CN117338956B - 一种用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物及其制备方法和应用 - Google Patents
一种用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物及其制备方法和应用,属于新型多功能纳米药物治疗技术领域。本发明利用二氧化硅为模板进行多巴胺包裹,利用氢氟酸刻蚀得到中空聚多巴胺,利用聚多巴胺的邻苯二酚结构容易被氧化成二醌结构,通过迈克尔加成反应表面修饰苯硼酸和透明质酸,从而实现双锚定铜绿假单胞菌以及炎症细胞。同时以中空聚多巴胺为载体载入甘草酸以及抗菌肽,聚多巴胺与甘草酸分别清除炎症级联反应早期产物活性氧(ROS)以及晚期产物细胞外高迁移率族蛋白B1(HMGB1),为临床防治铜绿假单胞菌性角膜炎等难治性角膜炎相关疾病提供新靶点和新策略。
Description
技术领域
本发明涉及新型多功能纳米药物治疗技术领域,尤其涉及一种用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物及其制备方法和应用。
背景技术
据世界卫生组织统计,角膜疾病是当今世界上导致视力丧失和盲的主要原因之一,全球至少有400万人受角膜疾病影响。细菌性角膜炎作为角膜疾病的主要原因,是一种常见的化脓性角膜炎,起病急、发展迅速,可在24~48h内完成角膜破坏。另外在细菌性角膜治疗过程中,如果短时间内细菌感染未得到有效控制,可导致角膜穿孔、感染扩散至邻近眼内组织,甚至引起化脓性眼内炎等严重后果,最终导致视力完全丧失。
目前,全身和眼局部抗生素治疗是细菌性角膜炎的一线治疗方法,但抗生素耐药和眼局部屏障的存在使治疗效果大打折扣。抗生素耐药性是目前感染性疾病药物治疗中不可回避的问题。在治疗细菌性角膜炎中常用的经验性抗生素如喹诺酮类药物、头孢菌素和氨基糖苷类抗生素已经存在明显的耐药趋势。此外,眼部特有的泪膜和角膜屏障也是导致抗生素生物利用度低不可避免的难点。泪膜屏障作用主要体现在持续的泪液周转(16%/min)导致的药物清除率高和药物稀释,使得药物在角膜前滞留时间只有短短的2min,并且8min后,药物会被稀释到1/1000,15~25min后,几乎所有的活性成分都消失。正是由于泪膜、角膜屏障的存在,仅有不到5%的药物能穿透角膜,生物利用度通常不到10%。因此,亟需一种新的方式来解决细菌性角膜炎的临床治疗中抗生素耐药和生物利用度低的难题。
近年来,纳米药物作为一种新的药物递送方法,在眼科疾病的治疗中崭露头角并广泛应用,为克服传统药物非特异性靶向、非选择性损伤机体组织以及生理屏障导致的药物生物利用度低的瓶颈问题提供了可能。纳米颗粒作为传导或输送药物的载体,是直径10~1000nm的固态胶体颗粒,一般由天然或合成高分子物质构成。与传统药物相比,纳米药物的首要优势体现在其粒径较小,能够更好地穿透生物膜、渗透细胞,提高药物在炎症组织中的浓度,增强治疗效果。其次,纳米药物可以利用带正电荷的纳米粒与带负电荷的角膜结合形成一种生物粘附,延长药物在眼内的滞留时间,实现长时间在病变组织维持最低有效治疗浓度。以上两个优势可以有效解决传统滴眼液角膜渗透性差、眼表停留时间短等难题。同时,纳米药物还可以通过靶向作用进一步提高药物的选择性,如利用表面修饰技术,将抗菌药物载体与靶向分子结合,特异性地定位于细菌感染部位,从而实现高效杀灭细菌,增强治疗效果。此外,针对细菌抗生素耐药性的棘手问题,有些纳米药物如金属及其氧化物纳米粒等自身便具备杀菌特性,例如,银纳米颗粒等金属纳米颗粒具有固有的抗炎、杀菌和抗血管生成功能,然而无机纳米材料虽然具有良好的杀菌效果且尺寸均匀、稳定性好,但它们的生物毒性仍然是一个挑战。因此如何利用有机纳米材料高度生物相容性的同时,实现良好的杀菌抗炎效果成为目前纳米药物研究的热点。
发明内容
有鉴于此,本发明的目的在于提供一种用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物及其制备方法和应用。本发明制得的级联免疫调控纳米药物能够实现滴眼液通过级联免疫调控、杀菌抗炎一体化促进铜绿假单胞菌性角膜炎的转归,且提出双向锚定理论,苯硼酸以及透明质酸分别实现了对细菌的捕杀结合以及对炎症的彻底清除。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物的制备方法,包括以下步骤:
将二氧化硅与多巴胺混合,得到多巴胺包裹的二氧化硅小球;
利用氢氟酸对所述多巴胺包裹的二氧化硅小球进行刻蚀,得到中空聚多巴胺;
将所述中空聚多巴胺、3-氨基苯硼酸和透明质酸混合进行迈克尔加成反应,得到修饰氨基苯硼酸和透明质酸的中空聚多巴胺;
将所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺、甘草酸和抗菌肽混合使甘草酸和抗菌肽负载于中空多巴胺内部,得到所述用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物。
优选地,所述二氧化硅与多巴胺的质量比为1:1~2。
优选地,所述中空聚多巴胺与3-氨基苯硼酸的质量比为1:4~5。
优选地,所述中空聚多巴胺与透明质酸的质量比为1:4~5。
优选地,所述迈克尔加成反应的时间为24~36h,温度为室温。
优选地,所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺与甘草酸的质量比为1:1~2。
优选地,所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺与抗菌肽的质量比为1:1~2。
优选地,所述抗菌肽为从林蛙皮中提取得到。
本发明还提供了上述技术方案所述制备方法制得的用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物。
本发明还提供了上述技术方案所述的用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物在制备治疗铜绿假单胞菌性角膜炎的药物中的应用。
本发明提供了一种用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物的制备方法,包括以下步骤:将二氧化硅与多巴胺混合,得到多巴胺包裹的二氧化硅小球;利用氢氟酸对所述多巴胺包裹的二氧化硅小球进行刻蚀,得到中空聚多巴胺;将所述中空聚多巴胺、3-氨基苯硼酸和透明质酸混合进行迈克尔加成反应,得到修饰氨基苯硼酸和透明质酸的中空聚多巴胺;将所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺、甘草酸和抗菌肽混合使甘草酸和抗菌肽负载于中空多巴胺内部,得到所述用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物。
与现有技术相比,本发明的有益效果如下:
本发明利用二氧化硅为模板进行多巴胺包裹,利用氢氟酸刻蚀得到中空聚多巴胺,利用聚多巴胺的邻苯二酚结构容易被氧化成二醌结构,通过迈克尔加成反应表面修饰苯硼酸和透明质酸,从而实现双锚定铜绿假单胞菌以及炎症细胞。同时以中空聚多巴胺为载体载入甘草酸以及抗菌肽,聚多巴胺与甘草酸分别清除炎症级联反应早期产物活性氧(ROS)以及晚期产物细胞外高迁移率族蛋白B1(HMGB1),最终制备出具备级联免疫调控杀菌-抗炎一体化的纳米药物。本发明通过级联免疫调控实现对铜绿假单胞菌性角膜炎病情发展的控制,同时兼具杀菌抗炎双重功效,苯硼酸和透明质酸双锚定的功效使其具备更加智能化的疗效,为临床防治铜绿假单胞菌性角膜炎等难治性角膜炎相关疾病提供新靶点和新策略;由于纳米药物的双向特异性锚定细菌及炎症细胞的功能,从而实现纳米药物在患病部位的智能长久停留,为克服临床用药生物利用度低、疗效差的问题提供新的解决思路与理论支持;同时纳米药物利用纳米尺寸优势更易穿透角膜屏障,提高生物利用度,且生物安全性好、易于操作。
附图说明
图1为SiO2和PDA@SiO2的SEM谱图;
图2为PDA@SiO2和HPDA的TEM谱图;
图3为为HPDA、HPDA-NBA和HPDA-NBA-HA的XPS谱图;
图4为甘草酸的标准曲线;
图5为不同浓度的HPDA-NBA-HA@GLA的药物释放曲线;
图6为抗菌肽的标准曲线;
图7为不同浓度的HPDA-NBA-HA@AMPS的药物释放曲线;
图8为不同浓度的HPDA-NBA-HA@GLA@AMPS的甘草酸释放曲线;
图9为不同浓度的HPDA-NBA-HA@GLA@AMPS的抗菌肽释放曲线;
图10为细菌铺板实验结果;
图11为不同药物浓度处理后,HCECs细胞内ROS含量的荧光显微镜图像;
图12为纳米药物滴眼液处理HCECs细胞后炎症因子Cox-2以及HMGB1的表达水平。
具体实施方式
本发明提供了一种用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物的制备方法,包括以下步骤:
将二氧化硅与多巴胺混合,得到多巴胺包裹的二氧化硅小球;
利用氢氟酸对所述多巴胺包裹的二氧化硅小球进行刻蚀,得到中空聚多巴胺;
将所述中空聚多巴胺、3-氨基苯硼酸和透明质酸混合进行迈克尔加成反应,得到修饰氨基苯硼酸和透明质酸的中空聚多巴胺;
将所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺、甘草酸和抗菌肽混合使甘草酸和抗菌肽负载于中空多巴胺内部,得到所述用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物。
在本发明中,若无特殊说明,使用的原料均为本领域市售商品。
本发明将二氧化硅与多巴胺混合,得到多巴胺包裹的二氧化硅小球。
在本发明中,所述二氧化硅与多巴胺的质量比优选为1:1~2。
在本发明中,所述混合优选为搅拌混合,所述搅拌混合的温度优选为室温,时间优选为24h,本发明对所述搅拌混合的转速没有特殊的限定。
在本发明中,所述混合完成后,本发明优选将所得混合物料离心得到样品,使用超纯水11000rpm,8min洗涤三次,然后冷冻真空干燥,得到所述多巴胺包裹的二氧化硅小球(PDA@SiO2)。
在本发明中,所述二氧化硅的粒径优选为90~130nm。
在本发明的具体实施例中,所述二氧化硅优选由包括以下步骤的方法制得:将350mL乙醇与7.5mL氨水和15mL硅酸四乙酯混合、搅拌5~6h后离心得到样品,使用甲醇11000rpm,8min洗涤三次,然后冷冻真空干燥,得到所述二氧化硅。
得到多巴胺包裹的二氧化硅小球后,本发明利用氢氟酸对所述多巴胺包裹的二氧化硅小球进行刻蚀,得到中空聚多巴胺。
在本发明中,所述多巴胺包裹的二氧化硅小球与氢氟酸的用量比优选为0.1g:300~600μL,所述氢氟酸优选为纯氢氟酸。
在本发明中,所述刻蚀的温度优选为室温,时间优选为1h。
所述刻蚀完成后,本发明优选将所得刻蚀后产物离心得到样品,使用超纯水11000rpm,8min洗涤三次,然后冷冻真空干燥,得到所述中空聚多巴胺。
得到中空聚多巴胺后,本发明将所述中空聚多巴胺、3-氨基苯硼酸和透明质酸混合进行迈克尔加成反应,得到修饰氨基苯硼酸和透明质酸的中空聚多巴胺。
在本发明中,所述中空聚多巴胺与3-氨基苯硼酸的质量比优选为1:4~5。
在本发明中,所述中空聚多巴胺与透明质酸的质量比优选为1:4~5。
在本发明中,所述迈克尔加成反应的时间优选为24~36h,温度优选为室温。本发明利用聚多巴胺的邻苯二酚结构容易被氧化成二醌结构,通过迈克尔加成反应表面修饰苯硼酸和透明质酸,从而实现双锚定铜绿假单胞菌以及炎症细胞。
在本发明中,所述迈克尔加成反应优选在水中进行。
所述迈克尔加成反应完成后,本发明优选将所得迈克尔加成产物离心得到样品,使用超纯水11000rpm,8min洗涤三次,然后冷冻真空干燥,得到所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺。
本发明优选先将所述中空聚多巴胺与3-氨基苯硼酸混合进行反应,再与透明质酸混合进行反应。
得到修饰氨基苯硼酸和透明质酸的中空聚多巴胺后,本发明将所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺、甘草酸和抗菌肽混合使甘草酸和抗菌肽负载于中空多巴胺内部,得到所述用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物。
在本发明中,所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺与甘草酸的质量比优选为1:1~2。
在本发明中,所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺与抗菌肽的质量比优选为1:1~2。本发明以所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺为载体载入甘草酸和抗菌肽,聚多巴胺与甘草酸分别清除炎症级联反应早期产物活性氧(ROS)以及晚期产物细胞外高迁移率族蛋白B1(HMGB1),最终制备出具备级联免疫调控杀菌-抗炎一体化的纳米药物。
本发明优选将所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺先与甘草酸在真空条件下在水溶液体系抽真空搅拌后,所得产物再与抗菌肽在真空条件下在水溶液体系抽真空搅拌。
在本发明中,所述抗菌肽优选为从林蛙皮中提取得到,所述抗菌肽具有杀菌作用,避免了抗生素耐药性这一临床难点。
本发明对所述抗菌肽的来源没有特殊的限定,采用市售商品即可。
在本发明中,所述混合优选在水中进行。
本发明优选将所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺、甘草酸和抗菌肽在真空条件下在水溶液体系抽真空搅拌24h,使甘草酸和抗菌肽负载于中空多巴胺内部,离心得到样品,使用超纯水11000rpm,8min洗涤三次,然后冷冻真空干燥,得到所述用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物(HPDA-NBA-HA@GLA@AMPS)。
本发明还提供了上述技术方案所述制备方法制得的用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物。
本发明还提供了上述技术方案所述的用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物在制备治疗铜绿假单胞菌性角膜炎的药物中的应用。
本发明对所述应用的具体方式没有特殊的限定,采用本领域技术人员熟知的方式即可。
下面将结合本发明中的实施例,对本发明中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1
一种用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物的制备方法,具体为以下步骤:350mL乙醇与7.5mL氨水、15mL硅酸四乙酯室温下混合、搅拌5h,离心得到样品,使用甲醇11000rpm,8min洗涤三次,冷冻真空干燥得到二氧化硅,二氧化硅与多巴胺按照质量比1:1室温下混合搅拌48h,离心得到样品,使用超纯水11000rpm,8min洗涤三次,冷冻真空干燥得到多巴胺包裹的二氧化硅小球(PDA@SiO2)。0.1g多巴胺包裹的二氧化硅小球使用300μL氢氟酸刻蚀1h,离心得到样品,使用超纯水11000rpm,8min洗涤三次,冷冻真空干燥得到中空聚多巴胺(HPDA),反应所得到的中空聚多巴胺和3-氨基苯硼酸按照1:4的质量比常温下在水溶液体系搅拌24h,离心得到固体(HPDA-NBA),然后将HPDA-NBA和透明质酸(中空聚多巴胺和透明质酸的质量比为1:4)常温下在水溶液体系搅拌24h,离心得到样品,使用超纯水11000rpm,8min洗涤三次,冷冻真空干燥得到修饰氨基苯硼酸和透明质酸的中空聚多巴胺(HPDA-NBA-HA),将修饰氨基苯硼酸和透明质酸的中空聚多巴胺与甘草酸按照1:1的质量比在真空条件下在水溶液体系抽真空搅拌24h,离心,得到的固体(HPDA-NBA-HA@GLA)与抗菌肽(修饰氨基苯硼酸和透明质酸的中空聚多巴胺与抗菌肽的质量比为1:1)在真空条件下在水溶液体系抽真空搅拌24h,离心得到样品,使用超纯水11000rpm,8min洗涤三次,冷冻真空干燥得到最终的级联免疫调控纳米药物(HPDA-NBA-HA@GLA@AMPS,纳米药物滴眼液)。
HPDA-NBA-HA@AMPS的制备:与HPDA-NBA-HA@GLA@AMPS的制备过程相同,区别仅在于不添加甘草酸。
级联免疫调控纳米药物(HPDA-NBA-HA@GLA@AMPS)杀菌性能测试
选用铜绿假单胞菌作为典型试验细菌。细菌菌株在37℃的条件下在液体培养基中生长12h。然后通过离心(3000rpm,6min)收集细菌,并用磷酸缓冲盐溶液洗涤三次。将得到的细菌重新分散在磷酸缓冲盐溶液中,并通过酶标仪调节细菌浓度,得到用于抗菌效果测试的细菌溶液,菌液浓度为107CFU/mL-1。
在测试之前,将所有样品(目标样品与对照样品)灭菌。随后菌液(107CFU/mL-1,铜绿假单胞菌,100μL)与五种不同合成阶段的纳米药物共培养4h,进行铺板实验。
胞内ROS清除研究
采用活性氧检测试剂盒检测纳米药物处理后细胞内ROS的含量。将HCECs细胞均匀分散在6孔细胞培养板中(密度为1×105个/孔)继续孵育24h。加入LPS(10μg/mL)刺激细胞12h,使细胞处于炎症状态。PBS清洗后加入不同浓度的HPDA-NBA-HA@GLA@AMPS(0、50、100、200μg/mL)处理细胞24h,不加任何刺激作为空白对照组。然后将细胞与DCFH-DA染色工作液(5μM)在37℃下避光孵育30min。用无血清细胞培养基充分洗涤细胞以去除多余染液,使用DAPI染色8min后标记细胞核,用倒置荧光显微镜采集荧光图像。
沉默HMGB1表达水平
蛋白提取和定量
HCECs细胞铺板贴壁后,使用LPS刺激12h后加入纳米药物处理24h,选择状态良好的细胞提取总蛋白。所有操作均在冰上进行。PBS清洗3遍后吸干,加入细胞裂解液(RIPA裂解液中分别加入1/100PMSF和磷酸酶抑制剂)冰上裂解10min,细胞刮刀刮取细胞转移至EP管中继续裂解30min,每10min振荡混匀一次。4℃,12000rpm离心30min后,获得的上清液即为目标蛋白。随后,在96孔板中加入含蛋白的BCA检测液(质量比蛋白:PBS:BCA工作液=1:24:200)后室温处理30min,用酶标仪采集562nm处吸光度。参考预先测定的标准曲线计算得出样品蛋白浓度。往剩余蛋白样品中加入5×Loading buffer混匀后放置于金属浴上加热(95℃,10min)使蛋白变性,待蛋白样品冷却后分装放入-80℃冰箱保存待用。
凝胶配制
仔细清洗1.5mm凝胶厚度用长、短玻璃板并晾干,组装在电泳夹上。加水检查组装是否紧密以防止液体渗漏,弃水并用滤纸吸干。吸取购买的试剂盒的下层胶溶液(4mL)、下层胶缓冲液(4mL)和促凝剂(80μL)混匀后配制成下层胶混合液,注入到玻璃板中合适位置,加入无水乙醇压平胶面,室温凝固10min,倒掉无水乙醇,滤纸吸干。随后,吸取购买的试剂盒的上层胶溶液(1mL)、上层胶缓冲液(1mL)和促凝剂(20μL)混合均匀,加入到玻璃板中,插入齿梳,注意防止气泡产生,静置10min后上层胶完全凝固,去除梳子,准备上样电泳。
上样和电泳
组装电泳装置,注入电泳缓冲液直到液面与电泳板上端相平。依次加入预染蛋白Marker和蛋白样品,准确连接电泳正负极。先将电泳仪电压调整至80V进行电泳,待预染蛋白Marker分开成清楚的蛋白条带后,更换电压为120V继续电泳直至蛋白条带跑到电泳槽底端。
转膜
准备所需大小的PVDF膜,于甲醇中浸泡激活,倒入少许转膜液到转膜托盘中。电泳完成后取下凝胶,裁掉上层胶后转移至托盘中平衡数秒。按照海绵垫→滤纸→PVDF膜→凝胶→滤纸→海绵垫的顺序正确安装转膜夹,去除各层间气泡后置于电转槽中,加入转膜液。电转槽周围覆盖足够的冰块降温,并调整电流参数为220mA,转膜75min。转膜前需要注意标记加样顺序。
封闭和抗原抗体结合
转膜完成后取出PVDF膜,加入封闭液振荡反应2h。用TBST溶液清洗3次,每次10min以充分去除残留封闭液。按照分子量裁剪PVDF膜至合适大小,并浸没在含有一抗稀释液稀释后的抗体孵育盒中,放于4℃摇床上摇晃过夜。次日,去除一抗后使用TBST清洗PVDF膜3次,加入二抗溶液并于室温下摇床上作用1h,去除二抗,TBST漂洗三次。
化学显影
取出PVDF膜,吸除多余液体后放置于显影板上,均匀滴加显影工作液,使用化学发光成像系统拍摄蛋白条带图。用Image J软件分析条带平均灰度值得到蛋白相对表达量。
图1为SiO2和PDA@SiO2的SEM谱图,证明了二氧化硅、聚多巴胺包裹二氧化硅的成功合成。
图2为PDA@SiO2和HPDA的TEM谱图,证明了聚多巴胺包裹二氧化硅、中空聚多巴胺的成功合成。
图3为HPDA、HPDA-NBA和HPDA-NBA-HA的XPS谱图,表1为HPDA、HPDA-NBA和HPDA-NBA-HA的XPS谱图数据,根据硼元素的峰值说明3-氨基苯硼酸的成功修饰。
表1HPDA和HPDA-NBA的XPS谱图数据
图4为甘草酸的标准曲线,图5为不同浓度的HPDA-NBA-HA@GLA的药物释放曲线,图6为抗菌肽的标准曲线,图7为不同浓度的HPDA-NBA-HA@AMPS的药物释放曲线,图8为不同浓度的HPDA-NBA-HA@GLA@AMPS的甘草酸释放曲线,图9为不同浓度的HPDA-NBA-HA@GLA@AMPS的抗菌肽释放曲线,通过药物释放曲线不仅证明了中空聚多巴胺内部成功负载甘草酸、抗菌肽,同时说明纳米药物具有缓释功效。
图10为细菌铺板实验结果,每一行分别为不同浓度纳米药物的铺板结果,每一纵列代表不同纳米药物处理组,通过细菌铺板可以证明最终合成的纳米药物具有良好的杀局功效。
图11为胞内ROS清除实验,不同药物浓度处理后,HCECs细胞内ROS含量的荧光显微镜图像,绿色:DCFH-DA标记的ROS,蓝色:细胞核,标尺为100μm,证明了纳米药物具有良好的ROS清除效果。
图12为纳米药物滴眼液处理HCECs细胞后炎症因子Cox-2以及HMGB1的表达水平,通过Western blot实验探究了角膜上皮细胞中的Cox-2和HMGB1的蛋白表达水平,证明纳米药物具有良好的抗炎效果以及沉默HMGB1的蛋白表达。
以上所述仅是本发明的优选实施方式,并非对本发明作任何形式上的限制。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物的制备方法,其特征在于,包括以下步骤:
将二氧化硅与多巴胺混合,得到多巴胺包裹的二氧化硅小球;
利用氢氟酸对所述多巴胺包裹的二氧化硅小球进行刻蚀,得到中空聚多巴胺;
将所述中空聚多巴胺、3-氨基苯硼酸和透明质酸混合进行迈克尔加成反应,得到修饰氨基苯硼酸和透明质酸的中空聚多巴胺;
将所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺、甘草酸和抗菌肽混合使甘草酸和抗菌肽负载于中空多巴胺内部,得到所述用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物。
2.根据权利要求1所述的制备方法,其特征在于,所述二氧化硅与多巴胺的质量比为1:1~2。
3.根据权利要求1所述的制备方法,其特征在于,所述中空聚多巴胺与3-氨基苯硼酸的质量比为1:4~5。
4.根据权利要求1或3所述的制备方法,其特征在于,所述中空聚多巴胺与透明质酸的质量比为1:4~5。
5.根据权利要求1所述的制备方法,其特征在于,所述迈克尔加成反应的时间为24~36h,温度为室温。
6.根据权利要求1所述的制备方法,其特征在于,所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺与甘草酸的质量比为1:1~2。
7.根据权利要求1或6所述的制备方法,其特征在于,所述修饰氨基苯硼酸和透明质酸的中空聚多巴胺与抗菌肽的质量比为1:1~2。
8.根据权利要求1所述的制备方法,其特征在于,所述抗菌肽为从林蛙皮中提取得到。
9.权利要求1~8任一项所述制备方法制得的用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物。
10.权利要求9所述的用于治疗铜绿假单胞菌性角膜炎的级联免疫调控纳米药物在制备治疗铜绿假单胞菌性角膜炎的药物中的应用。
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Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000009553A2 (en) * | 1998-08-14 | 2000-02-24 | Bionebraska, Inc. | Antimicrobial peptides isolated from the skin of american frogs |
CN104478996A (zh) * | 2014-12-08 | 2015-04-01 | 辽宁师范大学 | 一种新型阳离子抗菌肽及其应用 |
CN106139144A (zh) * | 2016-06-27 | 2016-11-23 | 华中科技大学同济医学院附属协和医院 | 一种具有协同抗肿瘤特性的透明质酸修饰的金‑碳纳米球及其制备方法与应用 |
CN111110652A (zh) * | 2020-01-06 | 2020-05-08 | 重庆医科大学 | 一种载药透明质酸聚多巴胺包覆介孔聚多巴胺纳米粒及其制备方法 |
CN113713081A (zh) * | 2021-10-11 | 2021-11-30 | 通化康元生物科技有限公司 | 林蛙抗菌肽在制备抗幽门螺旋杆菌药物中的应用 |
CN115812085A (zh) * | 2020-03-10 | 2023-03-17 | 国家科学研究中心 | 动态共价水凝胶、其前体及其用途 |
CN116270541A (zh) * | 2023-03-28 | 2023-06-23 | 中山大学 | 抗铜绿假单胞菌感染的纳米粒及制备方法和肺部吸入制剂 |
CN116327970A (zh) * | 2023-03-21 | 2023-06-27 | 北京积水潭医院 | 活性氧响应超润滑纳米载药体系的制备及应用 |
CN116813963A (zh) * | 2023-03-22 | 2023-09-29 | 山东第一医科大学附属眼科医院(山东省眼科医院) | 一种角膜塑形镜原材料表面修饰材料及其制备方法 |
CN116920165A (zh) * | 2023-06-02 | 2023-10-24 | 西北工业大学 | 一种原位产氧的水凝胶及其制备方法和应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110009352A1 (en) * | 2009-07-09 | 2011-01-13 | Minophagen Pharmaceutical Co., Ltd. | Restorative agent for antibacterial peptide production ability |
-
2023
- 2023-11-08 CN CN202311485625.7A patent/CN117338956B/zh active Active
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000009553A2 (en) * | 1998-08-14 | 2000-02-24 | Bionebraska, Inc. | Antimicrobial peptides isolated from the skin of american frogs |
CN104478996A (zh) * | 2014-12-08 | 2015-04-01 | 辽宁师范大学 | 一种新型阳离子抗菌肽及其应用 |
CN106139144A (zh) * | 2016-06-27 | 2016-11-23 | 华中科技大学同济医学院附属协和医院 | 一种具有协同抗肿瘤特性的透明质酸修饰的金‑碳纳米球及其制备方法与应用 |
CN111110652A (zh) * | 2020-01-06 | 2020-05-08 | 重庆医科大学 | 一种载药透明质酸聚多巴胺包覆介孔聚多巴胺纳米粒及其制备方法 |
CN115812085A (zh) * | 2020-03-10 | 2023-03-17 | 国家科学研究中心 | 动态共价水凝胶、其前体及其用途 |
CN113713081A (zh) * | 2021-10-11 | 2021-11-30 | 通化康元生物科技有限公司 | 林蛙抗菌肽在制备抗幽门螺旋杆菌药物中的应用 |
CN116327970A (zh) * | 2023-03-21 | 2023-06-27 | 北京积水潭医院 | 活性氧响应超润滑纳米载药体系的制备及应用 |
CN116813963A (zh) * | 2023-03-22 | 2023-09-29 | 山东第一医科大学附属眼科医院(山东省眼科医院) | 一种角膜塑形镜原材料表面修饰材料及其制备方法 |
CN116270541A (zh) * | 2023-03-28 | 2023-06-23 | 中山大学 | 抗铜绿假单胞菌感染的纳米粒及制备方法和肺部吸入制剂 |
CN116920165A (zh) * | 2023-06-02 | 2023-10-24 | 西北工业大学 | 一种原位产氧的水凝胶及其制备方法和应用 |
Non-Patent Citations (4)
Title |
---|
Jackie Fule Liu等."Smart" Matrix Microneedle Patch Made of Self-Crosslinkable and Multifunctional Polymers for Delivering Insulin On-Demand.Adv. Sci..2023,(第10期),第1-10页. * |
Song Yi Lee等.Ferrocene and glucose oxidase-installed multifunctional hydrogel reactors for local cancer therapy.Journal of Controlled Release.2022,(第349期),第617–633页. * |
陈家凯.基于苯硼酸敏感性和靶向性修饰的介孔硅载药递送系统.中国优秀硕士学位论文全文数据库(电子期刊) 工程科技Ⅰ辑.2018,(第09期),第B016-146页. * |
黄乔.基于苯硼酸和介孔二氧化硅的葡萄糖响应胰岛素释放体系的制备和性能研究.中国优秀硕士学位论文全文数据库(电子期刊) 工程科技Ⅰ辑.2022,(第1期),第B016-1850页. * |
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