CN117122677A - 含有至少2个硫原子的杂环化合物在制备纳米疫苗中的应用及制得的纳米疫苗 - Google Patents
含有至少2个硫原子的杂环化合物在制备纳米疫苗中的应用及制得的纳米疫苗 Download PDFInfo
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Abstract
本发明涉及免疫治疗或疫苗防治技术领域,尤其涉及含有两个或两个以上硫原子的杂环化合物及其制备纳米疫苗中的应用。本发明提供了含有至少两个硫原子且能够与多肽以共价或非共价连接的杂环化合物在制备纳米疫苗中的应用。该化合物与抗原自组装制得的纳米颗粒能够通过跨膜方式进入树突状细胞质内,提高对抗原及免疫佐剂的摄取效率。由于在进入细胞的过程中,能有效躲避或降低溶酶体内酶体对抗原或核酸佐剂生物降解,故而本发明的纳米疫苗能够高效激活树突状细胞,并提高对抗原的交叉呈递作用,进而有效激活CD8+T细胞,并促进T细胞增殖。因此,本发明的纳米疫苗能够利用高效的免疫激活与免疫调节作用预防肿瘤细胞增殖与病毒感染。
Description
本申请是2020年4月24日申请的发明名称为“含有至少2个硫原子的杂环化合物在制备纳米疫苗中的应用及制得的纳米疫苗”、申请号为202010330996.8的发明专利申请的分案申请。
技术领域
本发明涉及免疫治疗或疫苗防治技术领域,尤其涉及含有两个或两个以上硫原子的杂环化合物在制备纳米疫苗中的应用及制得的纳米疫苗。
背景技术
近年来,免疫疫苗的研制与开发在对于癌症及病毒感染的综合防治上已取得显著性进展,并在抗肿瘤或抗病毒感染的方面具备较高的临床应用价值。目前,各国已相继开发各类抗肿瘤或抗病毒疫苗用于疾病预防或治疗且已进入临床I、II期,有些已被美国和欧洲获批用于临床。然而此类疫苗在激发抗原呈递细胞成熟与增强交叉呈递方面仍面临一些挑战,例如简单混合的佐剂与抗原制剂被树突状细胞摄入效率不高,且被摄入的抗原或佐剂在进入胞内溶酶体等细胞器后,更易被酶降解而降低或失去对树突状细胞的激活,进而减弱抗原交叉呈递效应,无法有效激活机体免疫系统应对外界或体内肿瘤或病毒侵染,更无法应对特定类型肿瘤或病毒感染。
高效的抗原呈递及其激活免疫系统,发挥免疫调节作用,主要依赖于抗原呈递细胞是否能够高效摄入抗原,是否能被有效激活而发挥其对抗原的交叉呈递作用。现有纳米疫苗主要以胞吞-溶酶体途径进入树突状细胞内,即在被树突状细胞内吞后,会形成内吞小体或小泡,再与胞内溶酶体融合,而溶酶体内的核酸酶或蛋白水解酶会剪切或水解纳米疫苗释放出的核酸佐剂或抗原肽,降低核酸佐剂对树突状细胞的刺激与促激活作用,影响其对抗原肽的交叉呈递作用,与对未成熟T淋巴细胞的激活与促增殖作用,减弱机体对抗原肽所产生的细胞免疫应答,导致肿瘤免疫治疗效果差。
近年来,基于脂质体包埋技术开发的脂质体mRNA或多肽疫苗虽能在一定程度上降低溶酶体内酶体对其有效成份的降解,但仍然依赖于内吞-溶酶体途径摄入淋巴细胞内,无法躲避溶酶体内酶对其降解作用;且由于脂质体表面聚乙二醇能够有效抵抗或阻碍巨噬细胞等淋巴细胞对其内吞,进而降低了对抗原的摄取效率及淋巴细胞的激活比例,从而降低对抗原的交叉呈递作用。因此,迫切需要开发一种新的抗原摄取与呈递模式用于高效激活淋巴细胞与提高交叉呈递效率。
发明内容
有鉴于此,本发明要解决的技术问题在于提供含有二硫键的杂环化合物在制备纳米疫苗中的应用及制得的纳米疫苗,将硫醇类化合物与肿瘤新生多肽抗原或病毒多肽抗原以核酸佐剂,通过静电力,氢键与范德华力作用制备合成纳米疫苗,该疫苗能通过跨膜途径高效率进入树突状细胞胞质内部而降低其内部多肽抗原与核酸被溶酶体内蛋白酶或核酸酶降解。
本发明提供了式I化合物在制备纳米疫苗中的应用;
A-L-F
式I
其中,A为含有两个或两个以上S原子的杂环基;
L为无或为中间连接基团;
F为能够与多肽以共价或非共价连接的基团。
式I化合物中含有至少两个硫原子,其能够与核酸佐剂及新生抗原肽自组装形成的纳米疫苗。其中F基团与多肽连接,而A基团与细胞膜间进行硫醇交换,并介导纳米疫苗跨膜直接进入树突状细胞质。这种进入细胞内的方式不经胞吞-溶酶体途径,当其进入细胞后,在树突状细胞质中电解质缓冲液作用下,解体而释放其中的核酸佐剂与新生抗原肽。其释放的核酸佐剂能更有效的刺激树突状细胞成熟,增强树突状细胞对新生抗原肽的交叉呈递作用,增强对未成熟的T淋巴细胞的激活与促增殖能力,增强机体对新生抗原肽表达的肿瘤细胞特异性细胞免疫应答,增强T细胞依赖的肿瘤特异性细胞免疫治疗。
在本发明中,A中所述杂环基为4~8元环。
一些实施例中,所述A为:
在本发明中,L为-(CH2)n-、其中n=1~5;R为-H、C1~5的烷基、/>m=1~50。
一些实施例中,所述L为:
在本发明中,共价连接方式可选自:点击化学反应、交联反应;非共价连接的方式包括:静电作用、疏水相互作用、亲和作用。
其中,可通过点击化学反应与多肽连接的F基团包括:
可通过交联反应与多肽连接的F基团包括:
可通过其他反应方式与多肽形成共价连接的F基团包括:
可通过静电相互作用与多肽连接的F基团包括:
可通过疏水相互作用与多肽连接的F基团包括:
可通过亲和作用与多肽连接的F基团包括:
一些实施例中,所述F为:
一些实施例中,所述A为L为/> F为/>一些具体实施例中,所述式I化合物为/>
本发明提供的式I化合物都可以用于与多肽制备纳米颗粒,但实验表明,针对不同的多肽需要选择不同结构的式I化合物,否则形成的颗粒粒径过大,不能被细胞摄取。
本发明还提供了一种纳米颗粒,其原料包括式I化合物,核酸佐剂和抗原肽;
A-L-F
式I
其中,A为含有两个或两个以上S原子的杂环基;
L为无或为中间连接基团;
F为能够与多肽以共价或非共价连接的基团。
在本发明中,所述抗原肽选自肿瘤抗原、细菌抗原或病毒抗原。
本发明中,所述肿瘤抗原为肿瘤新生抗原,选自膀胱癌、血癌、骨癌、脑癌、乳腺癌、中枢神经系统癌症、宫颈癌、结肠癌、子宫内膜癌、食管癌、胆囊癌、胃肠道癌、外生殖器癌、泌尿生殖道癌、头癌、肾癌、喉癌、肝癌、肺癌、肌肉组织癌症、颈癌、口腔或鼻黏膜癌、卵巢癌、胰腺癌、前列腺癌、皮肤癌、脾癌、小肠癌、大肠癌、胃癌、睾丸癌和/或甲状腺癌的新生抗原;
本发明中,所述病毒抗原为流感病毒抗原、埃博拉病毒抗原、冠状病毒抗原、肝炎病毒抗原、、腮腺炎病毒抗原、水痘-带状疱疹病毒抗原、麻疹病毒抗原、风疹病毒抗原、流感病毒抗原和/或禽流感病毒抗原;
所述冠状病毒为新型冠状病毒COVID-19、SARS病毒;所述肝炎病毒为甲型肝炎病毒、乙型肝炎病毒或丙型肝炎病毒;所述流感病毒为甲型流感病毒、乙型流感病毒或丙型流感病毒,其中甲型流感病毒的血凝素突起的类型包括H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14或H15,神经氨酸酶突起类型为N1、N2、N3、N4、N5、N6、N7、N8或N9。
本发明中,所述细菌抗原为结合分枝杆菌抗原、葡萄球菌抗原、链球菌抗原、肺炎双球菌抗原、炭疽杆菌抗原、白喉杆菌抗原、变形杆菌抗原、百日咳杆菌抗原、霍乱弧菌抗原、脑膜炎双球菌抗原和/或伤寒杆菌抗原。
在一些具体实施例中,所述肿瘤抗原为肝癌抗原,具体为肝癌H22多肽抗原,更具体的,其抗原序列为HTDAHAQAFAALFDSMH。所述病毒抗原为冠状病毒抗原,具体为新型冠状病毒抗原;更具体的,其抗原序列为SYYSLLMPI、RYVLMDGSI、AYANSVFNI、TYASALWEI或GYLKLTDNV。
本发明实施例中,所述抗原的等电点<6.5。
本发明实施例中,所述核酸佐剂为CpG-ODN、Poly I:C。
在本发明中,在多肽抗原的N端添加Cy3荧光基团,在核酸佐剂的3’端标记FAM荧光基团。荧光基团用于定位纳米疫苗在树突状细胞中的位置。而实验表明添加荧光基团与否对于激活效果并无显著影响。
本发明实施例中,所述的纳米颗粒中,式I化合物,核酸佐剂和抗原肽的摩尔比为(50~100):(0.5~1):(20~50)。在一些具体实施例中,纳米颗粒中,式I化合物,核酸佐剂和抗原肽的摩尔比为50:0.5:20。
本发明所述纳米颗粒,通过带正电荷的式I化合物中的胍基与带负电荷的核酸佐剂中的磷酸根形成盐桥,并再与带负电荷的新生抗原肽的氨基酸,通过分子间的静电力相互作用,自组装制备合成。其中,核酸佐剂可以是CpG-ODN(鼠源或人源),也可以是Poly I:C(鼠源或人源);新生抗原肽的等电点(pI)小于6。
一些实施例中,所述纳米颗粒的制备方法包括:纳米疫苗制备方法:将式I化合物,核酸佐剂和多肽抗原与TM缓冲液(pH 7.4)混合,37℃,100rpm搅拌15min,后在去离子水中透析,制得含有纳米颗粒溶液。
式I化合物,核酸佐剂和多肽抗原溶液总体积与所述TM缓冲液的体积比为1:60~70。
具体实施例中,146.2mM的式I化合物甲醇溶液、100mM的核酸佐剂溶液、2.5mg/mL的多肽抗原溶液,按照体积比为1:1:1的比例混合,然后混合液总体积60~70倍的TM缓冲液(pH 7.4)混合。
本发明还提供了一种纳米疫苗,其包括本发明所述的纳米颗粒和疫苗中可接受的辅料。
所述疫苗中可接受的辅料包括:溶剂、渗透压调节剂。优选的,本发明制备的纳米疫苗包括本发明所述的纳米颗粒与氯化钠注射液。
本发明中所述纳米疫苗通过硫醇交换介导的跨膜方式被摄入树突状细胞质内,并因胞质内缓冲液而解体释放多肽抗原、核酸佐剂,进而辅助并高效激活树突状细胞,提高其抗原交叉呈递作用,有效激活CD8+T细胞。
本发明还提供了一种抗肿瘤的方法,其为给予本发明所述的纳米疫苗。
本发明还提供了一种抗病毒和/或细胞感染的方法,其为给予本发明所述的纳米疫苗。
本发明涉及免疫治疗或疫苗防治技术领域,尤其涉及含有两个或两个以上硫原子的杂环化合物及其制备纳米疫苗中的应用。本发明提供了含有至少两个硫原子且能够与多肽以共价或非共价连接的杂环化合物在制备纳米疫苗中的应用。该化合物与核酸佐剂、抗原自组装制得纳米颗粒。该纳米颗粒能够通过跨膜方式进入树突状细胞质内,提高对抗原及免疫佐剂的摄取效率。由于在进入细胞的过程中,能有效躲避或降低溶酶体内酶体对抗原或核酸佐剂生物降解,故而本发明的纳米疫苗能够高效激活树突状细胞,并提高对抗原的交叉呈递作用,进而有效激活CD8+T细胞,并促进T细胞增殖。因此,本发明的纳米疫苗能够利用高效的免疫激活与免疫调节作用预防肿瘤细胞增殖与病毒感染。
附图说明
图1示纳米疫苗制备与抗肿瘤示意图;
图2示实施例3中纳米疫苗的扫描电镜分析;
图3示实施例3中纳米疫苗和对照纳米疫苗的粒径分析;
图4示实施例3中纳米疫苗与树突状细胞共培养30min后,跨膜纳米疫苗摄入树突状细胞质内的共聚焦荧光成像图像,其中红光为多肽抗原标记荧光,绿光为核酸佐剂标记荧光,标尺为20μm;
图5示实施例3中纳米疫苗跨膜纳米疫苗摄入树突状细胞质内的共聚焦荧光成像图像,其中绿光为溶酶体指示剂,红光为多肽抗原标记荧光,标尺为20μm;
图6示实施例4中纳米疫苗与树突状细胞共培养30min后,跨膜纳米疫苗摄入树突状细胞质内的共聚焦荧光成像图像,其中红光为多肽抗原标记荧光,绿光为核酸佐剂标记荧光,标尺为10μm;
图7示实施例4中纳米疫苗跨膜纳米疫苗摄入树突状细胞质内的共聚焦荧光成像图像,其中绿光为溶酶体指示剂,红光为多肽抗原标记荧光,标尺为10μm;
图8示实施例5中纳米疫苗与树突状细胞共培养30min后,跨膜纳米疫苗摄入树突状细胞质内的共聚焦荧光成像图像,其中红光为多肽抗原标记荧光,绿光为核酸佐剂标记荧光,标尺为10μm;
图9示实施例5中纳米疫苗跨膜纳米疫苗摄入树突状细胞质内的共聚焦荧光成像图像,其中绿光为溶酶体指示剂,红光为多肽抗原标记荧光,标尺为10μm;
图10示实施例3制备的纳米疫苗与树突状细胞共培养3d后,树突状细胞的激活;
图11示实施例3制备的纳米疫苗中激活的树突状细胞与CD8+T细胞共培养2d后的增殖;
图12示实施例3制备的纳米疫苗对H22鼠源肝癌抗肿瘤作用的小鼠瘤体生长曲线。
具体实施方式
本发明提供了含有至少两个S原子的杂环化合物在制备纳米疫苗中的应用及制得的纳米疫苗,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明采用的试材皆为普通市售品,皆可于市场购得。
(1)化学试剂:合成所有核酸佐剂序列(序列见表1)由上海生工生物工程股份有限公司制备并利用HPLC纯化方法合成。LysoTracker和Hoechst33342购于美国赛默飞世尔科技公司。超纯水由Milli-Q水净化系统制备(18.2MΩ)。
(2)细胞系和细胞培养:鼠源肝癌细胞H22购于美国模式培养物保藏所(ATCC)。H22细胞培养于DMEM培养基(HyClone)添加终浓度10%胎牛血清(Gibco)和终浓度100IU/mL青霉素-链霉素,在37℃含5% CO2的气氛中进行培养。
(3)所需的鼠源核酸佐剂CpG-ODN作为激活树突状细胞的佐剂,碱基序列如下:
表1实施例中采用的CpG-ODN碱基序列表
名称 | 碱基序列(5’→3’) |
FAM荧光标记CpG-ODN | TCCATGACGTTCCTGACGTT-FAM |
无标记CpG-ODN | TCCATGACGTTCCTGACGTT |
化合物:
化合物1:于实施例1合成;
化合物2:于实施例2合成;
化合物3:购自麦克林生化科技有限公司。
下面结合实施例,进一步阐述本发明:
实施例1
将含有N,N'-羰基二咪唑与硫辛酸的二氯甲烷溶液,在0℃下逐步滴入乙二胺的二氯甲烷中,0℃搅拌1h以及置于室温再搅拌1h,并用无水硫酸钠除去水份,再利用减压浓缩而获得油状物。将油状物溶于含有1H-吡唑-1-甲脒盐酸盐的二氯甲烷溶液中,搅拌后,减压蒸馏,并将获得的沉淀物溶于甲醇,再利用乙醚洗涤沉淀,最终获得化合物1。
实施例2
分子1的无水CH2Cl2溶液中加入1,1-羰基二咪唑(CDI)活化,然后加入分子2进行缩合反应,得到分子3。在分子3的CH2Cl2溶液中加入三氟乙酸(TFA)脱去叔丁氧基羰基保护基团得到分子4。在无水CH2Cl2中,加入分子4、乙酰丙酸、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)、和N,N-二异丙基乙胺(DIPEA),通过缩合反应得到分子5。
实施例3
纳米疫苗制备方法:将实施例1制得的化合物1(浓度为146.2mM),100mM的核酸佐剂(CpG-ODN),2.5mg/mL的多肽抗原(小鼠肝癌细胞H22多肽抗原,氨基酸序列为HTDAHAQAFAALFDSMH,N端连接Cy3标记,等电点为5.71)按照体积比为1:1:1的比例混合,并投入其总体积60~70倍的TM buffer缓冲溶液中(pH 7.4),在37℃混合搅拌15min,并在去离子水中透析24h,去除未载入的核酸佐剂或多肽抗原,最终获得纳米疫苗。
纳米疫苗经震荡透析制备后,测其粒径与扫描电子显微镜表征纳米疫苗成功制备。结果显示,CpG-ODN、多肽抗原与化合物1在TM buffer(pH=7.5)缓冲液中可以形成球形纳米疫苗,其粒径约为100nm(如图2所示)其中有效成分CpG-ODN浓度为8.8μg/mL,多肽抗原浓度为6.11μg/mL。
对比例
将实施例1制得的化合物1(浓度为146.2mM),100mM的核酸佐剂(CpG-ODN),2.5mg/mL的对比多肽抗原(氨基酸序列为YKYRYLRHGKLR,N端连接Cy3标记,等电点为10.55)按照体积比为1:1:1的比例混合,并投入其总体积60~70倍的TM buffer缓冲溶液中(pH 7.4),在37℃混合搅拌15min,并在去离子水中透析24h,去除未载入的核酸佐剂或多肽抗原,最终获得对照纳米疫苗。经震荡透析制备后,应用纳米粒度仪表征纳米疫苗和对照疫苗,结果显示纳米疫苗的粒径约为100nm,而对照纳米疫苗的粒径约为600nm。表明由于多肽等电点的差异,形成的对照纳米疫苗的尺寸太大,难以实现高效细胞摄取。
实施例4
纳米疫苗制备方法:将实施例2制得的化合物2(浓度为146.2mM),100mM的核酸佐剂(CpG-ODN),2.5mg/mL的多肽抗原(新冠病毒抗原多肽1,氨基酸序列为FYYVWKSYV,N端连接Cy3标记,等电点为8.43)按照体积比为1:1:1的比例混合,并投入其总体积60~70倍的TMbuffer缓冲溶液中(pH 7.4),在37℃混合搅拌15min,并在去离子水中透析24h,去除未载入的核酸佐剂或多肽抗原,最终获得纳米疫苗。
纳米疫苗经震荡透析制备后,测其粒径与扫描电子显微镜表征纳米疫苗成功制备。其中有效成分CpG-ODN浓度为8.8μg/mL,多肽抗原浓度为6.11μg/mL。
实施例5
纳米疫苗制备方法:将化合物3(浓度为146.2mM),100mM的核酸佐剂(CpG-ODN),2.5mg/mL的多肽抗原(新冠病毒多肽抗原2,氨基酸序列为KYTQLCQYL,N端连接Cy3标记,等电点为8.5)按照体积比为1:1:1的比例混合,并投入其总体积60~70倍的TM buffer缓冲溶液中(pH 7.4),在37℃混合搅拌15min,并在去离子水中透析24h,去除未载入的核酸佐剂或多肽抗原,最终获得纳米疫苗。
纳米疫苗经震荡透析制备后,测其粒径与扫描电子显微镜表征纳米疫苗成功制备。其中有效成分CpG-ODN浓度为8.8μg/mL,多肽抗原浓度为6.11μg/mL。
实施例6
纳米疫苗制备方法:将实施例1制得的化合物1(浓度为146.2mM),100mM的核酸佐剂(CpG-ODN),2.5mg/mL的多肽抗原(新冠病毒多肽抗原3,氨基酸序列为SYYSLLMPI,等电点为5.55)按照体积比为1:1:1的比例混合,并投入其总体积60~70倍的TM buffer缓冲溶液中(pH 7.4),在37℃混合搅拌15min,并在去离子水中透析24h,去除未载入的核酸佐剂或多肽抗原,最终获得纳米疫苗。
纳米疫苗经震荡透析制备后,测其粒径与扫描电子显微镜表征纳米疫苗成功制备。其中有效成分CpG-ODN浓度为7.8μg/mL,多肽抗原浓度为7.41μg/mL。
实施例7
纳米疫苗制备方法:将实施例1制得的化合物1(浓度为146.2mM),100mM的核酸佐剂(CpG-ODN),2.5mg/mL的多肽抗原(新冠病毒多肽抗原4,氨基酸序列为RYVLMDGSI,等电点为6.21)按照体积比为1:1:1的比例混合,并投入其总体积60~70倍的TM buffer缓冲溶液中(pH 7.4),在37℃混合搅拌15min,并在去离子水中透析24h,去除未载入的核酸佐剂或多肽抗原,最终获得纳米疫苗。
纳米疫苗经震荡透析制备后,测其粒径与扫描电子显微镜表征纳米疫苗成功制备。其中有效成分CpG-ODN浓度为7.1μg/mL,多肽抗原浓度为5.82μg/mL。
实施例8
纳米疫苗制备方法:将实施例1制得的化合物1(浓度为146.2mM),100mM的核酸佐剂(CpG-ODN),2.5mg/mL的多肽抗原(新冠病毒多肽抗原5,氨基酸序列为AYANSVFNI,等电点为5.55)按照体积比为1:1:1的比例混合,并投入其总体积60~70倍的TM buffer缓冲溶液中(pH 7.4),在37℃混合搅拌15min,并在去离子水中透析24h,去除未载入的核酸佐剂或多肽抗原,最终获得纳米疫苗。
纳米疫苗经震荡透析制备后,测其粒径与扫描电子显微镜表征纳米疫苗成功制备。有效成分CpG-ODN浓度为9.1μg/mL,多肽抗原浓度为7.21μg/mL。
实施例9
纳米疫苗制备方法:将实施例1制得的化合物1(浓度为146.2mM),100mM的核酸佐剂(CpG-ODN),2.5mg/mL的多肽抗原(新冠病毒多肽抗原6,氨基酸序列为TYASALWEI,等电点为3.75)按照体积比为1:1:1的比例混合,并投入其总体积60~70倍的TM buffer缓冲溶液中(pH 7.4),在37℃混合搅拌15min,并在去离子水中透析24h,去除未载入的核酸佐剂或多肽抗原,最终获得纳米疫苗。
纳米疫苗经震荡透析制备后,测其粒径与扫描电子显微镜表征纳米疫苗成功制备。其中有效成分CpG-ODN浓度为7.1μg/mL,多肽抗原浓度为7.67μg/mL。
实施例10
纳米疫苗制备方法:将实施例1制得的化合物1(浓度为146.2mM),100mM的核酸佐剂(CpG-ODN),2.5mg/mL的多肽抗原(新冠病毒多肽抗原7,氨基酸序列为GYLKLTDNV,等电点为6.21)按照体积比为1:1:1的比例混合,并投入其总体积60~70倍的TM buffer缓冲溶液中(pH 7.4),在37℃混合搅拌15min,并在去离子水中透析24h,去除未载入的核酸佐剂或多肽抗原,最终获得纳米疫苗。
纳米疫苗经震荡透析制备后,测其粒径与扫描电子显微镜表征纳米疫苗成功制备。其中有效成分CpG-ODN浓度为9.12μg/mL,多肽抗原浓度为7.14μg/mL。
效果验证
A.纳米疫苗跨膜进入树突状细胞内的激光共聚焦荧光成像分析:
将经FAM与Cy3分别荧光染料标记的CpG-ODN与多肽抗原而制备好的纳米疫苗(实施例3~10)与未成熟的树突状细胞在共聚焦培养皿中共培养30min,然后利用Hoechst33342进行染色与激光共聚焦显微镜检测,观测其亚细胞定位。
将纳米疫苗与树突状细胞共培养30min,发现纳米疫苗能够快速进入树突状细胞内,其中,实施例3纳米疫苗进入细胞的效果如图4所示。为进一步验证疫苗其进入细胞内途径为硫醇交换介导的跨膜方式而非内吞途径,经LysoTracker溶酶体绿色染料,其标记有红色荧光的纳米疫苗能够与LysoTracker溶酶体染料有效区分,证明纳米疫苗能够通过硫醇交换跨膜而通过非内吞途径进入细胞,并预示其可显著降低溶酶体对CpG-ODN与多肽抗原的降解风险(图5)。
另外,实施例4纳米疫苗进入细胞的效果如图6~7所示;实施例5纳米疫苗进入细胞的效果如图8~9所示。
B.纳米疫苗能够高效激活树突状细胞成熟:
将树突状细胞接种于6孔板中并在37℃与PBS缓冲液、单独CpG-ODN、多肽抗原、CpG-ODN与多肽抗原混合液、纳米疫苗(实施例3)共培养3d,以LPS作为阳性对照。然后利用CD11c,CD80,CD86荧光标记抗体染色,并利用流式细胞仪进行表型分析。分析发现,纳米疫苗处理组能够有效激活DC细胞成熟,其成熟度显著高于其他处理组(PBS,单独CpG-ODN、多肽抗原、CpG-ODN与多肽抗原混合液),LPS为阳性对照组(如图10所示)。
C.纳米疫苗高效激活后的树突状细胞能有效激活T细胞成熟与增殖:
为证明纳米疫苗(实施例3)能够高效激活T细胞依赖的免疫反应,增强T细胞成熟与细胞增殖,将上述实验B中的经不同处理组后的树突状细胞与接种于6孔板中的未成熟的小鼠原代T细胞共培养2d,并利用CD8荧光标记抗体对经树突状细胞刺激后的T细胞进行染色,并利用流式细胞仪进行表型分析。此外,对CD8+T细胞的增殖进行评价,其主要利用CFSE染料标记经树突状细胞刺激T细胞,并继续共培养2d,利用利用流式细胞仪进行荧光定量分析。结果发现纳米疫苗处理后的树突状细胞能够高效的刺激T细胞成熟,并有效促进T细胞增殖,其表现为左侧荧光强度增强(P2)(如图11所示)。
D.纳米疫苗免疫后的小鼠能够有效预防肿瘤
为证明实施例3所合成的纳米疫苗免疫后的小鼠,能够预防肿瘤,将PBS缓冲液、单独CpG-ODN、多肽抗原、CpG-ODN与多肽抗原混合液、纳米疫苗经皮下注射BCLB/c雄性小鼠(5周龄),每5天免疫一次,共免疫5次后,然后将H22肿瘤移植入经PBS或纳米疫苗免疫后小鼠,每隔3天测定一次肿瘤大小,并绘制肿瘤生长曲线。如图12所示,其经纳米疫苗处理后的小鼠的肿瘤生长明显受到抑制,其抑制效率高于其他处理组。证明纳米疫苗能够更高效的抑制H22肝癌肿瘤生长,为肿瘤免疫防治提供研究依据。
因此,纳米疫苗具备良好的抗肿瘤活性,在肿瘤免疫治疗方面具有良好的临床转化与应用前景。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (24)
1.式I化合物在制备纳米疫苗中的应用;
A-L-F
式I
其中,A为含有两个或两个以上S原子的杂环基;
L为无或为中间连接基团;
F为能够与多肽以共价或非共价连接的基团。
2.根据权利要求1所述的应用,其特征在于,A中所述杂环基为4~8元环。
3.根据权利要求1或2所述的应用,其特征在于,
所述L为-(CH2)n-、其中n=1~5;R为-H、C1~5的烷基、 m=1~50。
4.根据权利要求1~3任一项所述的应用,其特征在于,所述F为:
5.根据权利要求1所述的应用,其特征在于,
所述A为:
所述F为:
所述L为:
6.根据权利要求1所述的应用,其特征在于,
所述A为:
所述F为:
所述L为:
7.根据权利要求1所述的应用,其特征在于,所述式I化合物为:
8.根据权利要求1所述的应用,其特征在于,所述式I化合物为时,所述多肽的等电点>6.5。
9.一种纳米颗粒,其原料包括式I化合物,抗原肽;
A-L-F
式I
其中,A为含有两个或两个以上S原子杂环基;
L为无或为中间连接基团;
F为能够与多肽以共价或非共价连接的基团。
10.根据权利要求9所述的纳米颗粒,其特征在于,所述抗原肽选自肿瘤抗原、细菌抗原或病毒抗原。
11.根据权利要求10所述的纳米颗粒,其特征在于,所述肿瘤抗原为肿瘤新生抗原,选自膀胱癌、血癌、骨癌、脑癌、乳腺癌、中枢神经系统癌症、宫颈癌、结肠癌、子宫内膜癌、食管癌、胆囊癌、胃肠道癌、外生殖器癌、泌尿生殖道癌、头癌、肾癌、喉癌、肝癌、肺癌、肌肉组织癌症、颈癌、口腔或鼻黏膜癌、卵巢癌、胰腺癌、前列腺癌、皮肤癌、脾癌、小肠癌、大肠癌、胃癌、睾丸癌和/或甲状腺癌的新生抗原;
所述病毒抗原为流感病毒抗原、埃博拉病毒抗原、冠状病毒抗原、肝炎病毒抗原、腮腺炎病毒抗原、水痘-带状疱疹病毒抗原、麻疹病毒抗原、风疹病毒抗原、流感病毒抗原和/或禽流感病毒抗原;
所述细菌抗原为结合分枝杆菌抗原、葡萄球菌抗原、链球菌抗原、肺炎双球菌抗原、炭疽杆菌抗原、白喉杆菌抗原、变形杆菌抗原、百日咳杆菌抗原、霍乱弧菌抗原、脑膜炎双球菌抗原和/或伤寒杆菌抗原。
12.根据权利要求9所述的纳米颗粒,其特征在于,所述纳米颗粒中还包括核酸佐剂,所述核酸佐剂为CpG-ODN、Poly I:C。
13.根据权利要求12所述的纳米颗粒,其特征在于,所述式I化合物,所述核酸佐剂和所述抗原肽的摩尔比为(50~100):(0~1):(20~50)。
14.根据权利要求9所述的纳米颗粒,其特征在于,
所述A为:
所述F为:
所述L为:
15.据权利要求9所述的纳米颗粒,其特征在于,所述式I化合物为:
16.根据权利要求9所述的纳米颗粒,其特征在于,所述式I化合物为时,所述抗原肽选自肿瘤抗原或病毒抗原。
17.根据权利要求9所述的纳米颗粒,其特征在于,所述式I化合物为时,所述抗原肽选自肝癌肿瘤抗原或新冠病毒抗原。
18.根据权利要求9所述的纳米颗粒,其特征在于,所述式I化合物为时,所述抗原肽选自病毒抗原。
19.根据权利要求9所述的纳米颗粒,其特征在于,所述式I化合物为时,所述抗原肽选自新冠病毒抗原。
20.包括权利要求12~13中任一项所述纳米颗粒的纳米疫苗的制备方法,其特征在于,包括:将所述式I化合物,所述核酸佐剂和所述抗原肽与TM缓冲液混合,37℃搅拌15min,然后在去离子水中透析,制得含有纳米颗粒的溶液。
21.一种纳米疫苗,其包括权利要求9~19任一项所述的纳米颗粒和疫苗中可接受的辅料。
22.权利要求21所述纳米疫苗在制备防治肿瘤、细菌感染和/或病毒感染的药物中的应用。
23.权利要求21所述纳米疫苗在制备防治肝癌肿瘤的药物中的应用。
24.权利要求21所述纳米疫苗在制备防治新冠病毒感染的药物中的应用。
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