CN111514315A - 一种肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法 - Google Patents
一种肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法 Download PDFInfo
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Abstract
本发明公开了一种肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法。首先以透明质酸(HA)为原料,采用高温脱水的方法制备部分碳化透明质酸荧光材料HA‑FCNs(HA fluorescence carbon nanoparticles);然后以硝酸钙为钙源,磷酸氢二铵为磷源,HA‑FCNs为标记物,采用共沉淀法制备了肿瘤靶向无定形磷酸钙荧光纳米复合材料(HA‑FCNs/ACP)。同时,将姜黄素(Cur)作为疏水药物模型,盐酸阿霉素(DOX)作为亲水药物模型,分别实现对两种药物的负载。本发明所制备的药物载体生物相容性好,具有明显的生物成像和pH响应性,对癌细胞(A549细胞)具有靶向性,在癌细胞内环境下有明显的酶促释放反应,显著降低了癌细胞存活率。
Description
技术领域
本发明涉及一种肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,属于生物材料技术领域。
背景技术
传统的剂型和给药方式,药物进入体内迅速释放,达到峰值后快速下降,使得血药浓度存在明显的峰-谷现象。当血药浓度过高时对机体带来损伤,低于一定浓度时达不到理想疗效。其次,传统给药方式也可能在达到治疗效果的同时对正常的组织器官造成损坏。因此,研发一种新型药物载体需以延长药物作用时间、提高药物对病灶部位的选择性、改善药物疗效为方向,进而达到药物缓释和控释的目的,这对人类健康有着极其重要的意义。在过去的研究中,人们已经研究了叶酸、透明质酸等的肿瘤靶向性。作为一种酸性粘多糖,透明质酸具有良好的生物相容性和肿瘤靶向性。无定形磷酸钙(ACP)具有优良的生物相容性、可生物降解性及pH敏感性,这使得其可作为抗癌药物理想载体的制备材料。然而,要实现诊疗一体化,还需对病灶部位进行明显成像。故而,考虑对透明质酸进行预处理,使其在保留部分透明质酸结构的基础上仍具有肿瘤靶向和成像的效果。
如果能解决上述技术问题,将能够有效拓宽透明质酸和ACP作为药物载体的应用范围。
发明内容
本发明是在磷酸钙纳米粒和部分碳化透明质酸的基础上,提出了一种肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,首先制备出无定形磷酸钙荧光纳米复合药物载体,实现肿瘤的靶向锚定和生物成像,以及在病灶部位的药物的pH响应和酶促可控释放。在此过程中,Ca2+首先通过静电吸附与HA-FCNs的羧基结合,然后PO4 3-与Ca2+和-COO-的配合物反应形成无定形磷酸钙荧光复合材料(HA-FCNs/ACP)。
本发明提供了一种肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,包括以下步骤:首先以透明质酸为原料,采用高温脱水的方法制备部分碳化透明质酸荧光材料;然后以硝酸钙为钙源,磷酸氢二铵为磷源,HA-FCNs为标记物,采用共沉淀法制备了肿瘤靶向无定形磷酸钙荧光纳米复合材料;在此基础上实现对亲水性药物或疏水性药物的负载。
以姜黄素Cur为疏水药物模型,采用上述方法对其进行负载,具体包括以下步骤:
(1)部分碳化透明质酸荧光纳米材料的制备:
首先,在油浴搅拌条件下,以透明质酸为原料,浓硫酸为脱水剂,高温脱水,透析三天,干燥,得到HA-FCNs荧光材料;
(2)负载Cur:
将上述HA-FCNs溶于水中,升温至25-40 ℃,缓慢滴加Ca(NO3)2溶液,共混搅拌10 min-1.5 h;配制5 mg/mL Cur水溶液,超声分散10 min,逐滴滴加到上述溶液中,共混搅拌1 h;随后,逐滴滴加(NH4)2HPO4溶液,同时用NaOH溶液调节pH,搅拌1 min-1 h后室温陈化10-24h至反应完全;离心,洗涤,干燥,得到Cur-HA-FCNs/ACP纳米粒。
以阿霉素DOX为亲水药物模型,采用上述方法对其进行负载,具体包括以下步骤:
(1)部分碳化透明质酸荧光纳米材料的制备:
首先,在油浴搅拌条件下,以透明质酸为原料,浓硫酸为脱水剂,高温脱水,透析三天,干燥,得到HA-FCNs荧光材料;
(2)负载DOX:
将上述HA-FCNs溶于水中,升温至25-40 ℃,缓慢滴加Ca(NO3)2溶液,共混搅拌10 min-1.5 h;配制0.6 mg/mL DOX水溶液,逐滴滴加到上述溶液中,共混搅拌1 h;随后,逐滴滴加(NH4)2HPO4溶液,同时用NaOH溶液调节pH,搅拌1 min-1 h后室温陈化10-24h至反应完全。离心,洗涤,干燥,得到DOX-HA-FCNs/ACP纳米粒。
对上述方法中工艺参数做出进一步说明如下:
所述部分碳化透明质酸荧光纳米材料的制备步骤中,透明质酸与水的质量比为0.02~0.04。
所述部分碳化透明质酸荧光纳米材料的制备步骤中,浓硫酸的用量为10~15 mL。
所述部分碳化透明质酸荧光纳米材料的制备步骤中,油浴温度为230~250 ℃,反应时间为1~2 min。
所述部分碳化透明质酸荧光纳米材料的制备步骤中,透析袋分子量为500~3500Da。
所述无定形磷酸钙荧光复合材料的制备步骤中,pH的范围为7~9。
所述无定形磷酸钙荧光复合材料的制备步骤中,HA-FCNs的浓度为600~1000 μg/mL。
负载的过程中,超声条件为:探头超声功率是200~500 W,超声2~4 s,间歇1~3 s。
进一步地,所述无定形磷酸钙荧光复合材料的制备步骤中,陈化的时间范围为10-24 h。
本发明的有益效果:
本发明提供的抗癌药物载体的制备方法所得的荧光复合材料具有良好的生物相容性和生物成像效果,对CD44受体过表达的肿瘤细胞具有靶向性,且载药纳米粒在肿瘤内环境中有明显的pH敏感性和酶促释放效果,实现了诊疗一体化。
附图说明
图1是本发明实施例1所制备的HA-FCNs荧光材料的荧光发射光谱和紫外吸收光谱图;
图2是本发明实施例1所制备的透明质酸和HA-FCNs荧光材料的核磁氢谱对比图;
图3是本发明实施例1-6不同HA-FCNs浓度下所制备的HA-FCNs/ACP荧光纳米粒的X射线衍射图谱;
图4是本发明实施例1所制备的HA-FCNs/ACP荧光纳米粒的扫描电镜图;
图5是本发明实施例1所制备的包封姜黄素纳米复合药物体系的药物释放曲线;
图6是本发明实施例2所制备的包封阿霉素纳米复合药物体系的药物释放曲线。
具体实施方式
下面通过实施例来进一步说明本发明,但不局限于以下实施例。
实施例1:
本实施例提供一种肿瘤靶向无定形磷酸钙荧光纳米复合材料HA-FCNs/ACP的方法,包括以下步骤:首先高温脱水合成部分碳化透明质酸(HA-FCNs),并将所制备的HA-FCNs加入到ACP的制备过程中,通过共沉淀法合成无定形磷酸钙荧光复合材料(HA-FCNs/ACP)。具体步骤如下:
(1)将0.3 g透明质酸溶解到10 mL去离子水中,在230 ℃油浴搅拌条件下,将10 mL浓硫酸加入透明质酸水溶液中,搅拌反应1.5 min,用分子量为500 Da的透析袋透析三天,冷冻干燥得到HA-FCNs荧光材料。
对所制备的HA-FCNs纳米粒进行荧光发射光谱和紫外光谱测试(图1),从图1中可以看出HA-FCNs在380 nm处呈现最大发射,且在UV-vis吸收的264 nm处显示出较为强烈的峰,这是因为降解的透明质酸片段和存在转化的双键。同时,在365 nm紫外灯照射下,HA-FCNs呈明亮的蓝色荧光。从核磁(图2)中可以看出HA-FCNs在一定程度上保留了透明质酸的部分结构。
(2)负载Cur的复合材料的制备:分别配制0.034 mol/L 29 mL硝酸钙水溶液、0.024 mol/L 29 mL磷酸氢二铵水溶液、1 mol/L 50 mL NaOH溶液、600 μg/mL 30 mL HA-FCNs水溶液和5 mg/mL 10 mL Cur水溶液,其中,超声分散Cur水溶液(超声条件为:探头超声功率500 W,超声2 s,间歇3 s)。在30 ºC条件下将HA-FCNs水溶液逐滴滴加到硝酸钙溶液中,共混搅拌1 h后,逐滴滴加Cur水溶液于上述混合液中,搅拌1 h。随后,将磷酸氢二铵溶液缓慢滴加到上述混合液中,并用NaOH溶液调节pH至8,搅拌10 min后,室温静置陈化12 h,离心除去上清液,洗涤,干燥,得到Cur-HA-FCNs/ACP纳米粒。
采用紫外分光光度计检测Cur在不同pH值(7.4, 6.5, 5.4)中释放量随时间的变化(见图5)。用紫外可见-分光光度计测定不同浓度Cur吸光度,根据吸光度和浓度绘制标准曲线,如Y=kx+b。
图5体现了负载疏水性药物Cur的载体在pH 5.4中的释药速率明显大于pH 6.5,且在pH 6.5中的释药速率大于pH 7.4的,这说明在酸性条件下载体的释药速率更快,也证明了药物载体具有明显的pH响应型释药性能。
实施例2
(1)将0.4 g透明质酸溶解到10 mL去离子水中,在230 ℃油浴搅拌条件下,将15 mL浓硫酸加入透明质酸水溶液中,搅拌反应2 min,用分子量为2000 Da的透析袋透析三天,冷冻干燥得到HA-FCNs荧光材料。
(2)分别配制0.034 mol/L 29 mL硝酸钙水溶液、0.024 mol/L 29 mL磷酸氢二铵水溶液、1 mol/L 50 mL NaOH溶液、700 μg/mL 30 mL HA-FCNs水溶液和0.6 mg/mL 10 mLDOX水溶液,其中,超声分散DOX水溶液(超声条件为:探头超声功率500 W,超声2 s,间歇3s)。在35 ºC条件下将HA-FCNs水溶液逐滴滴加到硝酸钙溶液中,共混搅拌1.5 h后,逐滴滴加DOX水溶液于上述混合液中,搅拌1 h。随后,将磷酸氢二铵溶液缓慢滴加到上述混合液中,并用NaOH溶液调节pH至8,搅拌10 min后,室温静置陈化24 h,离心去除上清液,洗涤,干燥,得到DOX-HA-FCNs/ACP纳米粒。
采用紫外分光光度计检测DOX在不同pH值(7.4, 6.5, 5.4)中释放量随时间的变化(见图6)。用紫外可见-分光光度计测定不同浓度DOX吸光度,根据吸光度和浓度绘制标准曲线,如Y=kx+b。从图6可以看出,释药速率趋势为:pH 5.4>pH 6.5>pH 7.4,这说明负载亲水性抗癌药物DOX的载体具有明显的pH响应型释药性能。
实施例3
(1)将0.2 g透明质酸溶解到10 mL去离子水中,在240 ℃油浴搅拌条件下,将15 mL浓硫酸加入透明质酸水溶液中,搅拌反应1 min,用分子量为3500 Da的透析袋透析三天,冷冻干燥得到HA-FCNs荧光材料。
(2)负载Cur的复合材料的制备:分别配制0.034 mol/L 29 mL硝酸钙水溶液、0.024 mol/L 29 mL磷酸氢二铵水溶液、1 mol/L 50 mL NaOH溶液、800 μg/mL 30 mL HA-FCNs水溶液和5 mg/mL 10 mL Cur水溶液,其中,超声分散Cur水溶液(超声条件为:探头超声功率400 W,超声3 s,间歇3 s)。在25 ºC条件下将HA-FCNs水溶液逐滴滴加到硝酸钙溶液中,共混搅拌1 h后,逐滴滴加Cur水溶液于上述混合液中,搅拌1 h。随后,将磷酸氢二铵溶液缓慢滴加到上述混合液中,并用NaOH溶液调节pH至9,搅拌1 min后,室温静置陈化12 h,离心除去上清液,洗涤,干燥,得到Cur-HA-FCNs/ACP纳米粒。
实施例4
(1)将0.4 g透明质酸溶解到10 mL去离子水中,在250 ℃油浴搅拌条件下,将10 mL浓硫酸加入透明质酸水溶液中,搅拌反应1.5 min,用分子量为2000 Da的透析袋透析三天,冷冻干燥得到HA-FCNs荧光材料。
(2)负载DOX的复合材料的制备:分别配制0.034 mol/L 29 mL硝酸钙水溶液、0.024 mol/L 29 mL磷酸氢二铵水溶液、1 mol/L 50 mL NaOH溶液、900 μg/mL 30 mL HA-FCNs水溶液和0.6 mg/mL 10 mL DOX水溶液,其中,超声分散DOX水溶液(超声条件为:探头超声功率500 W,超声3 s,间歇3 s)。在30 ºC条件下将HA-FCNs水溶液逐滴滴加到硝酸钙溶液中,共混搅拌30 min后,逐滴滴加DOX水溶液于上述混合液中,搅拌30 min。随后,将磷酸氢二铵溶液缓慢滴加到上述混合液中,并用NaOH溶液调节pH至7,搅拌30 min后,室温静置陈化10 h,离心去除上清液,洗涤,干燥,得到DOX-HA-FCNs/ACP纳米粒。
实施例5
(1)将0.3g透明质酸溶解到10 mL去离子水中,在240 ℃油浴搅拌条件下,将15 mL浓硫酸加入透明质酸水溶液中,搅拌反应1 min,用分子量为500 Da的透析袋透析三天,冷冻干燥得到HA-FCNs荧光材料。
(2)负载Cur的复合材料的制备:分别配制0.034 mol/L 29 mL硝酸钙水溶液、0.024 mol/L 29 mL磷酸氢二铵水溶液、1 mol/L 50 mL NaOH溶液、1000 μg/mL 30 mL HA-FCNs水溶液和5 mg/mL 10 mL Cur水溶液,其中,超声分散Cur水溶液(超声条件为:探头超声功率400 W,超声4 s,间歇3 s)。在40 ºC条件下将HA-FCNs水溶液逐滴滴加到硝酸钙溶液中,共混搅拌1.2 h后,逐滴滴加Cur水溶液于上述混合液中,搅拌40 min。随后,将磷酸氢二铵溶液缓慢滴加到上述混合液中,并用NaOH溶液调节pH至8,搅拌45 min后,室温静置陈化17 h,离心除去上清液,洗涤,干燥,得到Cur-HA-FCNs/ACP纳米粒。
对实施例1-5所制备的HA-FCNs/ACP纳米粒进行X射线衍射分析(图3),从图3中可以看出当HA-FCNs浓度不小于600 μg/mL时,复合纳米粒子在30º出现馒头峰,是ACP的特征峰,表明形成纳米粒子是非结晶状态。从扫描电镜(图4)中可以看出当HA-FCNs的浓度为600μg/mL时,制备所得的HA-FCNs/ACP纳米粒呈球形,粒径约100 nm,且分布均一。
实施例6
(1)将0.2 g透明质酸溶解到10 mL去离子水中,在250 ℃油浴搅拌条件下,将10 mL浓硫酸加入透明质酸水溶液中,搅拌反应2 min,用分子量为2000 Da的透析袋透析三天,冷冻干燥得到HA-FCNs荧光材料。
(2)负载DOX的复合材料的制备:分别配制0.034 mol/L 29 mL硝酸钙水溶液、0.024 mol/L 29 mL磷酸氢二铵水溶液、1 mol/L 50 mL NaOH溶液、1000 μg/mL 30 mL HA-FCNs水溶液和0.6 mg/mL 10 mL DOX水溶液,其中,超声分散DOX水溶液(超声条件为:探头超声功率500 W,超声3 s,间歇3 s)。在30 ºC条件下将HA-FCNs水溶液逐滴滴加到硝酸钙溶液中,共混搅拌30 min后,逐滴滴加DOX水溶液于上述混合液中,搅拌30 min。随后,将磷酸氢二铵溶液缓慢滴加到上述混合液中,并用NaOH溶液调节pH至8,搅拌45 min后,室温静置陈化20 h,离心去除上清液,洗涤,干燥,得到DOX-HA-FCNs/ACP纳米粒。
Claims (10)
1.一种肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,其特征在于包括以下步骤:
首先以透明质酸HA为原料,采用高温脱水的方法制备部分碳化透明质酸荧光材料HA-FCNs;然后以硝酸钙为钙源,磷酸氢二铵为磷源,HA-FCNs为标记物,采用共沉淀法制备了肿瘤靶向无定形磷酸钙荧光纳米复合材料;在此基础上实现对亲水性药物或疏水性药物的负载。
2.根据权利要求1所述的肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,其特征在于:所述药物为姜黄素Cur;具体包括以下步骤:
(1)部分碳化透明质酸荧光纳米材料的制备:
首先,在油浴搅拌条件下,以透明质酸为原料,浓硫酸为脱水剂,高温脱水,透析三天,干燥,得到HA-FCNs荧光材料;
(2)负载Cur:
将上述HA-FCNs溶于水中,升温至25-40 ℃,缓慢滴加Ca(NO3)2溶液,共混搅拌10 min-1.5 h ;配制5 mg/mL Cur水溶液,超声分散,逐滴滴加到上述溶液中,共混搅拌;随后,逐滴滴加(NH4)2HPO4溶液,同时用NaOH溶液调节pH,搅拌1 min-1 h后室温陈化10-24 h至反应完全;离心,洗涤,干燥,得到Cur-HA-FCNs/ACP纳米粒。
3.根据权利要求1所述的肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,其特征在于:所述药物为盐酸阿霉素DOX;具体包括以下步骤:
(1)部分碳化透明质酸荧光纳米材料的制备:
首先,在油浴搅拌条件下,以透明质酸为原料,浓硫酸为脱水剂,高温脱水,透析三天,干燥,得到HA-FCNs荧光材料;
(2)负载DOX:
将上述HA-FCNs溶于水中,升温至25-40 ℃,缓慢滴加Ca(NO3)2溶液,共混搅拌10 min-1.5 h ;配制0.6 mg/mL DOX水溶液,逐滴滴加到上述溶液中,共混搅拌1 h;随后,逐滴滴加(NH4)2HPO4溶液,同时用NaOH溶液调节pH,搅拌1 min-1 h后室温陈化10-24 h至反应完全;离心,洗涤,干燥,得到DOX-HA-FCNs/ACP纳米粒。
4.根据权利要求2或3所述的肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,其特征在于:所述部分碳化透明质酸荧光纳米材料的制备步骤中,透明质酸与水的质量比为0.02~0.04。
5.根据权利要求2或3所述的肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,其特征在于:所述部分碳化透明质酸荧光纳米材料的制备步骤中,浓硫酸的用量为10~15 mL。
6.根据权利要求2或3所述的肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,其特征在于:所述部分碳化透明质酸荧光纳米材料的制备步骤中,油浴温度为230~250℃,反应时间为1~2 min。
7.根据权利要求2或3所述的肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,其特征在于:所述部分碳化透明质酸荧光纳米材料的制备步骤中,透析袋分子量为500~3500 Da。
8.根据权利要求2或3所述的肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,其特征在于:所述无定形磷酸钙荧光复合材料的制备步骤中,pH的范围为7~9。
9.根据权利要求2或3所述的肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,其特征在于:所述无定形磷酸钙荧光复合材料的制备步骤中,HA-FCNs的浓度为600~1000 μg/mL。
10.根据权利要求2或3所述的肿瘤靶向无定形磷酸钙荧光纳米复合材料负载药物的方法,其特征在于:负载的过程中,超声条件为:探头超声功率是200~500 W,超声2~4 s,间歇1~3 s。
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