CN116589595A - 靶向mrsa嵌合抗原受体、药物递送体、car-巨噬细胞及应用 - Google Patents
靶向mrsa嵌合抗原受体、药物递送体、car-巨噬细胞及应用 Download PDFInfo
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- CN116589595A CN116589595A CN202310534129.XA CN202310534129A CN116589595A CN 116589595 A CN116589595 A CN 116589595A CN 202310534129 A CN202310534129 A CN 202310534129A CN 116589595 A CN116589595 A CN 116589595A
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Abstract
本发明属于生物医药技术领域,涉及靶向MRSA嵌合抗原受体、药物递送体、CAR‑巨噬细胞及应用。靶向MRSA嵌合抗原受体如SEQ ID NO.1所示。药物递送体由脂质纳米粒包覆RNA药物形成,所述RNA药物包括表达靶向MRSA嵌合抗原受体的mRNA和靶向CASP11的小干扰RNA;所述脂质纳米粒由可电离脂质、胆固醇、DOPE、PEG‑脂质自组装形成,PEG‑脂质为DMG‑PEG和DSPE‑PEG‑CRV的混合物。本发明通过特异性靶向MRSA嵌合抗原受体修饰巨噬细胞,实现对耐药菌MRSA的精准识别,同时提高巨噬细胞对耐药菌MRSA的吞噬活性和抗原呈递能力,提高对MRSA感染性疾病的治疗效果。
Description
技术领域
本发明属于生物医药技术领域,涉及靶向MRSA(耐甲氧西林金黄色葡萄球菌)嵌合抗原受体、药物递送体、CAR-巨噬细胞及应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
MRSA是临床常见的多重耐药菌,其造成的感染已成为世界三大难治感染性疾病之一。
巨噬细胞因天然的吞噬、消化、抗原提呈作用,是机体抗感染的第一道防线。然而,据发明人研究了解,MRSA能够通过表达蛋白和多糖抵抗由补体和抗体介导的调理作用,从而逃避巨噬细胞的吞噬;而且,MRSA能够诱导caspase-11/caspase-4(CASP11)蛋白在巨噬细胞内表达,CASP11可介导胞内MRSA逃逸线粒体活性氧(mtROS)的杀伤,进而实现MRSA在巨噬细胞内持久存活。因此,MRSA能够保护自身免受巨噬细胞清除。若能有效克服MRSA的免疫逃逸,则有利于巨噬细胞实现耐药菌MRSA的有效根除。
发明内容
为了解决现有技术的不足,本发明的目的是提供靶向MRSA嵌合抗原受体、药物递送体、CAR-巨噬细胞及应用,本发明通过特异性靶向MRSA嵌合抗原受体修饰巨噬细胞,能够实现对耐药菌MRSA的精准识别,同时能够提高巨噬细胞对耐药菌MRSA的吞噬活性和抗原呈递能力,从而提高对MRSA感染性疾病的治疗效果,增强机体抗菌免疫,降低MRSA感染的复发概率,改善患者预后。
为了实现上述目的,本发明的技术方案为:
一方面,一种靶向MRSA嵌合抗原受体,如SEQ ID NO.1所示。
本发明提供的靶向MRSA嵌合抗原受体能够特异性靶向MRSA,从而为实现对耐药菌MRSA的精准识别提供基础。
另一方面,一种编码上述MRSA嵌合抗原受体的核酸分子,所述核酸分子能够转录成表达靶向MRSA嵌合抗原受体的mRNA,所述表达靶向MRSA嵌合抗原受体的mRNA如SEQ IDNO.2所示,所述mRNA为线性或环状。
本发明通过提供所述核酸分子能够转录成表达靶向MRSA嵌合抗原受体的mRNA,从而实现靶向MRSA嵌合抗原受体的表达。
第三方面,一种药物递送体,由脂质纳米粒包覆RNA药物形成,所述RNA药物包括表达靶向MRSA嵌合抗原受体的mRNA(SasA-CAR mRNA)和靶向CASP11的小干扰RNA(siCASP11);
所述脂质纳米粒由可电离脂质、胆固醇、DOPE、PEG-脂质按照摩尔比(5~50):(5~60):(5~80):(0.2~25)自组装形成,PEG-脂质为DMG-PEG和DSPE-PEG-CRV的混合物,DMG-PEG和DSPE-PEG-CRV的摩尔比为(1~10):(1~10);
所述可电离脂质(YHS-12)的化学结构式如下所示:
通过特异性CAR修饰能够增强巨噬细胞对MRSA的吞噬活性,进而克服MRSA的细胞外免疫逃逸。但MRSA已经进化出巨噬细胞胞内免疫逃逸策略,能够避免在巨噬细胞内被消化杀伤,因此仅通过增强巨噬细胞对MRSA的吞噬活性无法彻底清除MRSA。鉴于CASP11蛋白在介导MRSA逃逸巨噬细胞胞内线粒体活性氧(ROS)杀伤中发挥关键作用,我们通过添加针对CASP11蛋白的siRNA(siCASP11)来有效沉默CASP11蛋白,实现基于线粒体活性氧介导的胞内MRSA杀伤。由于SasA-CAR mRNA与siCASP11的分子量、稳定性和分子构象等分子特征截然不同,因此将两者共同包载是本发明实现RNA药物递送的难点。本发明实验表明通过选择YHS-12、胆固醇、DOPE、DMG-PEG和DSPE-PEG-CRV共同形成的脂质纳米粒作为载体,能够大大提高对SasA-CAR mRNA与siCASP11的包封率,使其包封率可达90%以上,从而显著提高了RNA药物的利用效率。
第四方面,一种上述药物递送体的制备方法,通过乙醇预混法将可电离脂质、胆固醇、DOPE、PEG-脂质制成脂质体,然后通过微流控方法将所述脂质体与RNA药物进行复合包载。
第五方面,一种CAR-巨噬细胞,由上述靶向MRSA嵌合抗原受体修饰的巨噬细胞。
第六方面,一种CAR-巨噬细胞的制备方法,采用上述药物递送体转染巨噬细胞。
第七方面,一种上述药物递送体或CAR-巨噬细胞在制备MRSA感染性疾病的药物中的应用。
本发明的有益效果为:
1.本发明提供了靶向MRSA嵌合抗原受体,并利用靶向MRSA嵌合抗原受体修饰巨噬细胞,能够实现对耐药菌MRSA的精准识别,从而提高巨噬细胞对MRSA感染性疾病的治疗效果。
2.本发明通过脂质纳米粒包覆RNA药物形成药物递送体,并利用药物递送体转染巨噬细胞,实现了核酸药物的有效递送。通过选择YHS-12、胆固醇、DOPE、DMG-PEG和DSPE-PEG-CRV共同形成的脂质纳米粒作为载体包覆SasA-CAR mRNA与siCASP11,大大提高RNA药物的包封率,从而提高RNA药物的有效利用。另外,本发明通过乙醇预混法将YHS-12、辅助脂质与功能化脂质自组装制得脂质体,进一步通过微流控能够有效实现脂质纳米粒与RNA药物的复合,从而有效实现RNA药物的有效递送。
3.本发明通过药物递送体在体内外有效构建CAR-巨噬细胞。经验证,该CAR-巨噬细胞对MRSA具有显著的吞噬、胞内消化作用并能有效重建机体抗菌免疫,改善疾病治疗效果。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1是本发明实施例2中可电离脂质YHS-12的合成路线图;
图2是本发明实施例3的CRV-LNP-RNAs的粒径表征图;
图3是本发明实施例3中CRV-LNP-RNAs的透射电镜表征图;
图4是本发明实施例4中CRV-LNP-RNAs、LNP-RNAs包封率的测定结果图;
图5是本发明实施例5中CRV-LNP-RNAs对巨噬细胞活力影响测定结果图;
图6是本发明实施例6中CRV-LNP-RNAs对巨噬细胞转染效率测定结果图;
图7是本发明实施例7中CAR-巨噬细胞特异性吞噬SasA+聚苯乙烯颗粒共聚焦表征结果图,A为SasA+聚苯乙烯颗粒,B为CD133+聚苯乙烯颗粒;
图8是本发明实施例8中CAR-巨噬细胞的MRSA“胞杀”考察结果图;
图9是本发明实施例9中CAR-巨噬细胞体内抗菌结果图;
图10是本发明实施例9中不同制剂处理的脓毒血症免疫抑制-MRSA深部感染模型小鼠的生存期考察结果图。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
鉴于现有巨噬细胞难以有效清除MRSA,本发明提出了靶向MRSA嵌合抗原受体、药物递送体、CAR-巨噬细胞及应用。
本发明的一种典型实施方式,提供了一种靶向MRSA嵌合抗原受体,如SEQ ID NO.1所示。
本发明的另一种实施方式,提供了一种编码上述MRSA嵌合抗原受体的核酸分子,所述核酸分子能够转录成表达靶向MRSA嵌合抗原受体的mRNA,所述表达靶向MRSA嵌合抗原受体的mRNA如SEQ ID NO.2所示,所述mRNA为线性或环状。
本发明的第三种实施方式,提供了一种药物递送体,由脂质纳米粒包覆RNA药物形成,所述RNA药物包括表达靶向MRSA嵌合抗原受体的mRNA(SasA-CAR mRNA)和靶向CASP11的小干扰RNA(siCASP11);
所述脂质纳米粒由可电离脂质、胆固醇、DOPE、PEG-脂质按照摩尔比(5~50):(5~60):(5~80):(0.2~25)自组装形成,PEG-脂质为DMG-PEG和DSPE-PEG-CRV的混合物,DMG-PEG和DSPE-PEG-CRV的摩尔比为(1~10):
(1~10);
所述可电离脂质的化学结构式如下所示:
在一些实施例中,可电离脂质、胆固醇、DOPE、PEG-脂质的摩尔比为(10~25):(15~30):(10~40):(0.5~2.5),优选为15:10:25:2.5。
在一些实施例中,DMG-PEG和DSPE-PEG-CRV的摩尔比为5:(4~6)。
在一些实施例中,SasA-CAR mRNA与siCASP11的质量比为(1~10):(1~10),优选为5:(4~6)。
在一些实施例中,RNA药物与可电离脂质的质量比1:(5~20),优选为1:(9~11)。
本发明的第四种实施方式,提供了一种上述药物递送体的制备方法,通过乙醇预混法将可电离脂质、胆固醇、DOPE、PEG-脂质制成脂质体,然后通过微流控方法将所述脂质体与RNA药物进行复合包载。
在一些实施例中,将可电离脂质、胆固醇、DOPE、PEG-脂质溶于乙醇中,涡旋混匀自组装形成脂质体溶液;将RNA药物溶于酸性缓冲液中形成RNA药物水溶液,采用微流控将脂质体溶液与RNA药物水溶液混合,透析去除乙醇,即得。
在一些实施例中,酸性缓冲液的pH为3~5。
在一些实施例中,微流控将脂质体溶液与RNA药物水溶液混合的过程中,脂质体溶液与RNA药物水溶液体积比为1:2~4。
本发明的第五种实施方式,提供了一种CAR-巨噬细胞,由上述靶向MRSA嵌合抗原受体修饰的巨噬细胞。
本发明的第六种实施方式,提供了一种CAR-巨噬细胞的制备方法,采用上述药物递送体转染巨噬细胞。
具体地,将巨噬细胞与所述药物递送体加入至培养基中进行孵育。
本发明的第七种实施方式,提供了一种上述药物递送体或CAR-巨噬细胞在制备MRSA感染性疾病的药物中的应用。
具体地,所述MRSA感染性疾病包括但不限于MRSA引起的皮肤病、***、骨髓炎、肺炎、心内膜炎、脓毒血症等。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例详细说明本发明的技术方案。
实施例1 SasA-CAR mRNA的构建
SasA-CAR mRNA的构建使用体外转录方法进行,先设计靶向MRSA嵌合抗原受体(SasA-CAR);再构建编码SasA-CAR的基因;然后通过酶切实验以及纯化获得线性化DNA模板,在T7聚合酶作用下完成转录,进一步使用商业化试剂进行加帽,最后通过使用MEGAclear试剂盒对SasA-CAR mRNA进行纯化。SasA-CAR mRNA具体由上海吉荧生物技术有限公司提供。
SasA-CAR的氨基酸序列为:MASPLTRFLS LNLLLLGESI ILGSGEADVL MTQTPLSLPVSLGDQASISC RSSQSIVHSN GHTYLEWYLQ KPGQSPKLLI YKVSYRFSGV PDRFSGSGSG TDFTLKISRVEAEDLGIYYC FQGSHVPLTF GAGTKLELKG GGGSGGGGSG GGGSEVQLVE SGGGLVKPGG SLKLSCTVSGFTFSEYYMYW VRQTPEKRLE WVATISDAGS NINYPDSVKG RFTISRDNAK NNLYLQMNSL KSEDTAIYYCAREGYGPLFA YWGQGTLVTV SAMEQKLISE EDLTTTKPVL RTPSPVHPTG TSQPQRPEDC RPRGSVKGTGLDFACDIYIW APLAGICVAL LLSLIITLIR AKFSRSAETA ANLQDPNQLY NELNLGRREE YDVLEKKRARDPEMGGKQQR RRNPQEGVYN ALQKDKMAEA YSEIGTKGER RRGKGHDGLY QGLSTATKDT YDALHMQTLAPR,见SEQ ID NO.1。
SasA-CAR mRNA的核酸序列为:
AUGGCCAGCCCUCUGACCAGGUUCCUGAGCCUGAACCUGCUGCUGCUGGGCGAGAGCAUCAUCCUGGGCAGCGGCGAGGCCGAUGUUUUGAUGACCCAAACUCCACUCUCCCUGCCUGUCAGUCUUGGAGAUCAAGCCUCCAUCUCUUGUAGAUCAAGUCAGAGCAUUGUACAUAGUAAUGGACACACCUAUUUAGAAUGGUACCUGCAGAAACCAGGCCAGUCUCCAAAGCUCCUCAUCUACAAAGUUUCCUACCGAUUUUCUGGGGUCCCAGACAGGUUCAGUGGCAGUGGAUCAGGGACAGAUUUCACACUCAAGAUCAGCAGAGUGGAGGCUGAGGAUCUGGGAAUUUAUUAUUGCUUUCAAGGUUCACAUGUUCCUCUCACGUUCGGUGCUGGGACCAAACUGGAGCUAAAAUCUGGUGGCGGAGGCUCGGGCGGAGGUGGGUCGGGUGGCGGCGGAUCAAAGUGCAGCUGGUGGAGUCUGGGGGAGGCUUAGUGAAGCCUGGAGGGUCCCUGAAACUCUCCUGUACAGUCUCUGGAUUCACUUUCAGUGAAUAUUACAUGUAUUGGGUUCGCCAGACUCCGGAAAAGAGGCUGGAGUGGGUCGCAACCAUAAGUGAUGCUGGCAGUAACAUAAAUUAUCCAGACAGUGUGAAGGGACGAUUCACCAUUUCCAGAGACAAUGCCAAGAACAACCUUUACCUGCAAAUGAAUAGUCUGAAGUCGGAGGACACAGCCAUCUAUUAUUGUGCAAGAGAGGGGUAUGGUCCCCUGUUUGCUUACUGGGGCCAAGGGACUCUGGUCACUGUCUCUGCAAUGGAGCAGAAGCUGAUCAGCGAGGAGGACCUGACCACCACCAAGCCUGUGCUGAGGACCCCUAGCCCUGUGCACCCUACCGGCACCAGCCAGCCUCAGAGGCCUGAGGACUGCAGGCCUAGGGGCAGCGUGAAGGGCACCGGCCUGGACUUCGCCUGCGACAUCUACAUCUGGGCCCCUCUGGCCGGCAUCUGCGUGGCCCUGCUGCUGAGCCUGAUCAUCACCCUGAUCAGGGCCAAGUUCAGCAGGAGCGCCGAGACCGCCGCCAACCUGCAGGACCCUAACCAGCUGUACAACGAGCUGAACCUGGGCAGGAGGGAGGAGUACGACGUGCUGGAGAAGAAGAGGGCCAGGGACCCUGAGAUGGGCGGCAAGCAGCAGAGGAGGAGGAACCCUCAGGAGGGCGUGUACAACGCCCUGCAGAAGGACAAGAUGGCCGAGGCCUACAGCGAGAUCGGCACCAAGGGCGAGAGGAGGAGGGGCAAGGGCCACGACGGCCUGUACCAGGGCCUGAGCACCGCCACCAAGGACACCUACGACGCCCUGCACAUGCAGACCCUGGCCCCUAGG,见SEQ ID NO.2。
实施例2可电离脂质YHS-12的制备
YHS-12的合成路线如图1所示,首先将N-叔丁氧羰基-1,2-乙二胺(1.8g,11mmol)、1-溴十二烷(6.0g,24mmol)溶解于无水乙腈(40ml)中,随后加入碳酸钾(3.04g,22mmol)通过催化反应得到(2-(双十二烷基氨基)乙基)氨基甲酸叔丁酯(YHS-12-1);将YHS-12-1(2.0g,8mmol)溶于1,4-二氧六溶液(13mL)中,并加入盐酸的1,4-二氧六环溶液(13mL),室温反应条件下得到N1,N1-双十二烷基乙烷-1,2-二胺(YHS-12-2);最后,将延胡酰氯的二氯甲烷溶液缓慢滴加在YHS-12-2的二氯甲烷溶液中,在无催化剂条件下,冰浴反应24h得到目标化合物YHS-12。1H NMR(600MHz,Chloroform-d)δ6.83(s,0H),6.65(s,0H),3.32(q,J=5.8Hz,1H),2.51(t,J=6.0Hz,1H),2.35(t,J=7.6Hz,2H),1.34(t,J=7.6Hz,2H),1.25-1.18(m,5H),1.19(s,13H),0.81(t,J=7.0Hz,3H)。13CNMR(151MHz,Chloroform-d)δ164.11,132.89,53.87,52.35,37.20,31.88,29.62,29.54,29.31,27.48,26.96,22.64,14.04。
实施例3CRV-LNP-RNAs的制备及表征
将YHS-12、DOPE(二油酰磷脂酰乙醇胺)、胆固醇、DMG-PEG(二肉豆蔻酰聚乙二醇,麦克林公司;分子量:2509.2)与DSPE-PEG-CRV(由上海楚肽生物科技有限公司提供)以15:10:25:1.25:1.25的摩尔比溶于乙醇中,制得脂质有机相;将SasA-CAR mRNA和siCASP11(SasA-CAR mRNA与siCASP11质量比为1:1;SasA-CAR mRNA、siCASP11与YHS-12的质量比均为1:10)溶于pH=4,浓度为50mM的柠檬酸缓冲液,并使水相总体积为脂质有机相体积的3倍,通过微流控注射泵(Pump 11Elite型,美国Harvard Apparatus公司)按照3:1的流速比将核酸水相与脂质有机相快速混合,制得CRV/LNP-RNAs,并将CRV/LNP-RNAs在PBS缓冲液中,4℃透析过夜。利用动态光散射检测CRV/LNP-RNAs的纳米尺寸和多分散系数PDI,如图2所示,CRV/LNP-RNAs的粒径为156.9nm,PDI<0.3,表明制得大小均一、分散性良好的脂质纳米颗粒。通过透射电镜对其形貌进行表征,观察到CRV/LNP-RNAs形貌较为规整,为类球状颗粒,如图3所示。
实施例4LNP-RNAs、CRV/LNP-RNAs包封率考察
由于siRNA与mRNA分子特征(分子质量、稳定性、分子构象)截然不同,二者的共同包载递送也成为了一大难点。本实施例利用Quant-iT RiboGreen RNA定量试剂盒对实施例3提供的含五种脂质的脂质纳米粒CRV/LNP-RNAs和含四种脂质的脂质纳米粒LNP-RNAs(不含DSPE-PEG-CRV,YHS-12、DOPE、胆固醇、DMG-PEG摩尔比为15:10:25:2.5,制备方法同实施例3)的RNA包封率进行测定,结果如图4所示,可以看出CRV/LNP-RNAs与LNP-RNAs相比具有更高的RNA包载性能,包封率高达92.24%±0.76%,几乎将siCASP11和SasA-CAR mRNA完全包载。
实施例5 CRV-LNP-RNAs体外细胞毒性考察
为了检测CRV-LNP-RNAs的体外细胞毒性,将小鼠骨髓中提取诱导的原代巨噬细胞(BMDMs)接种于96孔板中并在细胞培养箱中培养过夜,接下来分别将含有不同浓度CRV-LNP-RNAs(100、250、500、1000、1250ng/ml,以SasA-CAR mRNA浓度进行计算)的DMEM培养基与BMDMs孵育,通过CCK-8试剂盒检测细胞活性。结果显示在所有浓度下,BMDMs的活性没有明显变化,如图5所示,表明实施例4提供的CRV/LNP-RNAs细胞毒性较低,具有优良的生物相容性。
实施例6 CRV-LNP-RNAs体外巨噬细胞转染实验
将BMDMs接种于6孔板中并在细胞培养箱中培养过夜,待细胞贴壁后,每孔加入2mL含有PBS、游离RNAs(Fr-RNAs)、CRV/LNP-RNAs的DMEM培养基(SasA-CAR mRNA浓度为0.5μg/mL),孵育6h后,弃去含有纳米制剂的培养基,加入2mL新鲜DMEM完全培养基继续培养18h,即得CAR-巨噬细胞。待细胞培养结束后,消化细胞,经FITC-anti-Myc标记后,通过流式细胞仪检测转染效率。由图6结果可知,本实施例提供的CRV/LNP-RNAs能有效转染原代巨噬细胞。
实施例7 CAR-巨噬细胞体外吞噬特异性测定
将实施例6制备的CAR-巨噬细胞分别与Cy5标记的CD133蛋白偶联的聚苯乙烯颗粒、Cy5标记的SasA蛋白偶联的聚苯乙烯颗粒共孵育,通过共聚焦进行表征。结果如图7所示,CAR-巨噬细胞吞噬了大量的SasA蛋白偶联的聚苯乙烯颗粒,但对于CD133蛋白偶联的聚苯乙烯颗粒仅吞噬了极少量,表明本发明制备的CAR-巨噬细胞能够特异性识别SasA抗原,并通过CAR胞内段信号传导激活巨噬细胞特异性吞噬作用,证明了实施例6制备的CAR-巨噬细胞用于靶向吞噬MRSA的可行性。
实施例8 CAR-巨噬细胞MRSA胞杀效果考察
将BMDMs接种于6孔板中并在细胞培养箱中培养过夜,待细胞贴壁后,每孔加入2mL含有PBS、游离RNAs(Fr-RNAs)、CRV/LNP-RNAs的DMEM培养基(SasA-CAR mRNA浓度为0.5μg/mL),孵育6h后,弃去含有纳米制剂的培养基,加入2mL新鲜DMEM完全培养基继续培养18h,随后用MRSA按照MOI 20:1的比例感染对照组BMDMs和CAR-巨噬细胞2h。感染结束后,加入含庆大霉素的培养基,进一步孵育1h以杀灭剩余的胞外游离细菌。分别于6h、24h用含0.1%Triton X-100的PBS裂解细胞,并通过平板菌落计数法计数MRSA的CFUs。结果显示,在不同时间点多功能CAR-巨噬细胞都显示了比PBS组更低的细菌负荷,如图8所示,表明多功能CAR-巨噬细胞具有强大的胞杀性能,有效克服MRSA胞内消化逃逸。
实施例9体内抗菌研究
脓毒血症是一种复杂难治且病情凶险的疾病,其晚期诱发的免疫抑制阶段进一步导致了患者的高死亡率,而MRSA已成为引发脓毒血症和继发性感染的常见原因。因此本实施例构建脓毒血症免疫抑制-MRSA深部感染模型对本发明制备的CAR-巨噬细胞体内抗菌治疗效果进行研究。
通过腹腔注射环磷酰胺溶液以及尾静脉接种MRSA构建脓毒血症免疫抑制-MRSA深部感染模型,随后将小鼠随机分为两组(每组5只),于MRSA接种后1h分别注射PBS(对照组)、CRV/LNP-RNAs,于48h后通过平板菌落计数法对小鼠外周血中的细菌负荷进行考察,并设置平行实验对不同制剂组小鼠的生存期进行考察。如图9所示,CRV/LNP-RNAs组小鼠外周血细菌负荷显著低于对照组小鼠,说明在体生成的CAR-巨噬细胞能够有效清除MRSA。此外,CRV/LNP-RNAs处理显著延长小鼠生存期,如图10所示。表明本发明制备的CAR-巨噬细胞能够有效清除MRSA,证明本发明制备的多功能CAR-巨噬细胞能够应用于MRSA感染性疾病的治疗。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种靶向MRSA嵌合抗原受体,其特征是,如SEQ ID NO.1所示。
2.一种编码权利要求1所述的MRSA嵌合抗原受体的核酸分子,其特征是,所述核酸分子能够转录成表达靶向MRSA嵌合抗原受体的mRNA,所述表达靶向MRSA嵌合抗原受体的mRNA如SEQ ID NO.2所示,所述mRNA为线性或环状。
3.一种药物递送体,其特征是,由脂质纳米粒包覆RNA药物形成,所述RNA药物包括表达靶向MRSA嵌合抗原受体的mRNA和靶向CASP11的小干扰RNA;
所述脂质纳米粒由可电离脂质、胆固醇、DOPE、PEG-脂质按照摩尔比(5~50):(5~60):(5~80):(0.2~25)自组装形成,PEG-脂质为DMG-PEG和DSPE-PEG-CRV的混合物,DMG-PEG和DSPE-PEG-CRV的摩尔比为(1~10):
(1~10);
所述可电离脂质的化学结构式如下所示:
4.如权利要求3所述的药物递送体,其特征是,可电离脂质、胆固醇、DOPE、PEG-脂质的摩尔比为(10~25):(15~30):(10~40):(0.5~2.5),优选为15:10:25:2.5;
或,DMG-PEG和DSPE-PEG-CRV的摩尔比为5:(4~6);
或,SasA-CAR mRNA与siCASP11的质量比为(1~10):(1~10),优选为5:
(4~6);
或,RNA药物与可电离脂质的质量比1:(5~20),优选为1:(9~11)。
5.一种权利要求3或4所述的药物递送体的制备方法,其特征是,通过乙醇预混法将可电离脂质、胆固醇、DOPE、PEG-脂质制成脂质体,然后通过微流控方法将所述脂质体与RNA药物进行复合包载。
6.如权利要求5所述的药物递送体的制备方法,其特征是,将可电离脂质、胆固醇、DOPE、PEG-脂质溶于乙醇中,涡旋混匀自组装形成脂质体溶液;将RNA药物溶于酸性缓冲液中形成RNA药物水溶液,采用微流控将脂质体溶液与RNA药物水溶液混合,透析去除乙醇,即得;
或,酸性缓冲液的pH为3~5;
或,微流控将脂质体溶液与RNA药物水溶液混合的过程中,脂质体溶液与RNA药物水溶液体积比为1:2~4。
7.一种CAR-巨噬细胞,其特征是,由权利要求1所述的靶向MRSA嵌合抗原受体修饰的巨噬细胞。
8.一种CAR-巨噬细胞的制备方法,其特征是,采用权利要求3或4所述的药物递送体转染巨噬细胞。
9.一种权利要求3或4所述的药物递送体或权利要求7所述的CAR-巨噬细胞在制备MRSA感染性疾病的药物中的应用。
10.如权利要求9所述的应用,其特征是,所述MRSA感染性疾病包括MRSA引起的皮肤病、***、骨髓炎、肺炎、心内膜炎或脓毒血症。
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