CN116392606A - 一种仿生生物膜修饰的多功能纳米治疗剂及其制备方法与应用 - Google Patents
一种仿生生物膜修饰的多功能纳米治疗剂及其制备方法与应用 Download PDFInfo
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- CN116392606A CN116392606A CN202310240457.9A CN202310240457A CN116392606A CN 116392606 A CN116392606 A CN 116392606A CN 202310240457 A CN202310240457 A CN 202310240457A CN 116392606 A CN116392606 A CN 116392606A
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Abstract
本发明公开了一种仿生生物膜修饰的多功能纳米治疗试剂,它是肿瘤细胞细胞膜修饰的多功能纳米治疗剂,多功能治疗剂是2,5‑双(6‑溴己基)‑3,6‑双(5‑(4‑(萘‑1‑基(苯基)氨基)苯基)呋喃‑2‑基)‑2,5‑二氢吡咯[3,4‑c]吡咯‑1,4‑二酮和原儿茶酸和在二硬脂酰基磷脂酰乙醇胺‑聚乙二醇作用下自助装而成的BPP纳米颗粒。本发明公开了仿生生物膜修饰的多功能纳米治疗剂在制备光治疗、化疗、抗血管生成协同治疗肿瘤药物的应用。本发明仿生生物膜修饰的多功能纳米治疗剂具有癌细胞膜的免疫逃逸和同源靶向的特殊能力,使得纳米治疗剂能够高效富集于肿瘤部位,发挥多模式肿瘤协同治疗能力,实现多模式肿瘤治疗。
Description
技术领域
本发明属于纳米医药领域,涉及仿生生物膜修饰的多功能纳米治疗剂及其制备方法,以及所述的多功能纳米治疗剂在协同治疗肿瘤上的应用。
背景技术
光治疗因其具有非侵入性、高选择性和可忽略的全身细胞毒性而得到了广泛关注。然而,肿瘤乏氧问题一直制约着光治疗,其中固体肿瘤中的氧分压远只有10mmHg汞柱,低于正常外周组织(40-60mmHg汞柱),严重阻碍了氧依赖性光动力疗法(PDT)的效果。此外,光热疗法(PTT)往往会诱发肿瘤组织中热休克蛋白的上调,导致肿瘤快速耐受,最终影响治疗效果。虽然PDT、PTT的协同作用可以通过提高血流速度来改善肿瘤组织的供氧,从而提高PDT的效率,PDT也可以反过来诱发肿瘤细胞的耐热性死亡,但不理想的激发光穿透深度和光热试剂的低特异性仍然阻碍着治疗效果。
肿瘤的发生往往伴随着新血管的形成,它既是肿瘤发生的前提条件,也是肿瘤生长、侵袭与转移的基础。因此,在抗癌时,针对肿瘤血管与针对肿瘤细胞一样关键。抗血管生成疗法通过中和或阻断血管内皮生长因子(VEGF)的表达,抑制新血管的生成,从而减少肿瘤组织的营养供应,最终导致肿瘤组织从内到外的凋亡或坏死。因此,将具有不同抗肿瘤机制的光疗和抗血管生成治疗结合起来,被认为是实现“1+2>3”效果和提高治疗效果的突破性策略。一方面,光疗可以根除由边缘正常血管提供营养的肿瘤细胞,而这种肿瘤细胞在抗血管生成治疗后仍然存在。另一方面,抗血管生成治疗可以破坏深部的肿瘤细胞,而这些细胞是光难以完全穿透的。同时,肿瘤血管抑制剂可以中和或阻断VEGF的作用,从而使肿瘤血管在一定时期内“正常化”。因此,光疗和抗血管生成治疗结合可以缓解局部肿瘤缺氧、改善局部运输以及治疗药物在肿瘤中的积累,从而最大限度地提高光疗的治疗效果。
原儿茶酸(3,4-二羟基苯甲酸,PCA)是一种广泛分布的天然酚类化合物,结构简单,生物活性高。此外,PCA已被确定为一种多靶点生物活性化合物,它不仅能阻断血管内皮细胞中VEGFR2依赖的Akt/MMP2和ERK途径以抗血管生成,而且对具有明显化疗毒性的各种肿瘤细胞具有良好的杀伤力。然而其在实际治疗中难以杀死全部肿瘤细胞,导致肿瘤复发和转移。
大多数无机纳米材料存在生物相容性差,难以代谢等缺点,而有机小分子吡咯并吡咯二酮(DPP)衍生物是易修饰、生物相容性高、毒性低的有机小分子染料,在有机电子器件以及生物探针领域有广泛应用。然而,在生物医药领域,DPP的水溶性和靶向性仍然是一个巨大的挑战。同时,在实际研究中也发现DPP产生的单纯光治疗和PCA介导的化疗和抗血管疗法一样并不能完全消融肿瘤细胞。为了克服这些缺点,将具有不同抗肿瘤机制的光疗,化疗和抗血管生成治疗结合起来,被认为是实现“1+2>3”效果和提高治疗效果的突破性策略。
然而由于体内的生物屏障,纳米粒子(NPs)作为外源性材料可能会被早期免疫系统识别,然后被肝脏和肾脏清除,这就限制了纳米粒子的进一步发展。
因此,设计并制备出仿生生物膜修饰的兼具光治疗、化疗、抗血管生成的能力的多功能纳米药物在肿瘤治疗领域具有广阔前景。
发明内容
本发明的目的旨在通过在纳米尺度上制备可用于抗肿瘤的仿生生物膜修饰的多功能纳米治疗剂,通过仿生生物膜修饰隐蔽纳米粒子,提高在肿瘤部位积累,并减少在健康组织中的滞留,有利于药物输送和肿瘤的局部治疗。。
本发明的目的通过以下技术方案实现的:
结构如式Ⅰ所示的光治疗剂2,5-双(6-溴己基)-3,6-双(5-(4-(萘-1-基(苯基)氨基)苯基)呋喃-2-基)-2,5-二氢吡咯[3,4-c]吡咯-1,4-二酮(BPA):
本发明的另一个目的是提供所述的2,5-双(6-溴己基)-3,6-双(5-(4-(萘-1-基(苯基)氨基)苯基)呋喃-2-基)-2,5-二氢吡咯[3,4-c]吡咯-1,4-二酮在制备肿瘤光治疗药物的应用。
本发明的另一个目的是提供一种仿生生物膜修饰的多功能纳米治疗剂,它是肿瘤细胞细胞膜修饰的多功能纳米治疗剂;所述的多功能治疗剂是所述的光治疗剂2,5-双(6-溴己基)-3,6-双(5-(4-(萘-1-基(苯基)氨基)苯基)呋喃-2-基)-2,5-二氢吡咯[3,4-c]吡咯-1,4-二酮(BPA)与化疗剂和抗血管生成剂原儿茶酸(PCA)在二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-mPEG2000)作用下自组装而成的BPP纳米颗粒(BPP NPs)。
所述的肿瘤细胞细胞膜为HeLa细胞。
所述的肿瘤细胞细胞膜是本领域技术人员通过常规方法制备得到的,具体的,可以采用以下方法:将肿瘤细胞细胞悬浮在含有苯甲基磺酰氟(1mM)的膜蛋白提取缓冲液A(碧云天细胞膜蛋白与细胞浆蛋白提取试剂盒)中,将混合物在冰浴中孵化15min以裂解细胞;之后,将细胞悬液在液氮中冷冻,在室温下解冻;冻融循环重复2次;在温度4℃下,700g离心10min去除沉淀,上清液进一步在温度4℃下高速离心30min,收集沉淀,即为肿瘤细胞细胞膜碎片。
本发明的另一个目的是提供一种所述的仿生生物膜修饰的多功能纳米治疗剂的制备方法,包括:
步骤(1)、在氮气气氛下,将4-(N-(萘-1-基)-N-苯基氨基)苯硼酸、双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮(DPP-1)、四(三苯基膦)钯(Pd(PPh3)4)和磷酸三钾加入到1,4-二氧六环中,加热搅拌;除去溶剂后得到粗产品,粗产品经分离得到2,5-双(6-溴己基)-3,6-双(5-(4-(萘-1-基(苯基)氨基)苯基)呋喃-2-基)-2,5-二氢吡咯[3,4-c]吡咯-1,4-二酮(BPA);
步骤(2)、将2,5-双(6-溴己基)-3,6-双(5-(4-(萘-1-基(苯基)氨基)苯基)呋喃-2-基)-2,5-二氢吡咯[3,4-c]吡咯-1,4-二酮(BPA)和原儿茶酸(PCA)溶于四氢呋喃中,得到混合溶液;在搅拌下,将混合溶液慢慢滴加到含有二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-mPEG2000)的去离子水中,滴加完毕,继续搅拌5~10分钟,氮气充分鼓吹除去反应液中的四氢呋喃,离心,取上清液,得到具有化疗、光治疗、抗血管生成功能的BPP纳米颗粒(BPP NPs)水分散体;
步骤(3)、将肿瘤细胞细胞膜溶解至去离子水中,超声破碎,得到肿瘤细胞细胞膜溶液;在搅拌下,慢慢将肿瘤细胞细胞膜溶液滴加到步骤(2)得到的BPP纳米颗粒(BPP NPs)水分散体中,避光孵育,离心,取上清液,得到仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒(mBPP NPs)。
步骤(1)中,DPP-1与4-(N-(萘-1-基)-N-苯基氨基)苯硼酸的摩尔比为1:2~1:10,优选为1:5;DPP-1与四(三苯基膦)钯的摩尔比为1:0.01~1:0.1,优选为1:0.05~1:0.06;DPP-1与磷酸三钾磷酸三钾的摩尔比为1:2~1:10,优选为1:4。
所述的加热搅拌的温度为25~120℃,加热搅拌的时间为8~36小时;优选的,所述的加热搅拌的温度为90~100℃,加热搅拌的时间为12~24小时。
粗产品经硅胶色谱柱分离纯化得到BPA,洗脱剂为石油醚:二氯甲烷=1:2V/V。
步骤(2)中,BPA和PCA溶解在四氢呋喃中即可,一般的,BPA在四氢呋喃中浓度为1~5mg/mL,PCA在四氢呋喃中浓度为1~5mg/mL。
BPA、PCA和DSPE-mPEG2000的质量比为1:1:0.5。
DSPE-mPEG2000在去离子水中浓度为0.1~0.5mg/mL。
混合溶液的滴加速度为1~30滴/分钟;搅拌的转速为500~2000转/分钟。
BPP纳米颗粒(BPP NPs)水分散体中BPP纳米颗粒的浓度是100~200μg/mL。
步骤(3)中,超声破碎的时间为2~10分钟,超声的功率为400W。
所述的肿瘤细胞细胞膜溶液中细胞膜浓度为0.1~1mg/mL。
所述的肿瘤细胞细胞膜和BPP纳米颗粒的质量比为1:1。
在搅拌速率为200~500转/分钟、温度为4~8℃下,慢慢将肿瘤细胞细胞膜溶液滴加到BPP纳米颗粒水分散体中。
所述的避光孵育在搅拌下进行,孵育的搅拌速率为200~500转/分钟,孵育温度为4~8℃,孵育时间为8~24小时。
本发明的另一个目的是提供所述的仿生生物膜修饰的多功能纳米治疗剂在制备光治疗、化疗、抗血管生成协同治疗肿瘤药物的应用。
与现有技术相比,本发明的有益效果:
本发明通过自组装和共孵育的方法得到仿生生物膜修饰的多功能纳米治疗剂,该多功能纳米治疗剂具有癌细胞膜修饰的同源靶向和免疫逃逸功能,并可协同光治疗、化疗、抗血管生成疗法等多种肿瘤治疗方式,具体表现为:
(1)、本发明仿生生物膜修饰的多功能纳米治疗剂合成工艺简单、产率高;
(2)、本发明仿生生物膜修饰的多功能纳米治疗剂具有精准的主、被动肿瘤靶向性,既可以通过仿生生物膜修饰实现同源靶向肿瘤细胞,还可以通过纳米颗粒的高渗透长滞留效应被动靶向肿瘤部位,同时仿生生物膜修饰也赋予了纳米颗粒免疫逃逸能力,避免其被免疫系统吞噬,最终使得纳米治疗剂更多的富集在肿瘤周围,提高疗效;
(3)、本发明仿生生物膜修饰的多功能纳米治疗剂可协同光治疗、化疗、抗血管生成疗法等多种方式,有效杀死肿瘤细胞,抑制肿瘤细胞再生和转移,实现多模式肿瘤治疗,应用前景广阔。
附图说明
图1为BPA的核磁1H-NMR图(400MHz,CDCl3),横坐标为化学位移,纵坐标为强度。
图2为多功能纳米治疗剂BPP纳米颗粒的透射电镜图。
图3为多功能纳米治疗剂BPP纳米颗粒细节透射电镜图。
图4为仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒的透射电镜图。
图5为仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒细节透射电镜图。
图6为仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒在水中的光热转换效率的检测结果,其中,曲线(a)为mBPP纳米颗粒的升降温曲线,曲线(b)为去离子水的升降温曲线,回归直线(c)为mBPP纳米颗粒降温的-ln值与时间的关系。
图7为仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒通过流式细胞仪分析不同浓度(0μM,10μM,20μM,30μM)下细胞凋亡机理图。
图8为多功能治疗剂BPP纳米颗粒及仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒通过荧光共聚焦显微镜观察的HeLa细胞内吞作用。
图9为多功能治疗剂BPP纳米颗粒及仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒通过荧光共聚焦显微镜观察的RAW264.7细胞内吞的免疫逃逸作用。
图10为仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒通过人脐静脉内皮细胞检测的抑制血管新生实验。
图11为仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒的裸鼠治疗瘤体积变化过程,其中横坐标表示时间,纵坐标表示肿瘤体积。
图12为仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒的裸鼠治疗后解剖肿瘤大小。
具体实施方式
下面通过实施例来进一步说明本发明的技术方案,以便较好的理解本发明的内容。
实施例1
BPA的合成
在氮气气氛下,将4-(N-(萘-1-基)-N-苯基氨基)苯硼酸(0.68g,2.00mmol)、DPP-1(0.30g,0.40mmol)四(三苯基膦)钯(0.026g,0.0225mmol)和磷酸三钾(0.34g,1.60mmol)加入到1,4-二氧六环(80mL)中,并在100℃下回流24h;除去溶剂后得到粗产品,粗产品采用硅胶色谱柱(洗脱剂为石油醚:二氯甲烷=1:2V/V),分离纯化得到2,5-双(6-溴己基)-3,6-双(5-(4-(萘-1-基(苯基)氨基)苯基)呋喃-2-基)-2,5-二氢吡咯[3,4-c]吡咯-1,4-二酮(BPA),产率96%。
BPA的核磁1H-NMR图(400MHz,CDCl3)见图1。1H NMR(400MHz,CDCl3,ppm):δ8.22(d,J=3.8Hz,2H),7.84(d,J=8.4Hz,4H),7.75(d,J=8.2Hz,2H),7.50-7.36(m,8H),7.34-7.25(m,4H),7.22-7.14(m,4H),7.10-7.03(m,4H),6.99-6.92(m,2H),6.91-6.85(m,4H),6.69(d,J=3.8Hz,2H),4.05(t,J=7.3Hz,4H),3.21(t,J=6.8Hz,4H),1.68(t,J=6.9Hz,8H),1.39-1.26(m,8H)13C NMR(100MHz,CDCl3,ppm):148.1,146.2,142.2,141.6,134.3,130.9,130.02,128.3,128.0,127.85,127.5,127.3,126.4,126.0,125.7,125.4,125.4,125.1,125.0,124.4,122.9,122.2,122.1,120.8,120.6,119.2,115.1,106.7,41.3,32.7,31.6,29.1,26.9,25.2.
BPP纳米颗粒(BPP NPs)的制备
1mg DSPE-mPEG2000超声溶解于5mL去离子水中,得到DSPE-mPEG2000溶液;将2mgBPA和2mg PCA溶于500μL THF中,得到混合溶液;维持DSPE-mPEG2000溶液在快速搅拌状态(转速为1000转/分钟),以20滴/分钟的滴加速度将混合溶液慢慢滴加至DSPE-mPEG2000溶液中;滴加完毕,继续搅拌5分钟,氮气充分鼓吹除去溶液中的四氢呋喃,然后离心,取上清液,即得多功能纳米治疗剂BPP纳米颗粒(BPP NPs)水分散体,BPP纳米颗粒的浓度是200μg/mL。
mBPP纳米颗粒(mBPP NPs)的制备
将培养收集到的5×107个HeLa细胞悬浮在1mL含有苯甲基磺酰氟(1mM)的膜蛋白提取缓冲液A(碧云天细胞膜蛋白与细胞浆蛋白提取试剂盒)中,将混合物在冰浴中孵化15min以裂解细胞;之后,将细胞悬液在液氮中冷冻,在室温下解冻;冻融循环重复2次;在温度4℃下,700g离心10min去除沉淀,得到的上清液进一步在温度4℃下高速离心30min,收集沉淀,即为肿瘤细胞细胞膜碎片。
将HeLa癌细胞膜溶于去离子水中(浓度为200μg/mL),400W超声破碎4min,得到HeLa癌细胞细胞膜溶液;在温度4℃下,维持的HeLa癌细胞细胞膜溶液搅拌速率为200转/分钟、按照肿瘤细胞细胞膜和BPP纳米颗粒的质量比为1:1,以20滴/分钟的滴加速度将HeLa癌细胞细胞膜溶液慢慢滴加到搅拌(转速为200转/分钟)的BPP纳米颗粒水分散体(浓度为200μg/mL)中;滴加完毕,在搅拌速率为200转/分钟、温度4℃下,避光孵育12h,离心,取上清液,得到仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒(mBPP NPs)。
多功能纳米治疗剂BPP纳米颗粒的透射电镜图见图2和图3,平均尺寸为90nm;仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒的透射电镜图见图4和图5,平均尺寸为101nm。通过透射电镜细节对比可以明显发现经仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒具有明显的核壳结构,且平均尺寸增加了约10nm。
实施例2
mBPP纳米颗粒的单线态氧及光热转换效率检测
mBPP纳米颗粒水分散体于660nm激光(1.3W/cm2)下照射10分钟,相同条件下检测去离子水作为空白对照。结果如图6,当温度达到平衡时,室温冷却;温度的大幅度上升表明mBPP纳米颗粒具有极强的光热转换能力。
实施例3
mBPP纳米颗粒体外细胞毒性检测
选取HeLa肿瘤细胞(购于GIBCO)进行体外定量毒性实验,测试mBPP纳米颗粒激光毒性。
具体实验步骤如下:mBPP纳米颗粒用DMEM培养基稀释成不同浓度(0μM,10μM,20μM,30μM)。HeLa肿瘤细胞被接种在6孔培养板,37℃下培养使其贴壁生长24小时,避光加药(500μL),且避光培养24小时,由660nm激光器分别照射各孔5分钟(1.3W/cm2),继续培养12小时,然后用Annexin V-FITC和propidium iodide(PI)进行染色。使用流式细胞分析仪计算绝对的细胞存活率,如图7所示,细胞存活率存在反浓度依赖性,给药浓度越大细胞存活率越低,IC50值约为23μM。
实施例4
mBPP纳米颗粒的体外细胞摄取实验和免疫逃逸实验
选取HeLa肿瘤细胞(购于GIBCO)进行体外细胞摄取实验。实验步骤如下:将HeLa肿瘤细胞接种到两个共聚焦小皿中,细胞贴壁后将培养基分别改为含BPP纳米颗粒(浓度为23μM)的DMEM培养基和含mBPP纳米颗粒(浓度为IC50浓度23μM)的DMEM培养基,孵育4小时后,用PBS洗三次细胞,用4%多聚甲醛固定,并用DAPI染色,用PBS彻底冲洗后,在共聚焦显微镜下观察细胞的摄取情况。如图8所示,与BPP纳米颗粒处理的HeLa细胞相比,经mBPP纳米颗粒处理的HeLa细胞表现出更明显的红色荧光,表明经癌细胞膜修饰的mBPP纳米颗粒对同种细胞具有更强的靶向性。
选取小鼠单核巨噬细胞白血病细胞(RAW264.7)细胞(购于GIBCO)进行体外免疫逃逸实验。实验步骤如下:将RAW264.7细胞接种到两个共聚焦小皿中,细胞贴壁后将DMEM培养基分别改为含BPP纳米颗粒(浓度为30μM)的DMEM培养基和含mBPP纳米颗粒(浓度为30μM)的DMEM培养基,孵育12小时后,用PBS清洗三次细胞,用4%多聚甲醛固定30分钟后,用DAPI染色,用PBS彻底冲洗后,在共聚焦显微镜上观察免疫细胞的摄取情况。如图9所示,经mBPP纳米颗粒处理的免疫细胞RAW264.7显示出较弱的红色荧光信号,这意味着mBPP纳米颗粒能够被免疫细胞更少的摄取,癌细胞膜的免疫逃逸能力得到充分体现。
实施例5
mBPP纳米颗粒抑制新生血管生成检测实验
将基质胶在冰上解冻,滴加到预先冷却的血管生成载玻片上(10μL/孔),在37℃下孵育1h;然后将人脐静脉内皮细胞(HUVECs,2×105细胞/孔)与用DMEM培养基配制的不同浓度(0、10、20、30μM)的mBPP纳米颗粒混合后接种,细胞在二氧化碳恒温培养箱中培养,在倒置的荧光显微镜下观察内皮细胞的血管结构并拍照观察。如图10所示,空白组(0μM)的血管随着时间的推移逐渐形成密集的血管网,而给药组血管网的形成受到明显的抑制,并呈现浓度依赖性。
实施例6
mBPP纳米颗粒体内肿瘤治疗性能
选择将HeLa肿瘤细胞注入腋下的裸鼠作为肿瘤模型,24只裸鼠随机分为6组。当肿瘤体积约为100mm3时,第一组(Control)通过尾静脉注射生理盐水,第二组(BPA NPs)小鼠通过尾静脉注射BPA纳米颗粒(100μg/mL,100μL,生理盐水配制)、第三组(BPA纳米颗粒激光光照组,BPA NPs+L)小鼠通过尾静脉注射BPA纳米颗粒(100μg/mL,100μL)、第四组(PCA组)小鼠通过尾静脉注射PCA溶液(100μg/mL,100μL,生理盐水配制)、第五组(mBPP NPs)小鼠通过尾静脉注射mBPP纳米颗粒(100μg/mL,100μL,生理盐水配制)、第六组(mBPP纳米颗粒激光光照组,mBPP NPs+L)小鼠通过尾静脉注射mBPP纳米颗粒(100μg/mL,100μL)。12小时后,第三、六组小鼠的肿瘤在660nm(1W/cm2)激光器各照射5分钟,其他组不进行光照。每2天测量一次肿瘤大小,结果如图11所示,第六组在第4天就基本消除掉了肿瘤组织。14天后处死小鼠,观察并测量肿瘤组织,如图12所示,第六组可以有效地彻底消除肿瘤,其他组均有不同程度的残留。
Claims (10)
2.权利要求1所述的2,5-双(6-溴己基)-3,6-双(5-(4-(萘-1-基(苯基)氨基)苯基)呋喃-2-基)-2,5-二氢吡咯[3,4-c]吡咯-1,4-二酮在制备肿瘤光治疗药物的应用。
3.一种仿生生物膜修饰的多功能纳米治疗试剂,其特征在于:它是肿瘤细胞细胞膜修饰的多功能纳米治疗剂;所述的多功能治疗剂是权利要求1所述的2,5-双(6-溴己基)-3,6-双(5-(4-(萘-1-基(苯基)氨基)苯基)呋喃-2-基)-2,5-二氢吡咯[3,4-c]吡咯-1,4-二酮和原儿茶酸和在二硬脂酰基磷脂酰乙醇胺-聚乙二醇作用下自助装而成的BPP纳米颗粒。
4.一种权利要求3所述的仿生生物膜修饰的多功能纳米治疗试剂的制备方法,其特征在于:包括:
步骤(1)、在氮气气氛下,将4-(N-(萘-1-基)-N-苯基氨基)苯硼酸、双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮、四(三苯基膦)钯(Pd(PPh3)4)和磷酸三钾加入到1,4-二氧六环中,加热搅拌;除去溶剂后得到粗产品,粗产品经分离得到2,5-双(6-溴己基)-3,6-双(5-(4-(萘-1-基(苯基)氨基)苯基)呋喃-2-基)-2,5-二氢吡咯[3,4-c]吡咯-1,4-二酮;
步骤(2)、将2,5-双(6-溴己基)-3,6-双(5-(4-(萘-1-基(苯基)氨基)苯基)呋喃-2-基)-2,5-二氢吡咯[3,4-c]吡咯-1,4-二酮和原儿茶酸溶于四氢呋喃中,得到混合溶液;在搅拌下,将混合溶液滴加到含有二硬脂酰基磷脂酰乙醇胺-聚乙二醇的去离子水中,滴加完毕,继续搅拌5~10分钟,氮气充分鼓吹除去四氢呋喃,离心,取上清液,得到BPP纳米颗粒水分散体;
步骤(3)、将肿瘤细胞细胞膜溶解至去离子水中,超声破碎,得到肿瘤细胞细胞膜溶液;在搅拌下,将肿瘤细胞细胞膜溶液滴加到BPP纳米颗粒水分散体中,避光孵育,离心,取上清液,得到仿生生物膜修饰的多功能纳米治疗剂mBPP纳米颗粒。
5.根据权利要求4所述的仿生生物膜修饰的多功能纳米治疗试剂的制备方法,其特征在于:步骤(1)中,双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮与4-(N-(萘-1-基)-N-苯基氨基)苯硼酸的摩尔比为1:2~1:10;双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮与四(三苯基膦)钯的摩尔比为1:0.01~1:0.1;双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮与磷酸三钾磷酸三钾的摩尔比为1:2~1:10。
6.根据权利要求5所述的仿生生物膜修饰的多功能纳米治疗试剂的制备方法,其特征在于:步骤(1)中,双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮与4-(N-(萘-1-基)-N-苯基氨基)苯硼酸的摩尔比是1:5;双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮与四(三苯基膦)钯的摩尔比为1:0.05~1:0.06;双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮与磷酸三钾磷酸三钾的摩尔比为1:4。
7.根据权利要求4所述的仿生生物膜修饰的多功能纳米治疗试剂的制备方法,其特征在于:步骤(1)中,所述的加热搅拌的温度为25~120℃,加热搅拌的时间为8~36小时;优选的,所述的加热搅拌的温度为90~100℃,加热搅拌的时间为12~24小时。
8.根据权利要求4所述的仿生生物膜修饰的多功能纳米治疗试剂的制备方法,其特征在于:步骤(2)中,BPA、PCA和DSPE-mPEG2000的质量比为1:1:0.5;DSPE-mPEG2000在去离子水中浓度为0.1~0.5mg/mL。
9.根据权利要求4所述的仿生生物膜修饰的多功能纳米治疗试剂的制备方法,其特征在于:步骤(3)中,所述的肿瘤细胞细胞膜溶液中细胞膜浓度为0.1~1mg/mL;所述的肿瘤细胞细胞膜和BPP纳米颗粒的质量比为1:1;
所述的避光孵育在搅拌下进行,孵育的搅拌速率为200~500转/分钟,孵育温度为4~8℃,孵育时间为8~24小时。
10.权利要求3所述的仿生生物膜修饰的多功能纳米治疗剂在制备光治疗、化疗、抗血管生成协同治疗肿瘤药物的应用。
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