CN105963277A - 一种具有pH和葡萄糖双重响应的纳米球及其制备和应用 - Google Patents
一种具有pH和葡萄糖双重响应的纳米球及其制备和应用 Download PDFInfo
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Abstract
本发明属于纳米材料制备和生物医学领域,具体涉及一种具有pH和葡萄糖双重响应的纳米球及其制备和应用。所述的纳米球是PAA‑PBA‑Glu‑PAA(PPGP)聚合物,其制备过程为:由3‑氨基苯硼酸修饰聚合物分子PAA获得PAA‑PBA,然后由葡萄糖胺修饰PAA制得PAA‑Glu,再将PAA‑PBA和PAA‑Glu通过硼酸酯键进行交联,在水/异丙醇混合液中进行自组装,获得PPGP纳米球。所制得的PPGP纳米球对抗癌药物阿霉素及核磁成像剂Gd3+具有很高的负载率,且针对pH及葡萄糖具有刺激响应释放特性,具有诊断、治疗一体化功能等优点。
Description
技术领域
本发明属于纳米材料制备和生物医学领域,具体涉及一种具有pH和葡萄糖双重响应的纳米球及其制备和应用。
背景技术
钆核磁成像造影剂是一类T1-加权MRI成像造影剂,且钆造影剂是医疗过程中常用的一类造影剂,具有水溶性好、毒性低或没有毒性、体内稳定、能够完全被排出体外、弛豫效能高、具有组织选择性等优点。但是其在体内呈非特异性分布,且静脉注射后迅速漏到血管外并被肾脏清除,因而检查所需剂量高,特别是对于肝、脾等组织病变及肿瘤缺乏特异性,有时为了诊断的目的,不得不加大用药剂量,从而增加了机体出现不良反应的风险。临床上常用的Gd-DTPA造影剂r1值较小(一般3.7 mM-1·s-1 ) ,而根据最近的研究结果表明,如果把钆离子做成纳米颗粒时,其MRI性能会得到提高。所以,发展基于钆离子纳米颗粒制备可降解型多功能药物载体,同时能提高钆造影剂的造影性能,对推动其临床应用具有非常明显的意义。
理想的纳米药物释放系统应具有生物相容性、生物可降解性、较长的血液循环时间、肿瘤靶向性、高载药率、药物刺激响应释放等特性。因此,利用纳米材料作为抗肿瘤药物的载体,构建新型的智能响应体系,实现定时、定量的将药物导入病变部位,提高药物的利用率,在临床治疗中具有潜在的应用前景。聚合物胶束(Polymeric Micelles,PMs)是近20多年来被广泛关注和快速发展的一种新型纳米载体。响应性聚合物胶束可对药物进行靶向可控缓释,在提高药效的同时减少药物的副作用,同时,能在体内降解为小分子化合物,从而被机体代谢、吸收或排泄,对人体无毒副作用,因而已成为药物传递系统研究的热点。
另一方面,低分子量的聚丙烯酸盐(PAA)能在水/异丙醇混合液中自组装成球,这种PAA纳米球因包含大量的羧基基团,所以对亲水性带正电的抗癌药物具有很高的负载率。但是,由于PAA纳米球良好的水溶性,易在水溶液中溶胀破裂,造成药物预泄露很严重,不利于其作为药物载体的后续应用。因此,采用化学手段增强PAA纳米球的交联,达到稳定其结构的目的;同时,又能赋予PAA纳米球新功能(如核磁成像、CT成像等),发展多功能刺激响应型药物释放体系,实现诊治一体化目的,具有极高的研究意义。
发明内容
本发明的目的在于提供一种具有pH和葡萄糖双重响应的纳米球及其制备和应用。本发明所制得PAA-PBA-Glu-PAA(PPGP)聚合物纳米球具有在肿瘤部位受pH及葡萄糖双重响应药物释放特性,同时,PPGP纳米球对抗癌药物阿霉素及核磁成像剂Gd3+具有很高的负载率,可实现诊断、治疗一体化的目的。
为实现上述目的,本发明采用如下技术方案:
一种具有pH和葡萄糖双重刺激响应性的纳米球:其是由3-氨基苯硼酸(PBA)修饰聚丙烯酸钠(PAA)分子制得PAA-PBA;再由葡萄糖胺(Glu)改性聚丙烯酸钠(PAA)获得PAA-Glu,然后将PAA-PBA和PAA-Glu通过硼酸酯键进行交联与自组装,而制得的PAA-PBA-Glu-PAA纳米球。
一种制备如上所述的具有pH和葡萄糖双重刺激响应性的纳米球的方法,包括如下步骤:
a)PAA-PBA聚合物材料的合成:
将一定量的EDC和NHS加入至3-氨基苯硼酸溶液中,然后加入聚丙烯酸钠,在室温条件下反应并对所得产物进行透析、冷冻干燥后,获得PAA-PBA聚合物材料;
b)PAA-Glu聚合物材料的合成:
将一定量的EDC和NHS加入至葡萄糖胺溶液中,加入聚丙烯酸钠,在室温条件下反应并对所得产物进行透析、冷冻干燥后,获得PAA-Glu聚合物材料;
c)PAA-PBA-Glu-PAA纳米球的组装:
将步骤a)制得的PAA-PBA聚合物材料分散于pH=8.0的PBS缓冲液中,加入步骤b)制得的PAA-Glu聚合物材料,室温搅拌12 h,通过硼酸酯键的构建获得PAA-PBA-Glu-PAA聚合物溶液;再往聚合物溶液中逐滴加入一定体积的异丙醇使聚合物分子自组装,然后经甲醇离心、洗涤处理,获得PAA-PBA-Glu-PAA纳米球。
步骤a)中聚丙烯酸钠与3-氨基苯硼酸的反应摩尔比是7:1~2:1;步骤b)中聚丙烯酸钠与葡萄糖胺的反应摩尔比是7:1~2:1。
步骤c)中PAA-Glu聚合物材料与PAA-PBA聚合物材料的质量比是1:1;步骤c)中在PAA-Glu-PBA-PAA的自组装反应体系中水和异丙醇体积比是1:20。
步骤c)制备的PAA-PBA-Glu-PAA纳米球的平均直径为100±10 nm;表面带负电。
一种如上所述的方法制得的具有pH和葡萄糖双重刺激响应性的纳米球的应用:作为核磁成像剂Gd3+和抗癌药物的载体。
在上述应用中,所述的抗癌药物为阿霉素;阿霉素与纳米球复合后,在pH≤6、葡萄糖浓度为1~10 mM的双重刺激条件下释放阿霉素。
在上述应用中,PAA-PBA-Glu-PAA纳米球和核磁成像剂Gd3+复合时的摩尔比为1:0.25~1:2,所制得的PAA-PBA-Glu-PAA-Gd3+纳米聚合物球的平均直径为100±10 nm、表面带正电;更优的,PAA-PBA-Glu-PAA纳米球和核磁成像剂Gd3+复合时的摩尔比为1:1.5,此时,Gd3+的实际固定量达到74.76%。
与其他药物释放体系相比,本发明的显著优点在于:
(1)本发明的聚合物纳米球PPGP制备方法简单,合成颗粒尺寸较为均一,条件温和可控,易于规模化;可负载核磁成像剂Gd3+和抗癌药物,具有pH和葡萄糖双重刺激响应性;
(2)本发明的聚合物纳米球对核磁成像剂Gd3+和抗癌药物的负载率高,对抗癌药物的负载率可达到74.21%,因此可有效降低治疗过程中药物载体的使用量,达到减少副作用的目的;
(3)本发明所述的聚合物纳米球PPGP生物相容性优良,易于在细胞内降解,是可降解型药物载体;
(4)以本发明聚合物纳米球为载体的核磁成像剂PPGP-Gd3+在肿瘤细胞内易受酸性及葡萄糖双重刺激响应,硼酸酯键解离,释放出所负载钆离子,可实现对肿瘤或炎症组织的MRI成像功能。
附图说明
图1为PPGP纳米球的低倍透射电镜图(TEM)(A);PPGP-Gd3+纳米球的低倍透射电镜图(TEM)(B);
图2为PPGP纳米球的红外图谱;横坐标为波数,纵坐标为吸收强度;
图3为PAA纳米球与PPGP纳米球的粒径变化图;横坐标为不同时间组样品(1、甲醇;2、pH=7.4,PBS-0 h;3、pH=7.4,PBS-24 h;4、pH=7.4,PBS-72 h);
图4为PAA-DOX纳米球与PPGP-DOX纳米球的预泄漏实验;横坐标为释放时间,纵坐标为释放百分率;
图5为考察PPGP-Gd3+纳米球的生物相容性测试;横坐标为纳米球浓度,纵坐标为细胞存活率;
图6为考察PPGP-Gd3+纳米球的细胞毒性实验;横坐标为不同样品组,纵坐标为细胞存活率;1、Control 2、PPGP-Gd3+ 3、PPGP-Gd3+-DOX 4、Free DOX;
图7为PPGP-Gd3+纳米球在不同条件下的体外核磁共振成像对比图;横坐标为Gd3+浓度,纵坐标为每秒分之一。
具体实施方式
下面以具体实施示例对本发明的技术方案做进一步说明,但是不能以此限制本发明的范围。
实施例1
一种制备具有pH和葡萄糖双重刺激响应的聚合物纳米球的方法,具体步骤为:
a)PAA-PBA聚合物材料的合成:
取4 mg PAA溶解于2 mL去离子水中,按摩尔比4:1加入2.46 mg 3-氨基苯硼酸,2.73mg EDC与1.634 mg NHS,在室温条件下反应24 h,透析处理24 h,冷冻干燥12 h,获得PAA-PBA聚合物材料;
b)PAA-Glu聚合物材料的合成:
取4 mg PAA溶解于2 mL去离子水中,按摩尔比4:1加入3.06 mg葡萄糖胺,2.73 mg EDC与1.634 mg NHS,在室温条件下反应24 h,透析处理24 h,冷冻干燥12 h,获得PAA-Glu聚合物材料;
c)PPGP聚合物纳米球的组装:
将步骤a)制得的PAA-PBA材料分散于pH=8.0的PBS缓冲液中,按质量比为1:1加入步骤b)制得的PAA-Glu材料,室温搅拌12 h,通过硼酸酯键的构建获得PPGP聚合物材料;之后再加入16 mL异丙醇使其自聚合,进一步形成PPGP聚合物纳米球;
d)PPGP-Gd3+-DOX聚合物纳米球的组装:
将步骤c)制得的PPGP聚合物纳米球分散于甲醇中,按摩尔比为1:1.5加入六水硝酸钆,室温搅拌6 h,离心甲醇清洗处理;再按质量比1:2加入DOX,室温搅拌12 h,进一步离心处理得PPGP-Gd3+-DOX聚合物纳米球。通过ICP-MS 检测确认Gd3+的实际固定量达到74.76%;荧光数据表明DOX的吸附率是74.21%。
步骤c)制备的PPGP纳米球相比PAA纳米球具有更好的热稳定性,有利于药物的缓释性能;Zeta粒度分析仪数据表明:在pH=7.4缓冲液中,在0-72h内,PPGP纳米球的粒径从150 nm增大到367 nm,而PAA纳米球则从367 nm增大到1365 nm。在pH=7.4缓冲液中,PPGP-DOX在48 h内的预泄露率为24.18%,而PAA-DOX的预泄露率为98.7%。上述数据说明PPGP纳米球在水中的稳定性更高,更有利于药物的缓慢释放。
应用实施例1
将实施例1步骤c)制得的PPGP纳米球,取0.5 mg重新分散到0.875 mL甲醇溶液中,加入12.5 uL,0.1 M的六水硝酸钆甲醇溶液,室温反应6 h之后,离心处理得固体,继续用甲醇洗2次,最后获得PPGP-Gd3+纳米聚合物胶束。
体外核磁成像实验结果表明,在pH=5.0缓冲液中PPGP-Gd3+纳米球造影剂的r1值为14.93 mM-1·S-1;在葡萄糖=10 mM,pH=5.0的缓冲液中,PPGP-Gd3+纳米球造影剂的r1值最高可达到34.34 mM-1·S-1,说明PPGP-Gd3+聚合物纳米球可实现针对pH及葡萄糖双重刺激响应触发的T1加权MRI成像。
性能检测:
1、将实施例1制得PPGP纳米球甲醇溶液滴在铜网上,晾干后进行TEM扫描(见图1),结果见图1所示,从图1-A中可以看出PPGP胶束为球形颗粒,尺寸较为均一,分散性较好,平均粒径约为100±10 nm;将应用实施例1制得PPGP-Gd3+纳米球甲醇溶液滴在铜网上,从图1-B中可以看出PPGP-Gd3+纳米聚合物胶束仍为球形结构,尺寸均一,平均粒径约为100±10 nm;
2、将实施例1制得不同产物在60℃干燥成固体,取3 mg与溴化钾均匀混合,之后利用压片机将其压制成透明均匀的小薄片;红外测试结果如图2所示,其中图2为四种聚合物的叠加图,可知聚合物b和d在波数为3000-3700 cm-1左右有较大宽峰,主要由于此二者聚合物含有较多-OH,并且聚合物b、c、d均有氨基吸收峰,说明PAA-Glu、PAA-PBA、以及PPGP均已通过酰胺键成功交联;在聚合物c、d中波数在1350-1310 cm-1处具有硼氧键吸收峰,进一步证明了PAA- PBA与PPGP成功合成;并且由于PPGP上硼氧键受聚合物影响,导致其发生轻微的红移;
3、分别取PAA纳米球和PPGP(纳米球加入至PBS(pH=7.4)缓冲溶液中,配制浓度为1mg/mL,混匀。样品置于25℃恒温箱中,分别于0 h~72 h取样测粒径大小变化。结果如图3所示,PPGP纳米球的粒径从90 nm增大到367 nm,而PAA纳米球则从132 nm增大到1365nm,可知PAA纳米球在水中溶胀明显,而PPGP纳米球相对稳定,说明通过硼酸酯键的交联,能够提高这类纳米球的稳定性;
4、取2 ml 1 mg/ml 实施例1制得的PPGP纳米球加入至4 ml 1 mg/ml的DOX(阿霉素)充分混合,室温避光搅拌12 h,离心,水洗两次收集上清液。用荧光分光光度计(Ex=488 nm),记录波长在500 nm至800 nm间的发射光谱最大吸收值,并根据阿霉素标准曲线计算出每克PPGP纳米球可负载742.1 mg的盐酸阿霉素。考察负载了阿霉素的PPGP纳米球和PAA纳米球在pH=7.4 PBS缓冲溶液中的预泄漏效果,具体的实验步骤如下:将1 mg吸附药物后的PPGP纳米球和PAA纳米球分别分散于3 ml PBS (pH=7.4,10 mM) 溶液中,放入透析袋后将透析袋加入47 ml PBS溶液中透析48 h,每隔0 h,3 h,6 h,12 h,24 h,48 h取3ml溶液测荧光,原溶液再补入3 ml PBS,测试的结果如图4所示。两条曲线分别代表PAA-DOX纳米球和PPGP-DOX纳米球;从图4中可以看出在pH=7.4条件下PAA-DOX所释放出的DOX荧光强度最高,PPGP-DOX在48 h内的预泄露率为24.18%,而PAA-DOX的预泄露率为98.7%。说明PPGP纳米球在水中的稳定性更高,有利于药物的缓慢释放;
5、用HeLa细胞作为目标细胞评价PPGP纳米球的生物相亲性:取已消化成单分散的HeLa细胞悬浊液用培养液稀释,以100μL/孔的密度接种到96孔板,每孔的细胞个数控制约为105个。将96孔板置于37 ℃,5% CO2的培养箱中培养24 h后,移去培养液,加入含有不同浓度的PPGP纳米球培养液,每组设置4个重复孔。继续培养4h后,移去培养液,用PBS缓冲液(pH=7.4)清洗两次,加入100μL培养液继续培养20 h后,每孔分别加入10μL浓度为5 mg/mL的MTT,继续培育4 h,小心移去培养液,加入150μL DMSO,37oC培养25 min,震荡均匀后,在酶标仪上测490 nm处的吸收值,计算细胞存活率,评价PPGP纳米球生物相亲性。图5显示PPGP纳米球浓度在5~100μg/ml区间,细胞存活率均在90%以上,说明PPGP纳米球生物具有优异的生物相亲性,适于当药物载体;
6、取已消化成单分散的HeLa细胞悬浊液用培养液稀释,以100μL/孔的密度接种到96孔板,每孔的细胞个数控制约为105个,每组设置4个复孔作为重复组。将96孔板置于37 ℃,5%CO2的培养箱中培养24 h后,移去孔中培养液,分别加入含有1,Control 2,PPGP-Gd3+(6.7 μg/ml) 3,PPGP-Gd3+-DOX(6.7μg/ml,DOX浓度为5μg/ml) 4,Free DOX(5μg/ml),其中3组中DOX的浓度为5μg/ml,加入细胞培养液,继续培养4 h,移去培养液,用PBS缓冲液清洗两次,加入100μL培养液继续培养20 h后,每孔分别加入10μL浓度为5 mg/mL的MTT,继续培育4 h,小心移去培养液,加入150μL DMSO,37 ℃培养20 min,振荡均匀后,在酶标仪上测490 nm处的吸收值,计算细胞存活率,评价各组治疗效果。从图6可以看出PPGP-Gd3+-DOX对癌细胞在24 h内抑制率达到63.68 %,说明该载药体系能有效地用于肿瘤的治疗;
7、PPGP-Gd3+纳米聚合物胶束溶液的体外核磁共振成像:
(1)先取1 ml 0.5mg/mL PPGP-Gd3+纳米聚合物胶束加入透析袋中,放入49 ml 2wt%硝酸进行解离24 h,使其完全解离,再取一定量按需要稀释测ICP-MS,根据ICP-MS测试结果计算出1 mL PPGP-Gd3+纳米聚合物胶束溶液中含有Gd3+为0.942 mM;
(2)取0.212 mL PPGP-Gd3+制备液,分别加入到1ml pH=7.4、pH=5.0、pH=5.0+10 mM葡萄糖的PBS溶液中(相当于所含Gd3+浓度为0.2 mM),浸泡24h,离心后,取一定量上清液配成5个不同浓度,取出1 mL去测MRI。从图7可以看出:在pH=5.0和pH=5.0+10 mM葡萄糖条件下,随着解离出来钆离子的浓度增加,成像的亮度也逐渐变亮;而在pH=7.4条件下,成像的亮度基本没有变化,说明在pH=7.4缓冲液中PPGP-Gd3+纳米球比较稳定,钆离子解离的量较少。由图7可知:在pH=5.0和pH=5.0+10 mM葡萄糖条件下的r1=14.93 mM-1·S-1和r1=34.34 mM-1·S-1均大于pH=7.4条件下的r1=2.54 mM-1·S-1,这说明在酸性以及葡萄糖存在条件下,PPGP-Gd3+纳米球发生解构,释放出较多的Gd3+,即造影剂增多,进而MRI成像效果增强。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (9)
1.一种具有pH和葡萄糖双重刺激响应性的纳米球,其特征在于:其是由3-氨基苯硼酸修饰聚丙烯酸钠分子制得PAA-PBA;再由葡萄糖胺改性聚丙烯酸钠获得PAA-Glu,然后将PAA-PBA和PAA-Glu通过硼酸酯键进行交联与自组装,而制得的PAA-PBA-Glu-PAA纳米球。
2.一种制备如权利要求1所述的具有pH和葡萄糖双重刺激响应性的纳米球的方法,其特征在于:包括如下步骤:
a)PAA-PBA聚合物材料的合成:
将一定量的EDC和NHS加入至3-氨基苯硼酸溶液中,然后加入聚丙烯酸钠,在室温条件下反应并对所得产物进行透析、冷冻干燥后,获得PAA-PBA聚合物材料;
b)PAA-Glu聚合物材料的合成:
将一定量的EDC和NHS加入至葡萄糖胺溶液中,加入聚丙烯酸钠,在室温条件下反应并对所得产物进行透析、冷冻干燥后,获得PAA-Glu聚合物材料;
c)PAA-PBA-Glu-PAA纳米球的组装:
将步骤a)制得的PAA-PBA聚合物材料分散于pH=8.0的PBS缓冲液中,加入步骤b)制得的PAA-Glu聚合物材料,室温搅拌12 h,通过硼酸酯键的构建获得PAA-PBA-Glu-PAA聚合物溶液;再往聚合物溶液中逐滴加入一定体积的异丙醇使聚合物分子自组装,然后经甲醇离心、洗涤处理,获得PAA-PBA-Glu-PAA纳米球。
3.根据权利要求2所述的制备具有pH和葡萄糖双重刺激响应性的纳米球的方法,其特征在于:步骤a)中聚丙烯酸钠与3-氨基苯硼酸的反应摩尔比是7:1~2:1;步骤b)中聚丙烯酸钠与葡萄糖胺的反应摩尔比是7:1~2:1。
4.根据权利要求2所述的制备具有pH和葡萄糖双重刺激响应性的纳米球的方法,其特征在于:步骤c)中PAA-Glu聚合物材料与PAA-PBA聚合物材料的质量比是1:1;步骤c)中在PAA-Glu-PBA-PAA的自组装反应体系中水和异丙醇体积比是1:20。
5.根据权利要求2所述的制备具有pH和葡萄糖双重刺激响应性的纳米球的方法,其特征在于:步骤c)制备的PAA-PBA-Glu-PAA纳米球的平均直径为100±10 nm;表面带负电。
6.一种如权利要求2-5任一项所述的方法制得的具有pH和葡萄糖双重刺激响应性的纳米球的应用,其特征在于:作为核磁成像剂Gd3+和抗癌药物的载体。
7.根据权利要求6所述的应用,其特征在于:所述的抗癌药物为阿霉素;阿霉素与纳米球复合后,在pH≤6、葡萄糖浓度为1~10 mM的条件下释放阿霉素。
8.根据权利要求6所述的应用,其特征在于:PAA-PBA-Glu-PAA纳米球和核磁成像剂Gd3+复合时的摩尔比为1:0.25~1:2,所制得的PAA-PBA-Glu-PAA-Gd3+纳米聚合物球的平均直径为100±10 nm、表面带正电。
9.根据权利要求8所述的应用,其特征在于:PAA-PBA-Glu-PAA纳米球和核磁成像剂Gd3+复合时的摩尔比为1:1.5。
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