CN117070455B - 一种巨噬细胞治疗制剂及其制备方法和在肿瘤治疗中的应用 - Google Patents
一种巨噬细胞治疗制剂及其制备方法和在肿瘤治疗中的应用 Download PDFInfo
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Abstract
本发明公开了一种巨噬细胞,所述巨噬细胞装载包被载药脂质纳米粒的外膜囊泡,所述巨噬细胞为M1型巨噬细胞,所述巨噬细胞表面锚定DG12。巨噬细胞表面锚定GPC3靶向肽,增强M1型巨噬细胞肿瘤靶向能力,特异性吞噬和杀伤GPC3高表达的HCC细胞。而且外膜囊泡融合C16‑神经酰胺,RILO可通过小窝蛋白介导的内吞途径进入M1型巨噬细胞,避免溶酶体等强酸性环境对纳米药物的降解,并且可使载药巨噬细胞在肿瘤部位以非游离形式释放药物,释放的药物可被肿瘤细胞和TAM摄取,发挥调节TAM表型,持续增强T细胞活力的作用,触发强大抗肿瘤免疫反应。
Description
技术领域
本发明属于肿瘤靶向治疗技术领域,具体涉及一种巨噬细胞、制备方法及其在肿瘤治疗中的应用。
背景技术
细胞治疗是当前和未来肿瘤免疫治疗的主要研究和发展方向。其中细胞治疗鼻祖Carl H.June教授领导开发的基因工程嵌合抗原受体巨噬细胞,在早期临床试验中取得令人鼓舞的早期结果。但是,现有巨噬细胞疗法多采用基因转染手段进行制备,具有诱发插入突变风险和转染效率偏低、工艺流程耗时长的技术难点。
越来越多的研究表明,使用靶向因子对纳米颗粒表面功能化可显著提高体内靶向效率,但是靶向多肽能否增强细胞疗法的靶向效率,值得进一步研究。此外,巨噬细胞的吞噬作用在维持体内平衡和调节免疫功能方面具有重要作用。在肿瘤治疗中,巨噬细胞可直接吞噬肿瘤细胞,释放细胞因子,持续激活下游适应性免疫反应,发挥抗肿瘤治疗效果。与M2型巨噬细胞相比,M1型巨噬细胞可下调SIRPα表达,具有更强吞噬能力。因此,采用非基因工程策略,通过肿瘤相关靶向肽锚定在巨噬细胞细胞膜表面的方式,增强巨噬细胞肿瘤靶向性和肿瘤特异性吞噬杀伤能力,进而提高巨噬细胞治疗制剂的抗肿瘤治疗效果具有广阔的临床应用前景。
发明内容
本发明一方面提供了一种巨噬细胞,所述巨噬细胞装载包被载药脂质纳米粒的外膜囊泡,所述巨噬细胞为M1型巨噬细胞。
进一步地,所述巨噬细胞与DG12共孵育。
二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-G12(DSPE-PEG5k-G12,DG12)。
G12是一种GPC3靶向肽(D-Cys-DHLASLWWGTEL,G12)。
进一步地,包被载药脂质纳米粒之前的外膜囊泡融合C16-神经酰胺。
进一步地,包被载药脂质纳米粒之前的外膜囊泡由FimH阳性菌株中获得。优选,MG1655菌株。
进一步地,所述载药脂质纳米粒所载的药物为R848和INCB-024360。
本发明一方面提供了一种巨噬细胞的制备方法,M1型巨噬细胞与包被载药脂质纳米粒的外膜囊泡共孵育。
进一步地,所述巨噬细胞通过与DG12共孵育,实现所述巨噬细胞表面锚定二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-G12(DSPE-PEG5k-G12,DG12)。
进一步地,所述包被载药脂质纳米粒的外膜囊泡,由FimH阳性菌株中获得的外膜囊泡经过C16-神经酰胺共挤压,然后包被载药的脂质纳米粒。
本发明一方面提供了一种巨噬细胞在制备抗肿瘤药物中的应用。
进一步地,所述治疗具有靶向性,为靶向治疗。
本发明中重要的缩写具体的意义为:
OMV(Outer membrane vesicle,外膜囊泡,未包被脂质纳米粒)
HCC(Hepatocellular carcinoma,肝细胞癌)
RIL(共载R848和INCB脂质纳米粒)
RILO(包被共载R848和INCB脂质纳米粒的OMV)
RILOM1(载包被共载R848和INCB脂质纳米粒OMV的M1型巨噬细胞)。
巨噬细胞的功能性极化具有多样性,目前认为巨噬细胞极化是一个重叠的功能状态连续谱,拥有M1型和M2型两个极端状态以及一系列兼具M1型和M2型功能的中间状态。其中,M1型巨噬细胞高表达CD80分子,具有促炎和抗肿瘤的作用;M2型巨噬细胞高表达CD206分子,具有抗炎和促肿瘤的作用。我们选用的是M1型巨噬细胞进行试验。
RILOM1G(载包被共载R848和INCB脂质纳米粒OMV的G12修饰的M1型巨噬细胞,RILOM1G的制备示意图如图1所示)
与现有技术相比,本发明的有益效果是:
1、将GPC3靶向肽(D-Cys-DHLASLWWGTEL,G12)通过化学合成制得二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-G12(DSPE-PEG5k-G12,DG12)锚定在巨噬细胞表面,制得靶向肽修饰的RILOM1(RILOM1G),用来增强M1型巨噬细胞肿瘤靶向能力,没有修饰DG12的巨噬细胞,只能依靠巨噬细胞自身对炎症的固有趋向性到达肿瘤,加上DG12后,在巨噬细胞固有趋向性的基础上,并因为配体-受体相互作用,进一步增强带有DG12的巨噬细胞对高表达GPC3的肝癌细胞靶向性,进而特异性吞噬和杀伤GPC3高表达的HCC细胞。
2、巨噬细胞的功能性极化具有多样性,目前认为巨噬细胞极化是一个重叠的功能状态连续谱,拥有M1型和M2型两个极端状态以及一系列兼具M1型和M2型功能的中间状态。其中,M1型巨噬细胞高表达CD80分子,具有促炎和抗肿瘤的作用;M2型巨噬细胞高表达CD206分子,具有抗炎和促肿瘤的作用。
3、RILO可通过小窝蛋白介导的内吞途径进入M1型巨噬细胞,避免溶酶体等强酸性环境对纳米药物的降解。此外,OMV中融合的C16-神经酰胺可使载药巨噬细胞在肿瘤部位以非游离形式释放药物,释放的药物可被肿瘤细胞和TAM摄取,发挥调节TAM表型,持续增强T细胞活力的作用,引发强大抗肿瘤免疫反应。
4、OMV包裹的共载两种药物的纳米粒,两种药物药效发挥协同效果,通过调节肿瘤相关巨噬细胞表型和增强T细胞活力从而调控免疫抑制微环境,如果纳米药物的纳米结构提前被降解,再释放出来,就无法控制药物在肿瘤部位的浓度和比例,因此达到很好的治疗作用。
5、外膜囊泡由FimH阳性菌株中获得。本发明利用了自然界中FimH阳性菌株自身可以通过小窝蛋白进入细胞的现象。发现采用这种菌株的OMV制备的RILO,能够很好的通过小窝蛋白介导的内吞途径进入M1型巨噬细胞。
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:RILOM1G的制备示意图。
图2:BMDM的形态学鉴定(a)和特征蛋白表征(b)。
图3:RILOM1制备时孵育浓度和孵育时间对细胞活力和DL的影响:M1(RAW264.7)与RILO孵育时的细胞活力(a);M1与不同浓度RILO孵育2h的细胞活力(b);M1(RAW264.7)与RILO孵育时的DL(c)(n=3)。
图4:RILM1(RAW264.7)、RILOM1(RAW264.7)和RILOM1的DL(n=3;***P<0.001;ns,no significance)。
图5:DG12的结构与合成过程。
图6:G12(a)、DSPE-PEG5k-Mal(b)和DG12(c)的1H-NMR谱图。
图7:RILOM1G制备时孵育浓度和孵育时间对细胞活力和G12锚定量的影响:RILOM1(RAW264.7)与DG12孵育时的细胞活力(a);RILOM1与不同浓度DG12孵育20min的细胞活力(b);RILOM1(RAW264.7)与DG12孵育时的G12锚定量(c)(n=3)。
图8:RILOM1-FITC(RAW264.7)、RILOM1-free G12-FITC(RAW264.7)和RILOM1G-FITC(RAW264.7)的共聚焦图像(标尺10μm,a);RILOM1G(RAW264.7)制备24h和48h后的流式细胞仪分析(标尺10μm,b)。
图9:RILOM1G(RAW264.7)制备24h和48h后的流式细胞仪分析。
图10:Western blot实验检测肿瘤靶向相关蛋白(a);transwell法评价肿瘤靶向能力(比例尺200μm,b)。
图11:静脉注射不同制剂的H22荷瘤小鼠在预设时间间隔的体内生物分布图像(n=3)。
图12:24h时肿瘤和主要器官的离体图像(a)和平均荧光强度(b)(n=3,
***P<0.001,**P<0.01,*P<0.05)。
图13:不同制剂特异性吞噬肿瘤细胞的流式散点图(a)及统计分析(b)(n=3,***P<0.001)。
具体实施方式
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,以下介绍具体实施方式详细说明本发明的技术方案。
一、实验材料
1.试剂与药品
R848(上海阿拉丁生化科技股份有限公司);INCB-024360(INCB,美国TargetMol公司);二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-马来酰亚胺(DSPE-PEG5k-Mal,西安瑞禧生物科技有限公司);GPC3靶向多肽(D-Cys-DHLASLWWGTEL,G12)(南京Leon生物科技有限公司);胎牛血清(FBS,浙江天杭生物科技股份有限公司);小鼠单核细胞集落刺激因子(M-CSF,美国PeproTech公司);其他试剂均为分析纯,购自国药集团化学试剂有限公司。
2.仪器设备
动态光散射分析仪(Nano-ZS90型,英国Malvern公司);透射电镜(HT7700型,日本Hitachi公司);细胞成像多功能检测系统(Cytation 5型,美国BioTek公司);流式细胞仪(Accuri C6 Plus型,美国BD公司)。
3.细胞与动物
小鼠肝癌细胞株H22和H22-luc购自上海富衡生物科技有限公司,在含有10%FBS和1%青链霉素混合液的RPMI-1640培养基中培养;小鼠骨髓来源巨噬细胞(Mouse bonemarrow-derived macrophage,BMDM)来源于6~8周龄雌性BALB/c小鼠;小鼠单核巨噬细胞白血病细胞株(RAW264.7)购买于上海富衡生物科技有限公司,在含有10%FBS的DMEM培养基中培养。所有细胞均在标准条件(37、5%CO2)下培养。雌性BALB/c小鼠(6~8周龄)购买于北京斯贝福生物技术有限公司。
4.试剂配制
4.1一定质量分数吐温-80缓冲液的配置
精密称定吐温-80 0.250g,加入1×PBS缓冲液定容至100mL,超声分散溶解后,即得质量分数为0.25%的吐温-80缓冲液,质量分数为0.5、1.0、1.5%的吐温-80缓冲液同法配置。
4.2HCC条件培养基的制备
HCC条件培养基由等体积含有10%FBS的DMEM培养基和孵育H22细胞48h后的培养上清构成。
二、实验方法和实验结果
1.RILO的制备及工艺处方优化
1.1RIL的制备
采用纳米沉淀法制备RIL。将R848和INCB按1:1的质量比溶解在无水乙醇中,得一定药物浓度(药脂比15:100,即9mg/mL的R848和9mg/mLINCB)的有机相。将大豆卵磷脂溶于一定质量分数(0.5%)吐温-80缓冲液中,超声溶解,得一定磷脂浓度(大豆卵磷脂浓度12mg/mL)的水相。根据实验室前期研究基础,确定水相与有机相体积比为10:1。使用微量注射泵在冰浴磁力搅拌600rpm条件下以10mL/h滴加速度将有机相加入到水相中。于室温磁力搅拌400rpm条件下,搅拌1h使乙醇挥发且无醇味,使用0.22μm微孔滤膜除去游离药物,即得RIL。
1.2OMV的提取分离
采用超高速离心法提取分离OMV。将大肠杆菌MG1655添加到250mL LB培养基中,在37,200rpm条件下震荡孵育。待分光光度计测得600nm处光密度约为1.2时,取上清液在4、4000g条件下离心10min。经0.45μm水系微孔滤膜抽滤后,使用截留分子量为100kDa的超滤离心管在4/>、4000g条件下离心10min,得浓缩滤液50mL。使用超高速离心机在4/>、150000g条件下离心3h,将浓缩物重悬于1×PBS缓冲液中,于4/>、150000g条件下再次离心3h以去除OMV中杂蛋白,最后将OMV重悬于1mL 1×PBS缓冲液中,于-80/>保存供进一步使用。根据试剂盒说明书,使用二奎啉甲酸(Bicinchoninic acid,BCA)法,以总蛋白含量为指标对OMV进行定量。
1.3RILO的制备
具体地,将C16-神经酰胺(450μM)加入一定浓度(400μg/mL)的OMV中,于4超声处理2min,得到富含C16-神经酰胺的OMV。然后,将富含C16-神经酰胺的OMV与新鲜制备的RIL在涡旋条件下以1:1的体积比混合,过膜(50nm)挤压数次(12次),即得RILO。
2.RILOM1的制备及表征
2.1BMDM的提取与诱导方法和鉴定
从6~8周龄雌性BALB/c小鼠股骨和胫骨中分离骨髓细胞,然后在标准条件下,于BMDM生长培养基(DMEM+10%FBS+20ng/mL M-CSF)中培养骨髓细胞。第3天,将培养基更换为新鲜的BMDM生长培养基,并去除非贴壁细胞。第7天,使用Brilliant Violet 421TM-F4/80抗体和PerCP/Cyanine5.5-CD11b抗体进行免疫荧光双染色评估BMDM形成。
在需要极化BMDM为M1型巨噬细胞(M1)的情况下,在第7天将培养基更换为刺激培养基。即使用含有FBS(10%)、LPS(100ng/mL)和IFN-γ(20ng/mL)的DMEM培养基刺激24h,即得M1,立即用于后续实验。本发明少许实验使用RAW264.7进行,已进行标注,其余所有涉及巨噬细胞的实验均使用BMDM。
为了更接近临床转化,除非另有说明,RILOM1和RILOM1G等基于巨噬细胞的相关制剂制备时使用BMDM。由图2可知,根据BMDM形态学鉴定,第1天时,骨髓中分离出的髓源细胞呈圆形,悬浮于培养基中;第3天时,换液除去未贴壁细胞后贴壁细胞即为巨噬细胞,有的呈圆形有的呈不规则梭形;第7天时,细胞形态偏圆形,收集细胞进行鉴定。经流式细胞术鉴定,共表达CD11b和F4/80的细胞占比可达95%以上,表明BMDM具有较高纯度。
2.2RILOM1的制备及单因素试验考察
将M1与RILO共孵育得到RILOM1。根据不同的实验目的,分别采用150mm直径培养皿(30mL培养体系)、12孔板(1mL培养体系)、24孔板(600μL培养体系)和96孔板(100μL培养体系)进行试验。将一定浓度的RILO加入密度约为1×105个细胞/cm2的M1中,置于37、5%CO2条件下孵育一定时间,弃去上清,使用DMEM培养基清洗以除去未被摄取的RILO,即得RILOM1。同法制备RILOM1(RAW264.7)和RILM1(RAW264.7)。
采用单因素试验,以细胞活力和载药量(Drug loading,DL)为指标,分别考察孵育浓度(400、200、100、50、25μg/mL,根据R848的浓度进行定量)和孵育时间(1、2、4h)共2个因素的影响,确定最佳孵育浓度和孵育时间。
为了测定RILOM1的DL(μg/106),采用高效液相色谱法定量测定孵育前后DMEM培养基中R848和INCB含量。具体地,将孵育前后的DMEM培养基各加入5倍甲醇破乳,并用流动相稀释,经0.22μm微孔滤膜过滤后,进样测定,按下式计算RILOM1的DL。
DL(μg/106)=(W孵育前培养基中药物含量-W孵育后培养基中药物含量)/N被孵育细胞的数量×106
采用MTT法检测RILOM1的细胞活力。使用DMEM培养基将RILO稀释至预设浓度,并加入含有M1或M1(RAW264.7)的96孔板中(每孔100μL培养体系),置于37、5%CO2条件下孵育一定时间后,弃去上清,除去未被摄取的RILO,加入含10%FBS的DMEM培养基,继续孵育48h后,每孔中加入20μL的MTT溶液(5mg/mL)继续孵育4h。随后,弃去培养基,每孔加入200μL的二甲基亚砜,于570nm处测定并计算细胞活力。
以细胞活力和DL为指标,考察孵育浓度(400、200、100、50、25μg/mL,根据R848的浓度进行定量)和孵育时间(1、2、4h)共2个因素的影响。如图3,首先以M1(RAW264.7)为模型细胞进行条件筛选,通过测定M1(RAW264.7)与不同浓度RILO孵育不同时间时的细胞活力,确定孵育时间为1或2h时,RILOM1(RAW264.7)具有较高细胞活力;进而测定M1(RAW264.7)与不同浓度RILO孵育1或2h时的DL,确定孵育时间为2h时,RILOM1(RAW264.7)具有较高的DL。在上述条件筛选的基础上,将M1与不同浓度RILO孵育2h后测定细胞活力,结果表明M1与不高于200μg/mL的RILO孵育2h时,细胞活力较高,均高于90%。因此,选择最佳孵育浓度为200μg/mL,最佳孵育时间为2h,在此条件下制备生物相容性更好、DL更高的RILOM1。
RILM1(RAW264.7)、RILOM1(RAW264.7)或RILOM1由极化的RAW264.7细胞或BMDM与RIL或RILO在200μg/mL(根据R848的浓度进行定量)条件下于37孵育2h制得。由图4可知,RILOM1(RAW264.7)或RILOM1的载药量均显著高于RILM1(RAW264.7)的载药量(P<0.001),说明OMV的包被可显著提高巨噬细胞吞噬装载纳米粒的能力。同时,RILOM1对R848和INCB的DL分别为29.75±4.76μg/106和25.13±2.75%μg/106,与RILOM1(RAW264.7)的DL(29.07±1.55μg/106和24.67±1.08%μg/106)无显著性差异。
3.RILOM1G的制备及表征
3.1DSPE-PEG5k-G12的合成与表征
精密称定DSPE-PEG5k-Mal和G12分别溶解于4mL和2mL的1×合成缓冲液(pH 7.4,含1mM EDTA),超声至溶解,得1.5mM的DSPE-PEG5k-Mal反应液和6mM的G12反应液。在氮气保护条件下,将G12反应液滴入DSPE-PEG5k-Mal反应液中,室温搅拌12h。得到的溶液使用截留分子量为3.5kDa的透析袋经超纯水透析48h纯化后,冷冻干燥,即得DSPE-PEG5k-G12(DG12),DG12的结构与合成过程如图5所示。通过1H-NMR验证DG12的结构,并通过高效液相色谱法测定G12在DG12中的接枝率。必要时,按上述步骤使用DSPE-PEG5k-Mal与G12-FITC反应合成DG12-FITC,用于相关实验。
通过DSPE-PEG5k-Mal中的马来酰亚胺基团与G12中的巯基反应合成GPC3靶向材料DG12。G12、DSPE-PEG5k和DG12的1H-NMR谱图如图6所示,DG12中马来酰亚胺特征峰d消失,出现G12相关特征峰a、b、c,说明DG12已成功合成。同时,使用高效液相色谱法测定DG12中G12的接枝率为19.784±0.102%。3.2RILOM1G的制备及单因素试验考察
RILOM1与DG12共孵育得到RILOM1G,经过孵育DG12中DSPE部分的疏水长链能够插入细胞膜,PEG5k部分具有较强亲水性和空间位阻,从而将G12锚定在细胞表面。具体地,将一定浓度的DG12加入密度约为1×105个细胞/cm2新鲜制备的RILOM1中,置于37、5%CO2条件下平摇孵育一定时间,弃去上清,使用1×PBS缓冲液清洗以除去残留的DG12,即得RILOM1G。同法制备RILOM1G(RAW264.7)。
采用单因素试验,以细胞活力和G12锚定量为指标,分别考察孵育浓度(400、200、100、50、25μg/mL,根据DG12的浓度进行定量)和孵育时间(10、20、30min)共2个因素的影响,确定最佳孵育浓度和孵育时间。
根据“2.2”项下的方法采用MTT法测定细胞活力。为测定RILOM1G的G12锚定量(μg/106),采用高效液相色谱法定量测定与细胞孵育前后DMEM培养基中DG12含量。RILOM1G中G12锚定量(μg/106)的计算公式:
G12锚定量(μg/106)=(W孵育前培养基中DG12含量-W孵育后培养基中DG12含量)/N被孵育细胞的数量×106×接枝率
以细胞活力和G12锚定量为指标,考察孵育浓度(400、200、100、50、25μg/mL,根据DG12的浓度进行定量)和孵育时间(10、20、30min)共2个因素的影响。如图7(a,c),首先以RILOM1(RAW264.7)为模型细胞进行条件筛选,通过测定RILOM1(RAW264.7)与不同浓度DG12孵育不同时间时的细胞活力和G12锚定量,发现当孵育时间超过20min时,G12锚定量已不再增加。因此,在孵育时间为20min的条件下,测定RILOM1与不同浓度DG12孵育后的细胞活力,如图7(b)所示,当DG12浓度小于等于100μg/mL时,具有较高的细胞活力(超过90%)。最终,确定G12锚定过程的最佳孵育浓度为100μg/mL,最佳孵育时间为20min。在最佳孵育浓度和时间条件下,RILOM1G的G12锚定量为6.67±0.64μg/106,略高于RILOM1G(RAW264.7)的5.76±0.79μg/106,但两者之间无显著差异。
3.3RILOM1G表面DG12稳定性考察
在最佳孵育浓度和孵育时间条件下,进行如下表征。
为证实G12是通过DSPE-PEG5k被锚定在细胞表面,合成制备G12-FITC和DG12-FITC。根据“3.2”项下的方法,在最佳孵育浓度和孵育时间条件下,制备RILOM1G-FITC(RAW264.7)和RILOM1-free G12-FITC(RAW264.7),使用Alexa647-F4/80抗体对细胞膜进行染色和使用Hoechst 33342对细胞核进行染色,并使用激光共聚焦成像系统观察。进一步地,将使用含10%FBS的DMEM培养基孵育RILOM1G-FITC(RAW264.7)不同时间(0、24、48h)后,在相应时间使用1×PBS缓冲液洗涤后,通过共聚焦显微镜和流式细胞仪分析G12在细胞表面的稳定性。
为更好表征RILOM1G(RAW264.7),使用FITC标记G12进行试验。为证实G12是通过DSPE-PEG5k被锚定在细胞表面,合成G12-FITC和DG12-FITC,分别用来制备RILOM1-freeG12-FITC(RAW264.7)和RILOM1G-FITC(RAW264.7)。如图8(a)所示,细胞膜被Alexa647-F4/80抗体标记显示红色荧光,G12被FITC标记显示绿色荧光,在RILOM1G-FITC(RAW264.7)组观察到红色与绿色荧光共定位现象,而RILOM1-free G12-FITC(RAW264.7)组的绿色荧光已进入细胞内部。说明G12是通过DSPE-PEG5k产生的脂质锚定作用成功保留在细胞表面。进一步地,使用共聚焦显微镜和流式细胞仪考察DG12在细胞表面的稳定性。如图8(b)和图9所示,DG12在37/>含10%FBS的DMEM培养基中可稳定在细胞表面,在48h时,仍有47.92%的DG12存在于细胞表面。
4.RILOM1G体外肿瘤趋向能力考察
4.1Western blot实验
采用Western blot实验检测肿瘤靶向相关蛋白。使用细胞全蛋白提取试剂盒提取分离M1、RILOM1和RILOM1G中的蛋白组分。配制10%的PAGE凝胶,样品蛋白成分经加热变性后上样,以每样品20μg蛋白的上样量进行试验,以RILO和DG12作为对照,随后依次在70V、30min和120V,80min的条件下进行电泳分离,经转膜、孵育一抗(兔源CCR2抗体和兔源beta-Tubulin抗体)、孵育二抗后,显色以表征CCR2。同法经转膜、孵育一抗(兔源CCR2抗体和兔源beta-Tubulin抗体)、孵育二抗后,显色以表征alpha 4。
4.2Transwell实验
采用24孔Transwell板(孔径8μm)进行实验,根据“2.1”、“2.2”和“3.2”项下方法制备M1、RILOM1或RILOM1G,将M1、RILOM1或RILOM1G重悬于无血清DMEM培养基,以每孔3×104个细胞接种于Transwell板上室,下室为含有10%FBS的DMEM培养基或HCC条件培养基。在37、5%CO2条件下孵育16h后,取出上室,清除上室Transwell膜上表面细胞,将Transwell膜下表面细胞使用结晶紫染色,置于荧光显微镜下拍照。
为了考察装载RILO并修饰DG12后,是否会影响细胞的肿瘤趋向性。首先通过Western blotting实验检测M1、RILOM1和RILOM1G的肿瘤靶向相关蛋白(alpha 4和CCR2)。如图10(a)所示,与M1组相比,装载RILO并修饰DG12后并未影响肿瘤靶向相关蛋白的表达。在此基础上,采用Transwell实验体外考察RILOM1G的肿瘤趋向性,结果如图10(b)所示,当下室为DMEM培养基时,M1、RILOM1和RILOM1G均未发生明显的迁移现象;当下室为HCC条件培养基时,M1、RILOM1和RILOM1G的迁移数量明显增加,且与M1组相比,RILOM1和RILOM1G的迁移数量无明显变化。上述结果表明,RILOM1G保留了M1完整的肿瘤靶向能力。
5.RILOM1G体内生物分布与肿瘤蓄积
使用H22荷瘤BALB/c小鼠模型评价RILOM1G体内生物分布。选用DiR作为近红外示踪剂,根据“1.3”、“2.2”和“3.2”项下方法,制备游离DiR、DiR-LO、DiR-LOM1和DiR-LOM1G。为了构建H22荷瘤BALB/c小鼠模型,在6~8周龄雌性BALB/c小鼠右侧腋窝处皮下注射1×106个H22细胞。经过12天后,将荷瘤小鼠随机分为4组,每组3只,尾静脉注射给与单次剂量游离DiR、DiR-LO、DiR-LOM1或DiR-LOM1G(剂量为3.0×106个/只小鼠,相当于2.5mg/kg DiR)。在第2、4、8、12和24h时,使用异氟烷吸入麻醉小鼠,并用IVIS光谱成像系统成像。在最后一次体内成像后(给药后24h),将小鼠安乐死,取肿瘤和主要器官(包括:心、肝、脾、肺和肾),用于离体器官成像。
选择H22荷瘤小鼠模型评估RILOM1G的肿瘤蓄积能力。选用DiR作为近红外示踪剂,制备游离DiR、DiR-LO、DiR-LOM1和DiR-LOM1G。如图11,尾静脉注射DiR-LOM1G的H22荷瘤小鼠在预设时间间隔的体内生物分布图像显示,荧光信号在肿瘤组织中逐渐积聚。与游离DiR组、DiR-LO组和DiR-LOM1组相比,DiR-LOM1G组在肿瘤组织中表现出更高的荧光强度,这一结果说明M1,特别是DG12修饰的M1可以显著增强药物在肿瘤部位的蓄积。如图12所示,24h时离体图像和平均荧光强度来评估不同制剂在肿瘤和主要器官中的分布。与DiR-LO、DiR-LOM1组比较,DiR-LOM1G组肿瘤荧光信号显著升高(P<0.001,P<0.05),并且正常肝脏荧光信号强度降低(P<0.01,P<0.05)。上述这些结果表明,RILOM1G比普通纳米颗粒和未经靶向修饰的M1相比,具有更强的肿瘤蓄积能力
6.特异性吞噬肿瘤细胞能力评价
通过流式细胞术评价RILOM1G特异性吞噬肿瘤细胞能力。根据“2.1”、“2.2”和“3.2”项下方法,制备M1、M1G、RILOM1和RILOM1G,然后使用细胞增殖示踪荧光探针CFSE进行标记,同时使用细胞膜染料DiD标记H22细胞。对于竞争性抑制实验,将CFSE标记的H22细胞与游离G12预先孵育30min以饱和H22细胞表面的GPC3。将CFSE标记的M1、M1G、RILOM1和RILOM1G与DiD标记的H22细胞,以1:1的比例在37、5%CO2的条件下共孵育4h。M1、M1G、RILOM1或RILOM1G吞噬H22细胞的百分率通过流式细胞仪检测CFSE+DiD+细胞与CFSE+细胞的比例进行计算。
将基于M1制备的制剂与GPC3阳性小鼠肝癌细胞H22共孵育,通过流式细胞术检测不同制剂吞噬H22细胞的百分率,考察DG12的修饰对M1及RILOM1吞噬作用的影响。吞噬百分率通过使用流式细胞仪检测CFSE+DiD+细胞与CFSE+细胞的比例进行计算。如图13所示,与M1组相比,M1G组和RILOM1组具有微弱的增强吞噬作用。相反,RILOM1G组的吞噬百分率超过M1组吞噬百分率的2倍以上,且显著优于M1G和RILOM1吞噬H22细胞的能力(***P<0.001,***P<0.001)。同时,竞争性抑制实验显著降低了RILOM1G通过DG12修饰增强的吞噬作用(***P<0.001),这是由于H22细胞上绝大多数GPC3已与G12结合,影响了RILOM1G与H22细胞中GPC3的相互作用。这些结果表明,RILOM1G显著提高了M1的肿瘤特异性吞噬能力。
Claims (7)
1.一种巨噬细胞,其特征是,所述巨噬细胞装载包被载药脂质纳米粒的外膜囊泡,所述巨噬细胞为M1型巨噬细胞,所述巨噬细胞表面锚定二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-G12,包被载药脂质纳米粒之前的外膜囊泡融合C16-神经酰胺,包被载药脂质纳米粒之前的外膜囊泡由FimH阳性菌株中获得,所述二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-G12具体结构为:
。
2.如权利要求1所述的巨噬细胞,其特征是,所述FimH阳性菌株为MG1655菌株。
3.如权利要求1所述的巨噬细胞,其特征是,所述载药脂质纳米粒所载的药物为R848和INCB-024360。
4.如权利要求1所述的巨噬细胞的制备方法,其特征是,M1型巨噬细胞与包被载药脂质纳米粒的外膜囊泡共孵育,然后在巨噬细胞表面锚定二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000-G12。
5.如权利要求4所述的巨噬细胞的制备方法,其特征是,所述包被载药脂质纳米粒的外膜囊泡的制备方法为由FimH阳性菌株中获得的外膜囊泡经过C16-神经酰胺共挤压,然后包被载药的脂质纳米粒。
6.如权利要求5所述的巨噬细胞的制备方法,其特征是,所述载药脂质纳米粒所载的药物为R848和INCB-024360。
7.如权利要求1所述的巨噬细胞在制备抗肝癌的药物中的应用,其中所述肝癌高表达GPC3。
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