CN115990145A - 一种pH响应型靶向纳米递送系统及其制备方法和应用 - Google Patents
一种pH响应型靶向纳米递送系统及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种pH响应型靶向纳米递送系统及其制备方法和应用,涉及纳米材料领域。该pH响应型靶向纳米递送系统的制备方法,包括以下步骤:将QTX125、姜黄素和CaCl2加入溶剂中反应后,再加入NH4HCO3继续反应,之后离心,干燥,得到CaCO3@Cur@QTX125;所述CaCO3@Cur@QTX125溶解在去离子水中,再加入透明质酸,反应,离心,干燥,得到CaCO3@Cur@QTX125@HA,即为所述pH响应型靶向纳米递送系统。本发明将Cur和QTX125结合,制备出的pH响应型靶向纳米递送系统,可以提升药物的靶向性、生物相容性及药物在体内的半衰期,进而提高结直肠癌的治疗效果。
Description
技术领域
本发明涉及纳米材料领域,特别是涉及一种pH响应型靶向纳米递送系统及其制备方法和应用。
背景技术
结直肠癌(colorectal cancer,CRC)发病率逐年升高,是全球第三大癌症相关发病原因(约10.0%),同时也是癌症致死的第二大主要原因。目前,手术和化学疗法仍然是结直肠癌治疗的最主要方法。但对于有淋巴结转移的可切除结直肠癌或者不可切除的转移性结直肠癌,即使使用氟尿嘧啶、奥沙利铂、伊利替康等药物化疗以及联合靶向治疗、免疫治疗,仍存在预后差、5年生存率低等系列问题。近年来,分子靶向制剂和免疫疗法逐步用于直肠癌的治疗,多项临床研究证实靶向药物(贝伐珠单抗、西妥西单抗等)以及PD-1/PD-L1免疫检查点抑制剂(Nivolumab、Pembrolizumab、Atezolizumab等)可以改善转移性结直肠癌患者预后。但单一的药物往往治疗效果不佳。
组蛋白去乙酰化酶(histone deacetylases,HDACs)可能在肿瘤的发生及侵袭中起到关键作用,是一个很有前景的抗肿瘤靶点。已有研究证明HDACs的异常表达与多种肿瘤(慢性淋巴细胞白血病、胃癌、乳腺癌、结直肠癌、肝癌、髓母细胞瘤、非小细胞肺癌、淋巴癌、神经母细胞瘤、卵巢癌、胰腺癌、前列腺癌和肾癌)相关。目前,HDACs抑制剂QTX125与其它药物的联合在各类肿瘤疾病中作用已经得到证实,而姜黄素(Cur)是一种很好的天然抗癌剂,通过调节多条信号通路抑制结直肠相关疾病的发生和发展,其较低的毒性和良好的生物相容性等特点而受到广泛关注。因此,本研究开发了一种高效、低毒的纳米递药系统来用于结直肠癌的治疗研究。
发明内容
本发明的目的是提供一种pH响应型靶向纳米递送系统及其制备方法和应用,以解决上述现有技术存在的问题,本发明提供的pH响应型靶向纳米递送系统可以提升药物的靶向性、生物相容性及药物在体内的半衰期。
为实现上述目的,本发明提供了如下方案:
本发明提供一种pH响应型靶向纳米递送系统的制备方法,包括以下步骤:
(1)将QTX125、姜黄素和CaCl2加入溶剂中反应后,再加入NH4HCO3继续反应,之后离心,干燥,得到CaCO3@Cur@QTX125;
(2)所述CaCO3@Cur@QTX125溶解在去离子水中,再加入透明质酸,反应,离心,干燥,得到CaCO3@Cur@QTX125@HA,即为所述pH响应型靶向纳米递送系统。
进一步地,在步骤(1)中,所述溶剂为无水乙醇。
进一步地,在步骤(1)中,所述QTX125、所述姜黄素、所述CaCl2、所述NH4HCO3和所述溶剂的添加比例为(3-10)mg:(3-10)mg:(100-3000)mg:(3-20)g:(50-500)mL。
进一步地,在步骤(1)中,加入所述NH4HCO3继续反应的温度为28-40℃。
进一步地,在步骤(2)中,所述CaCO3@Cur@QTX125、所述透明质酸和所述去离子水的添加比例为(1-3)mg:(1-5)mg:(10-50)mL。
进一步地,在步骤(2)中,所述透明质酸的分子量为50kDa-200kDa。
本发明还提供一种根据上述的制备方法制备得到的pH响应型靶向纳米递送系统。
本发明还提供上述的pH响应型靶向纳米递送系统在制备治疗结直肠癌的药物中的应用。
本发明公开了以下技术效果:
本发明将Cur和QTX125结合,通过气相扩散法制备得到了CaCO3@Cur@QTX125@HA,利用乙醇挥发作为系统的流动相,(NH4)HCO3在一定温度下发生分解,形成的产物在蒸汽压的作用下和CaCl2反应,从而形成CaCO3纳米载体。CaCO3@Cur@QTX125@HA是一种高效、低毒的pH响应型靶向纳米递送系统,CaCO3载体可在H+的作用下分解,进而释放药物,其可以提升姜黄素和QTX125的靶向性、生物相容性及药物在体内的半衰期,进而提高结直肠癌的治疗效果。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为实施例1的制备过程实物图;
图2为透射电子显微镜扫描材料表征结果;其中,A为CaCO3,B为CaCO3@Cur@QTX125,C为CaCO3@Cur@QTX125@HA;A、B和C中标尺均为500nm;
图3为复合纳米粒子制备过程中的化学组成;
图4为复合纳米粒子制备过程中的结晶特征;
图5为复合纳米粒子粒径及Zeta电位评估(DLS);
图6为药物释放响应结果;其中A为标准曲线(431nm处的吸光度);B为CaCO3@Cur@QTX125的检测结果;C为CaCO3@Cur@QTX125@HA的检测结果;NaAc为pH=5.2的醋酸-醋酸钠缓冲液,PBS为PBS缓冲液;
图7为复合纳米粒子对Hela(A)、HT-29(B)和HCV-29(C)细胞活性的影响比较;*表示p<0.05;
图8为复合纳米粒子生物相容性检测结果;*表示p<0.05。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
以下实施例中人宫颈癌细胞(Hela)、人膀胱癌细胞(HSV-29)、人结肠癌细胞(HT-29)和大鼠小肠上皮细胞(IEC-6)均购自ATCC上海细胞库。
实施例1
将3mg的QTX125、10mg姜黄素和CaCl2(10mM,100mg)溶解在50mL无水乙醇中,并于常温下搅拌12小时,然后,加入6gNH4HCO3,密封28℃,过夜搅拌(如图1所示)。然后,通过离心(6000rpm,20min)用水洗涤分散体数次,最后冻干得到CaCO3@Cur@QTX125。
取上述所制备的CaCO3@Cur@QTX125(1mg)溶解在50mL去离子水中,然后再加入1mg透明质酸(HA,分子量为50kDa),搅拌过夜,离心,冷冻干燥获得最终产物CaCO3@Cur@QTX125@HA。
实施例2
将5mg的QTX125、5mg姜黄素和CaCl2(10mM,150mg)溶解在100mL无水乙醇中,并搅拌18小时,然后,加入4gNH4HCO3,密封30℃,过夜搅拌。然后,通过离心(6000rpm,20min)用水洗涤分散体数次,最后冻干得到CaCO3@Cur@QTX125。
取上述所制备的CaCO3@Cur@QTX125(2mg)溶解在20mL去离子水中,然后加入3mgHA(分子量为100kDa),搅拌过夜,离心,冷冻干燥获得最终产物CaCO3@Cur@QTX125@HA。
实施例3
将10mg QTX125、3mg姜黄素和CaCl2(10mM,300mg)溶解在150mL无水乙醇中,并搅拌24小时,然后,加入6gNH4HCO3,密封40℃,过夜搅拌。然后,通过离心(6000rpm,20min)用水洗涤分散体数次,最后冻干得到CaCO3@Cur@QTX125。
取上述所制备的CaCO3@Cur@QTX125(3mg)溶解在10mL去离子水中,然后加入5mgHA(分子量选用200kDa),搅拌过夜,离心,冷冻干燥获得最终产物CaCO3@Cur@QTX125@HA。
对比例1
同实施例3,区别仅在于,选用分子量为250kDa的HA。
对比例2
同实施例3,区别仅在于,选用分子量为40kDa的HA。
效果验证:
1.通过透射电子显微镜(TEM)测定实施例2制备的CaCO3、CaCO3@Cur@QTX125和CaCO3@Cur@QTX125@HA的形貌,结果见图2。CaCO3、CaCO3@Cur@QTX125及CaCO3@Cur@QTX125@HA纳米粒子具有较好的单分散性和均一性。CaCO3的纳米球的尺寸约为240nm(图2中A)、CaCO3@Cur@QTX125为500nm(图2中B),而HA修饰后形成的CaCO3@Cur@QTX125@HA纳米粒子尺寸约450nm(图2中C)。
2.利用傅里叶红外光谱仪(FT-IR)分析实施例2制备过程中的化学组成,结果见图3。CaCO3在负载药物及HA修饰前后CaCO3@Cur@QTX125及CaCO3@Cur@QTX125@HA的吸收峰未发生明显变化。而在1089cm-1附近有一个尖锐的吸收峰,这主要是HA中糖苷基的振动吸收峰所致。
3.利用X射线衍射仪(XRD)测定CaCO3、CaCO3@Cur@QTX125及CaCO3@Cur@QTX125@HA制备过程中结晶特征结果见图4。图中CaCO3纳米粒子在载药和HA修饰前后具有较好的晶型特征,且峰位置未发生明显变化。
4.利用动态光散射技术(DLS)测定不同纳米粒子的粒径和电位。分别取等量的CaCO3、CaCO3@Cur@QTX125和CaCO3@Cur@QTX125@HA,测定纳米药物的粒径大小及Zeta电位,以评估各纳米粒子的尺寸和稳定性,结果见图5。图中CaCO3、CaCO3@Cur@QTX125和CaCO3@Cur@QTX125@HA纳米粒子的粒径依次约为:410nm、550nm及770nm,引起纳米粒子尺寸变化的原因是其在水溶液中存在的水合粒径所致。另外由于CaCO3@Cur@QTX125@HA的亲水性进一步引起其粒径的增大。CaCO3、CaCO3@Cur@QTX125和CaCO3@Cur@QTX125@HA的Zeta电位分别为4.83,-5.66,-8.11mV。
5.体外药物释放行为。
测定CaCO3@Cur@QTX125和CaCO3@Cur@QTX125@HA递送体系中Cur的释放行为。取实施例2制备的CaCO3@Cur@QTX125和CaCO3@Cur@QTX125@HA置于pH=5.2的醋酸盐缓冲液中,保证温度恒定在37℃,搅拌速度为120rpm。在每个预定时间点,取出2mL等分试样并离心收集上清液。通过UV-Vis光谱分析获得的上清液中释放的Cur,结果见图6。CaCO3@Cur@QTX125在醋酸盐缓冲液中(pH=5.2)具有更快的释放速率,这主要是CaCO3在酸性条件下发生降解引起的药物释放。CaCO3@Cur@QTX125@HA24 h内在PBS中的释放较慢,表明修饰完HA后具有良好的稳定性,其在醋酸盐缓冲液中可实现较快的释放。
6.CCK-8测定Hela和HT-29细胞48h内的细胞活性比较
Hela及HT29细胞分别以5×103/孔的密度接种于96孔板中,培养24小时。而后吸出培养基,Hela分别在不同浓度(1.25、2.5、5、10、20μg/mL)的无定型CaCO3、CaCO3@QTX125(实施例2)和CaCO3@Cur@QTX125@HA(实施例2)及HSV-29和HT-29细胞分别在浓度(0.625、1.25、2.5、5、10、20和40μg/mL)的无定型CaCO3、CaCO3@QTX125(实施例2)和CaCO3@Cur@QTX125@HA(实施例2)处理48h。孵育结束后,使用标准CCK-8法测定各组Hela、HT-29和HCV-29细胞的细胞活力;同理,测定各药物组对Hela、HCV-29和HT-29细胞活力的影响,结果见图7。相较于HCV-29、Hela细胞,同浓度下的纳米粒子对HT29具有更好的抑制作用,表明纳米药物对肿瘤细胞具有特异性。
7.CCK-8测定IEC-6细胞活性评估实施例2制备的等浓度梯度(1.25、2.5、5、10、20μg/mL)CaCO3@Cur@QTX125和CaCO3@Cur@QTX125@HA的生物相容性,结果见图8。该结果表明,纳米粒子具有较好的生物相容性。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.一种pH响应型靶向纳米递送系统的制备方法,其特征在于,包括以下步骤:
(1)将QTX125、姜黄素和CaCl2加入溶剂中反应后,再加入NH4HCO3继续反应,之后离心,干燥,得到CaCO3@Cur@QTX125;
(2)所述CaCO3@Cur@QTX125溶解在去离子水中,再加入透明质酸,反应,离心,干燥,得到CaCO3@Cur@QTX125@HA,即为所述pH响应型靶向纳米递送系统。
2.根据权利要求1所述的制备方法,其特征在于,在步骤(1)中,所述溶剂为无水乙醇。
3.根据权利要求1所述的制备方法,其特征在于,在步骤(1)中,所述QTX125、所述姜黄素、所述CaCl2、所述NH4HCO3和所述溶剂的添加比例为(1-20)mg:(3-10)mg:(100-300)mg:(3-6)g:(50-150)mL。
4.根据权利要求1所述的制备方法,其特征在于,在步骤(1)中,加入所述NH4HCO3继续反应的温度为28-60℃。
5.根据权利要求1所述的制备方法,其特征在于,在步骤(2)中,所述CaCO3@Cur@QTX125、所述透明质酸和所述去离子水的添加比例为(1-3)mg:(1-5)mg:(10-50)mL。
6.根据权利要求1所述的制备方法,其特征在于,在步骤(2)中,所述透明质酸的分子量为50kDa-200kDa。
7.一种根据权利要求1-6任一项所述的制备方法制备得到的pH响应型靶向纳米递送系统。
8.一种如权利要求7所述的pH响应型靶向纳米递送系统在制备治疗结直肠癌的药物中的应用。
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