阳离子纳米乳液包封的视黄酸作为佐剂，促进 OVA 特异性全身和粘膜反应
Summary
在该方案中，我们开发了一种阳离子纳米乳液包封的视黄酸 （RA），用作佐剂以促进抗原特异性全身和粘膜反应。通过将 FDA 批准的 RA 添加到纳米乳液中，肌肉注射纳米乳剂后，抗原特异性 sIgA 在阴道和小肠中得到促进。
Abstract
阳离子纳米结构已成为一种佐剂和抗原递送系统，可增强树突状细胞成熟、ROS 生成和抗原摄取，然后促进抗原特异性免疫反应。近年来，视黄酸（RA）因其激活粘膜免疫反应的作用而受到越来越多的关注;然而，为了将RA用作粘膜佐剂，有必要解决其溶解、加载和输送的问题。在这里，我们描述了一种阳离子纳米乳包封的视黄酸（CNE-RA）输送系统，由阳离子脂质1,2-二油酰基-sn-甘油-3-磷酸胆碱（DOTAP）、视黄酸、角鲨烯作为油相、聚山梨醇酯80作为表面活性剂和脱水山梨糖醇三油酸酯85作为辅助表面活性剂组成。利用动态光散射和分光光度计对其物理和化学性质进行了表征。与单独使用OVA相比，用抗原（卵清蛋白，OVA）和CNE-RA的混合物免疫小鼠显着升高了小鼠阴道灌洗液和小肠灌洗液中抗OVA分泌免疫球蛋白A（sIgA）的水平。该协议描述了用于制备，表征和评估CNE-RA佐剂效果的详细方法。
Introduction
佐剂通常用于通过刺激免疫系统对疫苗做出更强烈的反应来增强疫苗的功效，从而提高对特定病原体的免疫力1.纳米乳液（NE）助剂是指通过乳化一定比例的油相和水相，产生油包水（W/O）或水包油（O/W）2形式的乳液，具有热力学稳定性的胶体分散体系。O/W纳米乳液佐剂可在注射部位产生细胞因子和趋化因子，诱导单核细胞、中性粒细胞、嗜酸性粒细胞等重要免疫细胞的快速聚集和增殖，增强免疫应答，提高抗原的免疫原性3。目前，三种纳米乳剂佐剂（MF59、AS03和AF03）已获准用于疫苗，并显示出良好的安全性和有效性4。
然而，目前在常规胃肠外疫苗接种中许可的佐剂制剂对粘膜免疫问题处理得很差5.据报道，维甲酸 （RA） 可诱导免疫细胞肠道归巢，但其极性低、在水中的溶解度差以及光稳定性和热稳定性差限制了其用于稳健的肠道疫苗接种。纳米乳液为提高高亲脂性药物的生物利用度提供了机会，而 O/W 乳液佐剂的油芯适用于溶解 RA6 等非极性物质。因此，纳米乳剂可以作为RA的载体，以达到全身免疫和黏膜免疫的双重反应效果。
与中性或阴离子递送系统相比，阳离子递送系统具有相对高效的抗原包封和递送能力，可以增强抗原的免疫原性7,8,9。各种佐剂体系的阳离子表面电荷对其佐剂效果很重要 10,11,12。阳离子电荷是延长疫苗在注射部位的保留时间、增加抗原呈递和延长体内细胞免疫刺激的重要因素12.
基于上述考虑，我们开发了一种阳离子纳米乳液，可有效共递送RA和抗原。利用动态光散射（DLS）法测定纳米乳液的粒径和zeta电位，并通过肌内注射13评价纳米乳液联合OVA的全身和粘膜免疫反应。
Protocol
Representative Results
Discussion
在该协议中，我们开发了一种阳离子纳米乳液包封的视黄酸，用作佐剂以促进抗原特异性全身和粘膜反应。与传统的NE佐剂相比，它具有以下两个优点。首先，一般来说，O/W NEs的表面具有高负电荷，这使得直接负载抗原变得困难。阳离子NEs能有效吸附肽或蛋白抗原，增强特异性免疫原性。其次，传统疫苗研究的经验表明，皮下注射或肌肉注射很难刺激粘膜反应5。通过将 FDA 批准?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本研究由重庆市自然科学基金重点项目（CSTC2020jcyj-zdxmX0027）和国家自然科学基金项目（第32270988号）资助。
Materials
| 1640 medium | GIBCO, USA | C11875500BT | |
| 450 nm Stop Solution for TMB Substrate | Abcam | ab171529-1000 mL | |
| Automated Cell Counter | Countstar, China | IC1000 | |
| BSA | Sigma-Aldrich, USA | B2064-100G | |
| Centrifuge 5810 R | Eppendorf, Germany | 5811000398 | |
| Danamic Light Scattering | Malvern | Zetasizer Nano S90 | |
| DOTAP | CordenPharma, Switzerland | O02002 | |
| ELISpot Plus: Mouse IFN-gamma (ALP) | mabtech | ab205719 | |
| Fetal Bovine Serum | GIBCO, USA | 10099141C | |
| Full-function Microplate Reader | Thermo Fisher Scientific, USA | VL0000D2 | |
| Goat Anti-Mouse IgG1(HRP) | Abcam | ab97240-1mg | |
| Goat Anti-Mouse IgA alpha chain (HRP) | Abcam | ab97235-1mg | |
| Goat Anti-Mouse IgG H&L (HRP) | Abcam | Ab205720-500ug | |
| Goat Anti-Mouse IgG2a heavy chain (HRP) | Abcam | ab97245-1mg | |
| High pressure homogenizer | ATS | ||
| MONTANE 85 PPI | SEPPIC, France | L12910 | |
| MONTANOX 80 PPI | SEPPIC, France | 36372K | |
| OVA257–264 | Shanghai Botai Biotechnology Co., Ltd. | NA | |
| OVA323-339 | Shanghai Botai Biotechnology Co., Ltd. | NA | |
| Phosphate buffer saline | ZSGB-bio | ZLI-9061 | |
| Red Blood Cell Lysis Buffer | Solarbio, China | R1010 | |
| retinoic acid | TCI, Japan | TCI-R0064-5G | |
| Squalene | Sigma, USA | S3626 | |
| T10 basic Ultra-Turrax | IKA, Germany | ||
| TMB ELISA Substrate | Abcam | ab171523-1000ml | |
| trypsin inhibitor | Diamond | A003570-0100 | |
| Tween-20 | Macklin, China | 9005-64-5 | |
| Ultraviolet spectrophotometer | Hitachi | U-3900 |
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