CN116270549A - 一种靶向结肠炎的氧化苦参碱的纳米粒及其制备方法和应用 - Google Patents
一种靶向结肠炎的氧化苦参碱的纳米粒及其制备方法和应用 Download PDFInfo
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- CN116270549A CN116270549A CN202310275271.7A CN202310275271A CN116270549A CN 116270549 A CN116270549 A CN 116270549A CN 202310275271 A CN202310275271 A CN 202310275271A CN 116270549 A CN116270549 A CN 116270549A
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- chitosan
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- matrine
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Abstract
一种靶向结肠炎的氧化苦参碱的纳米粒及其制备方法和应用,属于口服药物靶向治疗炎症领域,具体涉及一种靶向结肠的唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒,该纳米粒能够通过对氧化苦参碱的保护以及结肠炎部位的靶向聚集,解决了其在体内药物易降解、不稳定以及无法到达结肠炎病变部位等问题,在纳米载体的保护和递送下大部分药物能完整的通过胃和小肠到达病变结肠,并长时间的聚集和滞留在病变的结肠部位,缓慢释药,从而发挥更好的体内治疗效果。该纳米粒给药系统工艺简单,容易重复,原材料价廉易得,环境友好,生物相容性良好。本发明实现了结肠及结肠病变部位的靶向给药,为治疗结肠炎提供了研究方向。
Description
技术领域
本发明属于口服药物靶向治疗炎症领域,涉及水溶性荷正电活性成分纳米载体的制备方法和应用,具体涉及一种靶向结肠的唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒及其制备方法和应用。
背景技术
溃疡性结肠炎是一种病因不明的慢性复发性胃肠道疾病,病变多位于乙状结肠和直肠,也可延伸至降结肠,甚至整个结肠。病程漫长,且反复发作。患者临床多表现为远端结肠和直肠的弥漫性黏膜炎症,同时伴有直肠出血、腹泻、腹痛和体重减轻。近年来,其发病率以惊人的速度增加。目前,常用的治疗药物包括:(1)氨基水杨酸类药物如美沙拉嗪,是肠炎最常用的药物,适用于轻中型患者,然而此类药存在着许多不良反应,故不可长期使用。(2)糖皮质激素类药物如泼尼松,对于急性发作期有较好的疗效,适用于中重型活动期患者。(3)免疫抑制剂类药物如环孢素,这一类药物应用后需要注意它的副作用,主要包括骨髓抑制等。
纵观现有的治疗方法及制剂类型,几乎全部为全身性用药,尚无靶向制剂或靶向给药方法。这将对结肠外的健康组织器官带来毒副作用。
氧化苦参碱(oxymatrine)主要提取自槐属(Sophor)中的苦参(S.flave scens)、越南槐(S.tonkinensis,山豆根常为其地方名)、苦豆子(S.alopecuroides)、和白刺花(S.viciifo lia),具有广泛的生物活性,如具有保护心、肝、肺、肾、脑、血管作用,同时对心脏有正性肌力、负性频率、抗心律失常的作用,还有升高白细胞,平喘,抗溃疡,抗肝纤维化,镇静,催眠,镇痛,抗癫痫等中枢神经药理作用,也具有抗肿瘤,免疫调节,抗菌,抗病毒,抗炎等药理作用,是一个药理作用广泛且治疗效果良好的中药活性成分。经实验,其抗炎机制主要是通过上调微小RNA-211-5p表达,抑制TLR4/MyD88/NF-κB号通路,下调吞噬细胞、淋巴细胞和受损组织细胞的炎性细胞因子(如IL、TNF-α、抗体等)的表达和上调抗炎细胞因子IL-10的表达从而产生抗炎作用。氧化苦参碱还可通过抑制环氧化酶,抑制炎症介质前列腺素生物合成以及抑制花生四烯酸转化成白三烯,也可通过稳定细胞膜抑制肥大细胞释放组胺,产生抗炎作用。
关于氧化苦参碱虽然具有广泛的药理作用,但是将其制成口服靶向制剂用于结肠炎的治疗未见报道。分析原因如下:(1)氧化苦参碱是一种亲水性很强的弱碱性药物,较难透过生物膜屏障;(2)在机体内和胃肠道内很容易被代谢成为毒性更大的苦参碱;(3)氧化苦参碱半衰期短,分布和消除都迅速,往往到不了病灶部位就会被代谢消除。
若将其制成适当的制剂,会明显提高其病变部位的药物浓度,延长其在病变部位的滞留时间,从而很大程度上提高其药效。因此本发明将氧化苦参碱利用制成结肠炎靶向的口服纳米粒。
纳米制剂的优点有:(1)能够有效地将生物活性分子输送到病变部位,提高治疗效率;(2)由于其独特的纳米尺寸,基于增强的渗透和滞留(EPR)效应,可以积聚在溃疡性结肠炎的病变部位;(3)减少传统药物的副作用;(4)提高药物的生物利用度。碳水化合物大分子因其良好的生物相容性和生物降解性被广泛用作纳米载体。其中,果胶(Pectin)是一种天然高分子多糖,它广泛分布于植物的果实、根、茎、叶等几乎所有的部位。研究表明,果胶对人体的健康有许多积极的作用,如降低人体内的血糖、胆固量,提高人体免疫力,降低癌症发病率等。选择果胶作为载体材料是因为:首先,果胶本身对溃疡性结肠炎就有一定的治疗作用;其次,果胶是负电性很强的多糖,用果胶制备荷负电荷的纳米粒,有利于在带正电的结肠病变部位实现黏附。最后,果胶受胃肠道pH的影响很小,只在结肠中特殊菌群释放的果胶酶作用下才会降解。普通的果胶纳米粒一般能实现的结肠部位靶向聚集释药,唾液酸修饰的果胶纳米粒可以特异性靶向巨噬细胞,不但有利于提高药物对结肠炎部位的靶向和黏附作用,还有利于增加药物在病变部位的摄取量,提高了治疗效果。
综上,本发明利用壳聚糖-接枝-唾液酸物理交联果胶制备成负载氧化苦参碱的结肠靶向黏附纳米粒,纳米粒通过对氧化苦参碱的保护以及结肠炎部位的靶向聚集解决了其在体内药物易降解、不稳定以及无法到达病变部位等问题。
发明内容
本发明的一个目的在于提供一种靶向结肠的唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒,其以氧化苦参碱为活性药物,具有合适的粒径和较高的载药量与包封率。
其中,所述的结肠靶向纳米粒由荷正电的壳聚糖-接枝-唾液酸、含氧化苦参碱的氯化钙溶液与荷负电的果胶经静电复合作用自组装而成。所述的壳聚糖-接枝-唾液酸为唾液酸与壳聚糖通过酰胺键、酯键结合的产物,所述的壳聚糖重均分子量为1000-10000,脱乙酰度为40%–99%,所述的唾液酸接入率为5%–60%,所述的果胶重均分子量为20000-400000,酯化度为50%-99%,壳聚糖-接枝-唾液酸交联果胶纳米粒,包裹药物,通过保护药物在胃肠道内不降解不释放,以提高氧化苦参碱的在病变的结肠部位的聚集量。通过提高药物在病变部位的聚集量和黏附性实现氧化苦参碱对溃疡性结肠炎的治疗效果。
本发明的又一个目的在于提供一种靶向结肠的唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒在制备治疗溃疡性结肠炎药物、食品或保健品中的应用。
本发明所述的一种靶向结肠的唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒在制备治疗结肠炎的口服靶向制剂中的应用。
本发明针对溃疡性结肠炎制备的唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒的制备方法,采用的技术方案如下:
首先,唾液酸经过活化后与壳聚糖反应制得壳聚糖-接枝-唾液酸;其次,将氧化苦参碱溶于氯化钙溶液中形成含有氧化苦参碱的氯化钙溶液,而后将制备的含有氧化苦参碱的氯化钙溶液滴入果胶溶液中使其自组装形成负载氧化苦参碱的果胶纳米粒;最后,将壳聚糖-接枝-唾液酸的溶液加入到荷负电的负载氧化苦参碱的果胶纳米粒溶液中,通过静电复合作用进一步自组装压缩纳米粒形成结肠靶向的纳米粒。
上述制备方法中,所述的壳聚糖-接枝-唾液酸材料的制备,包括如下步骤:
以1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)为交联剂,将唾液酸与壳聚糖化学偶联:壳聚糖溶解于0.1M的盐酸水溶液中制成壳聚糖溶液,壳聚糖的重复氨基葡萄糖单元上的部分氨基经过质子化转化为亲水性的离子R-NH3 +形式;唾液酸溶解在去离子水中,将EDC和NHS加入到唾液酸水溶液中,将混合物在室温下搅拌以活化羧基,然后与制备的壳聚糖溶液混合,反应后,用截留分子量为500Da的透析袋透析纯化,取透析完后的液体离心,弃去沉淀,取上清液冷冻干燥得壳聚糖-接枝-唾液酸,储存待用。
所述负载氧化苦参碱的果胶纳米粒与所述壳聚糖-接枝-唾液酸材料静电复合形成结肠靶向纳米粒,具体制备步骤如下:
称取氧化苦参碱,加入已过0.22μm微孔滤膜的5%~50%的氯化钙溶液,超声溶解,制得含氧化苦参碱的氯化钙溶液,外观澄清透明。氧化苦参碱在氯化钙水溶液中的质量浓度为2.7%~20.0%,优选氧化苦参碱的质量浓度为10%~15%;优选氯化钙的质量浓度为10%~30%,更优选氯化钙的浓度为18%~23%。
称取果胶粉末,使其充分溶解于蒸馏水中,将其过0.22μm微孔滤膜得果胶溶液,外观澄清透明。制得果胶溶液的质量浓度为0.01%~0.1%;优选果胶溶液的质量浓度为0.03%~0.08%;更优选果胶溶液的质量浓度为0.06%~0.08%。
称取壳聚糖-接枝-唾液酸,使其充分溶解于蒸馏水中,将其过0.22μm微孔滤膜得壳聚糖-接枝-唾液酸溶液,外观为淡黄色透明溶液。制得壳聚糖-接枝-唾液酸溶液的质量浓度为0.01%~0.1%,优选壳聚糖-接枝-唾液酸溶液的质量浓度为0.02%~0.08%,更优选壳聚糖-接枝-唾液酸溶液的质量浓度为0.04%~0.06%。
在37℃的水浴下,将含氧化苦参碱的氯化钙溶液在搅拌的条件下逐滴滴入到制备的果胶溶液中,搅拌均匀,然后滴入壳聚糖-接枝-唾液酸溶液,搅拌,即得壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱的结肠靶向黏附纳米粒。
本发明制备方法制备得到的纳米粒,粒径分布为80nm~700nm,Zeta电位为-10mV~-30mV。
本发明制备方法制备得到的纳米粒,载药量为2%~30%,包封率为60%~90%。
本发明中所述的“治疗”是指,为了阻止和降低疾病的发生或发展,使疾病病程的发展或加重得以抑制、遏制、减轻、改善、减缓、停止、延迟或反转,所描述的保持和/或用药时的疾病的、紊乱的或病理学状态的各种指标包括减轻或减少症状或并发症,或治愈或消除疾病、紊乱或状况。
本发明的有益效果:
本发明利用壳聚糖-接枝-唾液酸物理交联果胶制备成负载氧化苦参碱的结肠靶向黏附纳米粒,该纳米粒能够通过对氧化苦参碱的保护以及结肠炎部位的靶向聚集,解决了其在体内药物易降解、不稳定以及无法到达结肠炎病变部位等问题,在纳米载体的保护和递送下大部分药物能完整的通过胃和小肠到达病变结肠,并长时间的聚集和滞留在病变的结肠部位,缓慢释药,从而发挥更好的体内治疗效果。此外,该纳米粒给药系统工艺简单,容易重复,原材料价廉易得,环境友好,生物相容性良好,因此,具有良好的潜力和广阔的前景。本发明实现了结肠及结肠病变部位的靶向给药,为治疗结肠炎提供了研究方向。
附图说明
图1为实施例1、2、3、4、5中的唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒的制备方法流程图;
图2为实施例4中的唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒的粒径图;
图3为实施例6中的壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱纳米粒的体外释放图;
图4为实施例7中的壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱纳米粒的体内黏附靶向图,其中1为正常动物+壳聚糖-接枝-唾液酸/FITC交联果胶纳米粒组,2为模型动物+FITC组,3为模型动物+FITC交联果胶纳米粒组,4为模型动物+壳聚糖-接枝-唾液酸/FITC交联果胶纳米粒组;
图5为实施例8壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱的纳米粒对DSS诱导的溃疡性结肠炎的体重的影响;
图6为实施例8的结肠长度图;
图7为实施例8的IL-1β的免疫组化图。
具体实施方式
实施例1
唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒的制备,制备流程如图1所示。
称取18mg的氧化苦参碱原料药,向其中加入已过0.22μm微孔滤膜的质量浓度为18%的氯化钙溶液0.6mL,超声5min溶解,即得含3.0%氧化苦参碱的氯化钙溶液,外观呈澄清透明。
先称取7.5mg的果胶粉末,使其充分溶解于4.5g的蒸馏水中,配置成质量浓度为0.167%的果胶溶液,再取1.35g的0.1667%的果胶溶液,加蒸馏水至9mL,最终得质量浓度为0.025%的果胶溶液,将其过0.22μm微孔滤膜储存待用。
先称取2mg的壳聚糖-接枝-唾液酸材料,使其充分溶解于2g的蒸馏水中,配置成质量浓度为0.1%的壳聚糖-接枝-唾液酸溶液,再取1.2g的0.1%的唾液酸-接枝-唾液酸溶液,加蒸馏水至6mL,最终得质量浓度为0.02%的壳聚糖-接枝-唾液酸溶液,将其过0.22μm微孔滤膜储存待用。
其中,壳聚糖-接枝-唾液酸材料得制备方法参考了相关文献方法(Rana et al.,2021).Rana,R.,Rani,S.,Kumar,V.,Nakhate,K.T.,Ajazuddin,&Gupta,U.(2021).SialicAcid Conjugated Chitosan Nanoparticles:Modulation to Target Tumour Cells andTherapeutic Opportunities.AAPS PharmSciTech,23(1),10.https://doi.org/10.1208/s12249-021-02170-z。
在37℃的水浴搅拌下,将0.6mL含3.0%氧化苦参碱的氯化钙溶液在870r/min搅拌的条件下逐滴滴入到提前溶解好的7.8mL质量浓度为0.025%果胶溶液中,再逐滴滴入质量浓度为0.02%壳聚糖-接枝-唾液酸溶液8mL,搅拌固化2h,即得壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱的结肠靶向黏附纳米粒。
实施例2
唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒的制备,制备流程如图1所示。
称取42mg的氧化苦参碱原料药,向其中加入已过0.22μm微孔滤膜的质量浓度为20%的氯化钙溶液0.6mL,超声5min溶解,即得含7.0%氧化苦参碱的氯化钙溶液,外观呈澄清透明。
先称取15mg的果胶粉末,使其充分溶解于4.5g的蒸馏水中,配置成质量浓度为0.333%的果胶溶液,再取1.35g的0.333%的果胶溶液,加蒸馏水至9mL,最终得质量浓度为0.05%的果胶溶液,将其过0.22μm微孔滤膜储存待用。
先称取4mg的壳聚糖-接枝-唾液酸材料,使其充分溶解于2g的蒸馏水中,配置成质量浓度为0.2%的壳聚糖-接枝-唾液酸溶液,再取1.2g的0.2%的唾液酸-接枝-唾液酸溶液,加蒸馏水至6mL,最终得质量浓度为0.04%的壳聚糖-接枝-唾液酸溶液,将其过0.22μm微孔滤膜储存待用。
在37℃的水浴搅拌下,将0.6mL含7.0%氧化苦参碱的氯化钙溶液在870r/min搅拌的条件下逐滴滴入到提前溶解好的7.8mL质量浓度为0.05%果胶溶液中,再逐滴滴入质量浓度为0.04%壳聚糖-接枝-唾液酸溶液8mL,搅拌固化2h,即得壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱的结肠靶向黏附纳米粒。
实施例3
唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒的制备,制备流程如图1所示。
称取60.0mg的氧化苦参碱原料药,向其中加入已过0.22μm微孔滤膜的质量浓度为23%的氯化钙溶液0.6mL,超声5min溶解,即得10%氧化苦参碱的氯化钙溶液,外观呈澄清透明。
先称取22.5mg的果胶粉末,使其充分溶解于4.5g的蒸馏水中,配置成质量浓度为0.5%的果胶溶液,再取1.35g的0.5%的果胶溶液,加蒸馏水至9mL,最终得质量浓度为0.075%的果胶溶液,将其过0.22μm微孔滤膜储存待用。
先称取6mg的壳聚糖-接枝-唾液酸材料,使其充分溶解于2g的蒸馏水中,配置成质量浓度为0.3%的壳聚糖-接枝-唾液酸溶液,再取1.2g的0.3%的唾液酸-接枝-唾液酸溶液,加蒸馏水至6mL,最终得质量浓度为0.06%的壳聚糖-接枝-唾液酸溶液,将其过0.22μm微孔滤膜储存待用。
在37℃的水浴搅拌下,将0.6mL含10%氧化苦参碱的氯化钙溶液在870r/min搅拌的条件下逐滴滴入到提前溶解好的7.8mL质量浓度为0.075%果胶溶液中,再逐滴滴入质量浓度为0.06%壳聚糖-接枝-唾液酸溶液8mL,搅拌固化2h,即得壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱的结肠靶向黏附纳米粒。
实施例4
唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒的制备,制备流程如图1所示。
称取90mg的氧化苦参碱原料药,向其中加入已过0.22μm微孔滤膜的质量浓度为20%的氯化钙溶液0.6mL,超声5min溶解,即得含15%氧化苦参碱的氯化钙溶液,外观呈澄清透明。
先称取30mg的果胶粉末,使其充分溶解于4.5g的蒸馏水中,配置成质量浓度为0.666%的果胶溶液,再取1.35g的0.666%的果胶溶液,加蒸馏水至9mL,最终得质量浓度为0.1%的果胶溶液,将其过0.22μm微孔滤膜储存待用。
先称取8mg的壳聚糖-接枝-唾液酸材料,使其充分溶解于2g的蒸馏水中,配置成质量浓度为0.4%的壳聚糖-接枝-唾液酸溶液,再取1.2g的0.4%的唾液酸-接枝-唾液酸溶液,加蒸馏水至6mL,最终得质量浓度为0.08%的壳聚糖-接枝-唾液酸溶液,将其过0.22μm微孔滤膜储存待用。
在37℃的水浴搅拌下,将0.6mL含15%氧化苦参碱的氯化钙溶液在870r/min搅拌的条件下逐滴滴入到提前溶解好的7.8mL质量浓度为0.1%果胶溶液中,再逐滴滴入质量浓度为0.08%壳聚糖-接枝-唾液酸溶液8mL,搅拌固化2h,即得壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱的结肠靶向黏附纳米粒。
制备的唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒的表面形态和粒径、电位通过扫描电镜和马尔文进行测量如图2所示。
因为本药物(氧化苦参碱)是水溶性药物,而本发明制备的纳米粒是不溶性物质,因此,包封率88%和载药量15%是通过超滤法来进行测定得到的。
实施例5
唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒的制备,制备流程如图1所示。
称取108mg的氧化苦参碱原料药,向其中加入已过0.22μm微孔滤膜的质量浓度为30%的氯化钙溶液0.6mL,超声5min溶解,即得含18%氧化苦参碱的氯化钙溶液,外观呈澄清透明。
先称取30mg的果胶粉末,使其充分溶解于4.5g的蒸馏水中,配置成质量浓度为0.666%的果胶溶液,再取1.35g的0.666%的果胶溶液,加蒸馏水至9mL,最终得质量浓度为0.1%的果胶溶液,将其过0.22μm微孔滤膜储存待用。
先称取10mg的壳聚糖-接枝-唾液酸材料,使其充分溶解于2g的蒸馏水中,配置成质量浓度为0.5%的壳聚糖-接枝-唾液酸溶液,再取1.2g的0.5%的唾液酸-接枝-唾液酸溶液,加蒸馏水至6mL,最终得质量浓度为0.1%的壳聚糖-接枝-唾液酸溶液,将其过0.22μm微孔滤膜储存待用。
在37℃的水浴搅拌下,将0.6mL含18%氧化苦参碱的氯化钙溶液在870r/min搅拌的条件下逐滴滴入到提前溶解好的7.8mL质量浓度为0.1%果胶溶液中,再逐滴滴入质量浓度为0.1%壳聚糖-接枝-唾液酸溶液8mL,搅拌固化2h,即得壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱的结肠靶向黏附纳米粒。
采用实施例4中制备得到的纳米粒进行实施例6-8的实验。
实施例6
壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱纳米粒的体外释放实验
体外释放通过透析袋法进行实验。具体步骤为:以不同pH值的缓冲液模拟胃(pH1.0-2.0)、小肠(pH6.0-6.8)和结肠(pH7.0-7.4)中相应pH的生理环境。向透析袋(截留分子量为8000-14000)中放入已知药量的1mL的负载氧化苦参碱的纳米混悬液,再加入2mL的pH1.2的模拟胃液,将透析袋浸入pH1.2的40mL的模拟胃液中,开始释放。当释放达到2h后,将透析袋从pH1.2的模拟胃液中取出,立即放入pH6.8的模拟小肠液中,继续释放2h后,将透析袋从pH6.8的模拟小肠液中取出,立即放入pH7.4的模拟结肠液中,继续释放48h,37℃振荡水浴(100r/min),实验一式三份进行。于0h、1h、2h、3h、4h、5h、6h、7h、8h、12h、24h、48h从释放介质中进行取样(3mL),同时将相应的释放介补充到释放介质中,之后使用紫外-可见分光光度计参照校准曲线分析等分试样在220nm处的吸光度。
如图3,测定结果显示,在pH1.2条件下经2h模拟释放,氧化苦参碱原料药的释放总量为56.3%,而负载氧化苦参碱的纳米粒释放总量仅为21.8%;在pH6.8条件下2h负载氧化苦参碱的纳米粒共释放39.2%,而pH7.4条件下68h负载氧化苦参碱的纳米粒共释放约57%。数据表明在模拟胃液的强酸性、或小肠液的弱酸性缓冲液环境下,该纳米粒稳定性较好,总释放量约为57.1%,即约42.9%的口服剂量能够到达结肠部位。
实施例7
壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱纳米粒的体内靶向黏附实验
随机选择12只体重18-22g的Balb/c雄鼠,随机分为正常-壳聚糖-接枝-唾液酸/FITC交联果胶纳米粒组1(n=3只),模型-FITC生理盐水组2(n=3只),模型-壳聚糖-接枝-FITC交联果胶纳米粒组3(n=3只),模型-壳聚糖-接枝-唾液酸/FITC交联果胶纳米粒组4(n=3只)。模型组按时造模,其余和正常组保持一致条件饲养。造模第八天早上四组开始禁食,但是可以自由饮水,禁食24h后口服灌胃,组1和组4给予壳聚糖-接枝-唾液酸/FITC交联果胶纳米制剂(0.50mg/mL),组2给予FITC生理盐水溶(1.00mg/mL),组3给予壳聚糖-接枝-FITC交联果胶纳米粒制剂(0.50mg/mL)。四组分别于0.75h、6h、12h每个时间点处死一只小鼠,取出整个胃肠道(包括胃、十二指肠、空肠、回肠、盲肠、结肠、直肠),整理平铺在样品托盘中,活体成像仪拍摄荧光照片来确定黏附和靶向作用(参见图4)。
结果表明,与模型-壳聚糖-接枝-FITC交联果胶纳米粒组相比,模型-壳聚糖-接枝-唾液酸/FITC交联果胶纳米粒组不论6h还是12h时结肠部位荧光强度都最强,且比正常-壳聚糖-接枝-唾液酸/FITC交联果胶纳米粒组荧光还要强,说明所制备的壳聚糖-接枝-唾液酸交联果胶纳米粒具有靶向黏附病变组织的功能。
实施例8
壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱的纳米粒对DSS诱导的溃疡性结肠炎的体内治疗作用
随机选择15只体重18-22g的Balb/c雄鼠,随机分成正常组(n=5只)、模型组(n=5只)、制剂组(n=5只),使用分子量为36000-50000的10%的葡聚糖硫酸钠(DextranSulphate Sodium,DSS),每日灌胃Balb/c小鼠,灌胃八天后造成溃疡性结肠炎模型。同时制剂组按180mg/kg剂量连续灌胃纳米混悬液8天,正常组和模型组灌胃等体积的生理盐水。每日测量小鼠体重来计算体重下降率,观察其大便状态和便隐血程度。治疗结束后,按脱颈方法处死小鼠,取其结肠测定结肠长度,同时取部分结肠组织石蜡切片,显微镜下观察HE染色和IL-1β的免疫组化,以分析其粘膜和肌层厚度及巨噬细胞浸润情况。
结果表明,经壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱纳米粒治疗后的小鼠体重仅有少量下降,其体重变化与正常组无显著性差异,模型组小鼠体重出现大幅下降(约27%),与治疗组(制剂组)比较有显著性差异(p<0.05),说明壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱纳米粒可有效缓解溃疡性结肠炎导致的体重下降(参见图5)。
类似地,制剂组小鼠结肠长度与正常组比较也无差异,但与模型组比较差异具有显著性(p<0.05)(参见图6)。
类似地,在模型组小鼠结肠组织中可见大量的IL-1β促炎因子浸润,阳性覆盖率高达76.23%±3.21%,而制剂组仅见少量的IL-1β炎症因子浸润,阳性覆盖率为43.54±2.51%。制剂组与模型组具有显著性差异(p<0.05)(参见图7)。
以上数据表明,壳聚糖-接枝-唾液酸交联果胶负载氧化苦参碱纳米粒,可有效缓解DSS模型中溃疡性肠肠炎的炎性程度,可达到有效的结肠靶向治疗效果。
Claims (8)
1.一种靶向结肠炎的氧化苦参碱的纳米粒,其特征在于,其为靶向结肠的唾液酸修饰的果胶/壳聚糖/氧化苦参碱纳米粒,所述纳米粒由荷正电的壳聚糖-接枝-唾液酸、含氧化苦参碱的氯化钙溶液与荷负电的果胶经静电复合作用自组装而成;其中,所述的壳聚糖-接枝-唾液酸为唾液酸与壳聚糖通过酰胺键、酯键结合的产物。
2.根据权利要求1所述的靶向结肠炎的氧化苦参碱的纳米粒,其特征在于,所述的壳聚糖重均分子量为1000-10000,脱乙酰度为40%-99%;所述的唾液酸接入率为5%-60%;所述的果胶重均分子量为20000-400000,酯化度为50%-99%。
3.一种权利要求1或2所述的靶向结肠炎的氧化苦参碱的纳米粒的制备方法,其特征在于,包括如下步骤:
(1)唾液酸经过活化后与壳聚糖反应制得壳聚糖-接枝-唾液酸;
(2)将果胶溶于蒸馏水中制备果胶溶液,将氧化苦参碱溶于氯化钙水溶液中制备含氧化苦参碱的氯化钙溶液,将含有氧化苦参碱的氯化钙溶液缓慢加入至果胶溶液中,搅拌使之混合均匀,自组装形成负载氧化苦参碱的果胶纳米粒;
(3)将制备的壳聚糖-接枝-唾液酸溶于水中,通过滤膜除菌及除去不溶性杂质,而后在搅拌下缓慢加入至步骤(2)中制得的混合溶液中,通过静电复合作用进一步自组装压缩纳米粒形成结肠靶向的纳米粒。
4.根据权利要求3所述的靶向结肠炎的氧化苦参碱的纳米粒的制备方法,其特征在于,所述步骤(2)中制得果胶溶液的质量浓度为0.01%~0.1%。
5.根据权利要求3所述的靶向结肠炎的氧化苦参碱的纳米粒的制备方法,其特征在于,所述步骤(2)中使用的氯化钙水溶液质量浓度为5%~50%;氧化苦参碱在氯化钙水溶液中的质量浓度为2.7%-20.0%。
6.根据权利要求3所述的靶向结肠炎的氧化苦参碱的纳米粒的制备方法,其特征在于,所述步骤(3)中壳聚糖-接枝-唾液酸溶液的质量浓度为0.01%~0.1%。
7.根据权利要求3所述的靶向结肠炎的氧化苦参碱的纳米粒的制备方法,其特征在于,制备的纳米粒的粒径分布为80nm~700nm,电位为-10mV~-30mV;纳米粒载药量为2%~30%,包封率为60%~90%。
8.权利要求1所述的靶向结肠炎的氧化苦参碱的纳米粒在制备治疗溃疡性结肠炎药品、食品或保健品中的应用。
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