CN115025244A - 一种转铁蛋白修饰的双靶向雷公藤甲素磁性纳米颗粒的制备方法及其应用 - Google Patents
一种转铁蛋白修饰的双靶向雷公藤甲素磁性纳米颗粒的制备方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种口服转铁蛋白修饰的双靶向雷公藤甲素(Triptolide,Tri)磁性纳米颗粒的制备方法及其应用。该纳米颗粒是以氨基功能化磁性纳米(MNP‑NH2)作为磁性靶向配体,转铁蛋白(Tf)作为肿瘤靶向配体,并与PEG‑PLGA偶联,制备转铁蛋白修饰的Tri包封的PEG‑PLGA磁性纳米颗粒(Tf‑MNP‑PEG‑PLGA‑Tri)。该纳米颗粒的平均粒径为137.39±3.72nm。本发明所制备的纳米颗粒,不仅可以提高Tri的口服生物利用度,并具有肿瘤靶向作用(主动靶向和被动靶向),从而提高肿瘤对MNP的摄取,增强Tri的抗肿瘤活性。
Description
技术领域
本发明涉及制药技术领域,特别涉及一种转铁蛋白修饰的双靶向雷公藤甲素磁性纳米颗粒的制备方法及其应用。
背景技术
癌症是世界上主要的死亡原因之一,严重危害人类的生存。如今,肺癌已成为全球癌症相关死亡的主要原因。肺癌是一种病因复杂的恶性疾病,可能涉及多种因素,如遗传、慢性感染、环境污染、生活方式等。目前,主要的治疗手段包括手术、放疗、靶向治疗和生物免疫治疗。化疗被认为是肺癌的主要治疗策略。一般情况下,由于化疗耐药和放疗疗效有限,癌症患者的预后较差。除了上述治疗方案的挑战外,紫杉醇、阿霉素和5-氟尿嘧啶还存在严重的全身副作用,即心脏毒性、神经毒性和肝毒性。目前,这些治疗方法受限于对肿瘤组织的非选择性和对正常组织的高毒性。因此,识别靶向肺癌组织的新型药物传递系统尤为重要。
PEG-PLGA是一种具有PEG亲水端和PLGA疏水端的两亲性嵌段聚合物,常用来制备纳米颗粒。PEG和PLGA都是FDA批准的药物载体。发现将PEG链引入到聚合物载体能逃脱间隙内皮网络系统,增加药物的亲水性,延长药物在体内的循环时间,提高载体的稳定性,以及实现被动靶向的目的。与其他聚合物相比,PLGA具有良好的组织相容性和生物降解性。因此,PEG-PLGA是制备MNP的理想载体材料。
肿瘤的主动靶向可通过转铁蛋白(Tf)来实现。转铁蛋白受体(TfR)是一种跨膜糖蛋白,与转铁蛋白相互作用,介导铁的吸收。通过Tf和TfR介导与肿瘤细胞特异性结合,可用于肿瘤靶向治疗。因此,通过Tf对MNP表面进行修饰,药物可靶向到肿瘤细胞,从而提高肿瘤对MNP的摄取,增强药物的抗肿瘤活性。
Tri是一种广谱、高抗肿瘤活性的环氧化二萜内酯,对肺癌、神经母细胞瘤和胆管癌等具有很好的抑制效果。现有研究进一步阐述了Tri的抗肿瘤作用及机制,证实了Tri在干预细胞周期、诱导肿瘤细胞凋亡中的确切作用。然而,由于其水溶性差、体内生物利用度低,阻碍了Tri在癌症治疗中的临床应用。为了解决这些挑战,Tri已经被整合到几种纳米载体中,包括但不限于纳米脂质载体、胶束、脂质体、纳米粒子和自微乳化给药系统。Tri的这些纳米配方极大地改善了药物的溶解度和生物利用度。然而,这些纳米载体在靶向肿瘤和一些低载药载体的纳米颗粒聚集方面表现出不足,尚未满足开发最小毒性载药纳米制剂(如三载纳米颗粒)的需求。此外,开发上述纳米颗粒的作者没有加入配体,而配体有能力增强纳米载体的靶向性。基于上述缺点,本研究试图探索改进Tri包封到纳米颗粒的可能性,以克服其在临床上抗肿瘤活性的限制。
发明内容
针对前述背景技术,本发明制备了一种Tf修饰的PEG-PLGA-MNP-Tri磁性纳米颗粒,借以解决Tri溶解度及生物利用度较低的问题,并增加其靶向性,增强抗肿瘤活性。
本发明的技术方案如下:
一种转铁蛋白修饰的双靶向Tri磁性纳米颗粒,由如下成分制得:Tri、MNP-NH2溶液、PEG-PLGA、Tf。
其中聚乙二醇-聚乳酸聚乙醇酸共聚物(PEG-PLGA)和Tri的质量比为5:1-10:1。
PEG-PLGA中,PEG和PLGA的分子量可选用1k-10k。
Tf和MNP-PEG-PLGA-Tri的摩尔比为1-4:4。
本发明所述的磁性纳米粒包括以下步骤:
(1)MNP-NH2的制备:
采用共沉淀法合成MNP-NH2。将FeCl3和FeCl2溶解于蒸馏水中,置于三颈瓶中。将混合溶液在氮气保护下加热至90℃,10min后加入氨溶液沉淀Fe3+/Fe2+金属离子,进行磁分离后干燥可得磁性纳米颗粒(MNPs)。然后,将3-氨丙基三乙氧基硅烷(APTES)作为胺化试剂,通过硅化作用与Fe3O4纳米颗粒表面结合,形成MNP-NH2。其具体步骤为:将MNPs和APTES溶于80mL乙醇水中,反应液在氮气保护下,40℃回流24h,用热去离子水和乙醇洗涤3次,真空干燥。
(2)MNP-PEG-PLGA-Tri纳米粒子的制备:
采用W/O/W复合乳液溶剂蒸发法制备载药纳米颗粒。将PEG-PLGA和Tri溶于二氯甲烷作为油相,MNP-NH2溶液作为水相。将水相滴加到油相中,冰浴超声,加入5%PVA溶液,得到W/O/W双乳溶液。将双乳溶液倒入0.5%PVA水溶液中,高速搅拌,使二氯甲烷蒸发。
(3)Tf-MNP-PEG-PLGA-Tri纳米粒子的制备:
将MNP-PEG-PLGA-Tri纳米颗粒在水浴中孵育1h后,加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS),搅拌混匀。加入Tf,37℃水浴3h,透析(10kDa)12h,除去游离Tri和Tf,冷冻干燥,即可得Tf-MNP-PEG-PLGA-Tri磁性复合纳米颗粒。
本发明还提出一种转铁蛋白修饰的双靶向Tri磁性纳米颗粒在抗肿瘤方面的应用及其靶向作用研究。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
图1:(A)MNP-PEG-PLGA-Tri的粒径分布。
(B)Tf-MNP-PEG-PLGA-Tri的粒径分布。
(C)MNP-PEG-PLGA-Tri的TEM图像。
(D)Tf-MNP-PEG-PLGA-Tri的TEM图像。
(E)MNP-PEG-PLGA-Tri的SEM图像。
(F)Tf-MNP-PEG-PLGA-Tri的SEM图像。
图2:(A)Tri体外HPLC色谱图。
(B)空白血浆在254nm检测波长下的高效液相色谱图。
(C)在254nm检测波长下,Tri和乙酰苯胺在体内的HPLC图谱。
(D)空白血浆在254nm检测波长下的高效液相色谱图。
(E)在217nm检测波长下,Tri和乙酰苯胺在体内的HPLC图谱。
(F)在含有1%吐温80的pH 7.4的PBS中,游离Tri、MNP-PEG-PLGA-Tri和Tf-MNP-PEG-PLGA-Tri的体外释放曲线。
(G)口服游离Tri和Tf-MNP-PEG-PLGA-Tri的血浆浓度-时间曲线。
图3:口服游离Tri和Tf-MNP-PEG-PLGA-Tri后,不同时间点ICR小鼠心、肝、脾、肺、肾组织中Tri的浓度,(A)0.25h.(B)1h.(C)6h.(D)12h.(E)24h.##P<0.01,#P<0.05,与游离Tri相比较图。
图4:(A)不同浓度的游离Tri和Tf-MNP-PEG-PLGA-Tri对HKF细胞的体外细胞毒性研究。(B)不同浓度的游离Tri和Tf-MNP-PEG-PLGA-Tri对A549细胞的体外抗肿瘤作用。##P<0.01,与游离Tri相比较图。
图5:体外A549细胞摄取MNP-PEG-PLGA-Tri(A)和Tf-MNP-PEG-PLGA-Tri(B)。
图6:给予MNP-PEG-PLGA-Tri(A:1h,B:24h,C:48h)和Tf-MNP-PEG-PLGA-Tri(D:1h,E:24h,F:48h)后,在不同时间点对荷瘤裸鼠进行体内成像图。
图7:(A-E)肿瘤裸鼠经不同处理后的体内肿瘤形态(A:模型对照组,B:游离Tri组,C:空白纳米颗粒组,D:Tf-MNP-PEG-PLGA-Tri组,E:5-FU组)。
(F)14天内不同治疗方法对肿瘤裸鼠的肿瘤生长的影响曲线。
图8:不同治疗方法下裸鼠心脏、肝脏、脾脏、肺、肾及肿瘤的组织病理学图像(A:模型对照组,B:游离Tri组,C:空白纳米颗粒组,D:Tf-MNP-PEG-PLGA-Tri组,E:5-FU组)。
具体实施方式
下面结合具体实例对本发明做进一步说明,但不作为本发明权利要求范围限制。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“及/或”包括一个或多个相关的所列项目的任意的和所有的组合。
实施例1:按照以下方案制备Tf-MNP-PEG-PLGA-Tri磁性纳米颗粒
(1)制备MNP-NH2:
采用共沉淀法合成NP-NH2。将FeCl3(162.20g/mol)和FeCl2(126.75g/mol)按摩尔比2:1(质量比2.559:1)溶解于20mL蒸馏水中,置于三颈瓶中。将混合溶液在氮气保护下加热至90℃,10min后加入6mL氨溶液沉淀Fe3+/Fe2+金属离子,进行磁分离后干燥可得磁性纳米颗粒(MNPs)。然后,APTES作为胺化试剂,通过硅化作用与Fe3O4纳米颗粒表面结合,形成MNP-NH2。其具体步骤为:将MNPs和APTES(221.37g/mol)按摩尔比2:1溶于80mL乙醇水(v/v,1:1)溶液中,反应液在氮气保护下,40℃回流24h,用热去离子水和乙醇洗涤3次,在真空干燥箱中60℃干燥。
(2)MNP-PEG-PLGA-Tri纳米粒子的制备:
采用W/O/W复合乳液溶剂蒸发法制备载药纳米颗粒。将PEG-PLGA(200mg)和定量Tri(10,15,20,25和30mg)溶于2mL二氯甲烷(DCM,10%wt/vol)中作为油相,2mL MNP-NH2溶液(6%)作为水相。将水相滴加到油相中,在一定的超声功率下,冰浴超声30s,在初乳溶液中加入5%PVA溶液,得到W/O/W双乳溶液。将双乳溶液倒入2mL 0.5%PVA水溶液中,室温下在高速分散器中搅拌,使二氯甲烷蒸发。微球经0.22μm滤膜过滤,离心洗涤3次后冷冻干燥保存。采用Marvin ZS90粒度分析仪测定样品的粒径和ζ电位。每个实验进行三次。通过粒径、ζ电位和多分散性指数(PDI)测定得到了最佳配方。
(3)Tf-MNP-PEG-PLGA-Tri纳米粒子的制备:
将MNP-PEG-PLGA-Tri纳米颗粒在水浴中孵育1h后,然后将EDC和NHS按EDC:NHS=1:1(mol/mol)的比例加入纳米颗粒。然后,MNP-PEG-PLGA-Tri纳米颗粒在室温搅拌10分钟后被活化。基于1:4(mol/mol)的Tf:MNP-PEG-PLGA-Tri比率,将Tf添加到混合物中,然后在37℃的水浴中孵育3小时,然后将其添加到经处理的透析袋(10kDa)中。透析12h后,除去游离Tri和Tf,冷冻干燥,即可得Tf-MNP-PEG-PLGA-Tri磁性复合纳米颗粒。采用Marvin ZS90粒度分析仪测定样品的粒径和ζ电位。每个实验进行三次。
电镜下观察到Tf成功键合到纳米粒上,且纳米粒呈较好的圆形,大小均匀(137.39±3.72nm),PDI为0.224±0.010。
(4)测定Tf的共轭效率(CE%)
根据试剂盒说明书建立Tf的浓度标准曲线。精确吸取Tf-MNP-PEG-PLGA-Tri溶液(300μL)。然后加入甲醇(400μL)和氯仿(200μL),进行旋涡混合。然后在上述混合物中加入蒸馏水(300μL),涡旋混合,9000r/min离心3min,仔细去除上层。在氯仿相和沉淀蛋白层中加入甲醇(300μL),在9000r/min的涡流离心3min得到沉淀蛋白。去除上清液,在氮气下干燥蛋白。将获得的蛋白溶于200μL的PBS(pH 7.4)中,与10倍体积的BCA工作液混合,37℃孵育30min。用BCA蛋白定量试剂盒在560nm处测定吸光度。根据建立的标准曲线计算Tf-MNP-PEG-PLGA-Tri中Tf的CE%。CE%计算如下:
计算得到Tf-MNP-PEG-PLGA-Tri中Tf的CE%为87.45±1.17%。高CE%可能是由于选择EDC和NHS作为交联剂所致。这是因为EDC可以激活羧基,形成氨基反应的中间体。针对中间体在水溶液中的不稳定性,加入NHS,通过氨基反应将中间体转化为NHS酯,使中间体稳定水解,从而大大提高EDC介导的缩合反应效率和Tf的CE%
实施例2:Tf-MNP-PEG-PLGA-Tri的体外释放特性
分别取一定数量(5mL)的Tf-MNP-PEG-PLGA-Tri、MNP-PEG-PLGA-Tri和游离Tri悬浮液,并放置在沸腾的透析袋中。用密封夹密封透析袋,悬浮于200mL溶解介质中(pH7.4PBS含1%吐温80)。溶出仪的水浴温度为37℃,转速为100rpm。在0.5、1、2、3、4、6、8、10、12、24、36、48、72h时吸取溶解介质2mL。通过0.22μm滤膜过滤后,采用高效液相色谱法测定峰面积,计算各组Tri的累积释放率(Q%),绘制Tf-MNP-PEG-PLGA-Tri、MNP-PEG-PLGA-Tri和游离Tri的溶解曲线。
由图2F所示,与游离Tri相比,Tf-MNP-PEG-PLGA-Tri、MNP-PEG-PLGA-Tri在溶解介质中释放更多的Tri。游离Tri组72h内累积释放率约为24.79%。在MNP-PEG-PLGA-Tri和Tf-MNP-PEG-PLGA-Tri组中,Tri在前24h从纳米颗粒表面快速释放。24h后进入缓释期,MNP-PEG-PLGA-Tri和Tf-MNP-PEG-PLGA-Tri累计释放率分别约为83.51%和82.40%。此外,MNP-PEG-PLGA-Tri和Tf-MNP-PEG-PLGA-Tri的结果表明,Tf修饰并没有改变纳米粒子的药物释放。从MNP-PEG-PLGA-Tri和Tf-MNP-PEG-PLGA-Tri中增加和持续释放Tri可能是由于PEG-PLGA的基质影响疏水性药物的溶解和释放。疏水性PLGA和亲水性PEG以合适的比例组合形成共聚物,具有控制几种药物释放的潜力。
实施例3:大鼠体内药代动力学研究
12只大鼠在实验室环境中自适应3天后,随机分为游离Tri和Tf-MNP-PEG-PLGA-Tri两组,每组6只。实验前12小时保证大鼠禁食和自由饮水。两组大鼠均尾静脉注射给予相同剂量(300mg/kg)的游离Tri(CMC-Na混悬液)和Tf-MNP-PEG-PLGA-Tri溶液。分别于给药后1min、5min、10min、15min、30min、45min、1h、2h、4h、6h、8h、12h和16h,通过毛细血管从大鼠眼眶采集0.5mL血样。血样经处理后,萃取血样中的Tri,HPLC检测浓度。如图2G所示,Tf-MNP-PEG-PLGA-Tri比游离Tri具有更好的吸收。本实验确定的达到最大血药浓度时间(Tmax)、最大血药浓度(Cmax)、半衰期(t1/2)、浓度-时间曲线下面积(AUC)、平均停留时间(MRT)等药代动力学参数如表1所示。口服Tf-MNP-PEG-PLGA-Tri后,Tri血药浓度迅速升高,Cmax为6.07±0.52μg/mL,游离Tri组为3.22±0.23μg/mL。AUC从1.05±0.23上升到2.59±0.09h·μg/mL(p<0.01),而相对口服生物利用度为246.67%。我们观察到,Tf-MNP-PEG-PLGA-Tri显著提高了Tri的吸收和口服生物利用度,这可能与Tri的高溶解性和小粒径有关。Tf-MNP-PEG-PLGA-Tri的t1/2和MRT分别为0.23±0.01h和1.98±0.09h,游离Tri的t1/2和MRT分别为0.14±0.02h和1.21±0.07h。Tf-MNP-PEG-PLGA-Tri可以促进Tri的口服吸收,这可能有助于增强体内抗肿瘤活性。
Table 1.Pharmacokinetic parameters offree Tri and Tf-MNP-PEG-PLGA-Tri(n=5).
##P<0.01,compared with free Tri group.
实施例4:小鼠组织分布
100只雄性ICR小鼠分为2组,50只小鼠灌胃Tri(200mg/kg),50只小鼠灌胃Tf-MNP-PEG-PLGA-Tri(200mg/kg)。分别于给药后0.25、1、6、12、24h处死小鼠。组织(心、肝、脾、肺、肾)用生理盐水洗涤,滤纸干燥,称重,-70℃冷冻。将冷冻后的心、肝、脾、肺、肾组织放入均质管中解冻,加入1.0mL生理盐水均质。10000rpm离心10min后,取组织匀浆上清(200μL),按血液样本处理方法处理,用于HPLC检测。
图3所示,Tf-MNP-PEG-PLGA-Tri可促进组织中Tri的积累,尤其是在肝脏和脾脏中。
实施例5:细胞毒性试验
采用CCK-8法检测Tf-MNP-PEG-PLGA-Tri和游离Tri对体外HKF细胞的细胞毒性。HKF在96孔板(2.5×105细胞/mL)中进行对数生长。实验组(Tri含量分别为40、20、10、5、2.5、1.25和0.625μg/mL)在37℃,5%CO2和适宜饱和湿度条件下孵育过夜。同时设置空白对照组和阴性对照组,每组设5个多孔。孵育48h后,弃96孔板培养液,每孔加入100μL新鲜CCK-8溶液(CCK-8:DMEM=1:9)。然后,细胞在培养箱中连续培养至棕色。用酶联免疫吸附法(ELISA)测定490nm处的吸光度,计算细胞存活率。
如图4A,随着Tri浓度的增加,各组细胞存活率均略有下降。各浓度下,游离Tri组与Tf-MNP-PEG-PLGA-Tri组细胞存活率无显著差异(p>0.05)。但当Tri浓度达到最大值40μg/mL时,两组细胞存活率仍超过82%,说明制备的载体系统Tf-MNP-PEG-PLGA-Tri表现出良好的细胞相容性,几乎对HKF细胞的生长没有影响。结果证明,实施例1制备的Tf-MNP-PEG-PLGA-Tri具有良好的生物相容性,对正常细胞几乎没有毒性。
实施例6:体外肿瘤靶向性评价
利用激光扫描共聚焦显微镜(LSCM)研究了Tf-MNP-PEG-PLGA-Tri在A549细胞中的摄取情况。与上述制备方法相同,只是在加入Tri的同时加入红色荧光物质Cy5(2mL,10μg/mL),制备负载Cy5的Tf-MNP-PEG-PLGA-Tri和MNP-PEG-PLGA-Tri。
使用MTT测定法确定Tf-MNP-PEG-PLGA-Tri和游离Tri对A549细胞的抑制活性。选择5-FU作为阳性对照。简而言之,将A549细胞以每孔2×104的细胞密度接种在96孔培养板中。培养24小时后,弃去培养基,并用DMEM将200μL的游离Tri和Tf-MNP-PEG-PLGA-Tri稀释至0.625、1.25、2.5、5、10、20和40μg·mL将其分别加入到每个孔中,并培养48小时。以5mg/mL的浓度添加等分试样(20μL)的MTT溶液,孵育4小时。吸出培养基后,加入200μL二甲基亚砜(DMSO)。将板摇动10分钟,并用酶联免疫吸附测定仪在490nm处测量吸光度。肿瘤细胞的存活率计算如下:抑制率(%)=(1-A sample/A blank)×100%,其中A sample为实验组的吸光度,Ablank为空白对照组的吸光度。
如图4B所示,我们发现free Tri和Tf-MNP-PEG-PLGA-Tri均能抑制A549细胞的增殖,且呈剂量依赖性。令人惊讶的是,Tf-MNP-PEG-PLGA-Tri对A549细胞的抑制率显著高于游离Tri(p<0.01),这与5-FU的作用几乎一致。当Tri浓度达到最大值(40μg/mL)时,Tf-MNP-PEG-PLGA-Tri对A549细胞生长的抑制作用达到94%。如图5所示,纳米粒和A549细胞处理24h后,与MNP-PEG-PLGA-Tri相比,Tf-MNP-PEG-PLGA-Tri组有大量红色荧光物质明显穿过细胞膜进入细胞,然后扩散到细胞核内。在Tf-MNP-PEG-PLGA-Tri成功释放Tri后,A549细胞的摄取率较高。通过Tf-MNP-PEG-PLGA-Tri提高肿瘤细胞的靶向作用,可促进肿瘤细胞对药物的摄取,从而提高药物的传递效率和治疗效果。图5说明Tf-MNP-PEG-PLGA-Tri磁性纳米颗粒增加了肿瘤细胞的靶向作用,可促进肿瘤细胞对药物的摄取,从而提高给药效率和治疗效果,增强Tri对肿瘤细胞的杀伤作用。
实施例7:体内抗肿瘤活性评价
(1)模型建立及分组给药
BALB/C-nu裸鼠35只(4-6周龄,体重18-22g,公母各占一半),在没有特定病原体(SPF)的单独笼子中饲养。将处于对数生长期的A549细胞皮下注射到裸鼠体内。每只小鼠细胞数为1×107个/0.2mL。接种后定期观察裸鼠的精神状态、饮食和排便情况。当肿瘤体积达到75-150mm3时,进行后续实验。25只裸鼠随机分为5组,即模型对照组(0.9%生理盐水)、游离Tri组(10mg/kg)、空白纳米颗粒组(非载药)、Tf-MNP-PEG-PLGA-Tri组(10mg/kg)和阳性对照组(5-fu)。各组分别于第2、4、6、8、10、12、14天进行尾静脉注射。
(2)体内成像
10只裸鼠随机分为两组。然后分别经尾静脉注射Tf-MNP-PEG-PLGA-Tri和游离Tri0.2mL。注射剂量相当于2mg/kg Tri,各组饲养条件相同,体重无差异。在体内成像之前,裸鼠腹腔注射0.06mL 2.5%戊巴比妥钠使其进入麻醉状态。然后分别于1、24、48h分别使用动物体内成像仪进行体内成像。
(3)测量肿瘤的生长
用游标卡尺测量肿瘤的长(a)、宽(b),根据公式V=1/2(a×b2)计算肿瘤体积(V/mm3)。并计算各组间肿瘤大小的变化趋势,即相对肿瘤体积(RTV)=Vx/Vi,其中Vx和Vi分别表示治疗第x天和第1天的肿瘤大小。药物的抗肿瘤作用通过肿瘤抑制率(IR)来表达,其计算公式为:IR(%)=(1-RTV1/RTV2)100%,其中RTV1为实验组,RTV2为对照组。基于以上结果,分析肿瘤形成过程、肿瘤体积和肿瘤生长时间之间的关系。
治疗结束后,切除裸鼠肿瘤,HE染色,在显微镜下观察其组织病理状态。
从图6A&D可以看出,MNP-PEG-PLGA-Tri和Tf-MNP-PEG-PLGA-Tri在1h后在肿瘤部位大量积累,聚集量逐渐增加。与MNP-PEG-PLGA-Tri相比(图6A),Tf-MNP-PEG-PLGA-Tri在荷瘤裸鼠肿瘤部位具有更强的荧光强度。24小时后,荷瘤裸鼠MNP-PEG-PLGA-Tri的荧光强度显著降低(图6B),而Tf-MNP-PEG-PLGA-Tri的荧光强度仍主要集中在肿瘤部位(图6E)。48小时后,注射到荷瘤裸鼠体内的MNP-PEG-PLGA-Tri在肿瘤部位几乎无荧光(图6C),而注射到荷瘤裸鼠体内的Tf-MNP-PEG-PLGA-Tri在肿瘤部位有部分荧光。而内脏器官则明显下降甚至完全消失(图6F),说明Tf-MNP-PEG-PLGA-Tri在肿瘤部位存活时间更长,可以更有效地靶向肿瘤细胞,而不需要在内脏器官长期积累。结果证明,Tf-MNP-PEG-PLGA-Tri可以在肿瘤部位存活较长时间,有良好的肿瘤靶向性,能有效一致肿瘤生长,且无明显的全身毒性。
在肿瘤生长曲线中(Figure 7A-E),观察到除空白纳米颗粒组外,模型组肿瘤体积明显大于其他治疗组,而Tf-MNP-PEG-PLGA-Tri治疗组肿瘤体积明显小于其他治疗组,与图7F中肿瘤生长曲线一致。
HE切片染色也表明,Tri和Tf-MNP-PEG-PLGA-Tri对裸鼠重要器官无损伤。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (7)
1.一种转铁蛋白修饰的配体双靶向磁性纳米颗粒的制备方法,其特征在于:由Tri、PEG-PLGA、转铁蛋白和氨基化磁性纳米颗粒组成。
2.根据权利要求1所述的转铁蛋白修饰的双靶向雷公藤甲素磁性纳米颗粒的制备方法,其特征在于所述纳米颗粒的粒径范围为100-150nm。
3.根据权利要求1所述的转铁蛋白修饰的双靶向雷公藤甲素磁性纳米颗粒的制备方法,其特征在于,所述方法包括如下步骤:
(1)MNP-NH2的制备:
采用共沉淀法合成NP-NH2。将FeCl3和FeCl2溶解于蒸馏水中,置于三颈瓶中;
将混合溶液在氮气保护下加热至90℃,10min后加入氨溶液沉淀Fe3+/Fe2+金属离子,进行磁分离后干燥可得磁性纳米颗粒(MNPs);
然后,将3-氨丙基三乙氧基硅烷(APTES)作为胺化试剂,通过硅化作用与Fe3O4纳米颗粒表面结合,形成MNP-NH2。其具体步骤为:将MNPs和APTES溶于80mL乙醇水中,反应液在氮气保护下,40℃回流24h,用热去离子水和乙醇洗涤3次,真空干燥;
(2)MNP-PEG-PLGA-Tri纳米粒子的制备:
采用W/O/W复合乳液溶剂蒸发法制备载药纳米颗粒。将PEG-PLGA和Tri溶液二氯甲烷作为油相,MNP-NH2溶液作为水相;
将水相滴加到油相中,冰浴超声,加入5%PVA溶液,得到W/O/W双乳溶液;
将双乳溶液倒入0.5%PVA水溶液中,在室温下用高速分散器搅拌,使二氯甲烷蒸发;用0.22μm滤膜过滤,离心后,冷冻干燥保存;
(3)Tf-MNP-PEG-PLGA-Tri纳米粒子的制备:
将MNP-PEG-PLGA-Tri纳米颗粒在水浴中孵育1h后,加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS),搅拌混匀;
加入Tf,37℃水浴3h,透析(10kDa)12h,除去游离Tri和Tf,冷冻干燥,即可得Tf-MNP-PEG-PLGA-Tri磁性复合纳米颗粒。
4.根据权利要求3所述的转铁蛋白修饰的双靶向雷公藤甲素磁性纳米颗粒的制备方法,其特征在于,在MNP-NH2的制备过程中,加入FeCl3和FeCl2的摩尔比为1-5:1;加入氨溶液的体积为5-15mL;MNPs和APTES的摩尔比为1-3:1;乙醇水的体积比为1-5:1。
5.根据权利要求3所述的转铁蛋白修饰的双靶向雷公藤甲素磁性纳米颗粒的制备方法,其特征在于,在MNP-PEG-PLGA-Tri纳米粒子的制备过程中,聚乙二醇-聚乳酸聚乙醇酸共聚物(PEG-PLGA)和Tri的质量比为5:1-10:1;MNP-NH2溶液的浓度为5%-10%。
6.根据权利要求3所述的转铁蛋白修饰的双靶向雷公藤甲素磁性纳米颗粒的制备方法,其特征在于,在Tf-MNP-PEG-PLGA-Tri纳米粒子的制备过程中,EDC和NHS的摩尔比为1:1-3:1;Tf和MNP-PEG-PLGA-Tri的摩尔比为1-4:4。
7.一种如权利要求1-6任一项所述的转铁蛋白修饰的双靶向雷公藤甲素磁性纳米颗粒的制备方法法制备的纳米颗粒的体内外抗肿瘤活性研究及其靶向抗肿瘤中的应用。
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