CN110898032B - 一种近红外光致异构纳米药物传递体系的制备方法 - Google Patents
一种近红外光致异构纳米药物传递体系的制备方法 Download PDFInfo
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Abstract
本发明公开一种近红外光致异构纳米药物传递体系的制备方法,胆红素与3‑氨基苯硼酸进行缩合反应生成BR‑ABA,再通过硼酸酯键将胆红素接到羟乙基淀粉上,组装形成纳米组装体BRNPs。胆红素在作为纳米载体的同时自身亦可作为癌症治疗药物,提高癌症治疗效果;羟乙基淀粉具有良好的生物相容性,其作为亲水端在纳米粒子表面在提高纳米载体溶解性的同时提高血液循环稳定性。当纳米药物载体进入细胞之后通过近红外光照射使得胆红素光致异构,由疏水结构变为亲水结构,纳米粒子的结构被破坏,可释放药物。
Description
技术领域
本发明属于生物医用材料领域,更加具体地说,具体涉及一种近红外光致异构纳米药物传递体系的制备方法。
背景技术
纳米载体依据小尺寸效应,比表面积大和宏观量子隧道效应等优势,与生物医学有机结合,促进了疾病检测、分子成像和疾病诊治等学科的发展。近年来,科学家们对纳米载体运送化疗药物进行了大量的研究报道并取得了良好进展。
纳米材料相对于传统材料而言,具有吸附能力强、毒性低、扩散性好、催化能力高以及渗透能力强等多重优点。合物纳米粒子在药物传递方面拥有巨大的潜能吸引着科研界和医药产业界的广泛关注,主要原因有如下几点:(1)聚合物具有多种分子结构,可以根据需要设计和选择分子结构;(2)聚合物链在形成纳米粒子前后都可以进行化学修饰和改性;(3)可以采用不同工艺制备不同形式的纳米粒子;(4)纳米剂型能提高药物的稳定性,防止其降解,延长血液循环时间。
纳米材料相对于传统材料而言,具有吸附能力强、毒性低、扩散性好、催化能力高以及渗透能力强等多重优点。用于投递化疗药物的纳米载体经历了从简单设计、功能单一到精心设计、功能多样化,从依赖EPR效应的被动靶向到配体-受体介导的主动靶向的发展过程。某些化合物收到某种外界刺激而产生亲水和疏水的变化,以其为原材料设计的纳米载体可以保护它们所载的药物不受血液系统的降解,并在肿瘤组织中特异释放,这样可以大大降低系统毒性。
发明内容
本发明的目的在于克服现有技术的不足,提供一种近红外光致异构纳米药物传递体系的制备方法,将胆红素(BR)与3-氨基苯硼酸(ABA)进行缩合反应合成胆红素纳米自组装体(BR-ABA),通过硼酸酯键将BR链接到羟乙基淀粉(HES)上,自组装形成纳米组装体(BRNPs)。
本发明的技术目的通过下述技术方案予以实现。
一种近红外光致异构纳米药物传递体系的制备方法,按照下述步骤进行:
(1)胆红素纳米自组装体BR-ABA的合成
(2)BRNPs纳米粒子的制备
步骤1,将胆红素和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐置于溶剂中并均匀分散,在惰性保护气体氛围下室温避光搅拌反应,再向其中加入氨基苯硼酸和三乙胺在惰性保护气体氛围下室温避光搅拌继续反应,即可得到胆红素纳米自组装体(BR-ABA);
在步骤1中,惰性保护气体为氮气、氦气或者氩气。
在步骤1中,加入氨基苯硼酸和三乙胺继续反应之后,将所得产物加入搅拌中的30—50mL甲醇溶液中静置30—40min后在5000—6000rpm/min的条件下离心10—15min,弃去得到的混合溶液的上清液,取沉淀在20—30℃下真空干燥40—48h,得到产物BR-ABA。
在步骤1中,溶剂为二甲基亚砜、二甲基甲酰胺、二甲基乙酰胺或者四氢呋喃。
在步骤1中,胆红素和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量比为(160—180):(130—150),优选(170—180):(138—145),在室温20—25摄氏度下搅拌反应30—60min,优选40—60min,选择机械搅拌,搅拌速度为每分钟100—300转。
在步骤1中,胆红素、氨基苯硼酸和三乙胺的质量比为(160—180):(140—150):(100—110),优选(170—180):(146—150):(105—108),加入氨基苯硼酸和三乙胺之后在室温20—25摄氏度下搅拌反应1—6小时,优选4—6小时,选择机械搅拌,搅拌速度为每分钟100—300转。
步骤2,将步骤1得到的胆红素纳米自组装体和羟乙基淀粉在溶剂中进行混合,在避光惰性保护气体保护下搅拌反应,得到纳米组装体。
在步骤2中,溶剂为二甲基亚砜、二甲基甲酰胺、二甲基乙酰胺或者四氢呋喃。
在步骤2中,惰性保护气体为氮气、氦气或者氩气。
在步骤2中,反应产物用滤纸进行过滤,得到纳米组装体BRNPs。
在步骤2中,胆红素纳米自组装体、羟乙基淀粉的质量比为(1—10):100,优选(2—6):100。
在步骤2中,在室温20—25摄氏度下搅拌反应8—16小时,优选10—12小时,选择机械搅拌,搅拌速度为每分钟100—300转。
在步骤2中,选择将胆红素纳米自组装体溶于溶剂中形成BR-ABA溶液,使用微型注射器缓慢滴加到搅拌中的羟乙基淀粉溶液中,避光搅拌进行反应,采用匀速滴加,滴加时间为5—15min。
在本发明技术方案中,质量比的基础单位为mg。
依据本发明制备的近红外光致异构纳米药物传递体系,将胆红素(BR)与3-氨基苯硼酸(ABA)进行缩合反应合成胆红素纳米自组装体(BR-ABA),通过硼酸酯键将BR链接到羟乙基淀粉(HES)上,自组装形成纳米组装体(BRNPs)。
依据本发明制备的近红外光致异构纳米药物传递体系在制备肿瘤自靶向光致异构载药纳米粒子中的应用,将阿霉素装载到纳米组装体,再以HeLa细胞膜碎片包覆于纳米组装体表面以得到HeLa细胞膜包覆的纳米载体DBC3NPs,即本发明的肿瘤自靶向光致异构纳米载体。
利用纳米组装体BRNPs进行肿瘤自靶向光致异构纳米载体的制备方法,按照下述步骤进行:
步骤1,将阿霉素装载到纳米组装体,形成纳米自组装体DOX@BR NPs(DBNPs)
将均匀分散阿霉素的溶液滴加到搅拌状态的纳米组装体的溶液中,避光搅拌反应,得到自组装体DOX@BR NPs(DBNPs)
在步骤1中,溶剂为二甲基亚砜、二甲基甲酰胺、二甲基乙酰胺或者四氢呋喃。
在步骤1中,在室温20—25摄氏度下搅拌反应16小时,优选4—6小时,选择机械搅拌,搅拌速度为每分钟100—300转。
在步骤1中,经反应得到的产物透析20—24h后,得到纳米自组装体DOX@BRNPs(DBNPs)。
在步骤1中,使用微型注射器将DOX脱盐溶液缓慢滴加到搅拌中的BRNPs溶液中,采用匀速滴加,滴加时间为10—30min,优选10—20min。
步骤2,将HeLa细胞膜碎片包裹步骤1得到的纳米自组装体,即细胞膜包裹的纳米自组装体DOX@BR@CCCMs NPs(DBC3NPs)的制备
HeLa细胞膜碎片(CCCMs)均匀分散在水中,经冰水浴超声处理得到分散均匀的纳米级细胞膜碎片溶液,将均匀分散步骤1纳米自组装体的溶液和分散均匀的纳米级细胞膜碎片溶液进行涡旋混合均匀,静置后进行水相滤头过滤,所得溶液为HeLa癌细胞膜包裹的纳米自组装体DBC3NPs。
在步骤2中,室温20—25摄氏度下静置30—60min,依次经过1μm,0.85μm和0.45μm的水相滤头分别过滤3次。
在步骤2中,步骤1纳米自组装体和HeLa细胞膜碎片的质量比为(8—15):(3—6),优选(10—15):(4—6)。
在步骤2中,选择水作为溶剂,形成均匀分散步骤1纳米自组装体的溶液。
在制备方案中,以疏水性的胆红素为纳米载体的原材料,通过再沉淀法将其和亲水性的羟乙基淀粉相结合的同时形成亲水的纳米自组装体。胆红素在作为纳米载体的同时自身亦可作为癌症治疗药物,可提高癌症治疗效果;羟乙基淀粉具有良好的生物相容性,其作为亲水端在纳米粒子表面可以在提高纳米载体溶解性的同时提高血液循环稳定性。当纳米药物载体进入细胞之后通过近红外光照射使得胆红素光致异构,由疏水结构变为亲水结构,纳米粒子的结构被破坏,可释放药物。本发明制备方法简单,材料来源广泛,设计巧妙,实用性强。
近红外光致异构DBC3NPs纳米粒子兼具肿瘤自靶向和定点光诱导肿瘤高效治疗。疏水性的胆红素在作为纳米载体的同时,其自身也是癌症治疗药物,可提高癌症治疗效果;HES在血清α-淀粉酶作用下降解生成葡萄糖并诱发免疫反应,可使T细胞聚集辅助癌症治疗;纳米粒子负载抗癌药物阿霉素,能够使得纳米粒子对癌细胞的毒性显著增加;纳米粒子表面包覆的HeLa细胞膜显著增强了纳米粒子的自靶向性;近红外光照射使胆红素发生光致异构现象,由疏水结构变为亲水结构,加速释放抗癌药物;通过MTT法检测所得纳米制剂的肿瘤细胞杀伤能力。通过激光共聚焦显微镜(CLSM)检测HeLa细胞膜包覆的纳米组装体的同源细胞靶向性;通过体外细胞实验证明该纳米粒子可以很好的被细胞摄取,且在近红外光的诱导下可将药物快速释放并有效杀死癌细胞。本发明制备方法简单,材料来源广泛,实用性强,实验过程针对性强且效果明显。
附图说明
图1是本发明中BRNPs粒径分布图。
图2是本发明中BRNPs透射电镜照片。
图3是本发明中BRNPs紫外可见光谱图。
图4是本发明中BRNPs纳米粒子在455nm激发波长下的荧光发射光谱。
图5是本发明中DBC3NPs粒径分布图。
图6是本发明中DBC3NPs的透射电镜照片。
图7是本发明中在pH=7.4条件下,BRNPs与HeLa细胞共培养48h后,在有无近红外光照射情况下,在HeLa细胞内的生物毒性测试曲线图。
图8是本发明中在pH=7.4条件下DBC3NPs分别与HeLa细胞和L929细胞共培养48h后,在有无近红外光照射情况下,在两种细胞内的生物毒性测试曲线图。
图9是本发明中L929细胞和HeLa细胞对DBC3NPs的内吞行为的激光共聚焦荧光成像图,标尺:25μm。
具体实施方式
下面结合具体实例进一步说明本发明的技术方案,结合一下实例进一步说明本发明,但这些实例并不用来限制本发明。
药品表格(1)
药品表格(2)
<u>名称</u> | <u>厂商</u> | <u>规格</u> |
<u>DOX·HCl脱盐</u> | <u>天津市光复精细化工有限公司</u> | <u>分析纯</u> |
<u>人宫颈癌细胞(HeLa)</u> | <u>北京赛业科技有限公司</u> | <u>人源</u> |
<u>小鼠纤维母细胞(L929)</u> | <u>北京赛业科技有限公司</u> | <u>鼠源</u> |
<u>细胞培养基(DMEM)</u> | <u>北京赛业科技有限公司</u> | <u>高糖</u> |
<u>胎牛血清(FBS)</u> | <u>武汉普诺赛生物科技有限公司</u> | <u>特级</u> |
<u>噻唑蓝(MTT)</u> | <u>上海士锋生物科技有限公司</u> | <u>98%</u> |
仪器表格
<u>仪器名称</u> | <u>规格或型号</u> | <u>生产厂家</u> |
<u>优普系列超纯水机</u> | <u>UPR-II-10T</u> | <u>四川优普超纯科技有限公司</u> |
<u>电子分析天平</u> | <u>AS 220.R2</u> | <u>苏州陪科实验室仪器科技有限公司</u> |
<u>超声波清洗器</u> | <u>KQ-300E</u> | <u>昆山市超声仪器有限公司</u> |
<u>真空干燥箱</u> | <u>DZF-6020</u> | <u>天津星科仪器有限公司</u> |
<u>紫外-可见分光光度计</u> | <u>TU-1801</u> | <u>北京普析通用仪器有限责任公司</u> |
<u>纳米粒度及Zeta电位仪</u> | <u>NanoZS</u> | <u>英国马尔文仪器公司</u> |
<u>透射电子显微镜</u> | <u>JEM-2100F</u> | <u>日本JEOL公司</u> |
<u>X荧光谱仪</u> | <u>nF900</u> | <u>英国爱丁堡仪器公司</u> |
<u>超速冷冻离心机</u> | <u>TGL-16M</u> | <u>长沙湘仪离心机仪器有限公司</u> |
<u>光栅型多功能酶标仪</u> | <u>M200pro</u> | <u>帝肯(上海)贸易有限公司</u> |
<u>激光扫描共聚焦显微镜</u> | <u>TCS SP8</u> | <u>德国Leica公司</u> |
将180mg的胆红素(BR)和138mg EDC加入到5mL二甲基亚砜(DMSO)溶液中使其完全溶解,在氮气保护下室温避光搅拌40min。向溶液中加入146mg氨基苯硼酸(ABA)和150μL三乙胺(TEA)溶液,在氮气保护下室温避光搅拌4h。将所得产物加入搅拌中的30mL甲醇溶液中静置30min后在6000rpm/min的条件下离心10min。弃去得到的混合溶液的上清液,取沉淀在30℃下真空干燥48h,得到产物BR-ABA。取2mg BR-ABA溶于200μl四氢呋喃中配成10mg/mL的BR-ABA溶液,用微型注射器缓慢滴加到搅拌中的10mL的10mg/mL羟乙基淀粉(HES)溶液(四氢呋喃)中,避光搅拌12h,产物用滤纸进行过滤,得到纳米组装体BRNPs。
在25℃下取1mLBRNPs溶液、近红外光照射后的BRNPs溶液,检测粒径大小及分布情况,然后取1mL BRNPs溶液检测电势大小并计算其电势平均值。从图1中可以看出BRNPs的粒径为100±10nm左右,而近红外光照射后的BRNPs几乎无法测出粒径,这表明经过近红外光照射,BRNPs中的胆红素发生了构型的改变,生成了可溶性异构体,因此BRNPs纳米粒子结构被破坏;同时BRNPs的电势为-4.8mV,有助于在血液中循环。通过透射电镜(TEM)对纳米粒子的形貌进行观察。测试前,将50μL新制备的BRNPs溶液滴于碳支持膜上。待样品完全干燥后,在透射电子显微镜下观察样品的形貌并拍照,从图2中可以看出BRNPs为球形纳米粒子,粒径为80±10nm左右,且均匀分散于视野之中,这证明了BRNPs纳米粒子已成功形成。
通过荧光光谱仪对新制备的BRNPs进行荧光强度检测,激发波长为455nm,光谱宽度为480-650nm,得到样品的荧光光谱。从图3中可以明显看到在400-500nm之间有连续的吸收峰,其最高峰位于455nm左右,这与胆红素的特征吸收峰形状和峰值均吻合。另外,在280nm左右有一个尖锐的吸收峰,这与氨基苯硼酸的特征吸收峰值相吻合。这两个峰值亦可表明BRNPs纳米粒子已经生成。通过紫外-可见分光光度计对BRNPs溶液进行200-700nm波长范围的光谱扫描,得到样品的紫外吸收峰。从图4中可以看出,在波长为510nm和520nm处有两个明显的胆红素荧光发射峰,最大发射峰位于520nm附近。这进一步证明BRNPs已经形成。
HeLa细胞膜碎片的提取:取对数期生长的HeLa细胞,采用细胞刮刀将细胞小心刮下,收集到离心管内,700g离心5min得到细胞沉淀,加入等渗预冷的PBS溶液中(pH=7.4)重悬得到细胞悬浮液,再一次600g离心5min,弃去上清液,随后600g离心1min,以沉淀离心管壁上的残留液体,尽最大努力吸尽残留液体。最后,加入低渗溶液在冰浴条件下静置15min。15min后,将上述溶液在室温与液氮中反复冻融数次直至细胞完全破碎。最后,700g离心10min后小心得到上清液,以去除细胞核、细胞器和未破碎细胞。将上述上清液在14000g条件下离心30min得到沉淀即为HeLa细胞膜碎片(CCCMs)。上述细胞膜碎片-80℃冻干保存备用。
经胆红素(BR)、3-氨基苯硼酸(ABA)和羟乙基淀粉(HES)自组装形成纳米组装体BRNPs。将HeLa细胞膜碎片(CCCMs)通过连续挤出法包覆于载有阿霉素(Doxorubicin,DOX)的BRNPs表面以得到HeLa细胞膜包覆的纳米载体DBC3NPs,如下化学式所示。
纳米自组装体DOX@BR NPs(DBNPs)的制备:使用微型注射器将0.6mL 10mg/mL的DOX脱盐DMSO溶液缓慢滴加到搅拌中的10mL含有BRNPs的溶液中,避光搅拌4h,产物透析24h,得到纳米自组装体DOX@BR NPs(DBNPs)。
细胞膜包裹的纳米自组装体DOX@BR@CCCMs NPs(DBC3NPs)的制备:HeLa细胞膜碎片(CCCMs)溶于纯水中,经冰水浴超声处理得到分散均匀的纳米级细胞膜碎片溶液。然后,将10mL DBNPs溶液与3mL的2mg/mL的HeLa细胞膜碎片溶液涡旋混合均匀,静置30分钟,依次经过1μm,0.85μm和0.45μm的水相滤头分别过滤3次,所得溶液即为HeLa癌细胞膜包裹的纳米自组装体DBC3NPs。
细胞毒性试验:将人源宫颈癌细胞(HeLa细胞)以6000个/孔的细胞密度接种于含有培养基的96孔板内在37℃,5%的CO2培养箱培养24h。随后,加入不同浓度的BRNPs、DBC3NPs溶液,继续培养48h后,将培养基吸去,加入200μL新鲜的培养基继续培养4h。另取一块经过同样处理的板子在换液后对其进行近红外光照处理(5W/cm2,1min)。之后,每孔中重新加入200μL含有10%FBS以及20μL噻唑蓝(MTT)溶液(5mg/mL)的新鲜培养基继续在37℃,5%的CO2培养箱中培养4h。最后吸出所有培养基,每孔加入150μL DMSO,通过酶标仪在570nm波长处测定各孔的吸光度。
激光共聚焦显微镜观察纳米粒子的靶向和细胞内吞:分别将HeLa细胞和L929细胞接种于直径20mm的共聚焦培养皿内,其密度为2.5×105个/孔。在培养箱中培养24h后吸除培养基,将含有DBC3NPs溶液分别加入到培养皿内。2h后,吸除培养基并用PBS缓冲液反复冲洗,并加入1mL新鲜培养基。各取HeLa细胞和L929细胞组其中一个加有DBC3NPs材料的培养皿进行近红外光照处理(5W/cm2,5min),继续培养2h后吸去培养基,避光加入1mL Hoechst33254(10μL/mL)的DMEM溶液,对细胞核进行染色处理。15min后,吸去培养基用PBS缓冲液洗涤3次,用激光共聚焦显微镜观察拍照(激发波长为488nm)。
在25℃下取1mLBRNPs溶液、近红外光照射后的BRNPs溶液和DBC3NPs溶液。将人源宫颈癌细胞(HeLa细胞)以6000个/孔的细胞密度接种于含有培养基的96孔板内在37℃,5%的CO2培养箱培养24h。加入不同浓度的BRNPs、DBC3NPs溶液,继续培养48h后,将培养基吸去,加入200μL新鲜的培养基继续培养4h。为了证明材料的近红外光响应性,取另一块经过同样处理的板子在换液后对其进行近红外光光照处理(5W/cm2,1min)。近红外光照过后,将96孔板在暗处培养4h。得到上述孔板后,每孔中重新加入200μL新鲜的包含10%FBS以及20μL的噻唑蓝(MTT)溶液(5mg/mL)的培养基继续在37℃,5%的CO2培养箱中培养4h。将培养基更换为150μL DMSO溶液。最后吸出所有培养基后每孔加入150μL DMSO,避光摇床摇晃30min,通过酶标仪在570nm波长处测定各孔的吸光度。
分别将HeLa细胞和L929细胞接种于直径20mm的共聚焦培养皿内,其密度为2.5×105个/孔。在培养箱中培养24h后吸除培养基,将含有DBC3NPs溶液与培养基按照1:1分别加入到四个培养皿内。2h后,吸除培养基并用PBS缓冲液反复冲洗,并加入1mL新鲜培养基。各取HeLa细胞和L929细胞组其中一个加入DBC3NPs材料的培养皿进行近红外光照射(5W/cm2,5min),再继续培养2h,之后吸去培养基再用PBS缓冲液反复冲洗。避光加入1mL Hoechst33254(10μL/mL)的DMEM溶液,对细胞核进行染色处理。15min后,吸去培养基用PBS缓冲液多次冲洗,加入1mL多聚甲醛固定液对细胞进行固定处理,20min后吸除固定剂并再次用PBS溶液冲洗数次,再放入4℃冰箱留存用于激光共聚焦显微镜观察拍照(激发波长为488nm)。
从图5—6所示,平均粒径为220±20nm左右,同时测量其电势为-16.3mV,基本与CCCMs的电势保持一致(-24.8mV),证明CCCMs包覆在DBNPs表面。从图可以看出DBC3NPs呈球形均匀分布于视野中,粒径为180±20nm左右,从右上角的放大图中可以直观看出纳米粒子表面存在一层灰色膜层结构,进一步证明了CCCMs已成功包覆在纳米载体表面,以上的数据均与粒径仪所测数据吻合。
从图7所示,BRNPs在有无近红外光照射情况下的细胞毒性数据,看出细胞毒性随着BR药物浓度的增大都呈现增加的趋势,当引入近红外光照射时,BRNPs的细胞毒性明显增强,说明BRNPs在近红外光照射下发生了光致异构转变使得纳米粒子结构被破坏,释放出更多的BR药物分子。从图8所示,DBC3NPs在有无近红外光照射情况下与L929细胞共培养的细胞毒性数据,可以看出在无近红外光照射时DBC3NPs对L929细胞几乎没有细胞毒性,细胞存活率均高于90%;引入近红外光照射后,药物浓度在0-2mg/mL区间内的细胞毒性变化不大,且细胞存活率均高于90%;而当药物浓度进一步增大后,细胞毒性显著增强,表明近红外光的照射促使了光致异构的转变,加快了BR和DOX药物分子的释放。DBC3NPs在有无近红外光照射情况下与HeLa细胞共培养的细胞毒性数据,DBC3NPs对于HeLa细胞的细胞毒性明显高于L929细胞,当药物浓度为4.76mg/mL时,HeLa细胞的存活率仅为25%左右,而加入近红外光照射之后其细胞毒性进一步增强,最大药物浓度时细胞存活率为10%左右。这一结果证明了DBC3NPs对HeLa细胞具有同源靶向性,且近红外光的照射触发了纳米粒子的光致异构转变,使得大量药物分子释放出来,有望实现光致异构诱导的高效癌症治疗。
图9进一步证明包裹了HeLa细胞膜的DBC3NPs材料对于同源癌细胞具有自靶向作用。将HeLa细胞、L929细胞对DBC3NPs的内吞情况通过激光共聚焦显微镜进行了定性分析,可以看到L929细胞组中DOX的荧光强度明显弱于HeLa细胞组中DOX的荧光强度,这表明包裹了CCCMs的DBC3NPs材料具有明显的自靶向性,这是因为同种细胞膜的特异性蛋白识别作用,以及癌细胞所大量表达的黏附分子使癌细胞易于相互聚集有关。最后证明了DBC3NPs材料在近红外光的照射下可以发生光致异构,促进BR和DOX的释放。图9显示,从两组(L929细胞和HeLa细胞的近红外光光照组)有无近红外光照的对照组可以看到,在近红外光照射后,DOX的荧光强度均有了较明显的增强。这是由于DBC3NPs中的胆红素在近红外光的照射下发生光致异构,纳米粒子的结构发生改变,加速了DBC3NPs中的DOX释放。其结果与细胞毒性实验的结果一致。
根据本发明内容进行工艺参数的调整,均可实现BRNPs纳米粒子和肿瘤自靶向光致异构纳米载体的制备,经测试表现出与本发明基本一致的性能。以上对本发明做了示例性的描述,应该说明的是,在不脱离本发明的核心的情况下,任何简单的变形、修改或者其他本领域技术人员能够不花费创造性劳动的等同替换均落入本发明的保护范围。
Claims (14)
1.一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,按照下述步骤进行:
步骤1,将胆红素和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐置于溶剂中并均匀分散,在惰性保护气体氛围下室温避光搅拌反应,再向其中加入氨基苯硼酸和三乙胺在惰性保护气体氛围下室温避光搅拌继续反应,即可得到胆红素纳米自组装体;
步骤2,将步骤1得到的胆红素纳米自组装体和羟乙基淀粉在溶剂中进行混合,在避光惰性保护气体保护下搅拌反应,得到纳米组装体。
2.根据权利要求1所示的一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,在步骤1中,溶剂为二甲基亚砜、二甲基甲酰胺、二甲基乙酰胺或者四氢呋喃,惰性保护气体为氮气、氦气或者氩气。
3.根据权利要求1所示的一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,在步骤1中,胆红素和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量比为(160—180):(130—150),在室温20—25摄氏度下搅拌反应30—60min,选择机械搅拌,搅拌速度为每分钟100—300转。
4.根据权利要求1所示的一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,在步骤1中,胆红素和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量比为(170—180):(138—145),在室温20—25摄氏度下搅拌反应40—60min,选择机械搅拌,搅拌速度为每分钟100—300转。
5.根据权利要求1所示的一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,在步骤1中,胆红素、氨基苯硼酸和三乙胺的质量比为(160—180):(140—150):(100—110),加入氨基苯硼酸和三乙胺之后在室温20—25摄氏度下搅拌反应1—6小时,选择机械搅拌,搅拌速度为每分钟100—300转。
6.根据权利要求1所示的一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,在步骤1中,胆红素、氨基苯硼酸和三乙胺的质量比为(170—180):(146—150):(105—108),加入氨基苯硼酸和三乙胺之后在室温20—25摄氏度下搅拌反应4—6小时,选择机械搅拌,搅拌速度为每分钟100—300转。
7.根据权利要求1所示的一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,在步骤2中,溶剂为二甲基亚砜、二甲基甲酰胺、二甲基乙酰胺或者四氢呋喃;惰性保护气体为氮气、氦气或者氩气。
8.根据权利要求1所示的一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,在步骤2中,胆红素纳米自组装体、羟乙基淀粉的质量比为(1—10):100。
9.根据权利要求1所示的一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,在步骤2中,胆红素纳米自组装体、羟乙基淀粉的质量比为(2—6):100。
10.根据权利要求1所示的一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,在步骤2中,在室温20—25摄氏度下搅拌反应8—16小时,选择机械搅拌,搅拌速度为每分钟100—300转。
11.根据权利要求1所示的一种近红外光致异构纳米药物传递体系的制备方法,其特征在于,在步骤2中,在室温20—25摄氏度下搅拌反应10—12小时,选择机械搅拌,搅拌速度为每分钟100—300转。
12.依据权利要求1—11之一所述的制备方法制备的近红外光致异构纳米药物传递体系,其特征在于,将胆红素与3-氨基苯硼酸进行缩合反应合成胆红素纳米自组装体,通过硼酸酯键将胆红素链接到羟乙基淀粉上,自组装形成纳米组装体。
13.依据权利要求1—11之一所述的制备方法制备的近红外光致异构纳米药物传递体系在制备肿瘤自靶向光致异构载药纳米粒子中的应用。
14.根据权利要求13所述的应用,其特征在于,将阿霉素装载到纳米组装体,再以HeLa细胞膜碎片包覆于纳米组装体表面以得到HeLa细胞膜包覆的纳米载体DBC3NPs。
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