图2B为DSF-EP在不同溶质中的吸光度;图2C为粒径分布图;图2D为电位分布图;
图3为DSF-EP促进糖尿病伤口愈合,其中图3A为伤口不同时间的愈合情况;图3B为不同组别愈合面积随时间变化的变化;
图4为DSF-EP降低各炎性因子表达情况。图4A至4E分别代表IL-1α、IL-1β、IL-6、TNF-α、IL-18。
具体实施方式
下面将结合本发明实施例和附图,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行制备。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实施例1:DSF-EP的制备,包括以下步骤:
一、间充质干细胞膜提取
(1)收集长至70%-80%融合的(约108个)MSC细胞,1000g,5分钟离心,用1×PBS洗3次,最终所得细胞沉淀用3ml低渗缓冲液(1mmol/L NaHCO30.2mmol/L EDTA、1mmol/LPMSF)重悬;在4℃条件下裂解过夜;
(2)取(1)中的细胞重悬液装入Dounce杜恩斯匀浆机,将均浆机置于冰上,研磨细胞20次;
(3)取(2)中的重悬液以3200×g,5分钟离心以清除大的碎片,收集上层清液;
(4)取(3)中的上清液20000g,25分钟离心,丢弃沉淀,收集上层清液,100000g×35分钟离心,离心后,丢弃上层清液,收集白色沉淀(即细胞膜),用500μl 1×PBS重悬;
(5)取(4)中的重悬液检测膜蛋白含量。
为了验证本发明仿生间充质干细胞纳米颗粒的可行性,发明人对仿生间充质干细胞纳米颗粒表面膜蛋白及其相关受体蛋白进行了表征。发明人首先对仿生巨间充质干细胞纳米颗粒表面蛋白进行检测,通过蛋白电泳检测,实验结果如图2A显示,仿生间充质干细胞纳米颗粒的条带和间充质干细胞外泌体条带一致,而与间充质干细胞裂解液蛋白条带不同,进一步证明其表面覆盖的是间充质干细胞来源的囊泡,由此证明了本发明利用仿生间充质干细胞膜可发挥间充质干细胞促进伤口修复潜能的可行性。
二、PLGA载药纳米内核的合成
(1)将双硫仑按照质量浓度比4:1溶于1ml丙酮中;
(2)往(1)中加入聚(D,L-乳酸-co-乙醇酸),药物和聚(D,L-乳酸-co-乙醇酸)质量浓度比为3:10;
(3)将(2)中的混合液用1ml注射器快速打入高速旋转的3ml ddH2O中;
(4)将(3)中的混合液于搅拌器上自然蒸发4h(使丙酮完全蒸发),则获得PLGA载药纳米内核。
三、仿生间充质干细胞膜纳米载药***的制备及评估
本发明仿生间充质干细胞纳米颗粒的构建体系的关键点在于PLGA纳米内核稳定支持其表面膜材料,发明人设置比较在不同溶质中载药纳米颗粒的吸光度,通过检测其在水、油和1xPBS中的吸光度,对比在不同质量比下的材料的稳定性,确定膜生间充质干细胞膜纳米载药***是否成功制备。
(1)取步骤一中的间充质干细胞膜提取物和步骤二中PLGA载药纳米内核液混合在一起(膜蛋白和PLGA纳米颗粒质量比为1:2);
(2)将(1)中混合物100W水浴超声3min,即得仿生间充质干细胞膜纳米载药颗粒。
(3)将(2)中制备出的纳米颗粒分别溶解于水、油和1xPBS中,充分搅拌并于室温下静置10min,检测吸光度。
(4)海藻酸钠水凝胶的合成:海藻酸钠混合于水中,4°摇床混合溶解过夜,加入氯化钙后充分混匀,即得海藻酸钠水凝胶;
(5)将(2)的仿生间充质干细胞膜纳米载药颗粒与(4)的海藻酸钠水凝胶4°下充分混合。
由图2B可见,仿生间充质干细胞膜纳米颗粒与单独的双硫仑分别溶解于水、油和1xPBS中后,吸光度存在差异,在PBS中显示DSF-EP吸光度降低,表明包裹有仿生间充质干细胞膜的纳米颗粒其降解减少。由图2C可见,合成的仿生间充质干细胞膜纳米颗粒在电镜下较为均一,粒径约为150nm左右,其纳米颗粒表面有明显的完整双分子层结构,证明其生物膜覆盖。单独的PLGA纳米颗粒水合粒径约为180nm,电位为-35mV,单独的膜囊泡140nm左右,电位为-40mV,膜包裹PLGA内核后,和单独膜相比水合粒径降低至150nm,电位降低到-30mV,侧面证明膜修饰在PLGA纳米内核表面,膜包裹纳米颗粒制备较为稳定(图2C,图2D所示)。
实施例2DSF-EP促进糖尿病小鼠皮肤伤口愈合,实验过程如下:
(1)构建糖尿病小鼠模型
①C57随机选取3-4周雄性小鼠适应性饲养一周
②高脂高糖饲养6-8周,测量体重及空腹血糖水平
③小鼠禁食不禁水过夜(12h以上),称重并测量小鼠血糖水平,按动物数目和注射剂量来计算,将链脲佐菌素室温避光放置10min彻底解冻,称取所需的STZ(冻干粉),放到一干燥避光的无菌瓶内,用铝箔或锡箔纸包好;
④将柠檬酸缓冲液和装STZ的瓶子提前置于冰浴中,一起带到动物房。按照1%(w/v)10mg/ml浓度加入适量的柠檬酸缓冲液溶解STZ,充分溶解。按照动物空腹体重腹腔注射相应体积的STZ,需在30min内注射完毕;
⑤继续高糖饲料喂养,于打药后第2、3、5、7天检测血糖水平,连续两次空腹血糖水平大于11.1mmol/L的小鼠,认定为糖尿病小鼠。
(2)构建糖尿病小鼠皮肤损伤模型
①选取(1)中合格糖尿病小鼠称重麻醉后,剃去背部皮毛;
②每只小鼠均在背部制造一个1×1cm大小的圆形伤口
③每组小鼠分别给予PBS、PLGA、Exo-PLGA、DSF(双硫仑)、DSF-EP 100ul水凝胶伤口处涂抹,并与伤口生成后第0、3、6、9、12、15天观察伤口闭合情况。
由图3A可见每组小鼠伤口愈合情况,并由图3B愈合面积统计图可见,DSF-EP组(复合水凝胶)小鼠愈合速度最快且具有统计学意义,证明DSF-EP(复合水凝胶)可促进糖尿病小鼠皮肤伤口愈合。
实施例3DSF-EP抑制中性粒细胞NETs产生并作用于炎性因子,实验过程如下:
(1)分离糖尿病小鼠骨髓中性粒细胞
①取糖尿病小鼠胫骨股骨,用生理盐水吹出骨髓中的细胞并定容至5mL;
②取中性粒细胞提取试剂盒A液4mL于15mL离心管,小心滴加2mL C液于上层。此时A液和C液之间可见液面分隔,将骨髓细胞和生理盐水混合物滴加到最上层,950g离心20min;
③离心后液体分层,将含有中性粒细胞的分离液层转入新的15mL离心管中,加入生理盐水至12mL,950g离心10min,弃上清;
④加入1ml红细胞裂解液10min后用生理盐水终止,离心1800rpm,5min,弃上清;
⑤生理盐水或者无血清培养基重,计数;
(2)DSF-EP阻断中性粒细胞刺激产生NETs
①原代中性粒细胞铺板:将状态良好的中性粒细胞铺于12孔板中,5×105个细胞每孔,加入1mlRIPA培养基,分别加入1ulPBS、PMA(1mg/ml)刺激物后37℃孵育1h;
②DSF-EP处理原代中性粒细胞:每孔加入100ulPBS、PLGA、DSF、EXO-PLGA、DSF-EP,37℃孵育3h;
③收集②中培养上清,-30℃保存。
(3)DSF-EP培养上清抑制巨噬细胞分泌炎性因子,实验过程如下:
①原代巨噬粒细胞铺板:将新鲜的原代巨噬细胞铺于24孔板中,每孔含有3×105个中性粒细胞,培养液体积400μl;
②将(2)中的孵育液18000g离心15min取上清,加入①的巨噬细胞孔板中,补足培养基,使每孔培养液终体积为500μl,血清含量为10%;
③待②中的巨噬细胞37℃共孵育2h后,加入LPS后37℃共孵育2h;
④弃去③中上清,每孔加入200ul Trizol处理5min收集细胞,后提取细胞RNA后检测炎性因子表达。
结果如图4所示,经DSF-EP培养上清处理后的巨噬细胞,活化程度明显下降,DSF-EP培养上清能够抑制巨噬细胞分泌炎性因子,DSF-EP培养上清处理组巨噬细胞表达的IL-6及IL-1β显著下降,揭示DSF-EP在糖尿病皮肤创面处用于抗炎治疗的潜力,由此表示本发明在糖尿病皮肤创面中的可行性。
本发明的上述实施例仅仅是为了清楚地说明本发明技术方案的所作的举例,而并非是对本发明的具体实施方式的限定。凡在本发明权利要求书的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (10)
1.一种复合水凝胶,含有核-壳结构的纳米颗粒和凝胶成分,其特征在于,所述核由双硫仑和PLGA共聚物组成;所述壳为间充质干细胞膜;所述凝胶成分为海藻酸钠。
2.根据权利要求1所述复合水凝胶,其特征在于,所述纳米颗粒的直径为120~200nm。
3.根据权利要求1所述复合水凝胶,其特征在于,所述PLGA和双硫仑的质量比为10:0.5~3。
4.根据权利要求1所述复合水凝胶,其特征在于,所述壳核材料的混合质量比为0.125~0.5:1。
5.根据权利要求1所述复合水凝胶,其特征在于,所述核-壳结构的纳米颗粒与海藻酸钠的质量比为5:1。
6.一种权利要求1所述复合水凝胶的制备方法,其特征在于,包括以下步骤:
S1、提取间充质干细胞膜;
S2、PLGA载药纳米内核的合成:将PLGA和药物双硫仑混合,搅拌即得;
S3、将步骤S1的间充质干细胞膜和步骤S2的PLGA载药纳米内核混合,超声后即得仿生间充质干细胞膜纳米载药颗粒;
S4、海藻酸钠水凝胶的合成:海藻酸钠混合于水中制备海藻酸钠水凝胶;
S5、将步骤S3的仿生间充质干细胞膜纳米载药颗粒与步骤S4的海藻酸钠水凝胶4℃下混合即得。
7.根据权利要求6所述的复合水凝胶的制备方法,其特征在于,步骤S2磁力搅拌的条件为300~500rpm,温度为4~25℃,时间为4~48h。
8.根据权利要求6所述的复合水凝胶的制备方法,其特征在于,步骤S3所述水浴超声的功率为50W~100W,温度为4~25℃,时间为3~30分钟。
9.权利要求1至5任一所述复合水凝胶在制备用于糖尿病足或糖尿病皮肤损伤相关的药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述复合水凝胶用于促进糖尿病足或糖尿病皮肤伤口愈合,减少糖尿病足或糖尿病皮肤伤口愈合时间,减少糖尿病足或糖尿病皮肤损伤伤口处中性粒细胞NETs表达,调节免疫抑制伤口处IL-1α、IL-1β和/或IL-6。
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