CN115581660B - 一种微针抗肿瘤复合递药系统及其制备方法和应用 - Google Patents
一种微针抗肿瘤复合递药系统及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种微针抗肿瘤复合递药系统及其制备方法和应用,微针抗肿瘤复合递药系统将NO供体偶联的pH敏感性纳米递药系统和肿瘤疫苗纳米粒共同负载在聚乙烯吡咯烷酮微针中,NO供体偶联的pH敏感性纳米递药系统为纳米胶束,外壳由NO供体偶联的pH响应性共聚物构成,内核由化疗药物组成,肿瘤疫苗纳米粒是通过静电结合作用将蛋白抗原和免疫佐剂聚二甲双胍结合形成;本发明的递药系统通过可溶性微针递药系统穿透皮肤角质层直接递送药物,避免了肝脏首过效应,利用化学治疗、气体治疗、免疫治疗三种治疗手段协同治疗肿瘤,提高了药物的生物利用度,减少了药物的副作用。
Description
技术领域
本发明涉及一种复合递药系统,尤其涉及一种微针抗肿瘤复合递药系统,还涉及所述递药系统的制备方法及应用。
背景技术
皮肤癌是一种皮肤表皮发生恶性病变引起的恶性肿瘤,皮肤癌具有高转移能力,且常规技术手段难以使药物准确抵达肿瘤病灶,导致治愈率低、病变进展快。目前,气体治疗成为肿瘤治疗领域的新兴手段,其中,一氧化氮(Nitric oxide,NO)作为调节剂来调节免疫抑制性肿瘤微环境(tumor microenvironment,TME)被广泛应用于气体疗法,常用于储存和释放NO的供体为S-亚硝基谷胱甘肽(S-nitrosoglutathione,GSNO)。然而,GSNO在水溶液中的稳定性低,且对肿瘤组织无选择性,难以高效且长期的提供肿瘤靶向性。
发明内容
发明目的:本发明的目的是提供一种强靶向性、高效抑制肿瘤生长的微针抗肿瘤复合递药系统,第二目的是提供上述递药系统的制备方法及应用。
技术方案:本发明所述的微针抗肿瘤复合递药系统,是将NO供体偶联的pH敏感性纳米递药系统和肿瘤疫苗纳米粒共同负载在聚乙烯吡咯烷酮微针中;所述NO供体偶联的pH敏感性纳米递药系统为纳米胶束,外壳由NO供体偶联的pH响应性共聚物构成,内核由化疗药物组成。
优选的,所述NO供体偶联的pH敏感性纳米递药系统,是通过一氧化氮供体在pH响应性聚合物PLA-PEOz表面进行酯键连接GSNO形成两亲性偶联物,并通过疏水作用包载化疗药物构建的纳米递药系统;所述化疗药物为紫杉醇、阿霉素或卡莫司汀中的一种。
优选的,所述的pH响应性共聚物为聚乳酸-聚(2-乙基-2-唑啉),所述酯键的反应基团为嵌段共聚物中PEOz的羟基与NO供体中的羧基,其中NO供体为含有-COOH结构的S-亚硝基谷胱甘肽。
优选的,所述免疫佐剂肿瘤疫苗纳米粒,是通过静电结合作用将蛋白抗原和免疫佐剂聚二甲双胍结合形成。
所述复合递药系统的制备方法,包括如下步骤:
(1)NO供体偶联的pH响应性共聚物GSNO-PLA-PEOz的制备:
将含羧基的NO供体与活化剂溶于水或有机溶剂,避光条件下冰浴搅拌活化羧基;向反应体系中加入嵌段共聚物PLA-PEOz,继续室温搅拌;反应结束后除去反应溶剂;加水重新溶解并透析,冻干,即得GSNO-PLA-PEOz;
(2)NO供体偶联的pH敏感性纳米递药系统的制备:
将GSNO-PLA-PEOz和化疗药物溶于有机溶剂,减压旋蒸除去有机溶剂,接着加水水化得纳米粒混悬液,过滤即得形态为纳米胶束的NO供体偶联的pH敏感性纳米递药系统;
(3)免疫佐剂肿瘤疫苗纳米粒的制备:
取蛋白抗原和免疫佐剂溶于水中,搅拌超声,过滤即得免疫佐剂肿瘤疫苗纳米粒,所述免疫佐剂为聚二甲双胍PM;
(4)微针抗肿瘤复合递药系统的制备:
将步骤(2)得到的NO供体偶联的pH敏感性纳米递药系统的纳米胶束和步骤(3)得到的纳米粒,溶于针尖PVP溶液中注入模具,真空排泡干燥,然后加入基底PVP溶液真空干燥得到微针抗肿瘤复合递药体系。
优选的,步骤(1)中,所述活化剂为N,N′-羰基二咪唑(CDI)、二环己基碳二亚胺(DCC)、4-二甲氨基吡啶(DMAP)或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)中一种或两种以上的组合;所述有机溶剂为甲酰胺、N,N-二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)或四氢呋喃(THF)中一种或两种以上的组合;
所述活化羧基的时间为20min-24h;含羧基的NO供体与嵌段共聚物的质量比为1:10~30,反应时间为24h。
优选的,步骤(2)中,所述有机溶剂为甲醇或氯仿的一种或两种组合;所述化疗药物与GSNO-PLA-PEOz的质量比为1:5~10,成膜时间为10-15min,成膜温度为40-60℃,水化时间为5-30min,水化温度为40-60℃。
优选的,步骤(3)中,所述免疫佐剂和蛋白抗原的质量比为1:1~3,搅拌时间为24-48h,超声时间为10-20min,所述免疫佐剂肿瘤疫苗纳米粒的粒径为100-200nm。
优选的,步骤(4)中,所述纳米胶束、纳米粒、针尖PVP溶液和基底PVP溶液的质量比为200~300:1:8000~10000:40000~80000;所述PVP溶液为PVP K30和PVP K12的混合溶液,PVP K30和PVP K12的质量比为4:1~6。
上述微针抗肿瘤复合递药系统在制备抗肿瘤药物中的应用。
发明原理:本发明首先制备了一种pH响应聚合物胶束的两亲性嵌段共聚物聚(2-乙基-2-噁唑啉)-聚乳酸(PLA-PEOz),即GSNO-PLA-PEOz,利用该pH敏感性两亲性聚合物递送化疗药物GSNO及NO气体,制成NO供体偶联的pH敏感性纳米递药系统为纳米胶束,该纳米胶束是NO供体偶联的pH敏感性两亲性共聚物,并包载了脂溶性化疗药物构建了pH敏感性纳米给药系统;提高疗效的同时,可以降低毒副作用。其中,GSNO-PLA-PEOz聚合物胶束通过内吞作用内化后的细胞内运输通常通过pH梯度的内体溶酶体途径进行,充分利用实体肿瘤和正常组织之间细胞外基质的相对酸度存在差异,通过高通透性和滞留效应使纳米粒被动靶向于肿瘤组织,被溶酶体吞噬后,pH降低,PEOz内部发生质子转移,由弯曲状态变为直链状态,导致药物释放。在微针主动靶向的基础上进一步提高抗癌药物在肿瘤组织局部的药物浓度,增强抗肿瘤效果、减轻毒副作用。
接着,选择OVA作为肿瘤疫苗,并加入免疫佐剂polymet(PM),利用静电作用合成了PM/OVA纳米粒,促进激活机体免疫反应,进一步杀伤肿瘤。OVA呈现负电,与PM结合后可以增强OVA的细胞摄取能力,同时可以促进TAM M2复极化为M1,同时PM结构中的利用可溶性微针递药系统将抗原和佐剂共同递送至表皮层释放活性成分,诱导抗原特异性免疫,增强免疫效果,产生显著的抗肿瘤活性。
最后,将以上两种纳米粒共同负载于生物相容性好且在体内能够完全降解的聚乙烯吡咯烷酮微针PVP MNs中,将药物安全可控的递送至肿瘤部位。本发明的递药系统,一方面可以保证化疗药物最大程度的在肿瘤组织中蓄积,减小化疗药物对正常组织的毒性;另一方面可以实现NO的定点释放,保证NO在肿瘤组织中的最大起效浓度。NO可以促进化疗药物在肿瘤组织内的深层次渗透,增强化疗药物的抗肿瘤效果,同时可以促进M2型巨噬细胞复极化为M1型巨噬细胞。制备了纳米肿瘤疫苗,可以刺激机体B细胞产生特异性抗体,同时激活T细胞产生免疫反应杀伤肿瘤,纳米肿瘤疫苗中的免疫佐剂可在抗原的基础上进一步增强DC2.4细胞熟化,并且可以与NO共同重塑免疫抑制肿瘤微环境,增强肿瘤免疫。将两种纳米粒共同负载于微针当中,构建了微针抗肿瘤复合递药体系用于体表肿瘤的靶向治疗。该递药系统具有多疗法协同抗肿瘤的特点,能有效抑制肿瘤生长,促进肿瘤凋亡,具有良好的抗肿瘤效果,具有肿瘤精准靶向、肿瘤微环境双响应、化学治疗/气体治疗/免疫治疗三种手段协同治疗的特点。
有益效果:与现有技术相比,本发明具有如下优点:(1)本发明的微针抗肿瘤复合递药系统,能同时负载脂溶性化疗药物和肿瘤疫苗纳米粒,且能够将药物最大程度的截留在肿瘤部位,通过协调增效的效果减小用药剂量,降低药物对正常组织脏器的毒性;
(2)本发明微针抗肿瘤复合递药系统的制备方法,制备简单方便快速,与其他纳米载体相比具有较高的包封率和载药量,其负载量最高可达17.84%。
附图说明
图1为GSNO-PLA-PEOZ核磁氢谱图;
图2为GSNO-PLA-PEOz@PTX纳米粒透射电镜图(TEM);
图3为不同pH条件下GSNO-PLA-PEOz@PTX纳米粒粒径图;
图4为pH=5.0时GSNO-PLA-PEOz@PTX纳米粒TEM图;
图5为不同pH条件下GSNO-PLA-PEOz@PTX纳米粒体外药物释放图;
图6为PM/OVA纳米粒的TEM图;
图7为微针抗肿瘤复合递药体系不同时间溶解情况立体显微镜图;
图8为GSNO-PLA-PEOz@PTX及PLA-PEOz@PTX纳米粒对B16细胞的细胞毒性图;
图9为不同给药处理条件下的B16细胞凋亡实验数据图;
图10为不同给药处理条件下的B16细胞迁移实验数据图;
图11为DC2.4细胞对不同处理条件下的抗原摄取图;
图12为纳米肿瘤疫苗对DC2.4细胞熟化的实验数据图;
图13为复合纳米递药体系对RAW264.7细胞复极化的实验数据图;
图14为复合纳米递药体系对黑色素瘤荷瘤小鼠的肿瘤诱导坏死的实验数据图。
具体实施方式
下面结合附图及实施例对本发明作进一步描述。
实施例1:GSNO偶联的pH响应性两亲性共聚物GSNO-PLA-PEOz的合成称取2mg GSNO与活化剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)0.19mg溶于少量水中,将0.01mg 4-二甲氨基吡啶(DMAP)溶于4mL四氢呋喃(THF)中,将二者混合于避光条件下冰浴搅拌20min活化羧基;称取60mg嵌段共聚物PLA-PEOz室温搅拌反应24h;反应结束后,采用旋转蒸发仪除去反应溶剂,然后加入10mL超纯水重新溶解并转移至3500Da透析袋中去离子水透析27h;透析结束后,冻干,即得GSNO-PLA-PEOz产物。
实施例2:GSNO偶联的pH响应性两亲性共聚物GSNO-PLA-PEOz的结构表征
1.核磁氢谱(1H-NMR)表征:取适量GSNO、PLA-PEOz、GSNO-PLA-PEOz,溶于氘代DMSO,500MHz频率,采用核磁共振仪进行结构分析,结果见附图1,由结果可知,GSNO-PLA-PEOz合成成功。
2.NO键合率测定:将5mg GSNO-PLA-PEOz冻干物,溶于200μL纯水中,加2mg GSH孵育30min,按照NO检测试剂盒要求操作后,用酶标仪于λ=550nm下测定吸光度。测定的GSNO键合率为(16.7±0.008)%。
实施例3:GSNO偶联的pH响应性GSNO-PLA-PEOz@PTX纳米粒的制备
称取质量比为5:1的GSNO-PLA-PEOz和PTX溶解于5ml甲醇中,水浴条件下下减压旋蒸除去有机溶剂成膜,然后在一定温度水浴条件下加入纯水水化得纳米粒混悬液,用0.22μm微孔滤膜过滤后即得GSNO偶联的pH响应性GSNO-PLA-PEOz@PTX纳米粒。
为筛选最佳工艺条件,采用正交试验法,以PLA-PEOz及PTX为原料确定水化时间、水化温度、水化体积为主要影响的三个因素,并选择三个水平进行考察,以载药量、包封率、粒径及PDI为评价指标。正交试验因素水平表见表1;
表1、L9(33)正交试验水平表
按照L9(33)正交表安排试验1-9,具体工艺条件见表2所示:
表2、PLA-PEOz@PTX纳米粒正交试验工艺条件
试验1-9所得数据结果见表3所示
表3、正交试验数据表
用spss23.0软件分析对正交试验结果进行方差分析,结果如表4所示;
表4、正交试验方差分析表
对实验结果进行方差分析和直观分析可得,水化时间、水化温度、水化体积均对实验结果具有显著性差异(P<0.05),根据实验结果选定水化时间为5min,水化温度为60℃,水化体积为10mL为最佳实验条件,并以此条件作为GSNO-PLA-PEOz@PTX纳米粒的制备条件。
实施例4:GSNO偶联的pH响应性GSNO-PLA-PEOz@PTX纳米粒的表征
1.粒径及电位表征
用激光粒径仪测定GSNO-PLA-PEOz@PTX的水合粒径和Zeta电位。结果显示,粒径为(51.73±3.41)nm,PDI为(0.126±0.043),Zeta电位为(-6.05±0.08)mV。
2.透射电镜观察
取适量GSNO-PLA-PEOz@PTX,滴加在铜片上烘干,再滴加等量的磷钨酸染色3min,烘干,用透射电镜拍摄,结果见附图2,可见GSNO-PLA-PEOz@PTX纳米粒呈现圆形。
3.包封率与载药量测定
取1mg GSNO-PLA-PEOZ@PTX冻干产物溶于CH3OH中,超声20min,过0.45μm有机膜,将液体转入10mL容量瓶中,补足体积到10mL,进样至高效液相色谱(HPLC)中分析。结果显示,纳米粒包封率为(88.25%±1.21),纳米粒载药量为(17.65%±0.19)。
4.pH响应性验证
分别将GSNO-PLA-PEOz@PTX纳米粒按照1:10的比例置于pH=7.4、pH=6.8、pH=5.0的磷酸盐缓冲液中,常温放置12h,测定纳米粒的粒径、PDI。结果表明,随着pH的降低,GSNO-PLA-PEOZ@PTX的粒径增大,且PDI增加,结果见附图3。另外,通过TEM观察,结果见附图4,说明GSNO-PLA-PEOZ@PTX随着pH的变小,结构被破坏,整体变得松散。
5.GSNO-PLA-PEOZ@PTX纳米粒体外释药特性
取1mg GSNO-PLA-PEOZ@PTX纳米粒冻干产物溶于1mL纯水中后置于3500Da的透析袋中;释放介质为50mL含0.5%的吐温80的PBS溶液;置于水浴恒温摇床中摇荡(37℃,100rpm),分别于30min,1h,2h,4h,8h,12h,24h,48h取1mL样品,并补加1mL释放介质。将样品于HPLC下测定释放的PTX量。结果见附图5。
由结果可知药物在不同pH下均可释放,但在酸性环境中释放较快且释放量较多。在48h内,PTX在pH=5.0的条件下药物率达到80%以上,PTX在pH=7.4的条件下药物率接近65%,证明材料具有pH响应性在酸性条件下释放速率较快且释放量较多。
6.GSNO-PLA-PEOZ@PTX纳米粒的稳定性探究
制备三批GSNO-PLA-PEOz@PTX,测定其粒径及PDI,于4℃冰箱放置1周,观察粒径与PDI。发现PDI均<0.3,且粒径变化不超过4nm,说明GSNO-PLA-PEOz@PTX纳米粒稳定性良好。
实施例5:PM/OVA纳米粒的制备
分别称量不同比例OVA与PM置于西林瓶中,加入8ml水,超温搅拌反应一定时间后停止反应,将所得到的溶液与离心机中以4000rpm的速度离心10min去除不溶物;将上清液于超声细胞破碎仪中超声一定时间(200W,超2s,停2s);超声完过0.22μm水系滤膜,冻干即得PM/OVA NPs,根据预实验结果,选定3个因素进行考察,分别为OVA与PM比例、搅拌时间、超声时间。具体制备工艺条件见表5所示。
表5、PM/OVA单因素考察
数据结果如表6所示:
表6、PM/OVA单因素考察试验结果
经过单因素考察,最终确定OVA:PM的质量比为1:1,超声时间为20min,搅拌时间为48h。
实施例6:PM/OVA纳米粒的表征
1.粒径及电位表征
用激光粒径仪测定PM/OVA的水合粒径和Zeta电位。结果显示,粒径为(175.8±5.3)nm,PDI为(0.017±0.005),Zeta电位为(37.6±3.8)mV。
2.纳米粒包封率:取4ml PM/OVA纳米粒置于4mL 100KDa的超滤管中离心,离心25min,收集外管液体,用BCA蛋白试剂盒测定游离OVA量,然后如下公式计算OVA包封率,
结果得OVA包封率为79.47%
实施例7:“一体式”阵列微针抗肿瘤复合递药体系的制备
称取3mg的GSNO-PLA-PEOz@PTX纳米粒、40μg PM/OVA纳米粒溶于150μL 40%的聚乙烯吡咯烷酮(PVPK12:PVP K30=2:3)溶液中,充分溶解后注入微针模具中;放入真空干燥器中常温干燥过夜,用刮刀刮去针尖多余部分。注入400μL 40%的PVP K12:PVP K30=1:4的溶液填充微针基底,放入真空干燥器中室温干燥过夜,脱模取出即得。
实施例8:微针抗肿瘤复合递药体系的表征
1.微针抗肿瘤复合递药体系的溶解动力学考察
将微针插入小鼠皮肤内,在0min、3min、5min、10min、15min后取出,用立体显微镜观察微针溶蚀情况,并记录微针完全溶解时长。结果见附图7
由结果可知,微针插入小鼠皮肤后逐渐开始溶解,15min时针尖全部溶解。
2.微针抗肿瘤复合递药体系的载药量及体内释放量分析
用小刀刮下微针针尖,溶解后加入0.1mL纯水溶解后,再加入0.9mL甲醇超声破乳30min,过0.22μm微孔滤膜后用HPLC测定PTX的负载量。
将微针插入小鼠背部,待微针全部溶解后拔出(15min),全部溶解后,将基质溶解,过0.22μm微孔滤膜后用HPLC测定PTX的负载量。结果表7所示,由结果可知,微针平均针尖载药量为PTX 27.42μg,药物体内释放量为25.65μg。
表7、微针插入小鼠前后载药量分析
实施例9:微针抗肿瘤复合递药体系的抗肿瘤应用
1.细胞毒性实验
采用四甲基偶氮唑盐(MTT)法,分别考察PP@PTX、GPP@PTX纳米粒对小鼠黑色素瘤细胞(B16)的细胞毒性作用。实验结果表明,GPP@PTX纳米粒对B16细胞的IC50为37.16μg/mL,PP@PTX NPs对B16细胞的IC50为67.32μg/mL。表明连接NO供体可进一步促进化疗药物对B16细胞的杀伤效果。
2.细胞凋亡实验
采用Annexin V-EGFP/PI凋亡检测试剂盒,控制为PTX为5μg/mL后,分别测定PTX、PP@PTX NPs、GPP@PTX NPs体外诱导B16细胞凋亡的能力。如附图9所示,GPP@PTX NPs组相比于其他组对B16细胞具有最强的凋亡诱导效果。
3.细胞迁移实验
在六孔板中接种B16细胞,控制PTX的量为3μg/mL后,分别测定PTX、PP@PTX NPs、GPP@PTX NPs对B16迁移率的影响,并设置对照组,将24h后的划痕面积采用Image J软件分析。实验结果如图10,对照组的B16细胞迁移率约为66.40%,PTX组为33.41%,PP@PTX组为15.21%,GPP@PTX组为9.15%,说明GPP@PTX NPs能够显著抑制B16细胞的迁移。
4.免疫佐剂PM对抗原OVA摄取的促进作用
在共聚焦皿中接种树枝状细胞(DC2.4),FITC标记OVA,控制OVA的用量为10μg/mL,结果如图11所示,PM与OVA结合形成纳米粒后可以增加抗原呈递细胞DC2.4对OVA的摄取量。且随着时间推移摄取量呈上升状态,不同时间点DC2.4细胞更易摄取PM/FITC-OVA NPs,荧光强度大于FITC-OVA组;同时2h与1h相比,细胞的对OVA的摄取量更多,荧光强度增强。
5.纳米肿瘤疫苗对DC2.4细胞熟化效果
在六孔板中接种DC2.4细胞,控制OVA的用量为10μg/mL,并用CD86与CD80流式荧光抗体标记,用流式细胞仪检测DC2.4的熟化效果。结果如图12所示,证明免疫佐剂PM可以在抗原OVA的基础上进一步促进DC2.4细胞的熟化。
6.复合纳米递药体系对RAW264.7细胞复极化的效果
在六孔板中接种RAW264.7细胞,待其贴壁后用IL-4诱导36h,使得RAW264.7细胞极化成M2型巨噬细胞,接着考察不同组的复极化效果,控制OVA的用量为10μg/mL,GPP用量为100μg/mL。CD206是M2型巨噬细胞的特征受体,CD86是M1型巨噬细胞的特征受体,利用荧光抗体标记两种受体后利用流式细胞仪考察两种表型的巨噬细胞含量,进而考察不同纳米粒复合物对于M2型巨噬细胞的复极化效果。结果如图13所示,NO与免疫佐剂可以协同促进M2型巨噬细胞复极化为M1型巨噬细胞。
7.复合纳米递药体系对黑色素瘤荷瘤小鼠的肿瘤诱导坏死的情况
在6-8周龄的C57小鼠皮下注射B16肿瘤细胞,待肿瘤体积达到50-100mm3时分别采用不同制剂组进行给药,给药五次后处死小鼠,取肿瘤组织用4%多聚甲醛固定,石蜡包埋,切片厚度为5μm,用H&E进行染色后用扫描仪分析。结果如图14所示,PTX、PP@PTX、GPP@PTX三个微针组都能引起肿瘤细胞坏死,其中GPP@PTX微针的效果最佳,说明NO可以在一定程度上促进PTX的化疗效果。PM/OVA微针组与OVA微针组相比肿瘤坏死面积增大,说明免疫佐剂PM的加入能够促进抗原OVA引起的免疫反应。微针复合纳米递药系统(PM/OVA+GPP@PTX)相比于单独的免疫治疗(PM/OVA)或化学治疗(GPP@PTX)具有最佳诱导肿瘤坏死的作用,说明化学疗法、气体疗法与免疫疗法三者协同能达到更好的抗肿瘤效果。且微针相比于皮下注射组(i.h.)效果更佳,说明了微针这种给药方式可以将药物最大程度的输送至肿瘤部位,药物利用率高,实现最佳的治疗效果。
Claims (6)
1.一种微针抗肿瘤复合递药系统,其特征在于,所述复合递药系统是将NO供体偶联的pH敏感性纳米递药系统和肿瘤疫苗纳米粒共同负载在聚乙烯吡咯烷酮微针上;所述NO供体偶联的pH敏感性纳米递药系统为纳米胶束,外壳由NO供体偶联的pH响应性共聚物构成,内核由化疗药物组成;所述NO供体偶联的pH敏感性纳米递药系统,是通过一氧化氮供体在pH响应性聚合物PLA-PEOz表面进行酯键连接NO供体形成两亲性偶联物,并通过疏水作用包载化疗药物构建的纳米递药系统;所述化疗药物为紫杉醇;所述的pH响应性共聚物为聚乳酸-聚(2-乙基-2-唑啉),所述酯键的反应基团为嵌段共聚物中PEOz的羟基与NO供体中的羧基,其中NO供体为含有-COOH结构的S-亚硝基谷胱甘肽;所述肿瘤疫苗纳米粒,是通过静电结合作用将蛋白抗原和免疫佐剂聚二甲双胍结合形成;
所述复合递药系统由以下制备方法制得:
(1)NO供体偶联的pH响应性共聚物GSNO-PLA-PEOz的制备:
将含羧基的NO供体与活化剂溶于水或有机溶剂,避光条件下冰浴搅拌活化羧基;向反应体系中加入嵌段共聚物PLA-PEOz,继续室温搅拌;反应结束后除去反应溶剂;加水重新溶解并透析,冻干,即得GSNO-PLA-PEOz;
(2)NO供体偶联的pH敏感性纳米递药系统的制备:
将GSNO-PLA-PEOz和化疗药物溶于有机溶剂,减压旋蒸除去有机溶剂,接着加水水化得纳米粒混悬液,过滤即得形态为纳米胶束的NO供体偶联的pH敏感性纳米递药系统;
(3)肿瘤疫苗纳米粒的制备:
取蛋白抗原和免疫佐剂溶于水中,搅拌超声,过滤即得肿瘤疫苗纳米粒,所述免疫佐剂为聚二甲双胍PM;
(4)微针抗肿瘤复合递药系统的制备:
将步骤(2)得到的NO供体偶联的pH敏感性纳米递药系统的纳米胶束和步骤(3)得到的纳米粒,溶于针尖PVP溶液中注入模具,真空排泡干燥,然后加入基底PVP溶液真空干燥得到微针抗肿瘤复合递药体系。
2.根据权利要求1所述的递药系统,其特征在于,步骤(1)中,所述活化剂为N,N′-羰基二咪唑、二环己基碳二亚胺、4-二甲氨基吡啶或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐中一种或两种以上的组合;所述有机溶剂为甲酰胺、N,N-二甲基甲酰胺、二甲基亚砜或四氢呋喃中一种或两种以上的组合;所述活化羧基的时间为20min-24h;含羧基的NO供体与嵌段共聚物的质量比为1:10~30,反应时间为24h。
3.根据权利要求1所述的递药系统,其特征在于,步骤(2)中,所述有机溶剂为甲醇或氯仿的一种或两种组合;所述化疗药物与GSNO-PLA-PEOz的质量比为1:5~10,成膜时间为10-15min,成膜温度为40-60℃,水化时间为5-30min,水化温度为40-60℃。
4.根据权利要求1所述的递药系统,其特征在于,步骤(3)中,所述免疫佐剂和蛋白抗原的质量比为1:1~3,搅拌时间为24-48h,超声时间为10-20min,所述免疫佐剂肿瘤疫苗纳米粒的粒径为100-200nm。
5.根据权利要求1所述的递药系统,其特征在于,步骤(4)中,所述纳米胶束、纳米粒、针尖PVP溶液和基底PVP溶液的质量比为200~300:1:8000~10000:40000~80000;所述PVP溶液为PVP K30和 PVP K12的混合溶液,PVP K30和 PVP K12的质量比为4:1~6。
6.权利要求1所述的微针抗肿瘤复合递药系统在制备抗肿瘤药物中的应用。
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