CN105749296A - 一种溃疡性结肠炎组织靶向分子及其应用 - Google Patents
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Abstract
本发明公开了一种溃疡性结肠炎组织靶向分子及其应用。采用的靶向分子是透明质酸,它是一种呈线性带负电的糖胺聚糖,由重复的双糖单位葡糖醛酸与<i>N</i>?乙酰葡糖胺通过<i>β</i>?1,3或1,4糖苷键连接而成。透明质酸作为靶向部分,它对CD44受体的特异性粘附功能较强,而CD44在溃疡性结肠炎组织的结肠上皮细胞和巨噬细胞均呈过量表达状态。本发明利用透明质酸对纳米粒子进行表面修饰,使其能被溃疡性结肠炎治疗相关的关键细胞(结肠上皮细胞和巨噬细胞)特异性吞噬,从而介导纳米粒子在结肠炎组织部位的富集。
Description
技术领域
本发明属于纳米药物载体技术领域,具体涉及一种溃疡性结肠炎组织靶向分子的开发及其应用。
背景技术
溃疡性结肠炎是一种慢性炎症性结肠疾病,其主要表现是结肠部位上皮屏障损伤和免疫平衡损害。传统的治疗方法一般采用抗炎药物和免疫抑制剂。虽然它们可以控制炎症,但是缺乏长效性而且存在很大的安全隐患。近年来,靶向给药制剂得到了开发和应用。靶向给药又称定位释放药物,通过一定方法使药物聚结于靶区,对病变组织实行靶向给药,靶向给药可减少用药剂量,增强药物对靶组织定位的特异性,从而提高疗效和减少药物的毒副作用。
透明质酸是细胞外基质中广泛存在的多糖成分,由D一葡萄糖醛酸和N一乙酰葡萄糖胺基团构成,具有良好的生物相容性。CD44在溃疡性结肠炎组织中的结肠上皮细胞和巨噬细胞上过表达,同时透明质酸对CD44具有很高的亲和性。将透明质酸应用于纳米药物释放体系,可以介导纳米粒子通过CD44受体介导的胞吞作用而进入靶细胞中。透明质酸是溃疡性结肠炎组织靶向药物递送的一个潜在靶向分子。
发明内容
有鉴于此,本发明的目的之一在于提供一种溃疡性结肠炎组织的靶向分子;本发明的目的之二在于提供上述靶向分子功能化纳米粒子的制备方法和应用。
为实现上述发明目的,技术方案为:
透明质酸功能化纳米粒子的制备方法包括以下步骤:
1)将100mg聚乳酸-羟基乙酸共聚物和8mg香豆素6共同溶解在2mL的二氯甲烷-甲醇中,制成油相;
2)将该油相逐滴加至聚乙烯醇和壳聚糖的混合水相溶液中;
3)超声处理6次,每次10s以形成水包油乳液;
4)将该乳液迅速倒入含有聚乙烯醇和壳聚糖混合水相溶液中;
5)将有机溶剂在低真空条件下挥发;
6)离心收集纳米粒子,并用去离子水洗三次;
7)将纳米粒子涡旋分散在2-(N-吗啉)乙磺酸一水合物缓冲液中;
8)利用1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐/N-羟基琥珀酰亚胺对透明质酸的羧基基团进行活化;
9)将透明质酸溶液加入到纳米粒子悬浮液中,所得混合物在室温下搅拌2小时;
10)高速离心收集纳米粒子,并用去离子水洗三次;
11)冷冻干燥后储存在–20度的密闭容器中。
优选的,所述步骤1)中,二氯甲烷-甲醇溶液的比例为5:5~8:2。
优选的,所述步骤2)中,壳聚糖分子量为1.8×104。
优选的,所述步骤3)中,聚乙烯醇和壳聚糖水溶液的总体积为2~6mL。
优选的,所述步骤4)中,超声的参数为超声振幅30~80%。
优选的,所述步骤5)中,聚乙烯醇和壳聚糖水溶液的总体积为10~200mL。
优选的,所述步骤6)和10)中,离心速率为8000~20000rpm,离心时间为8~30min。
优选的,所述步骤11)中,冷冻干燥按照以下方法进行:在–80度冷冻3小时后置于冻干机中冻干12~36小时。
相对于现有的技术,本发明的优点在于:
1.本发明将透明质酸引入药物靶向递送系统,透明质酸是天然水溶性高分子,具有优良的生物相容性、非免疫原性、在体内可由酶作用而天然降解。
2.本发明透明质酸携带大量的一OH和一COOH等多种功能基团,可进行多种化学修饰,与药物通过醋化、氢键等结合,达到缓释、控释效果。
3.本发明利用透明质酸对溃疡性结肠炎组织中结肠上皮细胞和巨噬细胞上过表达的CD44具有很高的亲和性,从而达到药物的定位释放。
4.本发明对纳米粒子进行透明质酸表面修饰,得到透明质酸修饰纳米粒子,并对其进行体外和体内表征,具体是透明质酸功能化的纳米载药系统应用于溃疡性结肠炎的靶向纳米药物递送领域。
5.本发明首次提出、制备并开展透明质酸功能化纳米粒子制剂,这将有助于我们更好揭示纳米粒子表面功能化的机理和规律,同时为在分子水平上设计具有优异抗炎效果的载药纳米粒子提供理论依据。
附图说明
为了使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步的详细描述,其中:
图1为透明质酸修饰纳米粒子的形貌和粒径表征。图1a为透明质酸修饰纳米粒子的透射电子显微镜(TEM)图片,嵌入小图为单个纳米粒子的TEM图片。图1b为透明质酸修饰纳米粒子的粒径分布图。
图2为羧甲基纤维素修饰纳米粒子(对照组)和透明质酸修饰纳米粒子的细胞毒性实验。图2a图2b分别为纳米粒子处理结肠上皮细胞系(Colon-26细胞)和巨噬细胞系(Raw264.7)24h和48h的细胞活性。
图3为羧甲基纤维素修饰纳米粒子(对照组)和透明质酸修饰纳米粒子的细胞吞噬实验。图3a和图3b分别为结肠上皮细胞系(Colon-26细胞)和巨噬细胞系(Raw264.7)吞噬纳米粒子的流式细胞图,覆盖面积代表被细胞摄取的纳米粒子。图3c和图3d分别为结肠上皮细胞系(Colon-26细胞)和巨噬细胞系(Raw264.7)摄取纳米粒子的直方图。
图4为透明质酸修饰纳米粒子被溃疡性结肠炎组织靶向吞噬的实验。图4a为溃疡性结肠炎组织吞噬透明质酸修饰纳米粒子的荧光图片。图4b为溃疡性结肠炎组织中巨噬细胞吞噬透明质酸修饰纳米粒子的流式数据。
具体实施方式
以下将参照附图,对本发明的优选实施例进行详细的描述。
实施例1
实施例1透明质酸修饰纳米粒子的制备方法,包括以下步骤:
1)将100mg聚乳酸-羟基乙酸共聚物和8mg香豆素6共同溶解在2mL的二氯甲烷-甲醇(8:2)中,制成油相;
2)将该油相逐滴加至聚乙烯醇和壳聚糖的混合水相溶液(4mL)中,其中聚乙烯醇和壳聚糖的浓度分别为5%和0.5%。;
3)超声处理6次,每次10s以形成水包油乳液;
4)将该乳液迅速倒入含有聚乙烯醇和壳聚糖混合水相溶液(100mL)中,其中聚乙烯醇和壳聚糖的浓度分别为0.5%和0.05%;
5)将有机溶剂在低真空条件下挥发;
6)离心(12000rpm,15min)收集纳米粒子,并用去离子水洗三次;
7)将纳米粒子涡旋分散在2-(N-吗啉)乙磺酸一水合物缓冲液中;
8)利用1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐/N-羟基琥珀酰亚胺对透明质酸的羧基基团进行活化;
9)将透明质酸溶液加入到纳米粒子悬浮液中,所得混合物在室温下搅拌2小时;
10)高速离心(12000rpm,15min)收集纳米粒子,并用去离子水洗三次;
11)在-80℃冷冻3小时后,置于冻干机中-60℃冻干24小时,而后储存在–20℃的密闭容器中。
比较例1
比较例1羧甲基纤维素修饰纳米粒子的制备方法包括以下步骤:
1)将100mg聚乳酸-羟基乙酸共聚物和8mg香豆素6共同溶解在2mL的二氯甲烷-甲醇(8:2)中,制成油相;
2)将该油相逐滴加至聚乙烯醇和壳聚糖的混合水相溶液(4mL)中,其中聚乙烯醇和壳聚糖的浓度分别为5%和0.5%。;;
3)超声处理6次,每次10s以形成水包油乳液;
4)将该乳液迅速倒入含有聚乙烯醇和壳聚糖混合水相溶液(100mL)中,其中聚乙烯醇和壳聚糖的浓度分别为0.5%和0.05%;
5)将有机溶剂在低真空条件下挥发;
6)离心(12000rpm,15min)收集纳米粒子,并用去离子水洗三次;
7)将纳米粒子涡旋分散在2-(N-吗啉)乙磺酸一水合物缓冲液中;
8)利用1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐/N-羟基琥珀酰亚胺对羧甲基纤维素的羧基基团进行活化;
9)将羧甲基纤维素溶液加入到纳米粒子悬浮液中,所得混合物在室温下搅拌2小时;
10)高速离心(12000rpm,15min)收集纳米粒子,并用去离子水洗三次;
11)在-80℃冷冻3小时后,置于冻干机中-60℃冻干24小时,而后储存在–20℃的密闭容器中。
图1为透明质酸修饰纳米粒子的形貌和粒径表征。图1a为透明质酸修饰纳米粒子的TEM图片,嵌入小图为单个纳米粒子的TEM图片。图1b为透明质酸修饰纳米粒子的粒径分布图。
如图1所示,纳米粒子通过TEM观察形貌。如TEM图片显示,透明质酸修饰纳米粒子(Fig.1a)是球形的,平均直径大约128.2nm。结果显示用TEM和动态光散射(DLS)测定的粒径有一些差距,这是因为在不同的测试条件下纳米粒子的表面状态不同。用DLS测定时,纳米粒子是含水的,呈膨胀状态;相反,用TEM测定时,纳米粒子则呈完全脱水状态。
图2为羧甲基纤维素修饰纳米粒子(对照组)和透明质酸修饰纳米粒子的细胞毒性实验,图2a和图2b分别为纳米粒子处理结肠上皮细胞系(Colon-26细胞)和巨噬细胞系(Raw264.7)24h的细胞活性。细胞活性为处理后活细胞所占百分比。如图2所示,透明质酸修饰纳米粒子对所处理的两种细胞均没有明显毒性。
图3为对纳米粒子进行透明质酸修饰能增加了细胞对其摄取量。图3a和图3b分别为处理结肠上皮细胞系(Colon-26细胞)和巨噬细胞系(Raw264.7)吞噬纳米粒子的流式分析图,覆盖面积代表吞噬纳米粒子的细胞量。图3c和图3d分别为结肠上皮细胞系(Colon-26细胞)和巨噬细胞系(Raw264.7)摄取纳米粒子的直方图。
有效的细胞摄取对于保证药效是非常重要的。在此研究中,我们用了香豆素6固有荧光监测纳米粒子被两种细胞中吞噬情况,这两种细胞是溃疡性结肠炎治疗相关主要靶细胞(结肠上皮细胞和巨噬细胞)。结肠上皮细胞系(Colon-26细胞)和巨噬细胞系(Raw264.7)用透明质酸功能化纳米粒子或羧甲基纤维素(对照)修饰纳米粒子分别处理1~5小时,可以明显看出透明质酸功能化有显著增加细胞对其吞噬量。
图4为透明质酸修饰纳米粒子被溃疡性结肠炎组织靶向吞噬的实验。图4a为溃疡性结肠炎组织吞噬透明质酸修饰纳米粒子的荧光图片。图4b为溃疡性结肠炎组织中巨噬细胞吞噬透明质酸修饰纳米粒子的流式数据。如图4a所示,几乎所有结肠上皮细胞均吞噬了透明质酸纳米粒子。如图4b所示,大约75.8%巨噬细胞摄取了透明质酸修饰纳米粒子;相反,较少的巨噬细胞(65.2%)吞噬了羧甲基纤维素修饰纳米粒子。这表明透明质酸修饰纳米粒子对溃疡性结肠炎组织中结肠上皮细胞和巨噬细胞均具有很强的靶向能力,与体外实验结果(Fig.2)一致。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管通过参照本发明的优选实施例已经对本发明进行了描述,但本领域的普通技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离所附权利要求书所限定的本发明的精神和范围。
Claims (9)
1.一种溃疡性结肠炎组织靶向分子,其特征在于:通过溶剂挥发法制备载荧光探针分子(香豆素6)的聚酯纳米粒子,然后对其进行透明质酸表面功能化,包括以下步骤:
1)将聚乳酸-羟基乙酸共聚物和香豆素6共同溶解在二氯甲烷-甲醇中,制成油相;
2)将该油相逐滴加至聚乙烯醇和壳聚糖的混合水相溶液中;
3)超声处理6次,每次10s以形成水包油乳液;
4)将该乳液迅速倒入含有聚乙烯醇和壳聚糖混合水相溶液中;
5)将有机溶剂在低真空条件下挥发;
6)离心收集纳米粒子,并用去离子水洗三次;
7)将纳米粒子涡旋分散在2-(N-吗啉)乙磺酸一水合物缓冲液中;
8)利用1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐/N-羟基琥珀酰亚胺对透明质酸的羧基基团进行活化;
9)将透明质酸溶液加入到纳米粒子悬浮液中,所得混合物在室温下搅拌2小时;
10)高速离心收集纳米粒子,并用去离子水洗三次;
11)冷冻干燥后储存在–20度的密闭容器中。
2.根据权利要求1所述的一种溃疡性结肠炎组织靶向分子,其特征在于:所述步骤1)中,聚乳酸-羟基乙酸共聚物和香豆素6的质量比例为100mg:6mg~100mg:12mg。
3.根据权利要求1所述的一种溃疡性结肠炎组织靶向分子,其特征在于:壳聚糖分子量为1.8×104。
4.根据权利要求1所述的一种溃疡性结肠炎组织靶向分子,其特征在于:所述步骤1)中,二氯甲烷-甲醇溶液的比例为5:5~8:2。
5.根据权利要求1所述的一种溃疡性结肠炎组织靶向分子,其特征在于:所述步骤2)中,聚乙烯醇和壳聚糖混合水溶液的总体积为2~6mL,两者的浓度范围分别是2~5%和0.2~0.5%。
6.根据权利要求1所述的一种溃疡性结肠炎组织靶向分子,其特征在于:所述步骤3)中,超声的参数为超声振幅30~80%。
7.根据权利要求1所述的一种溃疡性结肠炎组织靶向分子,其特征在于:所述步骤4)中,聚乙烯醇和壳聚糖水溶液的总体积为10~200mL,两者的浓度范围分别是2~5%和0.2~0.5%。
8.根据权利要求1所述的一种溃疡性结肠炎组织靶向分子,其特征在于:所述步骤6)和10)中,离心速率为8000~20000rpm,离心时间为8~30min。
9.根据权利要求1所述的一种溃疡性结肠炎组织靶向分子,其特征在于:所述步骤11)中,冷冻干燥按照以下方法进行:在-80℃冷冻3小时后,置于冻干机中-60℃冻干12~36小时。
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