CN115227684A - 靶向M2型巨噬细胞CpG和ELE脂质体及制法和应用 - Google Patents
靶向M2型巨噬细胞CpG和ELE脂质体及制法和应用 Download PDFInfo
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- CN115227684A CN115227684A CN202210899297.4A CN202210899297A CN115227684A CN 115227684 A CN115227684 A CN 115227684A CN 202210899297 A CN202210899297 A CN 202210899297A CN 115227684 A CN115227684 A CN 115227684A
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- folic acid
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Abstract
本发明公开了靶向M2型巨噬细胞CpG和ELE脂质体及制法和应用。CpG脂质体为表面修饰叶酸且负载有CpG,每100重量份包括:1~5份磷脂、0.04~0.2份胆固醇、0.004~0.1份聚乙二醇衍生物、0.02~0.1份DSPE‑PEG‑叶酸、0.004~0.1份CpG。ELE脂质体为表面修饰叶酸和AS1411,负载有榄香烯,每100重量份包括:1~5份磷脂、0.04~0.2份胆固醇、0.004~0.1份聚乙二醇衍生物、0.15~0.75份ELE、0.02~0.1份DSPE‑PEG‑叶酸、0.0005~0.002份胆固醇‑AS1411。两种脂质体协同作用能够抑制肿瘤的生长和逆转肿瘤免疫微环境。
Description
技术领域
本发明属于医药化学技术领域,尤其是肿瘤化疗药物技术领域,具体涉及靶向M2型巨噬细胞的免疫激活剂(CpG)和榄香烯(ELE)脂质体及制备方法和应用。
背景技术
乳腺癌一直被认为是低免疫原性的“冷”肿瘤,由于缺乏浸润性T细胞并渗透性较低,是最难清除的肿瘤类型之一,与患者预后不良相关。乳腺癌微环境具有高度炎性特征,炎性乳腺癌患者高度表达的IL-8和生长调节癌基因(GRO)趋化因子,激活信号转导和转录活化因子3(STAT3),诱导免疫抑制M2型巨噬细胞和肿瘤间充质细胞的形成,促使其变“冷”。如何将“冷”肿瘤转变为“热”肿瘤,阐明免疫系统复杂正反馈调控机制是个关键问题。
目前,针对乳腺癌治疗方法包括化疗放疗、外科手术治疗和药物治疗的三大主要手段。乳腺癌至今治疗的难点还是肿瘤的转移,不仅与肿瘤的扩散、浸润和耐药等有关,还与肿瘤新生血管生成、较差的抗原呈递能力以及抑制性肿瘤微环境等紧密相关。现如今涌现出针对乳腺癌新型的治疗方法如免疫治疗、靶向治疗以及新型辅助化疗等联合治疗,不同于传统肿瘤治疗方法,免疫疗法并不直接针对肿瘤细胞,而是通过调动体内的免疫应答来间接清除肿瘤细胞。
免疫抑制性肿瘤微环境(ITME)是导致肿瘤耐药性和难治性的主要原因,逆转免疫抑制微环境是提高抗肿瘤治疗效果的重要策略。其中M2型肿瘤相关巨噬细胞(M2-TAMs)分泌大量免疫抑制性细胞因子,抑制T细胞的免疫应答和肿瘤浸润,促进肿瘤发展转移和新生血管的生成,塑造免疫抑制微环境促使肿瘤“变冷”。M1型巨噬细胞能够分泌大量的促炎性因子,招募和活化其他的免疫细胞,逆转肿瘤免疫抑制微环境,促使肿瘤“变热”,提高治疗效率。因此,将肿瘤组织中的M2型巨噬细胞极化为M1型巨噬细胞将是一种逆转免疫抑制微环境和提高抗肿瘤效果的重要手段。
免疫激活剂寡脱氧核苷酸(CpG-ODN)是一段具有免疫刺激活性的寡核苷酸序列(序列:TCCATGACGTTCCTGACGTT),能有效引发哺乳动物免疫反应。CpG-ODN无法穿透细胞膜,很容易被核酸酶清除,通过全身给药可能会在血清中引发炎症反应。由于缺乏有效的给药途径,免疫激活剂CpG难以输送到M2巨噬细胞,而限制了体内应用。
温郁金挥发油中提取的榄香烯(ELE)具有广谱抗肿瘤活性,效率高且毒性低,可穿透血脑屏障,除了杀伤肿瘤作用外,还具有免疫保护作用。
核仁素(nucleolin,NCL)是一种多功能核仁磷蛋白,在细胞增殖、生长、细胞因子和核生物发生中具有重要的作用。这种蛋白质存在于增殖细胞(正常和恶性)的细胞核中,也存在于细胞质中,特别是肿瘤细胞表面有利于核酸适体AS1411的转运和细胞内化。AS1411是一种合成的四链体核酸酶抗性DNA寡核苷酸适体,在多种癌细胞中诱导增殖停滞和细胞死亡,但对正常细胞几乎没有影响。AS1411对NCL的高亲和力和特异性使其成为一种合适的靶向工具,可用于治疗性药物载荷递送纳米系统的功能化,以选择性地靶向肿瘤细胞。AS1411可作为靶向肿瘤细胞的有效靶向分子,与其他靶向策略相比。AS1411可靶向多种类型肿瘤,其非免疫原性特性,已在人体试验中证明了安全性,作为一种有效的靶向配体能提高药物递送的效率。在不久的将来,AS1411适配体纳米递送系统可成为临床医生对癌症诊断和治疗有效策略之一。
发明内容
本发明的一个目的在于提供了一种靶向M2型巨噬细胞CpG和榄香烯脂质体,包括免疫激活剂CpG脂质体和榄香烯ELE脂质体。
所述的CpG脂质体为表面修饰叶酸且负载有免疫激活剂CpG的脂质体,按照重量份数,每100份CpG脂质体中包括:1~5份磷脂、0.04~0.2份胆固醇、0.004~0.1份聚乙二醇衍生物、0.02~0.1份DSPE-PEG-叶酸、0.004~0.1份免疫激活剂CpG,余量为水。
所述的ELE脂质体为表面修饰叶酸和核酸适体AS1411,且负载有榄香烯的脂质体,按照重量份数,每100份ELE脂质体中包括:1~5份磷脂、0.04~0.2份胆固醇、0.004~0.1份聚乙二醇衍生物、0.15~0.75份榄香烯ELE、0.02~0.1份DSPE-PEG-叶酸、0.0005~0.002份胆固醇-AS1411,余量为水。
所述的磷脂为大豆磷脂、氢化磷脂、蛋黄磷脂、合成磷脂中的一种或多种;
所述的聚乙二醇衍生物为聚乙二醇维生素E琥珀酸酯,或二硬脂酰基磷脂酰乙醇胺-聚乙二醇;
所述的DSPE-PEG-叶酸为DSPE-PEG1000-叶酸、DSPE-PEG2000-叶酸、DSPE-PEG3000-叶酸中的一种或多种。
本发明的另一个目的在于提供CpG脂质体和ELE脂质体的制备方法。具体是:
按比例称取磷脂、胆固醇、聚乙二醇衍生物、DSPE-PEG-叶酸溶于无水乙醇,在50~60℃下加热至溶解,并持续加热至乙醇完全除尽,得到第一有机相Ⅰ;将CpG溶于双蒸水,然后加入第一有机相Ⅰ,高速剪切后再进行超声破碎,得到CpG脂质体。
按比例称取磷脂、胆固醇、聚乙二醇衍生物、DSPE-PEG-叶酸溶于无水乙醇,在50~60℃下加热至溶解,并持续加热至乙醇完全除尽,然后按比例加入榄香烯,充分溶解得到第二有机相II;将胆固醇-AS1411溶于双蒸水,然后加入第二有机相II,高速剪切后再进行超声破碎,得到ELE脂质体。
高速剪切的速率为8000~15000r/min,剪切时间20~40min;超声破碎条件为230~240W。
本发明的第三个目的是提供利用以上方法制备的CpG脂质体和ELE脂质体的应用,二者联合使用,抑制肿瘤的生长和逆转肿瘤免疫微环境。
本发明克服了CpG用于逆转肿瘤微环境时缺乏有效的给药途径和毒性较大的问题,提供了靶向M2型巨噬细胞的CpG及ELE脂质体。CpG脂质体在体内体外可逆转M2型巨噬细胞转变为M1型巨噬细胞,逆转ITME,促进细胞毒性T细胞的浸润,并有效抑制肿瘤生长,ELE质体不仅能抑制肿瘤细胞增殖,还可选择性抑制M2型巨噬细胞,将CpG及ELE脂质体协同作用于M2型巨噬细胞,提高药物的利用率,疗效得到明显增强。同时,显著减少了CpG副作用和榄香烯的刺激性,显著提高了安全性,有效避免了对正常脑细胞及其他组织器官的副作用。CpG和ELE脂质体联合使用具有显著的协同抗肿瘤作用,重塑肿瘤微环境,为乳腺癌等的治疗提供了新方法。
附图说明
图1为本发明一实施例制得的CpG/Lipo-FA的扫描电镜图;
图2为本发明一实施例制得的ELE/Lipo-AS1411-FA的扫描电镜图;
图3为不同取样时间段的CpG/Lipo-FA的粒径和分散性指数示意图;
图4为不同取样时间段的ELE/Lipo-AS1411-FA的粒径和分散性指数示意图;
图5为不同浓度游离ELE、ELE/Lipo、ELE/Lipo-AS1411、FA-ELE/Lipo-AS1411对4T1细胞的细胞毒性示意图;
图6为不同浓度的ELE对M1与M2型巨噬细胞的细胞毒性示意图。
具体实施方式
以下结合具体实施例对本发明的技术方案做进一步说明:
本说明书实施例所述的内容仅仅是对发明构思的实现形式的列举,本发明的保护范围不应当被视为仅限于实施例所陈述的具体形式,本发明的保护范围也及于本领域技术人员根据本发明构思所能够想到的等同技术手段。
实施例1.
按照重量份数,称取1份大豆磷脂、0.1份胆固醇、0.1份聚乙二醇维生素E琥珀酸酯、0.1份DSPE-PEG1000-叶酸溶于无水乙醇,在50℃下加热至溶解,并持续加热至乙醇完全除尽,得到第一有机相Ⅰ;将0.1份CpG溶于98.6份双蒸水,然后加入第一有机相Ⅰ,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为8000r/min,剪切时间40min;超声破碎条件为240W。
实施例2.
按照重量份数,称取1份大豆磷脂、1份蛋黄磷脂、0.2份胆固醇、0.04份二硬脂酰基磷脂酰乙醇胺-聚乙二醇、0.01份DSPE-PEG1000-叶酸、0.01份DSPE-PEG2000-叶酸溶于无水乙醇,在52℃下加热至溶解,并持续加热至乙醇完全除尽,得到第一有机相Ⅰ;将0.04份CpG溶于97.7份双蒸水,然后加入第一有机相Ⅰ,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为10000r/min,剪切时间30min;超声破碎条件为230W。
实施例3.
按照重量份数,称取1份大豆磷脂、1份氢化磷脂、1份合成磷脂、0.04份胆固醇、0.008份聚乙二醇维生素E琥珀酸酯、0.044份DSPE-PEG3000-叶酸溶于无水乙醇,在55℃下加热至溶解,并持续加热至乙醇完全除尽,得到第一有机相Ⅰ;将0.008份CpG溶于96.9份双蒸水,然后加入第一有机相Ⅰ,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为12000r/min,剪切时间25min;超声破碎条件为235W。
实施例4.
按照重量份数,称取4份氢化磷脂、0.06份胆固醇、0.004份二硬脂酰基磷脂酰乙醇胺-聚乙二醇、0.032份DSPE-PEG2000-叶酸溶于无水乙醇,在56℃下加热至溶解,并持续加热至乙醇完全除尽,得到第一有机相Ⅰ;将0.004份CpG溶于95.9份双蒸水,然后加入第一有机相Ⅰ,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为15000r/min,剪切时间20min;超声破碎条件为238W。
实施例5.
按照重量份数,称取5份蛋黄磷脂、0.15份胆固醇、0.06份聚乙二醇维生素E琥珀酸酯、0.01份DSPE-PEG1000-叶酸、0.01份DSPE-PEG2000-叶酸、0.01份DSPE-PEG3000-叶酸溶于无水乙醇,在60℃下加热至溶解,并持续加热至乙醇完全除尽,得到第一有机相Ⅰ;将0.06份CpG溶于94.7份双蒸水,然后加入第一有机相Ⅰ,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为10000r/min,剪切时间35min;超声破碎条件为240W。
实施例6.
按照重量份数,称取3份合成磷脂、0.12份胆固醇、0.01份二硬脂酰基磷脂酰乙醇胺-聚乙二醇、0.03份DSPE-PEG1000-叶酸、0.03份DSPE-PEG3000-叶酸溶于无水乙醇,在54℃下加热至溶解,并持续加热至乙醇完全除尽,得到第一有机相Ⅰ;将0.01份CpG溶于96.8份双蒸水,然后加入第一有机相Ⅰ,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为9000r/min,剪切时间40min;超声破碎条件为230W。
实施例7.
按照重量份数,称取1份大豆磷脂、0.1份胆固醇、0.1份聚乙二醇维生素E琥珀酸酯、0.1份DSPE-PEG1000-叶酸溶于无水乙醇,在50℃下加热至溶解,并持续加热至乙醇完全除尽,然后加入0.3495份榄香烯,得到第二有机相II;将0.0005份胆固醇-AS1411溶于98.35份双蒸水,然后加入第二有机相II,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为8000r/min,剪切时间40min;超声破碎条件为240W。
实施例8.
按照重量份数,称取1份大豆磷脂、1份蛋黄磷脂、0.2份胆固醇、0.04份二硬脂酰基磷脂酰乙醇胺-聚乙二醇、0.01份DSPE-PEG1000-叶酸、0.01份DSPE-PEG2000-叶酸溶于无水乙醇,在52℃下加热至溶解,并持续加热至乙醇完全除尽,然后加入0.15份榄香烯,得到第二有机相II;将0.001份胆固醇-AS1411溶于97.589份双蒸水,然后加入第二有机相II,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为10000r/min,剪切时间30min;超声破碎条件为230W。
实施例9.
按照重量份数,称取3份合成磷脂、0.12份胆固醇、0.01份二硬脂酰基磷脂酰乙醇胺-聚乙二醇、0.03份DSPE-PEG1000-叶酸、0.03份DSPE-PEG3000-叶酸溶于无水乙醇,在54℃下加热至溶解,并持续加热至乙醇完全除尽,然后加入0.75份榄香烯,得到第二有机相II;将0.0015份胆固醇-AS1411溶于96.0585份双蒸水,然后加入第二有机相II,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为9000r/min,剪切时间40min;超声破碎条件为230W。
实施例10.
按照重量份数,称取4份氢化磷脂、0.06份胆固醇、0.004份二硬脂酰基磷脂酰乙醇胺-聚乙二醇、0.032份DSPE-PEG2000-叶酸溶于无水乙醇,在56℃下加热至溶解,并持续加热至乙醇完全除尽,然后加入0.402份榄香烯,得到第二有机相II;将0.002份胆固醇-AS1411溶于95.5份双蒸水,然后加入第二有机相II,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为15000r/min,剪切时间20min;超声破碎条件为238W。
实施例11.
按照重量份数,称取1份大豆磷脂、1份氢化磷脂、1份合成磷脂、0.04份胆固醇、0.008份聚乙二醇维生素E琥珀酸酯、0.044份DSPE-PEG3000-叶酸溶于无水乙醇,在55℃下加热至溶解,并持续加热至乙醇完全除尽,然后加入0.507份榄香烯,得到第二有机相II;将0.001份胆固醇-AS1411溶于96.4份双蒸水,然后加入第二有机相II,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为12000r/min,剪切时间25min;超声破碎条件为235W。
实施例12.
按照重量份数,称取5份蛋黄磷脂、0.15份胆固醇、0.06份聚乙二醇维生素E琥珀酸酯、0.01份DSPE-PEG1000-叶酸、0.01份DSPE-PEG2000-叶酸、0.01份DSPE-PEG3000-叶酸溶于无水乙醇,在60℃下加热至溶解,并持续加热至乙醇完全除尽,然后加入0.6592份榄香烯,得到第二有机相II;将0.0008份胆固醇-AS1411溶于94.1份双蒸水,然后加入第二有机相II,高速剪切后再进行超声破碎,得到CpG脂质体;高速剪切的速率为10000r/min,剪切时间35min;超声破碎条件为240W。
实施例13.
以100克计,CpG脂质体(CpG/Lipo-FA)原料组成具体是:2克大豆磷脂、0.1克胆固醇、0.004克DSPE-PEG2000、0.04克CpG、0.04克DSPE-PEG2000-叶酸、余量为水。
以100克计,ELE脂质体(ELE/Lipo-AS1411-FA)原料组成具体是:2克大豆磷脂、0.1克胆固醇、0.004克DSPE-PEG2000、0.25克榄香烯、0.04克DSPE-PEG2000-叶酸、0.002克胆固醇-AS1411,余量为水。
上述CpG/Lipo-FA及ELE/Lipo-AS1411-FA的制备方法如下:
(1)有机相制备:按比例称取磷脂、胆固醇、聚乙二醇衍生物、DSPE-PEG-叶酸溶于乙醇,在60℃下加热至溶解,并持续加热至乙醇完全除尽,得到有机相Ⅰ;按比例称取榄香烯溶于有机相Ⅰ,得到有机相II。
(2)水相制备:称取CpG溶于双蒸水,得到水相Ⅰ;称取胆固醇-AS1411溶于双蒸水,得到水相II。
(3)超声波分散:将上述有机相Ⅰ加入水相Ⅰ、有机相II加入水相II中,高速剪切后再进行超声破碎,即得CpG/Lipo-FA及ELE/Lipo-AS1411-FA。
1.扫描电子显微镜(TEM)表征:
将新制备的脂质体溶液浓度稀释至0.01mg/mL,取一滴稀释后脂质体溶液约10μL滴于大小合适的硅片上,使用氮气对溶液缓慢吹平,使其均匀分散并加速干燥,干燥后喷金处理,再通过SEM观察脂质体的微观形态。图1是本实施例制得的CpG/Lipo-FA的扫描电镜图,图2是ELE/Lipo-AS1411-FA的扫描电镜图,SEM观察是将脂质体干燥后的形态,因为粒子水分的流失,经过SEM法测量的脂质体粒径可能略微小于粒径仪所测量的结果。
2.粒径、PDI和Zeta电位测定:
按照本实施例的方法,分别制备不含DSPE-PEG-叶酸、胆固醇-AS1411和不含DSPE-PEG-叶酸的脂质体(ELE/Lipo及ELE/Lipo-AS1411)。
取本实施例制备的CpG/Lipo-FA、ELE/Lipo-AS1411-FA及上述ELE/Lipo、ELE/Lipo-AS1411,使用纳米粒径仪对脂质体的粒径和PDI进行测量,取50μL脂质体于粒径仪的吸收池中,按照一定比例稀释后对粒径、PDI和Zeta电位测量。PDI是衡量脂质体粒径均匀度的重要指标,PDI越小,表明粒子分散程度越均匀。表1中可以看出,该方法制得的ELE/Lipo-AS1411、ELE/Lipo-AS1411-FA粒径适中、粒径分布均匀。
3.包封率和载药量:
取本实施例制备的CpG/Lipo-FA、ELE/Lipo-AS1411-FA及上述ELE/Lipo、ELE/Lipo-AS1411各0.2mL,加入0.45μm的PVDF微滤离心管中,20℃、12000rpm离心10min,截留未载入到脂质体的榄香烯液滴。取下层液加乙腈至25mL,检测下层药物含量为A。取上层液加乙腈至25mL,超声30min,检测上层药物含量为B。过膜后,采用HPLC法检测。制备脂质体时加入的辅料与用药量为Z。同时,利用NanoDrop2000测定CpG/Lipo-FA的包封率。计算包封率和载药量:包封率(EE)=A/(A+B)×100%,载药量(DLC)=A/Z×100%。
利用高效液相色谱法对包载榄香烯的脂质体进行载药量和包封率的检测。在表1中三种榄香烯脂质体榄香烯的包封率基本达到90%以上,相关靶向分子对其包封率也未出现明显影响,均具有较高的载药量。CpG/Lipo-FA包封率为(74.07±0.9018)%。
表1.不同脂质体表征(n=3;mean±SD)
类别 | Size(nm)±SD | PDI±SD | ZP(mV)±SD | EE(%)±SD | DLC(%)±SD |
ELE/Lipo | 118.0±2.715 | 0.1467±0.0046 | 20.90±1.443 | 93.31±0.2566 | 17.74±0.04583 |
ELE/Lipo-AS1411 | 105.0±0.7506 | 0.0997±0.0131 | 23.74±1.850 | 98.63±0.1137 | 18.90±0.01528 |
ELE/Lipo-AS1411-FA | 144.0±3.439 | 0.0337±0.0285 | 23.41±2.227 | 95.37±1.1000 | 19.17±0.21940 |
CpG/Lipo-FA | 158.4±3.568 | 0.1990±0.0062 | 24.55±1.323 | 74.07±0.9018 | N/A |
4.脂质体稳定性检测
取本实施例制备的ELE/Lipo-AS1411,ELE/Lipo-AS1411-FA,储存于4℃,分别于不同时间1d、7d和30d进行取样,通过粒径仪对制备的不同时间段的脂质体粒径、分散性指数PDI和Zeta电位进行测定。结果如图3和4,两种脂质体在30d内粒径和PDI在较小的范围内波动,其外观状态也无明显变现,未出现沉淀,分层等现象,表现出较好的稳定性。
实施例14.脂质体靶向能力考察:
按照实施例13的方法,在脂质体制备过程中加入FITC荧光标记物制备得到Lipo-FITC、Lipo-FA-FITC和AS1411-Lipo-FA-FITC。
(1)M2型巨噬细胞纳米递送系统的靶向性研究:
为了对所制备纳米脂质体的M2型巨噬细胞特异性靶向能力进行考察,采用带有FITC荧光标记与靶向分子相同结构的脂质体进行巨噬细胞的摄取实验。
实验结果显示,当加入IL-4将RAW264.7细胞诱导为M2型巨噬细胞后,加入Lipo-FITC和Lipo-FA-FITC与M2型巨噬细胞孵育,未诱导的RAW264.7细胞加入Lipo-FA-FITC孵育后和IL-4+Lipo-FITC组细胞内出现少量绿色荧光。但Lipo-FA-FITC与M2型巨噬细胞孵育后,细胞内绿色荧光强度明显增加并与其他两组具有显著性差异,即M2型巨噬细胞对空白Lipo-FITC摄取较少,但其可大量摄取Lipo-FA-FITC。这一结果表明,Lipo-FA是经M2型巨噬细胞表面FA受体介导的内吞作用入胞,具有M2型巨噬细胞靶向性。
(2)4T1肿瘤细胞靶向纳米递送系统的靶向摄取研究:
1)适配体AS1411与4T1肿瘤细胞特异性结合:
4T1细胞分别与AS1411-FAM和C-AS1411-FAM(AS1411为对照组,C-AS1411为随机核苷酸序列)孵育1小时后,共聚焦显微镜下观察荧光强度。结果显示AS1411-FAM组荧光强度最强,进一步证明了AS1411能特异性靶向。肿瘤细胞,并非核酸特异性吸附。
2)4T1细胞与靶向脂质体特异性结合:
4T1细胞表面高表达的核仁素蛋白可与富含G序列的核酸适配体AS141形成一个富含G-四分体结构,可作为一种新的肿瘤靶点。结果显示Lipo-FITC、Lipo-FA-FITC和AS1411-Lipo-FA-FITC与4T1肿瘤细胞共同孵育,Lipo-FITC和Lipo-FA-FITC组胞内均只有少量绿色荧光出现,表明4T1肿瘤细胞对Lipo-FITC和Lipo-FITC的摄取较少。但AS1411-Lipo-FA-FITC与4T1细胞孵育后,胞内可观察到大量绿色荧光,即AS1411-Lipo-FA-FITC可被4T1肿瘤细胞大量摄取。此外AS1411-Lipo-FA-FITC的荧光强度明显高于Lipo-FA-FITC,这一结果表明,AS1411和FA同时靶向修饰能明显增加肿瘤细胞对脂质体的靶向摄取,并对肿瘤细胞具有靶向性。
实施例15.CpG脂质体极化M2型巨噬细胞为M1型巨噬细胞的能力研究:
通过采用Real-time PCR技术检测RAW264.7的M1和M2型标志性基因转录水平的变化,验证CpG脂质体极化M2型巨噬细胞为M1型巨噬细胞的能力。首先将IL-4(20ng/mL)作用于RAW264.7细胞诱导为M2型巨噬细胞,并用PBS、CpG/Lipo和CpG/Lipo-FA处理细胞,收集细胞后提取RNA并逆转录为cDNA,并用Real-time PCR检测RAW264.7相关基因mRNA水平的变化。以iNOS mRNA为例,经IL-4诱导后加入CpG/Lipo和CpG/Lipo-FA后iNOS mRNA显著上调至对照组的1.620±0.098和1.697±0.2558倍,CpG/Lipo-FA上调更为显著,并具有统计学上的差异(vs.IL-4,***P<0.001);而Arg-1mRNA显著降低至对照组的1.295±0.1061和0.5733±0.4343倍,CpG/Lipo-FA下调更为显著并具有统计学上的差异(vs.IL-4,**P<0.01)。以上结果证明,CpG/Lipo-FA更为显著极化M2型巨噬细胞为M1型巨噬细胞,也进一步证明FA可靶向M2型巨噬细胞。
采用双重免疫荧光的方法对CpG/Lipo-FA逆转极化M2型巨噬细胞的能力进行验证。经IL-4诱导的M2型巨噬细胞相关蛋白Arg-1高表达,而iNOS表达较少。在加入CpG/Lipo和CpG/Lipo-FA处理M2型巨噬细胞后,M2型巨噬细胞标志物Arg-1表达量均出现降低,其荧光强度显示显著降低,而M1型巨噬细胞标志物iNOS表达量增加,表示CpG/Lipo和CpG/Lipo-FA均能极化M2型巨噬细胞为M1型,并且CpG/Lipo-FA优于CpG/Lipo,表明CpG能显著逆转M2型巨噬细胞为M1型巨噬细胞,且CpG/Lipo-FA极化M2型巨噬细胞为M1型巨噬细胞的作用最强。
实施例16.肿瘤靶向脂质体的细胞毒性研究:
通过CCK-8法进行榄香烯对4T1乳腺癌细胞毒性研究。将4T1细胞(2×104个/孔)接种于96孔板中。培养24小时后,用含ELE、ELE/Lipo、ELE/Lipo-AS1411、FA-ELE/Lipo-AS1411的RMPI-1640培养液(药物浓度分别为10μg/mL、20μg/mL、40μg/mL、80μg/mL、和160μg/mL)处理细胞24小时。ELE使用含有1‰乙醇培养液溶解稀释,未处理的细胞作为对照。随后每个孔加入100μL10%的CCK-8溶液,孵育2小时。使用酶标仪测定450nm处吸光度值,采用Graphpad9.0计算分析各组药物的IC50值。不同浓度游离ELE、ELE/Lipo、ELE/Lipo-AS1411、FA-ELE/Lipo-AS1411对4T1细胞的细胞毒性示意图。n=3,x±SD,**P<0.01,***P<0.001,****P<0.0001(vs.ELE);#P<0.05,###P<0.001(vs.ELE/Lipo-AS1411)。如图5所示,各药物作用细胞24小时后,其IC50分别为921.4μg/mL,67.58μg/mL,44.78μg/mL和35.02μg/mL。由此可得出FA-ELE/Lipo-AS1411在4个浓度下对4T1细胞具有明显的细胞毒性,并具有显著性差异;在20μg/mL、40μg/mL以及80μg/mL浓度下FA-ELE/Lipo-AS1411能显著降低细胞的存活率(vs.ELE/Lipo-AS1411,***P<0.001),结果证实FA-ELE/Lipo-AS1411能增强ELE对肿瘤细胞的毒性作用。
通过CCK-8法进行榄香烯对M1和M2巨噬细胞毒性研究。预先对RAW264.7细胞进行处理使其极化为M1和M2型巨噬细胞,将M1和M2型巨噬细胞(2×105个/孔)接种在96孔板中。培养24小时后,用100μL含ELE的DMEM高糖培养液(药物浓度分别为10μg/mL、20μg/mL、40μg/mL、80μg/mL、和160μg/mL)处理细胞24小时。ELE使用含有1‰乙醇培养液溶解稀释,未处理的细胞作为对照。随后每个孔加入100μL的10%CCK-8溶液,孵育2小时。使用酶标仪测定450nm处吸光度值,采用Graphpad Prism9.0计算分析各组药物的IC50值。图6为不同浓度的ELE对M1与M2型巨噬细胞的细胞毒性示意图。n=3,x±SD,*P<0.05,***P<0.001,****P<0.0001。如图6所示,ELE在5种浓度下对M2型巨噬细胞具有选择性毒性作用,并具有统计学意义(均低于*P<0.05)。因此,间接证明ELE与CpG/Lipo-FA能够共同逆转和抑制肿瘤抑制性M2型巨噬细胞。
实施例17.M2型巨噬细胞靶向脂质体体内抗肿瘤药效研究:
将处于对数生长期的4T1乳腺癌细胞消化收集后,离心,重悬于PBS中并调整细胞密度,置于冰盒中备用。雌性BALB/c小鼠(雌性,4-6周龄,SPF级别)饲养后1周后,麻醉,待接种肿瘤部位脱毛并消毒,在该部位皮下注射100μL细胞悬浮液。
(1)CpG/Lipo-FA抗肿瘤药效:以接种肿瘤当天为第0d,接种第8d时将小鼠分为4组,每组4只小鼠,分别为Saline组、Lipo组、CpG/Lipo组和CpG/Lipo-FA组,CpG组,给药剂量为每只小鼠4μg,均为瘤内注射给药。给药时,隔天使用电子天平称量小鼠体重,并用游标卡尺测量肿瘤的最大直径记为A(mm)和最小直径记为B(mm),按照V=A×B2/2计算小鼠肿瘤体积V(mm3),最后一次给药后隔天取出肿瘤并进行称重。
结果显示,模型对照组生长速度快,在接种肿瘤第14d,肿瘤体积急速增长。在给药结束后第二天,与Saline组相比,CpG/Lipo-FA组肿瘤体积和肿瘤重量显著减少,具有明显的抗肿瘤治疗的效果,并对小鼠的体重无明显影响,表明无显著毒性作用。
(2)CpG/Lipo-FA联合AS1411-ELE/Lipo-FA抗肿瘤药效:以接种肿瘤当天为第0d,接种第8d时将小鼠分为6组,每组4只小鼠,分别为Saline组、Lipo组、CpG/Lipo-FA组、AS1411-ELE/Lipo-FA组、AS1411-ELE/Lipo-FA(7d)+CpG/Lipo-FA(7d)组(先连续注射AS1411-ELE/Lipo-FA七天,后注射CpG/Lipo-FA七天,称为方案A)以及AS1411-ELE/Lipo-FA(1d)+CpG/Lipo-FA(1d)组(先注射AS1411-ELE/Lipo-FA一天,后注射CpG/Lipo-FA一天,共7次,称为方案B)。ELE给药剂量为50mg/Kg(腹腔注射给药),CpG给药剂量为每只小鼠4μg(瘤内注射给药)。隔天用电子天平称量小鼠体重,并用游标卡尺测量肿瘤的最大直径记为A(mm)和最小直径记为B(mm),按照V=A×B2/2公式计算小鼠肿瘤体积V(mm3),同时计算各治疗组肿瘤抑制率,肿瘤抑制率(%)=(V对照-V)/V对照×100%。
结果显示,模型组小鼠肿瘤生长速度快,在接种12d左右进入快速生长期,肿瘤体积急速生长,在第14d肿瘤生长至1000mm3左右。与模型组相比,CpG/Lipo-FA组和AS1411-ELE/Lipo-FA组分别显示出一定地抗肿瘤作用,其肿瘤抑制率分别为52.58%和61.13%。ELE脂质体与CpG脂质体联合使用通过不同的给药间隔探究其抗肿瘤效果,结果显示,在结束治疗后AS1411-ELE/Lipo-FA(7d)+CpG/Lipo-FA(7d)(A)组与AS1411-ELE/Lipo-FA(1d)+CpG/Lipo-FA(1d)(B)组也均显示抗肿瘤特性,方案B效果更为明显,肿瘤抑制率分别为52.55%和75.24%。但是,肿瘤抑制率出现显著性差异,这可能与榄香烯抑炎作用相关,连续注射7d榄香烯虽然可以达到肿瘤治疗的作用,但是CpG/Lipo-FA不可逆转榄香烯抑炎作用。然而,结果显示两药交替给药可增强肿瘤治疗效果,也进一步验证CpG/Lipo-FA(4μg/per)可逆转间隔注射榄香烯导致的抑制性肿瘤微环境。
总之,榄香烯脂质体与CpG脂质体联合使用对乳腺癌具有较好的药效。同时应用榄香烯脂质体与CpG脂质体,协同作用于肿瘤细胞,可以抑制肿瘤的生长和逆转肿瘤免疫微环境,增强了疗效,显著减少了CpG副作用和榄香烯的刺激性,提高了安全性,对乳腺癌具有较好的临床转化应用前景。
Claims (7)
1.一种靶向M2型巨噬细胞的免疫激活剂CpG脂质体和榄香烯ELE脂质体,其特征在于:
所述的CpG脂质体为表面修饰叶酸且负载有免疫激活剂CpG的脂质体,按照重量份数,每100份CpG脂质体中包括:1~5份磷脂、0.04~0.2份胆固醇、0.004~0.1份聚乙二醇衍生物、0.02~0.1份DSPE-PEG-叶酸、0.004~0.1份免疫激活剂CpG,余量为水;
所述的ELE脂质体为表面修饰叶酸和核酸适体AS1411,且负载有榄香烯的脂质体,按照重量份数,每100份ELE脂质体中包括:1~5份磷脂、0.04~0.2份胆固醇、0.004~0.1份聚乙二醇衍生物、0.15~0.75份榄香烯ELE、0.02~0.1份DSPE-PEG-叶酸、0.0005~0.002份胆固醇-AS1411,余量为水。
2.如权利要求1所述的免疫激活剂CpG脂质体和榄香烯ELE脂质体,其特征在于:所述的磷脂为大豆磷脂、氢化磷脂、蛋黄磷脂、合成磷脂中的一种或多种。
3.如权利要求1所述的免疫激活剂CpG脂质体和榄香烯ELE脂质体,其特征在于:所述的聚乙二醇衍生物为聚乙二醇维生素E琥珀酸酯,或二硬脂酰基磷脂酰乙醇胺-聚乙二醇。
4.如权利要求1所述的免疫激活剂CpG脂质体和榄香烯ELE脂质体,其特征在于:所述的DSPE-PEG-叶酸为DSPE-PEG1000-叶酸、DSPE-PEG2000-叶酸、DSPE-PEG3000-叶酸中的一种或多种。
5.如权利要求1-4任一权利要求所述的免疫激活剂CpG脂质体和榄香烯ELE脂质体的制备方法,其特征在于:
按比例称取磷脂、胆固醇、聚乙二醇衍生物、DSPE-PEG-叶酸溶于无水乙醇,在50~60℃下加热至溶解,并持续加热至乙醇完全除尽,得到第一有机相Ⅰ;将CpG溶于双蒸水,然后加入第一有机相Ⅰ,高速剪切后再进行超声破碎,得到CpG脂质体;
按比例称取磷脂、胆固醇、聚乙二醇衍生物、DSPE-PEG-叶酸溶于无水乙醇,在50~60℃下加热至溶解,并持续加热至乙醇完全除尽,然后按比例加入榄香烯,充分溶解得到第二有机相Ⅱ;将胆固醇-AS1411溶于双蒸水,然后加入第二有机相Ⅱ,高速剪切后再进行超声破碎,得到ELE脂质体。
6.如权利要求5所述的制备免疫激活剂CpG脂质体和榄香烯ELE脂质体的方法,其特征在于:高速剪切的速率为8000~15000r/min,剪切时间20~40min;超声破碎条件230~240W。
7.如权利要求1-4任一权利要求所述的免疫激活剂CpG脂质体和榄香烯ELE脂质体联合使用,抑制肿瘤的生长和逆转肿瘤免疫微环境。
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