CN116327976A - 一种仿生纳米材料在制备多发性骨髓瘤治疗药物中的应用 - Google Patents
一种仿生纳米材料在制备多发性骨髓瘤治疗药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种仿生纳米材料在制备多发性骨髓瘤治疗药物中的应用,其是一种新的靶向仿生光热纳米药物,用于近红外诱导光热协同药物BTZ化疗以提升多发性骨髓瘤疗效。B细胞成熟抗原是非常理想的靶点,黑磷量子点优异的光热性能在肿瘤治疗中发挥着积极作用,且可起到药物载体作用,红细胞膜固有的仿生特性可以帮助纳米体系摆脱免疫***监视,提高纳米药物的体内循环。因此,选用BCMA抗体修饰在红细胞膜表面,并将核心治疗药BTZ和BPQDs包裹在红细胞膜内,制备成新的纳米药物,增强治疗药物在肿瘤部位的积累,并降低脱靶副作用。
Description
技术领域
本发明涉及一种协同光热/化疗纳米药物,具体涉及一种仿生纳米材料在制备多发性骨髓瘤治疗药物中的应用。属于医药技术领域。
背景技术
多发性骨髓瘤(MM)是一种骨髓浆细胞异常增殖引起的血液***恶性肿瘤。在过去的几十年里,MM的治疗取得了显著的进步。但由于MM的病理异质性,对不同的MM患者仍缺乏安全有效的治疗方法。硼替佐米(Bortezomib,BTZ)是临床上常用的一线治疗药物,具有良好的治疗效果。然而,由于药物耐受性的发展和周围神经毒性的增加,大多数患者仍然会遇到治疗的阻力和疾病的复发。因此,开发新的治疗策略,以打破耐药性和克服副作用是MM有效的治疗方向。
纳米材料有效递送药物技术的发展为提高MM的治疗效率和降低其耐药性提供了新思路。最近的研究表明,生物的免疫***可以通过网状内皮***(RES)吸收外来纳米颗粒,导致纳米药物在体内的快速消除。因此,仿生纳米颗粒,如细胞膜涂层纳米颗粒(CMC)的引入,为提高纳米药物的生物利用度和生物安全性提供了更好的解决方案。内源性生物膜来源的纳米颗粒,如红细胞(RBC)、血小板、癌细胞和干细胞,表现出对外源性颗粒的优势,如逃避免疫清除、延长循环时间和低免疫原性。既往研究表明,以富含膜蛋白为自身标记物的红细胞膜(EM)可将纳米颗粒伪装成宿主细胞,避免被免疫***消除,提高肿瘤靶向性的增强通透性和保留性(EPR)效应。此外,红细胞膜制备容易,已成为纳米粒子伪装涂层中应用最广泛的仿生策略。
B细胞成熟抗原(BCMA)在MM的发展过程中起着至关重要的作用,研究表明其在浆细胞恶性转化过程中选择性高表达。BCMA与临床状态的相关性支持其作为难以监测的传统患者群体的生物标志物。此外,NF-κB细胞内信号转导级联的激活可促进肿瘤生长、生存和耐药。TNF超家族成员13b(BAFF)是一种受NF-κB调控的重要细胞因子,可依次激活NF-κB并进一步调控Bcl-2等其他生长因子的表达,诱导MM细胞持续增殖和存活。此外,BAFF作为一种调节B淋巴细胞生存成熟的细胞因子,与BCMA特异性结合,因此劫持BCMA可视为阻断该通路激活的一种手段。
纳米材料对肿瘤细胞的多维杀灭和对肿瘤微环境的改善,为促进MM乃至血液恶性肿瘤治疗在深度和广度上的突破提供了有力手段。提出化疗与其他治疗方法的协同策略,以提高治疗效果,降低耐药性,其治疗效果已在一些研究中有所体现。光热疗法(Photothermal therapy,PTT)是一种利用光热剂辅助激光加热***的方法,在肿瘤治疗中得到了广泛的应用,近年来随着与化疗相结合具有良好的抗肿瘤作用而日益突出。在不同类型的光热纳米材料中,黑磷量子点(BPQDs)因其高的光热转换效率、固有的生物相容性和降解能力而备受关注,在肿瘤治疗方面显示出广阔的应用前景。
但是,黑磷量子点的稳定性较差,限制了其在临床药物中的应用。由于黑磷量子点暴露在血清及氧气存在的环境下非常容易降解,这就导致裸露的黑磷量子点无法在人体内环境中稳定发挥功能,因此需要利用一些表面改性手段来增强其体内长循环能力。本申请材料则利用了生物膜-红细胞膜来包裹黑磷量子点,不仅延长了纳米体系在体内的循环时间,也尽可能减少了纳米体系递送联合治疗剂至肿瘤部位前的损失。
发明内容
本发明的目的是为克服上述现有技术的不足,提供一种仿生纳米材料在制备多发性骨髓瘤治疗药物中的应用。
为实现上述目的,本发明采用下述技术方案:
一种仿生纳米材料在制备多发性骨髓瘤治疗药物中的应用,其中,所述仿生纳米材料为BTZ@BPQDs@EM@anti-BCMA,它是通过以下方法构建得到的:
(1)先将BCMAmAb与Traut’s试剂充分混合,反应,除盐,得到anti-BCMA-HS;
(2)接着向DSPE-PEG-Mal溶液中加入anti-BCMA-HS,搅拌孵育,得到DSPE-PEG-anti-BCMA;
(3)然后将黑磷量子点BPQDs与BTZ溶液充分混合孵育,得到BTZ@BPQDs,再将其与利用红细胞制成的EM囊泡混合,得到BTZ@BPQDs@EM,简称BBE;
(4)最后将DSPE-PEG-anti-BCMA与BTZ@BPQDs@EM混合孵育,即得。
优选的,所述仿生纳米材料在使用过程中配合NIR激光照射;NIR激光的功率密度为0.75-1.5W/cm2,波长为808nm。
优选的,步骤(1)的具体方法如下:先将BCMAmAb与Traut’s试剂分别利用PBS(1L的1×PBS含有10mMNa2HPHO4、2mMNaH2PO4、135mMNaCl、4.7mM KCl,pH7.3±0.1)在25℃-30℃配制成0.1-0.5mg/mLBCMAmAb溶液、0.1-0.5mg/mLTraut’s试剂溶液,接着将BCMAmAb溶液、Traut’s试剂溶液、5×10-3M PBS-EDTA缓冲液按照体积比500:27.5:20充分混合,然后转移至HS-3垂直混合仪(碧云天)中反应4-8h,使用脱盐柱清洗除去多余的Traut’s试剂,即得。
优选的,步骤(2)的具体方法为:先将DSPE-PEG-Mal利用PBS配制成DSPE-PEG-Mal溶液,接着向DSPE-PEG-Mal溶液中加入与DSPE-PEG-Mal等摩尔量的anti-BCMA-HS,充分混合,转移至垂直混合仪中,室温反应12~18h,利用超滤离心管除去多余的DSPE-PEG-Mal,PBS重悬即得。
进一步优选的,DSPE-PEG-Mal溶液的浓度为1mg/mL。
进一步优选的,超滤离心管的工艺参数如下:截留分子量10KD,4℃,14000g,20min。
优选的,步骤(3)中,黑磷量子点是通过下述方法制备得到的:先将黑磷晶体利用NMP(N-甲基吡咯烷酮)反复研磨,得到1mg/mL混悬液,接着将混悬液转移至离心管中,冰上超声得到分散液,一次离心取上清,再次离心取沉淀,NMP重悬,即得。
优选的,步骤(3)中,BTZ@BPQDs的制备方法如下:先将BPQDs对应5mM BTZ溶液充分混合,室温孵育24h,透析除去未反应的原料,即得;其中,BPQDs与BTZ溶液的配比为50μg:2mg。
优选的,步骤(3)中,EM囊泡是通过以下方法制备得到的:先将健康人外周血利用PBS稀释成稀释血样,接着将稀释血样滴加至人外周血淋巴分离液中,得到悬液,悬液离心得到红细胞沉淀,再向红细胞沉淀中加入1×PBS,离心清洗,加入0.25×PBS,充分混合,冰上裂解,4℃,12000rpm离心10min,小心吸弃上层,保留红色膜层,加入1×PBS重悬膜层,再次离心处理,直至上清液呈现无色,底部粉红色沉淀即为红细胞膜,超声处理,Avanti MiniExtruder脂质体挤出器(Avanti Polar Lipids)挤压,离心取上清,即得。
优选的,步骤(3)的具体方法如下:先将30μg/mLBTZ@BPQDs溶液与等质量EM囊泡混合,水浴超声处理,脂质体挤出器挤压处理,即得。
优选的,步骤(4)的具体方法为:将0.2mg/mL DSPE-PEG-anti-BCMA与30μg/mLBTZ@BPQDs@EM等体积混合,37℃孵育1h,离心除去未结合的原料,即得。
本发明的有益效果:
本发明构建获得一种靶向仿生光热纳米药物,用于近红外诱导光热协同药物BTZ化疗以提升多发性骨髓瘤疗效。由于B细胞成熟抗原(BCMA)在CAR-T和ADC等新治疗领域的出色表现,且在MM细胞及患者浆细胞表面高表达,是非常理想的靶点。同时,黑磷量子点(BPQDs)优异的光热性能也在肿瘤治疗中发挥着积极作用,且可起到药物载体作用。此外,红细胞膜(EM)固有的仿生特性可以帮助纳米体系摆脱免疫***监视,提高纳米药物的体内循环。因此,选用BCMA抗体修饰在红细胞膜表面,并将核心治疗药BTZ和BPQDs包裹在红细胞膜内,制备成新的纳米药物BTZ@BPQDs@EM@anti-BCMA,增强治疗药物在肿瘤部位的积累,并降低脱靶副作用。
具体分析如下:
1、本发明制备得到一种仿生纳米材料,具体是仿生红细胞膜包裹的黑磷量子点纳米载药颗粒BTZ@BPQDs@EM@anti-BCMA(简称BBE@anti-BCMA),相比于裸露的黑磷量子点,稳定性有较大的提高,为黑磷纳米材料在体内运用提供更大的可能,且药物施用剂量下降,可一定程度上降低药物副作用。
2、运用了主动靶向策略,偶联BCMA抗体,主动靶向B细胞成熟抗原,实现材料的特异性高效递送的同时还可以有效抑制抗原表达丰度。
3、运用新的治疗策略,首次将化疗与光热治疗相结合用于血液肿瘤,既能有效杀伤肿瘤细胞,又能克服动物模型对硼替佐米的耐药性。
4、本发明设计方法简单,反应条件温和,为多发性骨髓瘤的精准治疗提供了一种新的策略。
附图说明
图1为BTZ@BPQDs@EM@anti-BCMA的光热效应图。
图2为BTZ@BPQDs@EM@anti-BCMA光热稳定性示意图。
图3为BTZ@BPQDs@EM@anti-BCMA体外靶向抑制性示意图;其中,a为不同处理后流式检测MM.1S细胞表面BCMA的表达丰度变化,峰向左偏移表示BCMA的丰度受到BCMA抗体的屏蔽(抗体单独存在或修饰在纳米材料上),而阴性对照(IgG和BBE)基本不会改变BCMA的细胞表面丰度;b为IgG处理MM.1S后表面BCMA丰度变化的统计图;c为BCMA单克隆抗体单独处理MM.1S后表面BCMA丰度变化的统计图;d为BBE处理MM.1S后表面BCMA丰度变化的统计图;e为BBE@anti-BCMA处理MM.1S后表面BCMA丰度变化的统计图。
图4为BTZ@BPQDs@EM@anti-BCMA体外杀伤效果示意图。
图5为BTZ@BPQDs@EM@anti-BCMA体外凋亡效应WB图;其中,a为使用不同处理后细胞层面凋亡信号的变化WB条带图,b为a的统计图。
图6为BTZ@BPQDs@EM@anti-BCMA体内光热治疗热成像及温度变化曲线图。
图7为各组小鼠治疗后肿瘤离体示意图及治疗过程的肿瘤生长曲线;其中,a为不同处理组的小鼠经过五次治疗后离体肿瘤直观拍照图;b为不同处理组小鼠肿瘤经过五次治疗后的肿瘤体积统计图;c为不同处理组小鼠在五次治疗过程中的肿瘤体积变化统计图。
图8为各处理组小鼠体内安全性评估,主要器官H&E染色示意图。
图9为各处理组小鼠肿瘤组织免疫荧光染色图和体内凋亡效应的WB结果及其分析图,其中,a为不同处理组小鼠经过5轮治疗后肿瘤组织WB的检测凋亡相关分子变化的条带图;b为不同处理组小鼠经过5轮治疗后肿瘤组织WB的检测凋亡相关分子变化的统计图;c为不同处理组小鼠经过5轮治疗后肿瘤组织WB的检测凋亡相关分子变化的免疫荧光结果。(比例尺为5μm)。
具体实施方式
下面结合附图和实施例对本发明进行进一步的阐述,应该说明的是,下述说明仅是为了解释本发明,并不对其内容进行限定。
实施例1:一种仿生纳米材料(BTZ@BPQDs@EM@anti-BCMA)的构建方法
具体步骤如下:
S1:anti-BCMA-HS的合成:以BCMAmAb(圣克鲁斯生物技术(上海)有限公司)为原料,与Traut’s试剂(上海阿拉丁生化科技股份有限公司)充分混合,反应后除盐得到anti-BCMA-HS;
S2:DSPE-PEG-anti-BCMA的合成:DSPE-PEG-Mal溶液(上海阿拉丁生化科技股份有限公司)中加入anti-BCMA-HS,经搅拌孵育后得到DSPE-PEG-anti-BCMA;
S3:EM囊泡的制备:将1mL健康人外周血离心获得底层红细胞,离心洗涤后低温低渗裂解红细胞,裂解后的红细胞经多次离心得到红细胞膜层,经脂质体挤出器挤出获得粒径相对均一的EM囊泡,剩余红细胞膜通过离心除去;
S4:BPQDs的制备:取黑磷晶体(南京先丰纳米材料科技有限公司)进行研磨,后将研磨溶液超声水浴后离心得到超小黑磷量子点;
S5:BTZ@BPQDs的制备:取BPQDs与BTZ(上海源叶生物科技有限公司)溶液充分混合孵育24h,经水浴超声,透析除去未结合药物后得到BTZ@BPQDs;
S6:BTZ@BPQDs@EM的合成:将EM囊泡溶液离心,加入BTZ@BPQDs溶液充分混合,于脂质体挤出器中机械挤压制备获得BTZ@BPQDs@EM,简称BBE;
S7:BTZ@BPQDs@EM@anti-BCMA的制备:将DSPE-PEG-anti-BCMA于BTZ@BPQDs@EM混合,37℃孵育1h,离心得到BTZ@BPQDs@EM@anti-BCMA,简称BBE@anti-BCMA。
其中,步骤S1中,所述anti-BCMA-HS的合成具体步骤为:
S1.1:27.5μL Traut’s试剂(PBS,0.2mg/mL),20μL EDTA缓冲液(PBS,5×10-3M)与500μLBCMAmAb(PBS,0.2mg/mL)充分混合;
S1.2:S1.1中的混合液在垂直混合仪中反应4h,使用脱盐柱清洗除去多余的Traut’s试剂,得到anti-BCMA-HS;
步骤S2中,所述DSPE-PEG-anti-BCMA的合成具体步骤为:
S2.1:28μL DSPE-PEG-Mal(PBS,1mg/mL)与S1中反应得到的anti-BCMA-HS以1:1摩尔比混合;
S2.2:将S2.1中的混合液在垂直混合仪中反应过夜(室温);
S2.3:多余的DSPE-PEG-Mal通过超滤离心管除去,截留分子量为10KD,4℃,14000g,20min;
S2.4:离心后加入500μLPBS重悬得到0.2mg/mLDSPE-PEG-anti-BCMA;
步骤S3中,所述EM囊泡的制备具体步骤为:
S3.1:将1mL健康人外周血与2mL 1×PBS(含质量浓度0.5%BSA)混合稀释血样,滴加到3mL人外周血淋巴分离液上;
S3.2:将S3.1的悬液以300g离心20min,慢升慢降,获取底部红细胞沉淀;
S3.3:向红细胞沉淀加入2mL 1×PBS,2000rpm离心5min以清洗红细胞,重复3次;
S3.4:向清洗后的红细胞沉淀加入4mL预冷的0.25×PBS,充分混合,冰上裂解1h;
S3.5:裂解后的红细胞混悬液4℃,12000rpm离心10min,小心吸弃上层,保留红色膜层(可保留一部分液体);
S3.6:加入1mL 1×PBS重悬膜层,重复S3.5离心步骤,直至上清液呈现无色,底部粉红色沉淀即为红细胞膜;
S3.7:将S3.6步骤得到的红细胞膜通过探头超声5min,超声功率600W,频率19~25KHz,工作1s,间隔2s;
S3.8:将超声后的溶液过装有200nm膜的脂质体挤出器挤压20次,5000rpm离心5min除去大片段红细胞膜,上清液含有较高浓度的EM囊泡;
步骤S4中,BPQDs的制备具体步骤为:
S4.1:称取30mg黑磷晶体于玛瑙研钵,逐次加入2~3mLNMP,反复研磨,二者混悬液终体积为30mL;
S4.2:研磨后的混悬液转移至50mL离心管,冰上超声10h,超声功率600W,频率19~25KHz,工作2s,间隔4s,每隔1h换一次冰;
S4.3:将S4.2超声获得的分散液以10000rpm离心20min,收集上清;
S4.4:再将S4.3的上清以14000rpm离心20min,收集沉淀,并将其重悬于NMP中,即得到黑磷量子点分散液(BPQDs);
步骤S5中,BTZ@BPQDs的制备具体步骤为:
S5.1:离心获取50μg BPQDs,分别与2mg 1mM~5mM的BTZ(PBS)溶液充分混合;
S5.2:混合液置于垂直混合仪中室温孵育24h,未结合的药物通过透析除去,获得BTZ@BPQDs溶液,并通过280nm左右的BTZ吸收峰确定透析液中游离药物的含量;
步骤S6中,BTZ@BPQDs@EM的合成具体步骤为:
S6.1:将BTZ@BPQDs(30μg/mL)与EM囊泡等质量混合,水浴超声3min(42KHz,300W)
S6.2:超声后的溶液过装有100nm膜的Avanti Mini Extruder脂质体挤出器(Avanti Polar Lipids)挤压20次,制备获得BTZ@BPQDs@EM;
步骤S7中,BTZ@BPQDs@EM@anti-BCMA的制备具体步骤为:
S7.1:将S6.2制备获得的BTZ@BPQDS@EM与S2.4制备获得的DSPE-PEG-anti-BCMA等体积混合,37℃孵育1h;
S7.2:孵育后的产物经2000rpm离心5min除去未结合的抗体。
表1示出了实施例1中BTZ@BPQDs、BBE、BBE@anti-BCMA的分散性及载药能力表征。
表1分散性及载药能力表征
PDI为多分散系数,用于表征制剂中纳米颗粒的分散性,一般0.1以下为单分散;PDI分散性系数由粒径仪检测得到,药物封装和负载率由透析后游离药物在270-280nm处的吸收峰吸光值对应标曲药物浓度经计算获得。
EE(%)为药物封装效率,DLC(%)为药物负载率
试验例1:BTZ@BPQDs@EM@anti-BCMA的光热效应
所述BTZ@BPQDs@EM@anti-BCMA在使用过程中需配合NIR激光照射;所述NIR激光的功率密度为1.0W/cm2,波长为808nm。
取200μL PBS及相同浓度的BPQDs、BTZ@BPQDs@EM、BTZ@BPQDs@EM@anti-BCMA的水溶液置于96孔板中中,使用波长为808nm的激光器作为激发光源,以1.0W·cm-2的功率持续照射上述水溶液5min,记录温度变化数据并绘制温度变化曲线,见图1,BPQDs和BTZ@BPQDs@EM@anti-BCMA温度变化相当,表明修饰后并不会明显影响黑磷的光热转换效率。
试验例2:测定BTZ@BPQDs@EM@anti-BCMA的光热稳定性
取200μL BPQDs溶液及相同浓度的BTZ@BPQDs@EM和BTZ@BPQDs@EM@anti-BCMA的细胞培养液置于96孔板中,使用波长为808nm的激光器作为激发光源,以1.0W cm-2的功率持续照射上述溶液5min,冷却5min,记录循环中温度变化,并重复5次循环,进而比较各材料的光热稳定性。由图2可见,相比BPQDs,BTZ@BPQDs@EM@anti-BCMA表现出更高的光热效应,显示出BTZ@BPQDs@EM@anti-BCMA更高的稳定性。
试验例3:药物BTZ的负载效率
50μg BPQDs与不同浓度BTZ(1~5mM)反应后,3500MWCO透析袋透析8h,收集透析液,以消光光谱数据为基准,取280nm处溶液的吸收值,然后利用吸收值与BTZ浓度的关系式以及相应的溶液体积计算出不同浓度配比的黑磷的载药量,进而计算出载药率。
试验例4:BTZ@BPQDs@EM@anti-BCMA体外特异性靶向抑制能力
收集MM.1S细胞(ATCC,USA)并在冰上的结合缓冲液中与BTZ@BPQDs@EM@anti-BCMA一起孵育2h。用洗涤缓冲液冲洗三次后。用IgG孵育的MM.1S细胞用作对照。悬浮细胞用于流式细胞仪分析,BCMA特异性流式抗体检测处理后MM.1S细胞表面BCMA丰度变化。流式细胞仪分析显示(图3),MM.1S经抗体及BTZ@BPQDs@EM@anti-BCMA处理后BCMA丰度成浓度依赖性下降,但会达到饱和。
试验例5:BTZ@BPQDs@EM@anti-BCMA体外杀伤效果
采用CCK-8法和活死细胞双染法评估BTZ@BPQDs@EM@anti-BCMA体外肿瘤细胞杀伤效果。将MM.1S细胞培养在96孔板(1×104,100μL/孔)中,培养过夜。随后,将这些培养基替换为含有PBS、BPQDs@EM@anti-BCMA、BTZ、BTZ@BPQDs@EM@anti-BCMA、BPQDs@EM@anti-BCMA+NIR(808nm,1.0W·cm-2,5min)和BTZ@BPQDs@EM@anti-BCMA+NIR(808nm,1.0W·cm-2,5min)的新鲜1640培养基,并孵育24h。最后通过CCK-8测定法测量细胞活力。对于活死细胞染色实验,将MM.1S细胞(1×104/孔)接种在96孔板中并孵育过夜。然后,将BPQDs或BTZ@BPQDs@EM@anti-BCMA(浓度:10、20、40μg/mL)加入到相应的孔中并孵育4h,然后用808nm激光照射(1.0W·cm-2,5min)并孵育24h。用PBS洗涤后,根据制造商的方案对细胞进行染色,然后通过倒置显微镜拍摄。由图4可知,与其他组相比,联合光热与药物的肿瘤细胞杀伤效果最高。
试验例6:BTZ@BPQDs@EM@anti-BCMA体外凋亡效应
为了确定体外光热BTZ@BPQDs@EM@anti-BCMA颗粒诱导的细胞凋亡,通过WesternBlotting检测凋亡相关信号分子的表达。将MM.1S细胞(5×105细胞/孔,1mL)在6孔板中培养24h。随后,加入含有PBS、BPQDs@EM@anti-BCMA、BTZ、BTZ@BPQDs@EM@anti-BCMA、BPQDs@EM+NIR(808nm,1.0W cm-2,5min)、BPQDs@EM@anti-BCMA+NIR(808nm,1.0W·cm-2,5min)和BTZ@BPQDs@EM@anti-BCMA+NIR(808nm,1.0W·cm-2,5min)的新鲜培养基以孵育4h,并将NIR组暴露于808nm激光(1.0W cm-2,10min)。进一步培养24h后,用PBS洗涤细胞,并用RIPA裂解液冰上裂解细胞提取总蛋白。在用含5%脱脂牛奶的PBST封闭一小时后,将样本与兔单克隆抗体孵育过夜,并用抗兔IgG染色1h。最后,在多功能成像仪中显影蛋白变化。由图5可见,光热联合药物处理剪切体Caspase-3以及Bax信号明显高于对照组,而磷酸化p65则明显低于对照组,表明光热联合药物处理后,有明显的促细胞凋亡效应。
试验例7:BTZ@BPQDs@EM@anti-BCMA在多发性骨髓瘤治疗中的应用
NCG小鼠(NOD/ShiLtJGpt-Prkdcem26Cd52Il2r gem26Cd22/Gpt(NCG)mice,江苏集萃药康生物科技股份有限公司)皮下注射1×106个MM.1S细胞,当肿瘤体积达到50mm3左右时,小鼠被随机分配为7个治疗组(n=3):PBS(100μL),BPQDs@EM@anti-BCMA(30μg/mL,100μL),BTZ(0.5mg/kg,100μL),BTZ@BPQDs@EM@anti-BCMA(30μg/mL,100μL),BPQDs@EM+NIRlaser(30μg/mL,100μL,808nm,1.0W cm-2,5min),BPQDs@EM@anti-BCMA+NIR laser(30μg/mL,100μL,808nm,1.0W cm-2,5min),BTZ@BPQDs@EM@anti-BCMA(30μg/mL,100μL,808nm,1.0Wcm-2,5min)+NIR laser。纳米颗粒以及药物通过腹腔注射,隔天注射一次,共给药5次。使用公式计算肿瘤体积(mm3):肿瘤体积=(最短直径)2×(最长直径)×0.5,记录小鼠肿瘤体积变化,对纳米药物体内安全性进行评估,探究纳米药物对肿瘤血管的和体内免疫效应的影响。
图6为BTZ@BPQDs@EM@anti-BCMA体内光热治疗热成像及温度变化曲线图。
图7为各组小鼠治疗后肿瘤离体示意图及治疗过程的肿瘤生长曲线;各组肿瘤拍照示意图和肿瘤体积变化示意图,相比于其他组,BTZ@BPQDs@EM@anti-BCMA+NIR laser表现出显著增强的抑瘤效果。
图8为各处理组小鼠体内安全性评估,主要器官H&E染色示意图。
图9为各处理组小鼠肿瘤组织免疫荧光染色图和体内凋亡效应的WB结果及其分析图,相比于其他组,经BTZ,BTZ@BPQDs@EM@anti-BCMA和BTZ@BPQDs@EM@anti-BCMA+NIRlaser处理后,小鼠肿瘤抗凋亡信号被显著抑制,同时,BTZ@BPQDs@EM@anti-BCMA+NIRlaser处理组可以显著缓解由单药重复施用带来的耐药性信号紊乱。
综上,本发明开发了一种B细胞靶向仿生光热载药纳米体系,特异性靶向B细胞成熟抗原高度表达的肿瘤细胞,实现纳米药物的高效递送,结合光热效应,有效抑制多发性骨髓瘤的生长,抑制肿瘤内抗凋亡信号,并激发机体解除耐药性,为多发性骨髓瘤的精准治疗提供了一种新的策略,并为其他血液***恶性肿瘤的治疗提供新思路。
上述虽然结合附图对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
Claims (9)
1.一种仿生纳米材料在制备多发性骨髓瘤治疗药物中的应用,其特征在于,所述仿生纳米材料为BTZ@BPQDs@EM@anti-BCMA,它是通过以下方法构建得到的:
(1)先将BCMAmAb与Traut’s试剂充分混合,反应,除盐,得到anti-BCMA-HS;
(2)接着向DSPE-PEG-Mal溶液中加入anti-BCMA-HS,搅拌孵育,得到DSPE-PEG-anti-BCMA;
(3)然后将黑磷量子点BPQDs与BTZ溶液充分混合孵育,得到BTZ@BPQDs,再将其与利用红细胞制成的EM囊泡混合,得到BTZ@BPQDs@EM,简称BBE;
(4)最后将DSPE-PEG-anti-BCMA与BTZ@BPQDs@EM混合孵育,即得。
2.根据权利要求1所述的应用,其特征在于,所述仿生纳米材料在使用过程中配合NIR激光照射;NIR激光的功率密度为0.75-1.5W/cm2,波长为808nm。
3.根据权利要求1所述的应用,其特征在于,步骤(1)的具体方法如下:先将BCMAmAb与Traut’s试剂分别利用PBS在25℃-30℃配制成0.1-0.5mg/mLBCMAmAb溶液、0.1-0.5mg/mLTraut’s试剂溶液,接着将BCMAmAb溶液、Traut’s试剂溶液、5×10-3MPBS-EDTA缓冲液按照体积比500:27.5:20充分混合,然后转移至垂直混合仪中反应4-8h,使用脱盐柱清洗除去多余的Traut’s试剂,即得。
4.根据权利要求1所述的应用,其特征在于,步骤(2)的具体方法为:先将DSPE-PEG-Mal利用PBS配制成DSPE-PEG-Mal溶液,接着向DSPE-PEG-Mal溶液中加入与DSPE-PEG-Mal等摩尔量的anti-BCMA-HS,充分混合,转移至垂直混合仪中,室温反应12~18h,利用超滤离心管除去多余的DSPE-PEG-Mal,PBS重悬即得。
5.根据权利要求1所述的应用,其特征在于,步骤(3)中,黑磷量子点是通过下述方法制备得到的:先将黑磷晶体利用NMP反复研磨,得到1mg/mL混悬液,接着将混悬液转移至离心管中,冰上超声得到分散液,一次离心取上清,再次离心取沉淀,NMP重悬,即得。
6.根据权利要求1所述的应用,其特征在于,步骤(3)中,BTZ@BPQDs的制备方法如下:先将BPQDs对应5mMBTZ溶液充分混合,室温孵育24h,透析除去未反应的原料,即得;其中,BPQDs与BTZ溶液的配比为50μg:2mg。
7.根据权利要求1所述的应用,其特征在于,步骤(3)中,EM囊泡是通过以下方法制备得到的:先将健康人外周血利用PBS稀释成稀释血样,接着将稀释血样滴加至人外周血淋巴分离液中,得到悬液,悬液离心得到红细胞沉淀,再向红细胞沉淀中加入1×PBS,离心清洗,加入0.25×PBS,充分混合,冰上裂解,4℃,12000rpm离心10min,小心吸弃上层,保留红色膜层,加入1×PBS重悬膜层,再次离心处理,直至上清液呈现无色,底部粉红色沉淀即为红细胞膜,超声处理,挤压,离心取上清,即得。
8.根据权利要求1所述的应用,其特征在于,步骤(3)的具体方法如下:先将30μg/mLBTZ@BPQDs溶液与等质量EM囊泡混合,水浴超声处理,挤压处理,即得。
9.根据权利要求1所述的应用,其特征在于,步骤(4)的具体方法为:将0.2mg/mLDSPE-PEG-anti-BCMA与30μg/mLBTZ@BPQDs@EM等体积混合,37℃孵育1h,离心除去未结合的原料,即得。
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