CN113952449B - 一种水包油型碘化油纳米乳佐剂的制备方法及其应用 - Google Patents
一种水包油型碘化油纳米乳佐剂的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种水包油型碘化油纳米乳佐剂的制备方法及其应用,包括如下步骤:(1)将Tween‑80与超纯水混合均匀后加热;(2)将卵磷脂溶于无水乙醇中,逐滴加入其中,获得水相;(3)取碘油作为油相,逐滴加入水相中;(4)进行超声处理,获得乳剂;(5)将乳剂离心处理若干次;(6)使用过滤膜过滤步骤(5)所得的物料,即得。发明制得的水包油型碘化油纳米乳佐剂在作为佐剂的同时也可作为一种肿瘤特异性抗原负载平台,系统地将佐剂与肿瘤特异性抗原联合使用,克服了单纯肿瘤特异性抗原免疫原性低而造成对机体免疫反应激活不足的问题。
Description
技术领域
本发明属于疫苗佐剂技术领域,具体涉及一种水包油型碘化油纳米乳佐剂的制备方法及其应用。
背景技术
目前肿瘤疫苗是肿瘤预防与治疗的新方向之一,肿瘤纳米疫苗免疫治疗成功的一个关键策略在于免疫注射后保持疫苗的稳定性,使佐剂与抗原同时到达淋巴结并同时作用于DC,提高DC抗原交叉递呈效率,诱导有效的CD8+T细胞介导的抗肿瘤免疫应答。在肿瘤纳米疫苗的研发中,除了要求疫苗具有特异性、高效性和安全性以外,普遍适用原则即免疫佐剂和特异性抗原可灵活制备成疫苗,也是研发人员所追求的重要目标。弗氏佐剂是能引起细胞免疫反应的一种佐剂,由矿物油与乳化剂组成,与抗原物理混合后形成油包水乳剂,所产生的副作用限制了它的应用,因此,迫切地需要一种可代谢并且具有免疫调节作用的油剂合成乳佐剂用于疫苗的研发。
发明内容
本发明的目的在于克服现有技术缺陷,提供一种水包油型碘化油纳米乳佐剂的制备方法。
本发明的另一目的在于提供上述水包油型碘化油纳米乳佐剂的应用。
本发明的再一目的在于提供一种肿瘤纳米疫苗制剂。
本发明的技术方案如下:
一种水包油型碘化油纳米乳佐剂的制备方法,包括如下步骤:
(1)将Tween-80与超纯水混合均匀后加热至70-100℃;
(2)将卵磷脂溶于无水乙醇中获得卵磷脂乙醇溶液,然后在700-800rpm的搅拌速度下,将卵磷脂乙醇溶液逐滴加入到温度为70-100℃的步骤(1)所得的物料中,获得水相;
(3)取碘油作为油相,在700-800rpm的搅拌速度下将碘油逐滴加入到温度为70-100℃的步骤(2)所得的水相中,直至物料体积变为8-12mL;
(4)将步骤(3)所得的物料于2-4℃进行超声处理,获得乳剂;
(5)将步骤(4)所得的乳剂转移至90-110KD的超滤离心管中,于3000-4000rpm离心处理若干次,每次15-20min,收集超滤离心管中的超滤膜所截留的物料;
(6)使用孔径为0.2-0.3μm过滤膜过滤步骤(5)所得的物料,即得。
在本发明的一个优选实施方案中,所述步骤(1)中,所述Tween-80与超纯水的比例为180-250μL∶18-20mL。
进一步优选的,所述步骤(2)中,所述卵磷脂与无水乙醇的比例为100-120mg∶220-260μL。
更进一步优选的,所述碘油、Tween-80和卵磷脂的比例为500μL∶180-250μL∶100-120mg。
再进一步优选的,所述碘油、Tween-80和卵磷脂的比例为500μL∶200μL∶120mg。
在本发明的一个优选实施方案中,所述超声处理采用超声细胞破碎仪进行,其参数为:30min,750w,20KHz,20%,2s/2s。
在本发明的一个优选实施方案中,所述过滤膜的孔径为0.22μm。
本发明的另一技术方案如下:
上述制备方法制得的水包油型碘化油纳米乳佐剂在制备肿瘤纳米疫苗制剂中的应用。
本发明的再一技术方案如下:
一种肿瘤纳米疫苗制剂,由胆固醇修饰的肿瘤特异性抗原水溶液与上述制备方法制得的水包油型碘化油纳米乳佐剂混合制备而成,其中,该肿瘤纳米疫苗制剂所含的肿瘤特异性抗原浓度为100-600μg/mL。
在本发明的一个优选实施方案中,所述肿瘤特异性抗原包括OVA、B16-M27、B16-M33和TRP2180-188。
在本发明的一个优选实施方案中,所述胆固醇修饰的肿瘤特异性抗原水溶液与所述水包油型碘化油纳米乳佐剂的体积比为1∶0.1-5
本发明的有益效果是:
1、本发明制得的水包油型碘化油纳米乳佐剂在作为佐剂的同时也可作为一种肿瘤特异性抗原负载平台,系统地将佐剂与肿瘤特异性抗原联合使用,克服了单纯肿瘤特异性抗原免疫原性低而造成对机体免疫反应激活不足的问题。
2、本发明制得的水包油型碘化油纳米乳佐剂利用临床批准用药碘油制备,所含均为低毒性物质并在安全的使用剂量范围内,具有较高生物安全性。
3、本发明的制备方法可通过调节反应条件改变水包油型碘化油纳米乳佐剂的粒径,具备可控性和可重复性。
4、本发明的肿瘤纳米疫苗制剂通过化学基团间的相互作用将经两亲性脂类物质胆固醇修饰的肿瘤特异性抗原简便稳定的连接到水包油型碘化油纳米乳佐剂上制备成肿瘤纳米疫苗,具有较好的普适性。
5、本发明的肿瘤纳米疫苗制剂相比传统的佐剂与抗原物理混合制备形成的疫苗,能在注射后保持稳定性并聚集在淋巴结使佐剂与抗原同时作用于DC,在不降低安全性的情况下最大限度地提高疫苗效力,启动机体抗原特异性T细胞介导的抗肿瘤免疫应答。
6、本发明的肿瘤纳米疫苗制剂粘度低、性质稳定、粒径与形态均一,免疫注射后容易到达淋巴结,具有靶向性,且生物安全性好,粒径可调控切重复性好。
附图说明
图1为本发明实施例3中TEM照片,其中具体为,检测水包油型碘化油纳米乳佐剂的形貌和大小(A)以及粒径仪检测其粒径分布(B),肿瘤纳米疫苗制剂经4℃保存7天后TEM检测的形貌和大小(C)以及粒径仪检测其粒径分布(D);
图2为本发明实施例3中活体动物荧光成像检测肿瘤纳米疫苗制剂的淋巴结靶向性结果图。
图3为本发明实施例3中免疫荧光试验检测肿瘤纳米疫苗制剂对DCs抗原交叉的作用结果图。
图4为本发明实施例3中体内试验检测、分析抗原特异性CD8+T淋巴细胞的分化、增殖结果图。
图5为本发明实施例3中肿瘤纳米疫苗制剂抗肿瘤治疗效果图:肿瘤组织体积变化(A),小鼠生存期(B)。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
实施例1
(1)将200μLTween-80与18mL超纯水混合均匀后加热至80℃;
(2)将120mg卵磷脂溶于250μL无水乙醇中获得卵磷脂乙醇溶液,在750rpm的搅拌速度下,逐滴加入到80℃的步骤(1)所得的物料中,获得水相;
(3)取500μL碘油作为油相,在750rpm的搅拌速度下将碘油逐滴加入到温度为80℃的步骤(2)所得的水相中,直至物料体积变为约10mL,整个滴加时间为15-20min;
(4)将步骤(3)所得的物料于4℃进行超声处理,获得乳剂;超声处理采用超声细胞破碎仪进行,其参数为:30min,750w,20KHz,20%,2s/2s;
(5)将步骤(4)所得的乳剂转移至100KD的超滤离心管中,于3500rpm离心处理3次,每次15min,收集超滤离心管中的超滤膜所截留的物料;
(6)使用孔径为0.22μm过滤膜过滤步骤(5)所得的物料,即得水包油型碘化油纳米乳佐剂,其组成成份类似于弗氏佐剂,均由油与表面活性剂组成,因此将其命名为碘化油纳米-类弗氏佐剂(iodinated oil nano-Freund′s adjuvant,IONFA)。
实施例2
(1)将OVA作为模式抗原,使用胆固醇(CHO)进行官能团修饰,将DMSO(200μl)和DIEA(15μl)、OVA(5mg)、干燥的硝基苯胆固醇3mg混合,4℃搅拌反应混合物12h,反应完成后,在真空中去除溶剂,用CHCl3进行研磨而成,(以下用OVA257-264表示),称取1mg的OVA257-264溶于1mL超纯水中加到3mL的实施例1制得的IONFA中,在4℃环境下,磁力搅拌12h;
(2)将步骤(1)所得的物料装入100KD的透析袋中,在4℃环境下,用2L的PBS溶液透析48h;
(3)收集透析袋中物料,使用0.22μm无菌过滤膜过滤,得到无菌的IONFA纳米疫苗(即本发明中的肿瘤纳米疫苗制剂),并命名为IONFA-OVA257-264。
实施例3
1、水包油型碘化油纳米乳佐剂以及肿瘤纳米疫苗制剂的纳米颗粒形态学表征:使用透射电镜(TEM)检测实施例1制得的水包油型碘化油纳米乳佐剂以及实施例2制得的肿瘤纳米疫苗制剂的纳米颗粒大小和形貌,使用粒径仪检测其粒径分布。如图1所示纳米颗粒为乳圆状,粒径大小分布在50-100nm(A),粒径分布比较均一(B),使用水包油型碘化油纳米乳佐剂制备成肿瘤纳米疫苗制剂后,在4℃冰箱静置7天后检测,其纳米颗粒形貌、大小、粒径分布与水包油型碘化油纳米乳佐剂佐剂保持一致,结果说明了在制备与保存的过程中,纳米颗粒形态未发生改变。
2、肿瘤纳米疫苗制剂的体内稳定性与体内靶向检测:为了检测实施例2制得的IONFA与OVA257-264连接形成的肿瘤纳米疫苗制剂体内注射之后,是否能保持其稳定性到达淋巴结,使用Cy5.5标记OVA257-264、使用吲哚菁绿(ICG)标记佐剂后合成肿瘤纳米疫苗制剂后,皮下注射,Cy5.5标记的OVA257-264为对照组,使用动物活体成像系统检测。结果如图2所示,在24h时,检测到IONFA-OVA257-264组中小鼠左侧腹股沟淋巴结内(红色箭头指向)同时出现了Cy5.5与ICG的共定位信号,在对照组中,Cy5.5信号(抗原信号)依然停留在注射部位附近,说明了IONFA-OVA257-264在体内的稳定性以及IONFA可协同抗原同时到达淋巴结并富集于此,表明了实施例2制得的肿瘤纳米疫苗制剂具有靶向淋巴结的特性。
3、肿瘤纳米疫苗制剂对DCs抗原交叉递呈的作用评价:为了检测IONFA-OVA257-264是否能刺激DCs成熟并对OVA257-264进行交叉递呈,使用IONFA-OVA257-264刺激未成熟树突状细胞,OVA257-264为对照组,通过免疫荧光试验使用共聚焦荧光显微镜检测24h时DCs表面MHC-I-Ag复合物的表达情况,结果如图3所示,与OVA257-264组相比,IONFA-OVA257-264组的DCs细胞表面MHC-I-Ag复合物强阳性。表明了肿瘤纳米疫苗制剂能够促使DCs通过MHC-I分子途径对抗原进行递呈。
4、肿瘤纳米疫苗制剂体内免疫效果评价:为了检测IONFA-OVA257-264诱导抗原特异性T细胞免疫反应的功能,对小鼠进行了3次免疫(每周一次,连续三周,每次注射2.2μM),每次免疫前一天采集小鼠外周血并分离出外周血单个核细胞(PBMC),通过荧光四聚体法使用流式细胞仪检测CD8+T淋巴细胞表面复合物(SIINFEKL-H-2kb),结果如图4所示,在三次检测中,每一次的检测结果IONFA-OVA257-264组的抗原特异性CD8+T在PBMC中的占比均高于其他对照组,差异具有统计学意义(P<0.0001),且随着免疫的增强,其比例随之增加,表明了肿瘤纳米疫苗制剂可以有效诱导机体产生抗原特异性T细胞免疫反应。
5、肿瘤纳米疫苗制剂的抗肿瘤治疗效果的评价:将小鼠随机分组后,皮下接种Hep1-6-OVA肿瘤细胞,每只接种1×106个,在接种第10天后,经皮下注射,对小鼠进行3次免疫治疗,每周一次。从接种肿瘤细胞开始持续监测肿瘤体积以及小鼠生长情况,结果如图5所示,经IONFA-OVA257-264治疗之后,肿瘤体积逐渐变小直至肿瘤消退,而不使用纳米疫苗治疗的小鼠,肿瘤细胞持续生长(A),且经过纳米疫苗治疗后,可延长小鼠生存期(B),表明了肿瘤纳米疫苗制剂可以诱导机体发生免疫应答发挥抗肿瘤效果达到治疗作用。
以上所述,仅为本发明的较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
Claims (4)
1.一种肿瘤纳米疫苗制剂,其特征在于:由胆固醇修饰的肿瘤特异性抗原OVA水溶液与水包油型碘化油纳米乳佐剂混合制备而成,其中,该肿瘤纳米疫苗制剂所含的肿瘤特异性抗原浓度为100-600 μg/mL,该胆固醇修饰的方法为:将200 μl 的DMSO和15 μl 的DIEA、5mg 的OVA、3mg的干燥的胆固醇混合,4℃搅拌反应混合12h,反应完成后,在真空中去除溶剂,用CHCl3进行研磨而成;
其制备方法包括:
(1)胆固醇修饰的肿瘤特异性抗原OVA水溶液与所述水包油型碘化油纳米乳佐剂以1:0.1-5的体积比混合,在4 ℃环境下,磁力搅拌12 h;
(2)将步骤(1)所得的物料装入100 KD的透析袋中,在4 ℃环境下,用2 L的PBS溶液透析48 h;
(3)收集透析袋中物料,使用0.22 μm无菌过滤膜过滤,即得;
上述水包油型碘化油纳米乳佐剂的制备方法包括如下步骤:
(1)将Tween-80与超纯水混合均匀后加热至70-100℃,Tween-80与超纯水的比例为180-250 μL: 18-20 mL;
(2)将卵磷脂溶于无水乙醇中获得卵磷脂乙醇溶液,然后在700-800 rpm的搅拌速度下,将卵磷脂乙醇溶液逐滴加入到温度为70-100℃的步骤(1)所得的物料中,获得水相,卵磷脂与无水乙醇的比例为100-120 mg: 220-260 μL;
(3)取碘油作为油相,在700-800 rpm的搅拌速度下将碘油逐滴加入到温度为70-100℃的步骤(2)所得的水相中,直至物料体积变为8-12 mL,碘油、Tween-80和卵磷脂的比例为500 μL: 180-250 μL: 100-120 mg;
(4)将步骤(3)所得的物料于2-4 ℃进行超声处理,获得乳剂;
(5)将步骤(4)所得的乳剂转移至90-110 KD的超滤离心管中,于3000-4000 rpm离心处理若干次,每次15 -20 min,收集超滤离心管中的超滤膜所截留的物料;
(6)使用孔径为0.2-0.3μm过滤膜过滤步骤(5)所得的物料,即得。
2. 如权利要求1所述的一种肿瘤纳米疫苗制剂,其特征在于:所述碘油、Tween-80和卵磷脂的比例为500 μL: 200 μL: 120 mg。
3.如权利要求1所述的一种肿瘤纳米疫苗制剂,其特征在于:所述超声处理采用超声细胞破碎仪进行,其参数为:30 min,750 w,20 KHz,20 %,2 s/2 s。
4.如权利要求1所述的一种肿瘤纳米疫苗制剂,其特征在于:所述步骤(6)的过滤膜的孔径为0.22μm。
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