CN111888480A - 一种在活细胞表面锚定修饰纳米药物的方法 - Google Patents
一种在活细胞表面锚定修饰纳米药物的方法 Download PDFInfo
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Abstract
本发明公开了一种在活细胞表面锚定修饰纳米药物的方法。通过细胞膜锚定分子的疏水尾链将活性反应基团引入到活细胞表面,在纳米药物表面修饰对应反应基团,活细胞表面修饰的活性反应基团与纳米药物表面修饰的对应反应基团发生生物正交点击反应,从而将纳米药物锚定修饰到细胞表面得到修饰了纳米药物的活细胞。本发明方法简便、快捷、通用,可以应用到各种具有脂质膜结构的细胞包括原代细胞,并且经过这种改造后不会影响细胞自身的功能,为细胞改造提供了一个新的技术平台,具有非常广阔的应用前景。本发明公开的通过上述细胞改造技术得到的细胞药物,相比于单纯的细胞及单纯的纳米药物具有最佳的治疗效果,为多种疾病治疗提供新思路和新药物。
Description
技术领域
本发明属于生物技术领域,具体涉及一种在活细胞表面锚定修饰纳米药物的方法。
背景技术
随着纳米技术的发展,纳米药物在各种疾病治疗中的应用越来越广泛,1964年第一个纳米药物问世以来,其他类型的纳米药物例如聚合物胶束,白蛋白纳米粒等相继问世,迄今为止已有36个纳米药物上市。但是,纳米药物存在一定的局限性,从给药部位到靶部位需要克服层层生理屏障,包括血液、组织、细胞等,最终到达靶部位的药量仅有给药剂量的5%-8%,靶向效率较低,临床疗效不够理想。
为了提高纳米药物的靶向效率,应用内源性细胞作为递送纳米药物的工具得到了广泛研究。一方面内源性细胞可以帮助纳米药物逃避网状内皮系统(ReticuloendothelialSystem,RES)的识别,且提高纳米药物富集于特定组织的能力,从而提高其体内滞留时间及靶向效率;另一方面内源性细胞如T细胞(含嵌合抗原受体T细胞(CAR-T细胞)、T细胞受体基因工程改造的T细胞(TCR-T细胞))、自然杀伤细胞(Natural killer cells,NK)可用于过继性细胞治疗,通过选择不同纳米药物可与内源性细胞发挥协同治疗作用,从而实现最佳的治疗效果。因此,开发更多安全有效的内源性细胞递送系统对于提高纳米药物或过继性细胞治疗的疗效均具有重要意义。
目前,除了利用细胞吞噬天赋将纳米药物荷载到细胞内部的方法外,还可以在细胞表面修饰纳米药物,以构建细胞药物递送系统。常用的细胞表面荷载纳米药物的方式主要有以下几种。(1)化学方式:纳米药物直接与细胞表面的功能基团(如巯基或氨基)进行化学反应。但细胞表面不一定含有充足的游离巯基或氨基,并且这种直接利用细胞表面天然蛋白上的反应基团进行化学反应的方式,可能会影响细胞正常的生理功能。(2)糖基化方式:通过糖基工程化使细胞膜上表达叠氮基团(-N3),继而通过化学反应将纳米药物修饰到细胞表面。但糖基工程化所需时间较长,不适用于所有细胞种类。(3)基因工程化方式:通过基因工程技术使细胞表面表达含有环辛炔的糖蛋白,继而通过化学反应将纳米药物修饰到细胞表面。这种方式需要特定的生物学技术对细胞进行处理,且处理过程相对复杂,耗时而且成本较高。(4)物理方式:通过受体-配体相互作用或静电作用。这种方式容易发生胞吞,并受限于细胞表面过表达的受体,且长期占据细胞表面的受体也可能会干扰到细胞的正常生理功能。因此,研究新型的细胞表面荷载纳米药物方式具有广泛的应用前景和研究价值。
发明内容
本发明的目的是针对现有技术的上述不足,提供一种在细胞表面锚定修饰纳米药物的方法。
本发明的另一目的是提供按照该方法制备的修饰了纳米药物的活细胞。
本发明的又一目的是提供该修饰了纳米药物的活细胞的应用。
一种在细胞表面锚定修饰纳米药物的方法,通过细胞膜锚定分子的疏水尾链将活性反应基团引入到活细胞表面,在纳米药物表面修饰对应反应基团,活细胞表面修饰的细胞膜锚定分子的活性反应基团与纳米药物表面修饰的对应反应基团发生生物正交点击反应,从而将纳米药物锚定修饰到细胞表面得到修饰了纳米药物的活细胞。
其中,R1为常见脂质或烷烃链,如二硬脂酰基磷脂酰乙醇胺(DSPE)、二油酰磷脂酰乙醇胺(DOPE)、1,2-双十六烷基-3-甘油-磷酸乙醇胺(DHPE)、胆固醇、C链长度为6-20的长链烷烃等,优选二硬脂酰基磷脂酰乙醇胺(DSPE)。
n=8-200,优选n=20-100。
本发明提供一种上述细胞膜锚定分子的合成方法,合成路线如下:
(1)将四氮嗪酸(或叠氮酸,双环[6.1.0]壬炔酸,氮杂二苯并环辛炔酸)与N-叔丁氧羰基-L-赖氨酸(Boc-Lys-OH)溶于氯仿(或二氯甲烷,四氢呋喃)中,加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDCI)(或N,N-二环己基碳二亚胺(DCC)),N-羟基丁二酰亚胺(NHS)和三乙胺(TEA)(或4-二甲氨基吡啶(DMAP)),25℃-45℃反应10-20h,水洗,无水硫酸钠(或无水硫酸镁)干燥有机层并浓缩,二氯甲烷/甲醇柱层析,得到四氮嗪化(或叠氮化,双环[6.1.0]壬炔化,氮杂二苯并环辛炔化)衍生物合成反应式:
(2)将与分子量为400-10000的PEG衍生物溶于N,N-二甲基甲酰胺(DMF)(或二甲亚砜(DMSO))中,并依次加入六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBop)(或1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC),N-羟基丁二酰亚胺(NHS))、三乙胺(TEA)(或N,N-二异丙基乙胺(DIPEA)),25℃-45℃反应10-20h,透析24-48h,冻干,得到四氮嗪PEG化(或叠氮PEG化,双环[6.1.0]壬炔PEG化,氮杂二苯并环辛炔PEG化)衍生物合成反应式:
本发明选用的活细胞,优选人、动物等具有脂质膜结构的原代细胞或永生化细胞,包括肿瘤细胞、中性粒细胞、T细胞、间充质干细胞、造血干细胞、自然杀伤细胞、抗原递呈细胞、巨噬细胞等,进一步优选T细胞或中性粒细胞;所述的T细胞选自含嵌合抗原受体T细胞、T细胞受体基因工程改造的T细胞或普通未经修饰的T细胞。
本发明选用的纳米粒,可以为脂质体、纳米囊泡、固体脂质纳米粒、胶束等,优选脂质体。
本发明选用的治疗剂,可以为疏水性药物如阿伐麦布、紫杉醇、槲皮素、BAY 87-2243、TGF-β抑制剂、白皮杉醇等,亲水性药物如阿霉素、柔红霉素、丝裂霉素等,蛋白治疗药物如PD-1单抗、PD-L1单抗等,基因治疗药物如siRNA、mRNA、shRNA、质粒等,优选阿伐麦布、紫杉醇、PD-1单抗。
本发明还公开一类对应反应基团修饰剂,结构通式如下:
其中,R1为常见脂质或烷烃链,如二硬脂酰基磷脂酰乙醇胺(DSPE)、二油酰磷脂酰乙醇胺(DOPE)、1,2-双十六烷基-3-甘油-磷酸乙醇胺(DHPE)、胆固醇、C链长度为6-20的长链烷烃等,优选二硬脂酰基磷脂酰乙醇胺(DSPE)。
本发明提供了一种上述对应反应基团修饰的合成方法,合成路线如下:
(1)将羟基化(或氨基化)双环[6.1.0]壬炔(或四氮嗪,氮杂二苯并环辛炔,叠氮)与对硝基苯基氯甲酸酯溶于二氯甲烷(或氯仿,四氢呋喃)中,加入吡啶,25℃-40℃反应4-10h,反应液浓缩二氯甲烷/甲醇柱层析,得到对硝基苯基化双环[6.1.0]壬炔(或四氮嗪,氮杂二苯并环辛炔,叠氮)。合成反应式:
(2)将对硝基苯基化双环[6.1.0]壬炔(或四氮嗪,氮杂二苯并环辛炔,叠氮)与N-芴甲氧羰基-L-赖氨酸(Fmoc-Lys-OH)溶于氯仿(或二氯甲烷,四氢呋喃),加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)(或N,N-二环己基碳二亚胺(DCC)),N-羟基丁二酰亚胺(NHS)和三乙胺(TEA)(或4-二甲氨基吡啶(DMAP)),25℃-45℃反应10-20h,水洗,无水硫酸钠(或无水硫酸镁)干燥有机层并浓缩,二氯甲烷/甲醇柱层析,得到双环[6.1.0]壬炔化(或四氮嗪化,氮杂二苯并环辛炔化,叠氮化)衍生物合成反应式:
(3)将与氨基化(或羟基化)磷脂(或胆固醇,长链烷烃)衍生物溶于二氯甲烷(或氯仿,四氢呋喃)中,加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)(或N,N-二环己基碳二亚胺(DCC)),N-羟基丁二酰亚胺(NHS)(或1-羟基苯并三唑(HOBT))和三乙胺(TEA)(或N,N-二异丙基乙胺(DIPEA)),25℃-45℃反应3-24h,水洗,无水硫酸钠(或无水硫酸镁)干燥有机层并浓缩,二氯甲烷/甲醇柱层析,得到双环[6.1.0]壬炔化(或四氮嗪化,氮杂二苯并环辛炔化,叠氮化)磷脂(或胆固醇,长链烷烃)衍生物合成反应式:
(4)将溶于氯仿(或二氯甲烷,四氢呋喃)中,加入二乙胺(或哌啶),0℃-45℃反应2-24h,水洗,无水硫酸钠(或无水硫酸镁)干燥有机层并浓缩,二氯甲烷/甲醇柱层析,得到对应反应基团修饰剂Ⅱ。合成反应式:
本发明的纳米药物粒径在1-1000nm之间,优选10-500nm;治疗剂载药量在0.1%-20%之间,优选1%-15%;对应反应基团修饰剂与纳米粒的比例为1:150-1:3,优选1:50-1:5。
本发明所述的纳米药物表面的对应反应基团与细胞膜表面的活性反应基团之间的生物正交点击化学反应,包括酮/羟胺缩合,巯基或氨基与马来酰亚胺的迈克尔加成反应,环张力驱动的叠氮-炔环加成反应(SPAAC),高张力驱动逆电子需求的Dields-Alder环加成反应(SPIEDAC),优选SPAAC及SPIEDAC反应。
作为本发明方法的一种优选,将所述的细胞膜锚定分子与活细胞0-40℃共孵5-120min得到表面修饰细胞膜锚定分子的活细胞;所述的表面修饰对应反应基团的纳米药物与表面修饰细胞膜锚定分子的活细胞0-37℃共孵5-120min得到修饰了纳米药物的活细胞。
本发明所述的细胞膜锚定分子与活细胞共孵过程中,细胞膜锚定分子的浓度范围优选10-200μg/mL;孵育时间优选10-60min;孵育温度优选4-37℃。
本发明所述的纳米药物与修饰后的活细胞的共孵过程中,纳米药物的药物浓度范围优选5-200μg/mL,孵育时间优选10-60min;孵育温度优选4-37℃。
基于本发明公开的活细胞表面锚定修饰的新技术,本发明还公开了一类修饰了纳米药物的活细胞,含有活细胞、细胞膜锚定分子和纳米药物。首先将细胞膜锚定分子与活细胞共孵一段时间,制备活性反应基团修饰的活细胞。随后将纳米药物与修饰后的活细胞共孵,通过纳米药物表面的对应反应基团与细胞膜表面的活性反应基团发生生物正交点击反应,使纳米药物能稳定锚定在活细胞表面形成细胞药物(图1)。该细胞药物不仅可以利用活细胞的生理/病理特性延长纳米药物的体内循环时间,同时提高纳米药物对特定部位的靶向效率,还能使纳米药物与活细胞具有协同治疗作用。最终根据选用的活细胞类型和治疗剂,将细胞药物用于多种疾病的治疗。
作为本发明的优选,本发明请求保护按照所述的方法制备得到的修饰了纳米药物的T细胞;进一步优选按照本发明所述的方法制备得到的修饰了纳米药物的嵌合抗原受体T细胞、T细胞受体基因工程改造的T细胞。
本发明所述的细胞药物,其活细胞存活率>80%,载药量为0.1-20μg/106个细胞,且保持活细胞的正常生理功能,包括细胞增殖能力、细胞趋化能力、细胞活化能力等。
本发明所述的修饰了纳米药物的活细胞在制备治疗肿瘤或炎性相关疾病的药物中的应用。
所述的肿瘤选自黑色素瘤、脑胶质瘤、乳腺癌或卵巢癌;所述的炎性相关疾病选自脑卒中或关节炎。
本发明所述的细胞膜锚定分子在制备活细胞药物中的应用,所述的活细胞药物为表面修饰了纳米药物的活细胞。
本发明所述的对应反应基团修饰剂在制备活细胞药物中的应用,所述的活细胞药物为表面修饰了纳米药物的活细胞;优选表面修饰了纳米药物的T细胞;进一步优选表面修饰了纳米药物的嵌合抗原受体T细胞、T细胞受体基因工程改造的T细胞。
有益效果:
本发明开发了一种新型的细胞表面荷载纳米药物的方法。该方法模拟GPI锚的磷脂疏水尾链将化学反应基团引入到细胞膜的表面,随后将表面修饰对应反应基团的纳米药物经化学反应修饰到细胞表面,得到相应的细胞药物用于多种疾病的治疗。这种新型荷载方式是通过疏水作用将反应基团引入细胞表面,不干扰细胞的基因、代谢及天然存在的蛋白活性,对细胞影响相对较小,适用于具有脂质膜结构的任何细胞。综上所述,我们研究的新型细胞荷载技术具有安全、稳定、高效、广谱的特点,与其他方式相比,具有独特的优势;并且可根据荷载的纳米药物以及选用的细胞种类用于多种疾病的治疗。
本发明公开的这种细胞表面锚定技术简便、快捷、通用,可以应用到各种具有脂质膜结构的细胞包括原代细胞,如人源T细胞(实施例12,13)、人源CAR-T细胞(实施例14,15)、鼠源T细胞(实施例16)、鼠源TCR-T细胞(实施例17)、人源中性粒细胞(实施例18,19)、鼠源中性粒细胞(实施例20)、间充质干细胞(实施例21),肿瘤细胞,如肺癌细胞A549(实施例22),并且经过这种改造后不会影响细胞自身的功能(实施例25-27),为细胞改造提供了一个新的技术平台,具有非常广阔的应用前景。
本发明公开的通过上述细胞改造技术得到的细胞药物,相比于单纯的细胞及单纯的纳米药物具有最佳的治疗效果(实施例28-30),为多种疾病治疗提供新思路和新药物。
附图说明
图1是本发明细胞药物的制备流程图。
图2是本发明细胞膜锚定分子与对应反应基团修饰剂反应后的紫外光谱。
图3是本发明纳米药物的透射电镜图。
图4是本发明细胞药物的激光共聚焦图。
图5是本发明细胞药物的存活率检测。
图6是本发明细胞药物的增殖能力表征。
图7是本发明细胞药物的趋化能力表征。
图8是本发明细胞药物治疗原位黑色素瘤的抑瘤曲线及肿瘤组织图。
图9是本发明细胞药物治疗原位乳腺癌的抑瘤曲线。
图10是本发明细胞药物治疗原位脑胶质瘤的效果图。
具体实施方式
实施例1
细胞膜锚定分子二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-赖氨酸-四氮嗪(DSPE-PEG5k-Tre)的制备与表征
将4-(6-(嘧啶-2-基)-1,2,4,5-四嗪-3-基)苯甲酸(四氮嗪酸(Tre-COOH),80mg,0.29mmol)以及N-叔丁氧羰基赖氨酸盐酸盐(Boc-Lys-OH·HCl,126.42mg,0.26mmol)溶于三氯甲烷(30mL)中,加入N-羟基琥珀酰亚胺(NHS,35.68mg,0.31mmol)及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,59.43mg,0.31mmol),DIPEA(136.24μL,100.82mg,0.78mmol),室温反应过夜。水洗,无水硫酸钠干燥,浓缩有机层,二氯甲烷/甲醇柱层析,得到紫红色粉末状固体(N2-(叔丁氧羰基)-N6-(4-(6-(嘧啶-2-基)-1,2,4,5-四嗪-3-基)苯甲酰基)赖氨酸,90mg,61.9%)。将二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-氨基(50mg,0.01mmol)溶于DMF(5mL)中,依次加入六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBop,11.45mg,0.022mmol)、三乙胺(4.09μL,3.03mg,0.03mmol)及(N2-(叔丁氧羰基)-N6-(4-(6-(嘧啶-2-基)-1,2,4,5-四嗪-3-基)苯甲酰基)赖氨酸,10.52mg,0.02mmol),搅拌过夜。反应液置于透析袋中,二甲基亚砜作为透析介质透析48h,去离子水继续透析48h,冻干,得到紫红色棉絮状产物(二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-N2-(叔丁氧羰基)-N6-(4-(6-(嘧啶-2-基)-1,2,4,5-四嗪-3-基)苯甲酰基)赖氨酸,31.7mg,60.8%)。二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-N2-(叔丁氧羰基)-N6-(4-(6-(嘧啶-2-基)-1,2,4,5-四嗪-3-基)苯甲酰基)赖氨酸(31.7mg)溶于去离子水(5mL)中,加入三氟醋酸(TFA,50μL),搅拌过夜。之后将反应液转移至透析袋中,去离子水作为透析介质透析48h,冻干,得到紫红色棉絮状产物(二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-赖氨酸-四氮嗪,20mg)。
1H-NMR(300MHz,d6-DMSO):δ9.21(2H,d),8.68(1H,d),8.19(2H,d),7.51(2H,d),5.11-5.19(4H,m),4.57-4.52(7H,m),4.10-3.99(9H,m),3.77-3.68(8H,m),3.53-3.46(475H,m),2.32-2.19(5H,m),1.56-1.40(7H,m),1.25-1.20(45H,m),0.85(6H,t)。
实施例2
细胞膜锚定分子二油酰基磷脂酰乙醇胺-聚乙二醇2000-赖氨酸-巯基(DOPE-PEG2k-SH)的制备与表征
将巯基丙酸(SH-COOH,30mg,0.29mmol)及N-叔丁氧羰基赖氨酸盐酸盐(Boc-Lys-OH·HCl,126.42mg,0.26mmol)溶于三氯甲烷(30mL)中,加入N-羟基琥珀酰亚胺(NHS,35.68mg,0.31mmol)及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,59.43mg,0.31mmol),DIPEA(136.24μL,100.82mg,0.78mmol),室温反应过夜。水洗,无水硫酸钠干燥,浓缩有机层,二氯甲烷/甲醇柱层析,得到淡黄色固体(N2-(叔丁氧羰基)-N6-(3-巯基丙酰基)赖氨酸,82mg,85.4%)。将二油酰基磷脂酰乙醇胺-聚乙二醇2000-氨基(20mg)溶于DMF(5mL)中,依次加入六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBop,11.45mg,0.022mmol)、三乙胺(4.09μL,3.03mg,0.03mmol)及(N2-(叔丁氧羰基)-N6-(3-巯基丙酰基)赖氨酸,6.68mg,0.02mmol),搅拌过夜。反应液置于透析袋中,二甲基亚砜作为透析介质透析48h,去离子水继续透析48h,冻干,得到淡黄色棉絮状产物(二油酰基磷脂酰乙醇胺-聚乙二醇2000-N2-(叔丁氧羰基)-N6-(3-巯基丙酰基)赖氨酸,21.7mg,54.2%)。二油酰基磷脂酰乙醇胺-聚乙二醇2000-N2-(叔丁氧羰基)-N6-(3-巯基丙酰基)赖氨酸(21.7mg)溶于去离子水(5mL)中,加入三氟醋酸(TFA,50μL),搅拌过夜。之后将反应液转移至透析袋中,去离子水作为透析介质透析48h,冻干,得到淡黄色棉絮状产物(二油酰基磷脂酰乙醇胺-聚乙二醇2000-赖氨酸-巯基,10mg)。
1H-NMR(300MHz,d6-DMSO):δ5.26(4H,m),5.11-5.19(4H,m),4.57-4.52(9H,m),4.10-3.99(9H,m),3.62-3.56(8H,m),3.53-3.46(184H,m),2.52-2.29(7H,m),1.59-1.43(7H,m),1.25-1.20(45H,m),0.85(6H,t)。
实施例3
细胞膜锚定分子十八醇-谷氨酸-聚乙二醇1000-赖氨酸-叠氮(SA2-Glu-PEG1k-N3)的制备与表征
将叠氮丙酸(N3-COOH,33mg,0.29mmol)及N-叔丁氧羰基赖氨酸盐酸盐(Boc-Lys-OH·HCl,126.42mg,0.26mmol)溶于三氯甲烷(30mL)中,加入N-羟基琥珀酰亚胺(NHS,35.68mg,0.31mmol)及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,59.43mg,0.31mmol),DIPEA(136.24μL,100.82mg,0.78mmol),室温反应过夜。水洗,无水硫酸钠干燥,浓缩有机层,二氯甲烷/甲醇柱层析,得到白色固体(N2-(叔丁氧羰基)-N6-(3-叠氮丙酰基)赖氨酸,90mg,90.4%)。将十八醇-谷氨酸-聚乙二醇1000-氨基(20mg)溶于DMF(5mL)中,依次加入六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBop,11.45mg,0.022mmol)、三乙胺(4.09μL,3.03mg,0.03mmol)及(N2-(叔丁氧羰基)-N6-(3-叠氮丙酰基)赖氨酸,6.86mg,0.02mmol),搅拌过夜。反应液置于透析袋中,二甲基亚砜作为透析介质透析48h,去离子水继续透析48h,冻干,得到白色棉絮状产物(十八醇-谷氨酸-聚乙二醇1000-N2-(叔丁氧羰基)-N6-(3-叠氮丙酰基)赖氨酸,21.7mg,40.5%)。十八醇-谷氨酸-聚乙二醇1000-N2-(叔丁氧羰基)-N6-(3-叠氮丙酰基)赖氨酸(21.7mg)溶于去离子水(5mL)中,加入三氟醋酸(TFA,50μL),搅拌过夜。之后将反应液转移至透析袋中,去离子水作为透析介质透析48h,冻干,得到淡黄色棉絮状产物(十八醇-谷氨酸-聚乙二醇1000-赖氨酸-叠氮,10mg)。
1H-NMR(300MHz,d6-DMSO):δ5.37(4H,m),5.16-5.09(4H,m),4.38-4.22(9H,m),4.10-3.99(9H,m),3.62-3.56(8H,m),3.53-3.46(83H,m),2.62-2.33(7H,m),1.59-1.43(7H,m),1.27-1.22(69H,m),0.85(6H,t)。
实施例4
对应反应基团修饰剂二硬脂酰基磷脂酰乙醇胺-赖氨酸-环壬炔(DSPE-BCN)的制备与表征
将双环[6.1.0]壬-4-炔-9-基甲醇(350mg,2.33mmol)溶于二氯甲烷(30mL)中,加入对硝基苯基氯甲酸酯(1.17g,5.82mmol)及吡啶(Py,0.64g,8.15mmol),室温反应6h。反应液浓缩后经柱层析得白色粉末状固体(双环[6.1.0]壬-4-炔-9-基甲基-(4-硝基苯基)氨基甲酸酯,520mg,71.1%)。将双环[6.1.0]壬-4-炔-9-基甲基-(4-硝基苯基)氨基甲酸酯(360mg,1.14mmol)溶于5mL DMF中,依次加入N-芴甲氧羰基-L-赖氨酸(612mg,1.26mmol)、DIPEA(0.65mL,3.77mmol),反应4h,反应液用柠檬酸钠水溶液及饱和食盐水洗涤,无水硫酸钠干燥,浓缩后经柱层析纯化得白色油状固体(N2-(((9H-芴-9-基)甲氧基)羰基)-N6-((双环[6.1.0]壬-4-炔-9-基甲氧基)羰基)赖氨酸,320mg,51.6%)。N2-(((9H-芴-9-基)甲氧基)羰基)-N6-((双环[6.1.0]壬-4-炔-9-基甲氧基)羰基)赖氨酸(100mg,0.18mmol)、N-羟基琥珀酰亚胺(NHS,26mg,0.12mmol)及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,45mg,0.12mmol),二硬脂酰基磷脂酰乙醇胺(DSPE,137mg,0.202mmol)溶于三氯甲烷(20mL),加入DIPEA(106μL,0.30mmol),室温反应过夜。反应液用柠檬酸水溶液(2×80mL)及饱和食盐水(2×80mL)洗涤,收集有机相,无水硫酸钠干燥,减压蒸馏浓缩后经柱层析纯化得淡粉色粉末状固体(1-(((2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)-6-(((二环[6.1.0]壬-4-炔-9-基甲氧基)羰基)氨基)己酰氨基)乙氧基)(羟基)磷酰基)氧基)乙烷-1,2-二基二硬脂酸酯,200mg,88.5%)。在50mL茄型瓶中加入10mL的二氯甲烷,随后加入1-(((2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)-6-(((二环[6.1.0]壬-4-炔-9-基甲氧基)羰基)氨基)己酰氨基)乙氧基)(羟基)磷酰基)氧基)乙烷-1,2-二基二硬脂酸酯(100mg),充分溶解后,加入二乙胺,反应过夜。反应液经充分浓缩后柱层析纯化,最终得白色粉末状固体(二硬脂酰基磷脂酰乙醇胺-赖氨酸-环壬炔,50mg,61.3%)。
MS,ESI-,m/z:calcd for C58H106N3O11P(M-H)-1050.8found 1050.8,(M+H2O-H)-1068.8found 1068.8。1H-NMR(300MHz,CDCl3):δ5.42(1H,m),5.11(1H,m),4.40-4.26(1H,m),4.09-4.03(1H,m),3.90-3.77(6H,m),3.67-3.54(2H,m),3.07(2H,m),2.32-2.09(8H,m),1.78(4H,m),1.50-1.28(8H,m),1.28-1.17(58H,m),0.80(6H,t),0.61-0.55(3H,m)。
实施例5
对应反应基团修饰剂十四醇-谷氨酸-赖氨酸-马来酰亚胺(TA2-Glu-Lys-Mal)的制备与表征
将N-羟乙基马来酰亚胺(Mal-OH,328mg,2.33mmol)溶于二氯甲烷(30mL)中,加入对硝基苯基氯甲酸酯(1.17g,5.82mmol)及吡啶(Py,0.64g,8.15mmol),室温反应6h。反应液浓缩后经柱层析得固体(2-马来酰亚胺-(4-硝基苯基)氨基甲酸酯,520mg,73.2%)。将2-马来酰亚胺-(4-硝基苯基)氨基甲酸酯(347mg,1.14mmol)溶于5mL DMF中,依次加入N-芴甲氧羰基-L-赖氨酸(612mg,1.26mmol)、DIPEA(0.65mL,3.77mmol),反应4h,反应液用柠檬酸钠水溶液及饱和食盐水洗涤,无水硫酸钠干燥,浓缩后经柱层析纯化得白色油状固体(N2-(((9H-芴-9-基)甲氧基)羰基)-N6-((2-马来酰亚胺)氨基甲酸酯基)赖氨酸,320mg,68%)。N2-(((9H-芴-9-基)甲氧基)羰基)-N6-((2-马来酰亚胺)氨基甲酸酯基)赖氨酸(74.34mg,0.18mmol)、N-羟基琥珀酰亚胺(NHS,26mg,0.12mmol)及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,45mg,0.12mmol),十四醇-谷氨酸(TA2-Glu,109mg,0.202mmol)溶于三氯甲烷(20mL),加入DIPEA(106μL,0.30mmol),室温反应过夜。反应液用柠檬酸水溶液(2×80mL)及饱和食盐水(2×80mL)洗涤,收集有机相,无水硫酸钠干燥,减压蒸馏浓缩后经柱层析纯化得固体(十四醇-谷氨酸-N2-(((9H-芴-9-基)甲氧基)羰基)-N6-((2-马来酰亚胺)氨基甲酸酯基)赖氨酸,150mg,89%)。在50mL茄型瓶中加入10mL的二氯甲烷,随后加入十四醇-谷氨酸-N2-(((9H-芴-9-基)甲氧基)羰基)-N6-((2-马来酰亚胺)氨基甲酸酯基)赖氨酸(93.4mg,0.1mmol),充分溶解后,加入二乙胺,反应过夜。反应液经充分浓缩后柱层析纯化,最终得白色固体十四醇-谷氨酸-赖氨酸-马来酰亚胺,57mg,68%)。
MS,ESI-,m/z:calcd for C42H82N4O6S(M+H)+835.6115found 835.6024。1H-NMR(300MHz,CDCl3):δ7.86(2H,s),4.55(1H,m),4.20-4.06(4H,m),3.46(2H,t),3.25(1H,m),3.04(2H,m),2.82-2.39(6H,q),1.80-1.75(2H,m),1.62-1.17(52H,m),0.88(6H,t)。
实施例6
对应反应基团修饰剂胆固醇-赖氨酸-环辛炔(Chol-Lys-ADIBO)的合成及表征
将N-((3-羟基)-5,6-二氢二苯并[b,f]氮杂环辛炔(羟基化氮杂二苯并环辛炔,643mg,2.33mmol)溶于二氯甲烷(30mL)中,加入对硝基苯基氯甲酸酯(1.17g,5.82mmol)及吡啶(Py,0.64g,8.15mmol),室温反应6h。反应液浓缩后经柱层析得白色固体(1-(N-((3-羟基)-5,6-二氢二苯并[b,f]氮杂环辛炔)-(4-硝基苯基)氨基甲酸酯,830mg,80.7%)。将1-(N-((3-氨基)-5,6-二氢二苯并[b,f]氮杂环辛炔)(4-硝基苯基)氨基甲酸酯(500mg,1.14mmol)溶于5mL DMF中,依次加入N-芴甲氧羰基-L-赖氨酸(612mg,1.26mmol)、DIPEA(0.65mL,3.77mmol),反应4h,反应液用柠檬酸钠水溶液及饱和食盐水洗涤,无水硫酸钠干燥,浓缩后经柱层析纯化得白色油状固体(N2-(((9H-芴-9-基)甲氧基)羰基)-N6-(N-((3-羟基)-5,6-二氢二苯并[b,f]氮杂环辛炔)氨基甲酸酯)赖氨酸,520mg,68%)。N2-(((9H-芴-9-基)甲氧基)羰基)-N6-(N-((3-羟基)-5,6-二氢二苯并[b,f]氮杂环辛炔)氨基甲酸酯)赖氨酸(120mg,0.18mmol)、N-羟基琥珀酰亚胺(NHS,26mg,0.12mmol)及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,45mg,0.12mmol),胆固醇(Chol,78mg,0.202mmol)溶于三氯甲烷(20mL),加入DIPEA(106μL,0.30mmol),室温反应过夜。反应液用柠檬酸水溶液(2×80mL)及饱和食盐水(2×80mL)洗涤,收集有机相,无水硫酸钠干燥,减压蒸馏浓缩后经柱层析纯化得固体(胆固醇-N2-(((9H-芴-9-基)甲氧基)羰基)-N6-(N-((3-羟基)-5,6-二氢二苯并[b,f]氮杂环辛炔)氨基甲酸酯)赖氨酸,150mg,80.6%)。在50mL茄型瓶中加入10mL的二氯甲烷,随后加入胆固醇-N2-(((9H-芴-9-基)甲氧基)羰基)-N6-(N-((3-羟基)-5,6-二氢二苯并[b,f]氮杂环辛炔)氨基甲酸酯)赖氨酸(103mg,0.1mmol),充分溶解后,加入二乙胺,反应过夜。反应液经充分浓缩后柱层析纯化,最终得白色固体胆固醇-赖氨酸-环辛炔,56mg,68.5%)。
MS,ESI-,m/z:calcd for C52H73N3O4(M+H)+804.5634found 804.5665。1H-NMR(300MHz,CDCl3):δ7.63(1H,d),7.32(5H,m),7.21(2H,m),5.37(1H,m),5.18(2H,d),4.63(1H,m),,3.41(1H,m),3.22(2H,t),3.10(2H,t),2.66(2H,m),2.31(2H,m),1.99(4H,m),1.84(5H,m),1.53(8H,m),1.31(8H,m),1.12(8H,m),1.02(6H,m),0.92(4H,m),0.86(6H,m),0.68(3H,m)。
实施例7
细胞膜锚定分子与对应反应基团修饰剂之间的生物正交点击反应
以DSPE-PEG5k-Tre与DSPE-BCN为例,四氮嗪基团(Tre)在540nm左右有明显的特征吸收峰,当它与二环[6.1.0]壬炔(BCN)发生SPIEDAC反应后在540nm的紫外吸收峰会消失。将细胞膜锚定分子(DSPE-PEG5k-Tre)溶于氯仿中,随后加入对应反应基团修饰剂(DSPE-BCN)的氯仿溶液,室温反应,反应液利用紫外分光光度计进行波长扫描,同时对DSPE-PEG5k-Tre的氯仿溶液进行波长扫描,绘制吸收曲线。结果见图2。由图2可知DSPE-PEG5k-Tre与DSPE-BCN的反应液在540nm左右的四氮嗪的特征吸收峰消失,表明两者之间的生物正交点击反应基本完全,因此该细胞膜锚定分子与对应反应基团修饰剂之间可发生温和高效的点击化学反应。
实施例8
修饰对应反应基团的脂质体纳米药物(BCN-Ava-Lip)的制备与表征
取市售大豆磷脂(SPC)100mg,胆固醇15mg,阿伐麦布(Ava)3mg,加入对应反应基团修饰剂(DSPE-BCN)25mg,溶于氯仿及甲醇中。旋转蒸发5min除去有机溶剂,真空干燥过夜。37℃水合30min。探头超声10-30min,依次过0.80,0.45,0.22μm滤膜,得到修饰DSPE-BCN的脂质体(BCN-Ava-Lip)。经测定,修饰对应反应基团的纳米药物(BCN-Ava-Lip)的粒径为91.5±1.4nm,载药量为2.3%,包封率为89.1%。
实施例9
修饰对应反应基团的脂质体纳米药物(Mal-siRNA-Lip)的制备与表征
取SPC 15mg,对应反应基团修饰剂(TA2-Glu-Lys-Mal)8mg,阳离子脂质材料15mg,胆固醇9mg,溶于氯仿及甲醇中。旋转蒸发除去有机溶剂,真空干燥过夜。37℃水合30min。探头超声10-30min,依次过0.80,0.45,0.22μm滤膜,得到修饰TA2-Glu-Lys-Mal的空白脂质体(Mal-Lip)。取Mal-Lip(9.4mg/mL)14μL加入186μL超纯水稀释,同时取siRNA(0.5mg/mL)10μL加入190μL超纯水稀释,将两者涡旋混匀(此时N/P=5),室温静置孵育30min,得到修饰TA2-Glu-Lys-Mal并包载有siRNA的脂质体(Mal-siRNA-Lip)。经测定,修饰对应反应基团的纳米药物(Mal-siRNA-Lip)的粒径为117.3±2.8nm,包封率为100%。
实施例10
修饰对应反应基团的固体脂质纳米粒药物(ADIBO-PTX-NPs)的制备与表征
取泊洛沙姆3mg,超纯水溶解后加热至75℃,作为水相;精密称取紫杉醇(PTX)3mg,单硬脂酸甘油酯30mg,对应反应基团修饰剂(Chol-Lys-ADIBO)15mg,加入少量乙醇,75℃搅拌熔融,作为油相,待两相完全溶解且温度相同时,将水相倒入油相中快速搅拌以充分混合,混合液挥至无醇味,超声5min,室温冷却后得到修饰Chol-Lys-ADIBO的固体脂质纳米粒(ADIBO-PTX-NPs)。经测定,修饰对应反应基团的纳米药物(ADIBO-PTX-NPs)的粒径为165.3±1.1nm,载药量为5.6%,包封率为90%。
实施例11
纳米药物的电镜表征
以BCN-Ava-Lip为例,将纳米药物溶液稀释至一定浓度,滴加至铺有碳膜的铜网上,室温静置,用滤纸吸取多余的溶液,以0.1%磷钨酸钠溶液负染,洗去染液,室温干燥后应用HT-7700透射电子显微镜观察拍照(电压100kV)。透射电镜图像如图3。结果显示纳米药物BCN-Ava-Lip形状近球形,粒径均一。
实施例12
人源T细胞药物(BCN-Ava-Lip/hT细胞)的制备
将人外周血来源的T细胞(hT细胞)悬液的密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(DSPE-PEG5k-Tre),4℃共孵30min,离心(1500rmp,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的hT细胞。将纳米药物BCN-Ava-Lip调成等渗,稀释为阿伐麦布浓度为150μg/mL的溶液,将其与上述表面带有活性反应基团的hT细胞在25℃共孵20min,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的人源T细胞,即BCN-Ava-Lip/hT细胞药物。
实施例13
人源T细胞药物(ADIBO-PTX-NPs/hT细胞)的制备
将人外周血来源的T细胞(hT细胞)悬液的密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(SA2-Glu-PEG1k-N3),4℃共孵20min,离心(1500rmp,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的hT细胞。将纳米药物(ADIBO-PTX-NPs)调成等渗,稀释为紫杉醇浓度为100μg/mL的溶液,将其与上述表面带有反应基团的hT细胞在37℃共孵45min,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的人源T细胞,即ADIBO-PTX-NPs/hT细胞药物。
实施例14
CAR-T细胞药物(BCN-Ava-Lip/CAR-T细胞)的制备
悬浮六孔板每孔加入1mL的10μg/mL纤连蛋白,4℃包被过夜,PBS洗涤2次,去除未结合蛋白。每孔加入2×105个人外周血来源的T细胞(hT细胞),并加入1mL含8μg/mL聚丙烯和10ng/mL IL-2的ImmunoCult TM-XF T细胞培养基,随后加入107IU慢病毒包装的可以编码huGD2.CD28.4-1BB.z-CAR-GFP的质粒,1500g平板离心60min,每8h离心一次,共离心3次。此后将转染培养基替换为2mL新鲜的T细胞培养基,继续培养扩增。当CAR-T细胞的CAR蛋白表达阳性率大于30%时继续培养扩增并用于后续研究。将上述制备好的CAR-T细胞悬液的密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(DSPE-PEG5k-Tre),4℃共孵30min,离心(1500rmp,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的CAR-T细胞。将纳米药物BCN-Ava-Lip调成等渗,稀释为阿伐麦布浓度为150μg/mL的溶液,将其与上述表面带有活性反应基团的CAR-T细胞在25℃共孵20min,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的CAR-T细胞,即BCN-Ava-Lip/CAR-T细胞药物。
实施例15
CAR-T细胞药物(ADIBO-PTX-NPs/CAR-T细胞)的制备
按照实施例14中的方法制备CAR-T细胞,并将制备得到的CAR-T细胞悬液的密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(SA2-Glu-PEG1k-N3),4℃共孵20min,离心(1500rpm,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的hT细胞。将纳米药物(ADIBO-PTX-NPs)调成等渗,稀释为紫杉醇浓度为100μg/mL的溶液,将其与上述表面带有反应基团的CAR-T细胞在37℃共孵45min,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的CAR-T细胞,即ADIBO-PTX-NPs/CAR-T细胞药物。
实施例16
鼠源T细胞药物(BCN-Ava-Lip/mT细胞)的制备
将小鼠脾脏来源的T细胞(mT细胞)悬液的密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(DSPE-PEG5k-Tre),4℃共孵30min,离心(1500rmp,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的mT细胞。将纳米药物(BCN-Ava-Lip)调成等渗,稀释为阿伐麦布浓度为150μg/mL的溶液,将其与上述表面带有反应基团的mT细胞在25℃共孵20min,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的鼠源T细胞,即BCN-Ava-Lip/mT细胞药物。
实施例17
TCR-T细胞药物(BCN-Ava-Lip/TCR-T细胞)的制备
将Pmel-1或者OT-1小鼠脾脏来源的T细胞(TCR-T细胞)悬液的密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(DSPE-PEG5k-Tre),4℃共孵30min,离心(1500rmp,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的TCR-T细胞。将纳米药物(BCN-Ava-Lip)调成等渗,稀释为阿伐麦布浓度为150μg/mL的溶液,将其与上述表面带有反应基团的TCR-T细胞在25℃共孵20min,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的TCR-T细胞,即BCN-Ava-Lip/TCR-T细胞药物。
实施例18
人源中性粒细胞药物(BCN-Ava-Lip/hNEs)的制备
将人外周血来源的中性粒细胞(hNEs)悬液的密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(DSPE-PEG5k-Tre),4℃共孵30min,离心(1500rmp,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的hNEs。将纳米药物(BCN-Ava-Lip)调成等渗,稀释为阿伐麦布浓度为150μg/mL的溶液,将其与上述表面带有反应基团的hNEs在25℃共孵20min,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的人源中性粒细胞,即BCN-Ava-Lip/hNEs细胞药物。
实施例19
人源中性粒细胞药物(Mal-siRNA-Lip/hNEs)的制备
将人外周血来源的中性粒细胞(hNEs)悬液的密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(DOPE-PEG2k-SH),4℃共孵15min,离心(1500rmp,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的hNEs。将纳米药物(Mal-siRNA-Lip)调成等渗,稀释为siRNA浓度为200nM的溶液,将其与上述表面带有反应基团的hNEs在4℃共孵2h,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的人源中性粒细胞,即Mal-siRNA-Lip/hNEs细胞药物。
实施例20
鼠源中性粒细胞药物(BCN-Ava-Lip/mNEs)的制备
将小鼠骨髓来源的中性粒细胞(mNEs)悬液的密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(DSPE-PEG5k-Tre),4℃共孵30min,离心(1500rmp,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的mNEs。将纳米药物(BCN-Ava-Lip)调成等渗,稀释为阿伐麦布浓度为150μg/mL的溶液,将其与上述表面带有反应基团的mNEs在25℃共孵20min,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的鼠源中性粒细胞,即BCN-Ava-Lip/mNEs细胞药物。
实施例21
人源间充质干细胞药物(ADIBO-PTX-NPs/hMSC)的制备
将人脐带来源的间充质干细胞(hMSC细胞)悬液的密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(SA2-Glu-PEG1k-N3),4℃共孵20min,离心(1500rmp,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的hMSC细胞。将纳米药物(ADIBO-PTX-NPs)调成等渗,稀释为紫杉醇浓度为100μg/mL的溶液,将其与上述表面带有反应基团的mT细胞在37℃共孵45min,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的人源MSC细胞,即ADIBO-PTX-NPs/hMSC细胞药物。
实施例22
肿瘤细胞(BCN-Ava-Lip/A549细胞)的制备
将肺癌细胞A549的细胞悬液密度调整到1×106细胞/mL,每毫升细胞悬液加入一定量的细胞膜锚定分子(DSPE-PEG5k-Tre),4℃共孵30min,离心(1500rmp,5min),弃上清,PBS洗涤2-3次,重悬得到表面带有反应基团的mNEs。将纳米药物(BCN-Ava-Lip)调成等渗,稀释为阿伐麦布浓度为150μg/mL的溶液,将其与上述表面带有反应基团的A549细胞在25℃共孵20min,离心(1500rmp,5min),弃上清,PBS洗涤去除未发生反应的纳米药物,重悬得到表面修饰有纳米药物的肿瘤细胞,即BCN-Ava-Lip/A549细胞。
实施例23
细胞药物载药量的测定
将上述实施例12-22制备的十一种不同的细胞药物于1500rmp离心5min后,弃上清,向细胞沉淀中加入适量体积的SDS细胞裂解液,充分涡旋,4℃静置30min,加入4倍体积乙腈进行蛋白沉淀和药物萃取,4℃静置30min,1500rpm涡旋5min,12000rpm离心10min,取上清液进行HPLC或微孔板检测。结果显示BCN-Ava-Lip/hT细胞、BCN-Ava-Lip/CAR-T细胞、BCN-Ava-Lip/mT细胞、BCN-Ava-Lip/TCR-T细胞、BCN-Ava-Lip/hNEs、BCN-Ava-Lip/mNEs、ADIBO-PTX-NPs/hT细胞、ADIBO-PTX-NPs/CAR-T细胞、Mal-siRNA-Lip/hNEs、ADIBO-PTX-NPs/hMSC、BCN-Ava-Lip/A549细胞的载药量分别为4.92μg Ava/106个hT细胞,4.65μg Ava/106个CAR-T细胞,4.38μg Ava/106个mT细胞,4.42μg Ava/106个TCR-T细胞,4.14μg Ava/106个hNEs,3.61μg Ava/106个mNEs,10.82μg PTX/106个hT细胞,8.25μg PTX/106个CAR-T细胞,83nM siRNA/106个hNEs,8.36μg PTX/106个hMSC细胞,6.95μg Ava/106个A549细胞。
实施例24
细胞药物的激光共聚焦表征
取SPC 100mg,胆固醇15mg,DSPE-BCN 25mg,溶于氯仿及甲醇中,并加入罗丹明B-1,2-双十六烷基-3-甘油-磷酸乙醇胺三乙铵盐(RhoB-DHPE)(2mg/mL,25μL)。旋转蒸发除去有机溶剂,真空干燥过夜。37℃水合30min,探头超声10-30min,依次过0.80,0.45,0.22μm滤膜,得到荧光标记的纳米药物RhoB-BCN-Lip。按照上述细胞药物的制备方法,将荧光标记的纳米药物RhoB-BCN-Lip修饰到不同的细胞表面,得到五种荧光标记的细胞药物(RhoB-BCN-Lip/mT细胞、RhoB-BCN-Lip/hT细胞、RhoB-BCN-Lip/CAR-T细胞、RhoB-BCN-Lip/mNEs、RhoB-BCN-Lip/hNEs)。
将新鲜制备的荧光标记的细胞药物用细胞核染料Hoechst33342(1μg/mL)进行荧光标记,多聚甲醛(PFA)固定后,进行激光共聚焦拍摄(图4)。由图可知,罗丹明的红色荧光存在于细胞膜上,这说明荧光标记的纳米药物通过本发明公开的活细胞表面锚定修饰技术成功的修饰在了活细胞上。
实施例25
细胞药物的存活率检测
以鼠源T细胞为例,按照实施例16的方法制备得到BCN-Ava-Lip/mT细胞,之后在含有5μg/mL抗CD3抗体,2μg/mL抗CD28抗体以及10ng/mL白介素-2(IL-2)的培养基中对BCN-Ava-Lip/mT细胞进行培养扩增,并于培养扩增的第0、4、7、10天分别对细胞进行台盼蓝染色,倒置荧光显微镜计数,计算细胞在扩增过程中的存活率。以扩增培养的mT细胞作为阳性对照。存活率=未染色细胞数/细胞总数×100%。人源T细胞药物BCN-Ava-Lip/hT细胞及CAR-T细胞药物BCN-Ava-Lip/CAR-T细胞的存活率检测方法与BCN-Ava-Lip/mT细胞相同。存活率检测结果如图5。结果显示,细胞药物组的存活率与阳性对照组的存活率无显著性差异,并且细胞存活率均在80%以上,这说明本发明公开的活细胞表面锚定修饰技术制备的细胞药物不会影响细胞的存活。
实施例26
细胞药物增殖能力表征
以鼠源T细胞为例,按照实施例16的方法制备得到BCN-Ava-Lip/mT细胞,之后在含有5μg/mL抗CD3抗体,2μg/mL抗CD28抗体以及10ng/mL白介素-2(IL-2)的培养基中对BCN-Ava-Lip/mT细胞进行培养扩增,并于培养扩增的第0、4、7、10天分别进行细胞计数。以扩增培养的mT细胞作为对照。细胞的体外扩增倍数=刺激后的细胞数/刺激前的细胞数。BCN-Ava-Lip/hT细胞及BCN-Ava-Lip/CAR-T细胞的增殖表征方法与BCN-Ava-Lip/mT细胞相同。增殖能力见图6。结果显示,细胞药物组的增殖能力与阳性对照组的增殖能力无显著性差异,这说明本发明公开的活细胞表面锚定修饰技术制备的细胞药物不会影响细胞的增殖能力。
实施例27
细胞药物趋化能力的表征
以鼠源中性粒细胞为例,按照实施例17的方法制备得到BCN-Ava-Lip/mNEs,将BCN-Ava-Lip/mNEs以1×106个细胞铺于Transwell小皿的上室,下室加入终浓度为1nM,10nM,100nM的趋化三肽(fMLP)培养液,5%CO2、37℃孵育12h,取出小室,分别收集上室及趋化到下室的细胞,计数并计算趋化指数。以下室中加入不含有fMLP的培养液作为空白对照,其余操作相同。以上室中加入mNEs,下室加入终浓度为1nM,10nM,100nM的fMLP培养液作为阳性对照组,其余操作相同。趋化指数=(实验组细胞下层细胞数量-空白对照组下层细胞数量)/细胞总量。趋化能力结果见图7。结果显示,细胞药物组的趋化能力与阳性对照组的趋化能力无显著性差异,这说明本发明公开的活细胞表面锚定修饰技术制备的细胞药物不会影响细胞的趋化能力。
实施例28
细胞药物(BCN-Ava-Lip/mT细胞)的肿瘤治疗效果
以鼠源T细胞药物BCN-Ava-Lip/mT细胞对黑色素瘤的抑制效果为例,16只C57BL/6J小鼠右侧背部皮内接种2×106个/只的B16F10黑色素瘤细胞悬液构建原位黑色素瘤模型。接种后将小鼠置于清洁级的饲养室中饲养,给予充足的水及饲料,每天观察肿瘤的生长状况,应用游标卡尺测量肿瘤的直径,按照以下公式计算肿瘤体积:V=L×W×W/2,其中,L为肿瘤的长径,W为肿瘤的短径,当C57BL/6J小鼠的肿瘤体积达到50mm3后,将小鼠随机分为4组,每组4只,分别给予:1)生理盐水;2)BCN-Ava-Lip(Ava:2mg/kg);3)mT细胞(1×107个细胞/只);4)BCN-Ava-Lip/mT细胞(1×107个细胞/只,Ava:2mg/kg)。以第一次给药记为第0天,分别于0、3、6、9、12天进行瘤内注射给药,共给药5次。从给药的第0天开始,隔天测量肿瘤的长径与短径,计算出肿瘤体积,以时间(天)为横坐标,肿瘤体积(mm3)为纵坐标,绘制得到肿瘤的生长曲线。于给药后的第14天,将荷瘤小鼠安乐死后小心剥离肿瘤组织,拍照观察肿瘤大小,结果见图8。结果显示,相比于T细胞组及纳米药物组(BCN-Ava-Lip),细胞药物组(BCN-Ava-Lip/mT细胞)具有最佳的抑瘤效果。
实施例29
细胞药物(ADIBO-PTX-NPs/hT细胞)的肿瘤治疗效果
以人源T细胞药物ADIBO-PTX-NPs/hT细胞对乳腺癌的抑制效果为例,20只BALB/c小鼠右侧乳垫接种3×106个/只的人源乳腺癌细胞(4T1乳腺癌细胞)悬液构建原位乳腺癌模型。接种后将小鼠置于清洁级的饲养室中饲养,给予充足的水及饲料,每天观察肿瘤的生长状况,应用游标卡尺测量肿瘤的直径,按照以下公式计算肿瘤体积:V=L×W×W/2,其中,L为肿瘤的长径,W为肿瘤的短径,当BALB/c小鼠的肿瘤体积达到50mm3后,将小鼠随机分为4组,每组5只,分别给予:1)生理盐水;2)ADIBO-PTX-NPs(PTX:5mg/kg);3)hT细胞(1×107个细胞/只);4)ADIBO-PTX-NPs/hT细胞(1×107个细胞/只,PTX:5mg/kg)。以第一次给药记为第0天,分别于0、6、12天进行静脉注射给药,共给药3次。从给药的第0天开始,隔天测量肿瘤的长径与短径,计算出肿瘤体积,以时间(天)为横坐标,肿瘤体积(mm3)为纵坐标,绘制得到肿瘤的生长曲线,结果见图9。结果显示,相比于T细胞组及纳米药物组(ADIBO-PTX-NPs),细胞药物组(ADIBO-PTX-NPs/hT细胞)具有最佳的抑瘤效果。
实施例30
细胞药物(BCN-Ava-Lip/CAR-T细胞)的肿瘤治疗效果
以CAR-T细胞药物BCN-Ava-Lip/CAR-T细胞对脑胶质瘤的抑制效果为例,15只重症免疫缺陷小鼠(NSG小鼠)大脑接种2×105个/只的人源脑胶质瘤细胞(LN229脑胶质瘤细胞)悬液构建原位脑胶质瘤模型。接种后给予小鼠充足的水及饲料,活体成像观察肿瘤的生长状况,当NSG小鼠的脑胶质瘤模型构建成功后,将小鼠随机分为3组,每组5只,分别给予:1)生理盐水;2)CAR-T细胞(5×106个细胞/只);3)BCN-Ava-Lip/CAR-T细胞(5×106个细胞/只,Ava:1mg/kg)。以第一次给药记为第0天,分别于0、6、12天进行脑原位注射给药,共给药3次。从给药的第0天开始,活体成像观察小鼠的肿瘤生长情况,结果见图10。结果显示,相比于CAR-T细胞组,细胞药物组(BCN-Ava-Lip/CAR-T细胞)具有最佳的抑瘤效果。
Claims (23)
1.一种在细胞表面锚定修饰纳米药物的方法,其特征在于通过细胞膜锚定分子的疏水尾链将活性反应基团引入到活细胞表面,在纳米药物表面修饰对应反应基团,活细胞表面修饰的细胞膜锚定分子的活性反应基团与纳米药物表面修饰的对应反应基团发生生物正交点击反应,从而将纳米药物锚定修饰到细胞表面得到修饰了纳米药物的活细胞。
3.根据权利要求2所述的方法,其特征在于所述的常见脂质选自二硬脂酰基磷脂酰乙醇胺、二油酰磷脂酰乙醇胺、1,2-十六烷基-3-甘油-磷酸乙醇胺或胆固醇;优选二硬脂酰基磷脂酰乙醇胺。
4.根据权利要求2所述的方法,其特征在于所述的n=20-100。
6.根据权利要求2所述的方法,其特征在于所述的活细胞选自人或动物具有脂质膜结构的原代细胞或永生化细胞,优选肿瘤细胞、中性粒细胞、T细胞、间充质干细胞、造血干细胞、自然杀伤细胞、抗原递呈细胞、巨噬细胞中的任意一种,进一步优选T细胞或中性粒细胞;所述的T细胞选自嵌合抗原受体T细胞、T细胞受体基因工程改造的T细胞或普通未经修饰的T细胞。
7.根据权利要求2所述的方法,其特征在于所述的纳米药物为载有治疗剂的纳米粒;所述的纳米粒为粒径为1-1000nm的脂质体、纳米囊泡、固体脂质纳米粒或胶束。
8.根据权利要求7所述的方法,其特征在于所述的治疗剂为治疗肿瘤或炎性相关疾病的药物,选自小分子化学药物、蛋白治疗药物或基因治疗药物中的一种或多种;所述的小分子化学药物优选疏水性药物或亲水性药物,所述的疏水性药物选自阿伐麦布、紫杉醇、槲皮素、BAY87-2243、TGF-β抑制剂、白皮杉醇中的任意一种或多种,所述的亲水性药物选自阿霉素、柔红霉素、丝裂霉素中的任意一种或多种;所述的蛋白治疗药物优选PD-1单抗、PD-L1单抗,所述的基因治疗药物优选siRNA、mRNA、shRNA、质粒。
9.根据权利要求8所述的方法,其特征在于所述的治疗剂选自阿伐麦布、紫杉醇或PD-1单抗。
11.根据权利要求1所述的方法,其特征在于所述的生物正交点击反应选自酮/羟胺缩合,巯基或氨基与马来酰亚胺的迈克尔加成反应,环张力驱动的叠氮-炔环加成反应或高张力驱动逆电子需求的Dields-Alder环加成反应,优选环张力驱动的叠氮-炔环加成反应或高张力驱动逆电子需求的Dields-Alder环加成反应。
12.根据权利要求1~11中任一项所述的方法,其特征在于将所述的细胞膜锚定分子与活细胞0-40℃共孵5-120min得到表面修饰细胞膜锚定分子的活细胞;所述的表面修饰对应反应基团的纳米药物与表面修饰细胞膜锚定分子的活细胞0-37℃共孵5-120min得到修饰了纳米药物的活细胞。
13.按照权利要求1~11中任一项所述的方法制备得到的修饰了纳米药物的活细胞;优选按照权利要求1~11中任一项所述的方法制备得到的修饰了纳米药物的T细胞;进一步优选按照权利要求1~11中任一项所述的方法制备得到的修饰了纳米药物的嵌合抗原受体T细胞、T细胞受体基因工程改造的T细胞。
14.权利要求13所述的修饰了纳米药物的活细胞在制备治疗肿瘤或炎性相关疾病的药物中的应用。
15.根据权利要求14所述的应用,其特征在于所述的肿瘤选自黑色素瘤、脑胶质瘤、乳腺癌或卵巢癌;所述的炎性相关疾病选自脑卒中或关节炎。
19.权利要求16所述的细胞膜锚定分子在制备活细胞药物中的应用,所述的活细胞药物为表面修饰了纳米药物的活细胞。
21.根据权利要求20所述的对应反应基团修饰剂,其特征在于所述的常见脂质选自二硬脂酰基磷脂酰乙醇胺、二油酰磷脂酰乙醇胺、1,2-十六烷基-3-甘油-磷酸乙醇胺或胆固醇;优选二硬脂酰基磷脂酰乙醇胺。
23.权利要求20所述的对应反应基团修饰剂在制备活细胞药物中的应用,所述的活细胞药物为表面修饰了纳米药物的活细胞;优选表面修饰了纳米药物的T细胞;进一步优选表面修饰了纳米药物的嵌合抗原受体T细胞、T细胞受体基因工程改造的T细胞。
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