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。

动物实验按照《实验动物使用和护理指南》进行，并经第三军医大学实验动物福利和伦理委员会批准。
1.纳米乳液（NEs）的制备
<ol><li>对于水相制备，在40°C下搅拌时，将0.15g聚山梨酯80溶解在28.2mL磷酸盐缓冲盐水（PBS）中。</li>
	<li>对于油相制备，使用 表1所示的纳米乳液的油相配方。在40°C下搅拌时，将sornitan trileate 85，DOTAP和RA溶解在角鲨烯中。</li>
	<li>对于初级 O/W 乳液制备，以 1400 rpm 的转速磁性搅拌油相，同时以 1 mL/min 的速率缓慢滴加水性水溶液。使用高剪切乳化剂以 30,000 rpm 的速度预乳化混合物 6 分钟。</li>
	<li>对于高压均质化 （HPH），用高压均质器在 900 bar 下处理上述制备的初级 O/W 乳液 12 个循环，以获得 160-190 nm 范围内的均相纳米乳液。</li>
	<li>在过程结束时，将乳液收集在 50 mL 烧瓶中，并通过 0.2 μm PES 滤膜对乳液进行过滤灭菌。</li>
	<li>通过导管将烧瓶连接到舒伦克管线，旋转三通旋塞阀将系统连接到真空管，然后打开真空泵以在烧瓶中产生真空。然后，旋转三通旋塞阀，将系统连接到氩气管并用氩气填充。重复此步骤 3 次，以确保烧瓶充满氩气。</li>
	<li>更换软木塞并用paraffine薄膜密封，用锡箔包裹烧瓶并将其储存在4°C。</li>
</ol>2. 纳米乳液的表征
<ol><li>粒径和 zeta 电位检测<ol><li>打开仪器并等待 30 分钟，让激光光源稳定下来。</li>
		<li>用超纯水将 NE 稀释 50 倍，并将 1 mL 样品添加到相应的样品池中。</li>
		<li>要测量颗粒大小，请将温度设置为25°C，选择水作为分散介质，并选择一次性塑料盘子作为测量池。加载样品并自动测量。将每个样本的三次重复测量的平均值报告为最终结果。</li>
		<li>要测量 zeta 电位，请将温度设置为 25 °C。 由于选择水相作为分散介质，选择Smoluchowsi模型，折叠毛细管池作为测量池。加载样品并自动测量。将每个样品的三次重复测量的平均值报告为最终结果。</li>
	</ol></li>
	<li>包封率和载药率的测定<ol><li>为了确定 NE 中负载的 RA 量，使用无水乙醇破乳并从 NE 中提取 RA。向 5 μL 样品中加入 995 μL 乙醇，并使用 355 nm 的分光光度计测定溶液的 RA 含量。将吸光度与标准曲线进行比较。使用以下公式： 包封 = 实际 RA 载量/添加药物的重量 载药率=实际RA载量/样品质量</li>
	</ol></li>
</ol>3. 免疫程序和样本采集
<ol><li>免疫程序和分组<ol><li>根据以下实验组，将年龄为6-8周龄，体重约为18-21g，每组5只的雌性Balb / C小鼠分组，在第0天，第14天，第28天进行免疫：（1）PBS组，（2）卵清蛋白（OVA）组，（3）OVA+ NE-RA（OVA/NE-RA）组，（4）OVA + CNE-RA（OVA/CNE-RA）组。</li>
		<li>通过肌肉注射到股四头肌上部用200μL的不同疫苗制剂免疫所有小鼠：100μL乳剂和15μgOVA吸收在100μLPBS中，在给药前混合。注射 100 μL/后腿。对于对照组的小鼠，单独给予PBS。</li>
		<li>安乐死：在完成三次免疫后2周，通过腹膜内注射100mg / kg的1%戊巴比妥钠对所有小鼠实施安乐死。</li>
		<li>样本采集和处理：安乐死后，用剪刀切开小鼠的胸部，将注射器直接插入心脏，吸出约0.5毫升的全血。让血液在室温下静置2小时，然后在4°C下以1800× g 离心5分钟。 收集血清，等分试样并储存在-80°C以进行进一步分析。</li>
		<li>收集全血、脾脏、阴道灌洗液 （VLF）、支气管肺泡灌洗液 （BALF）、鼻灌洗液 （NLF） 和小肠灌洗液 （SILF）。</li>
	</ol></li>
	<li>脾淋巴细胞的分离<ol><li>将小鼠浸泡在75%（v / v）的乙醇中进行短暂灭菌，将小鼠转移到干净的工作台上。用剪刀剪开左腹部的皮肤，露出并切除脾脏。</li>
		<li>将脾脏放入含有 3 mL PBS 的培养皿中，研磨并使用筛子（200 目，70 μm）和研磨棒过滤到 15 mL 离心管中。</li>
		<li>在室温下以650× g 离心细胞5分钟。弃去上清液，用 3 mL 红细胞裂解液悬浮沉淀物，在室温下孵育 5 分钟。</li>
		<li>向试管中加入 10 mL PBS 并摇匀以终止反应，然后在室温下以 650 x g 离心 5 分钟。</li>
		<li>弃去上清液，将细胞重悬于 10 mL PBS 中，将 20 μL 细胞稀释液加入自动细胞计数仪进行细胞计数。</li>
		<li>在室温下以650× g 离心细胞5分钟。弃去上清液，用 1640 完全培养基（1% 青霉素 - 链霉素，10% 胎牛血清）将细胞稀释至 1 x 106 个细胞/mL。</li>
	</ol></li>
	<li>VLF收集：用75μL 1%BSA / PBST冲洗阴道4次，混合灌洗溶液并收集在1.5mL离心管中，在4°C下以5000× g 离心20分钟，并用移液管收集上清液并储存在-80°C。</li>
	<li>BALF 和 NLF 集合<ol><li>处死后用75%（v / v）乙醇消毒小鼠。抬起鼠标肚子并伸直脖子。用剪刀在小鼠颈部的皮肤上做一个纵向切口，然后用镊子分离肌肉并充分暴露气管。</li>
		<li>通过一个小的横向开口切开气管，将软管插入肺部，将注射器连接到软管上，将0.5mL PBS注射到肺部并退出，重复冲洗3次，并在4°C下以650× g 离心5分钟，将上清液（BALF）储存在-80°C。</li>
		<li>将软管反转到鼻腔，将注射器连接到软管上并注射 0.5 mL PBS，液体将通过鼻腔流入 1.5 mL 离心管。从离心管中吸出洗涤液并重复冲洗2x，然后在4°C下以650× g 离心5分钟，将上清液（NLF）储存在-80°C。</li>
	</ol></li>
	<li>SILF 系列<ol><li>制备含有0.1 mg/mL胰蛋白酶抑制剂，50 mM / L EDTA-2Na，1 mM / L苯甲基磺酰氟（PMSF）的PBS溶液作为小肠灌洗液。在室温下搅拌溶解并储存在4°C。</li>
		<li>沿肠管切开小肠，浸入 3 mL 小肠灌洗液中。通过在4°C下以15rpm垂直振荡混合30分钟。 在4°C下以1440× g 离心20分钟，并用移液管收集上清液，然后在4°C下以5000× g 离心20分钟。将上清液（SILF）储存在-80°C。</li>
	</ol></li>
</ol>4.肌内给药后OVA特异性抗体反应的评估
<ol><li>通过酶联免疫吸附测定 （ELISA） 测定血清 IgG、IgG1、IgG2a 亚类以及 VLF、BALF、NLF 和 SILF 中特异性 sIgA 的水平。</li>
	<li>对于抗原包被，用包被缓冲液将OVA稀释至5μg/ mL，并在4°C下用每孔100μLova溶液包被96孔ELISA板过夜。</li>
	<li>用 PBST 洗涤 ELISA 板 5 次，并通过在 37 °C 下与 250 μL 1%BSA/PBST 孵育封闭 2 小时。</li>
	<li>用PBST洗涤ELISA板5次，然后加入100μL稀释的样品，如下所述进行测试，并在37°C下孵育1小时。用 0.5% BSA/PBST 稀释血清、VLF、BALF、NLF，用 20% 胎牛血清 （FCS）/PBST 稀释 SILF。<ol><li>首先使用两步稀释程序将血清稀释 1000 倍：将 20 μL 血清稀释至 1 mL，然后将 25 μL 稀释的血清稀释至 500 μL。 使用这种稀释的血清检测 IgG1、IgG2a。</li>
	</ol></li>
	<li>将 200 μL 以 1000x 稀释的血清加入到包被板的第一行中，并将 100 μL 0.5% BSA/PBST 加入到除第一行以外的所有孔中。将 100 μL 从第一排转移到第二排，并用移液器混合 10x。重复此步骤至第 8 行并丢弃 100 μL 液体，已获得不同浓度的血清用于检测 IgG。</li>
	<li>进行 BALF、NLF 和 SILF 的 1：1 稀释以检测 IgA。对 VLF 使用与血清相同的稀释程序。</li>
	<li>用 PBST 洗涤 ELISA 板 5 次。将 100 μL HRP 偶联的山羊抗小鼠 IgG/IgG1/IgG2a/IgA（用 PBST 1：10000 稀释）加入适当的孔中，并在 37 °C 下孵育 40 分钟。</li>
	<li>用 PBST 洗涤 ELISA 板 5 次，加入 100 μL TMB ELISA 底物（高度敏感），并将板转移到避光的地方 5 分钟。立即加入 100 μL 450 nm 终止溶液用于 TMB 底物以停止反应。</li>
	<li>测量每个孔在450nm处的吸光度（OD），并通过取空白组小鼠血清值的2.1倍作为阳性反应值来计算抗体滴度。</li>
</ol>5. ELISpot 检测
<ol><li>按照 ELISpot Plus 的指南，使用试剂盒中的小鼠 IFN-γ （ALP） 进行检测。</li>
	<li>从密封包装中取出板，用无菌 PBS（200 μL/孔）洗涤 4 次。用 1640 完全培养基（200 μL/孔）调节板，并在室温下孵育 30 分钟。</li>
	<li>取出培养基，将步骤3.2.2中制备的调节浓度的淋巴细胞悬浮液（100μL/孔）加入板中，然后加入100μL刺激物，即含有20μg/ mL OVA257~264或OVA323~339或1640完全培养基的1640完全培养基。将板置于37°C，5%CO2 的加湿培养箱中48小时。用铝箔包裹板，以避免蒸发。</li>
	<li>弃去细胞悬浮液并用 PBS（200 μL/孔）洗涤板 5 次。在含有0.5%胎牛血清（PBS-0.5%FCS）的PBS中将检测抗体（R4-6A2-生物素）稀释至1μg/ mL。加入 100 μL/孔，在室温下孵育 2 小时。</li>
	<li>按照步骤 5.2 清洗板。在PBS-0.5%FCS中稀释链霉亲和素-ALP（1：1000），加入100μL/孔，在室温下孵育1小时。</li>
	<li>按照步骤 5.2 清洗板。通过 0.45 μm 过滤器过滤即用型底物溶液 （BCIP/NBT-plus），并加入 100 μL/孔。发展直到出现明显的斑点。</li>
	<li>在自来水中大量清洗以停止发色，取下柔软的塑料，让板子晾干。</li>
	<li>在 ELISpot 读数仪中检查和计数斑点。</li>
</ol>6. 统计分析
<ol><li>采用单因素方差分析法分析4组数据间的差异，然后进行Tukey多重比较后测。将所有结果表示为均值 ± SD.，小于 0.05 的 P 值被认为显著。</li>
</ol>

评估纳米乳化包埋的维甲酸在小鼠中的免疫原性

<ol>
	<li>Pulendran, B., Arunachalam, P. S., O'Hagan, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+concepts+in+the+science+of+vaccine+adjuvants.">Emerging concepts in the science of vaccine adjuvants.</a> Nat Rev Drug Discov. 20 (6), 454-475 (2021).</li><li>Pandey, P., Gulati, N., Makhija, M., Purohit, D., Dureja, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoemulsion:+A+novel+drug+delivery+approach+for+enhancement+of+bioavailability.">Nanoemulsion: A novel drug delivery approach for enhancement of bioavailability.</a> Recent Pat Nanotech. 14 (4), 276-293 (2020).</li><li>Chen, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disintegration+and+cancer+immunotherapy+efficacy+of+a+squalane-in-water+delivery+system+emulsified+by+bioresorbable+poly(ethylene+glycol)-block-polylactide.">Disintegration and cancer immunotherapy efficacy of a squalane-in-water delivery system emulsified by bioresorbable poly(ethylene glycol)-block-polylactide.</a> Biomaterials. 35 (5), 1686-1695 (2014).</li><li>Iwasaki, A., Omer, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+and+how+vaccines+work.">Why and how vaccines work.</a> Cell. 183 (2), 290-295 (2020).</li><li>Spadoni, I., Fornasa, G., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+protection+mediated+by+cooperation+between+vascular+and+epithelial+barriers.">Organ-specific protection mediated by cooperation between vascular and epithelial barriers.</a> Nat Rev Immunol. 17 (12), 761-773 (2017).</li><li>Singh, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoemulsion:+Concepts,+development+and+applications+in+drug+delivery.">Nanoemulsion: Concepts, development and applications in drug delivery.</a> J Cont Release. 252, 28-49 (2017).</li><li>Yan, W. L., Chen, W. S., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+adjuvant+activity+of+cationic+liposome:+Phosphorylation+of+a+MAP+kinase,+ERK+and+induction+of+chemokines.">Mechanism of adjuvant activity of cationic liposome: Phosphorylation of a MAP kinase, ERK and induction of chemokines.</a> Mol Immunol. 44 (15), 3672-3681 (2007).</li><li>Korsholm, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adjuvant+mechanism+of+cationic+dimethyldioctadecylammonium+liposomes.">The adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes.</a> Immunology. 121 (2), 216-226 (2007).</li><li>Agger, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+liposomes+formulated+with+synthetic+mycobacterial+cordfactor+(CAF01):+A+versatile+ddjuvant+for+vaccines+with+different+immunological+requirements.">Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): A versatile ddjuvant for vaccines with different immunological requirements.</a> Plos One. 3 (9), e3116 (2008).</li><li>Slutter, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+vaccination+with+N-trimethyl+chitosan+and+PLGA+based+nanoparticles:+Nanoparticle+characteristics+determine+quality+and+strength+of+the+antibody+response+in+mice+against+the+encapsulated+antigen.">Nasal vaccination with N-trimethyl chitosan and PLGA based nanoparticles: Nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen.</a> Vaccine. 28 (38), 6282-6291 (2010).</li><li>Nochi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanogel+antigenic+protein-delivery+system+for+adjuvant-free+intranasal+vaccines.">Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines.</a> Nat Mater. 9 (8), 685-685 (2010).</li><li>Henriksen-Lacey, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+cationic+charge+and+antigen+adsorption+are+important+properties+for+the+efficient+deposition+of+antigen+at+the+injection+site+and+ability+of+the+vaccine+to+induce+a+CMI+response.">Liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a CMI response.</a> J Control Release. 145 (2), 102-108 (2010).</li><li>Zhong, X. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanovaccines+mediated+subcutis-to-intestine+cascade+for+improved+protection+against+intestinal+infections.">Nanovaccines mediated subcutis-to-intestine cascade for improved protection against intestinal infections.</a> Small. 18 (1), e2105530 (2022).</li><li>Mora, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+gut-homing+IgA-secreting+B+cells+by+intestinal+dendritic+cells.">Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells.</a> Science. 314 (5802), 1157-1160 (2006).</li><li>Iwata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+imprints+gut-homing+specificity+on+T+cells.">Retinoic acid imprints gut-homing specificity on T cells.</a> Immunity. 21 (4), 527-538 (2004).</li><li>Hammerschmidt, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+induces+homing+of+protective+T+and+B+cells+to+the+gut+after+subcutaneous+immunization+in+mice.">Retinoic acid induces homing of protective T and B cells to the gut after subcutaneous immunization in mice.</a> J Clin Invest. 121 (8), 3051-3061 (2011).</li><li>Burger, C., Shahzad, Y., Br&#252;mmer, A., Gerber, M., du Plessis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traversing+the+skin+barrier+with+nano-emulsions.">Traversing the skin barrier with nano-emulsions.</a> Curr Drug Deliv. 14 (4), 458-472 (2017).</li><li>Lodaya, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+design,+optimization+and+evaluations+of+an+&#945;-tocopherol-containing+self-emulsified+adjuvant+system+using+inactivated+influenza+vaccine.">Formulation design, optimization and evaluations of an &#945;-tocopherol-containing self-emulsified adjuvant system using inactivated influenza vaccine.</a> J Cont Release. 316, 12-21 (2019).</li><li>Carmona-Ribeiro, A. M., P&#233;rez-Betancourt, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+nanostructures+for+vaccines+design.">Cationic nanostructures for vaccines design.</a> Biomimetics. 5 (3), 32 (2020).</li><li>Lam, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=trialkyl+ionizable+lipids+are+versatile+lipid-nanoparticle+components+for+therapeutic+and+vaccine+applications.">trialkyl ionizable lipids are versatile lipid-nanoparticle components for therapeutic and vaccine applications.</a> Adv Mater. 35 (15), e2209624 (2023).</li><li>Nie, T. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+coating+approach+to+overcome+mucosal+entrapment+of+DNA+nanoparticles+for+oral+gene+delivery+of+glucagon-like+peptide+1.">Surface coating approach to overcome mucosal entrapment of DNA nanoparticles for oral gene delivery of glucagon-like peptide 1.</a> Acs Appl Mater Inter. 11 (33), 29593-29603 (2019).</li><li>Lou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+self-amplifying+mRNA+vaccines+by+cationic+lipid+nanoparticles:+The+impact+of+cationic+lipid+selection.">Delivery of self-amplifying mRNA vaccines by cationic lipid nanoparticles: The impact of cationic lipid selection.</a> J Cont Release. 325, 370-379 (2020).</li></ol>

作者声明他们在这项工作中没有利益冲突。

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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1640 medium</td>
			<td>GIBCO, USA</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>450 nm Stop Solution for TMB Substrate</td>
			<td>Abcam</td>
			<td>ab171529-1000 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Countstar, China</td>
			<td>IC1000</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich, USA</td>
			<td>B2064-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Eppendorf, Germany</td>
			<td>5811000398</td>
			<td></td>
		</tr>
		<tr>
			<td>Danamic Light Scattering</td>
			<td>Malvern</td>
			<td>Zetasizer Nano S90</td>
			<td></td>
		</tr>
		<tr>
			<td>DOTAP</td>
			<td>CordenPharma, Switzerland</td>
			<td>O02002</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISpot Plus: Mouse IFN-gamma (ALP)</td>
			<td>mabtech</td>
			<td>ab205719</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO, USA</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Full-function Microplate Reader</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>VL0000D2</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG1(HRP)</td>
			<td>Abcam</td>
			<td>ab97240-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgA alpha chain (HRP)</td>
			<td>Abcam</td>
			<td>ab97235-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG H&#38;L (HRP)</td>
			<td>Abcam</td>
			<td>Ab205720-500ug</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat&#160;Anti-Mouse IgG2a heavy chain (HRP)</td>
			<td>Abcam</td>
			<td>ab97245-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure homogenizer</td>
			<td>ATS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MONTANE 85 PPI</td>
			<td>SEPPIC, France</td>
			<td>L12910</td>
			<td></td>
		</tr>
		<tr>
			<td>MONTANOX 80 PPI</td>
			<td>SEPPIC, France</td>
			<td>36372K</td>
			<td></td>
		</tr>
		<tr>
			<td>OVA257&#8211;264</td>
			<td>Shanghai Botai Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>OVA323-339</td>
			<td>Shanghai Botai Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>ZSGB-bio</td>
			<td>ZLI-9061</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Solarbio, China</td>
			<td>R1010</td>
			<td></td>
		</tr>
		<tr>
			<td>retinoic acid</td>
			<td>TCI, Japan</td>
			<td>TCI-R0064-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Squalene</td>
			<td>Sigma, USA</td>
			<td>S3626</td>
			<td></td>
		</tr>
		<tr>
			<td>T10 basic Ultra-Turrax</td>
			<td>IKA, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TMB ELISA Substrate</td>
			<td>Abcam</td>
			<td>ab171523-1000ml</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>Diamond</td>
			<td>A003570-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Macklin, China</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet spectrophotometer</td>
			<td>Hitachi</td>
			<td>U-3900</td>
			<td></td>
		</tr>
	</tbody>
</table>

cationic nanoemulsion-encapsulated retinoic acid as an adjuvant to promote ova&#45;specific systemic and mucosal responses

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。

Adjuvants are often used to enhance the efficacy of a vaccine by stimulating the immune system to respond more strongly to the vaccine, thereby increasing immunity to a particular pathogen1. Nanoemulsion (NE) adjuvant refers to a colloidal dispersion system with thermodynamic stability by emulsifying a certain proportion of oil phase and aqueous phase to produce an emulsion in the form of water-in-oil (W/O) or oil-in-water (O/W)2. O/W nanoemulsion adjuvant can produce cytokines and chemokines at the injection site, induce the rapid aggregation and proliferation of important immune cells such as monocytes, neutrophils, and eosinophils, and enhance the immune response, and improve the immunogenicity of antigens3. Currently, three nanoemulsion adjuvants (MF59, AS03, and AF03) have been licensed for use in vaccines and have shown good safety and efficacy4.
However, mucosal immunity has been poorly addressed by the currently licensed adjuvant formulations in conventional parenteral vaccination5. Retinoic acid (RA) has been reported to induce intestinal homing of immune cells, but its low polarity, poor solubility in water, and poor light and thermal stability limit its use for robust enteric vaccination. Nanoemulsions offer opportunities to increase the bioavailability of highly lipophilic drugs, and the oil core of O/W emulsion adjuvants is suitable for dissolving non-polar substances such as RA6. Therefore, nanoemulsions can be used as carriers for RA in order to achieve the dual response effect of systemic immunity and mucosal immunity.
Compared to neutral or anionic delivery systems, cationic delivery systems have relatively efficient antigen encapsulation and delivery capabilities, which can enhance the immunogenicity of antigens7,8,9. The cationic surface charge of a variety of adjuvant systems is important for their adjuvant effects10,11,12. The cationic charge is an important factor in prolonging vaccine retention at the injection site, increasing antigen presentation and prolonging the stimulation of cellular immunity in the body12.
Based on the above considerations, we developed a cationic nanoemulsion to effectively co-deliver RA and antigens. The particle size and zeta potential of the nanoemulsion were determined using dynamic light scattering (DLS), and the systemic and mucosal immune responses of the nanoemulsion combined with OVA were evaluated by intramuscular injection13.

作者聚焦：用于粘膜疫苗接种的阳离子纳米乳封装视黄酸系统的开发和评估

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

总共制备了四种纳米乳液制剂，其粒径（图1）、zeta电位和包封效率均进行了表征，如 表2所示。粒径集中在160-190nm附近，DOTAP的加入逆转了纳米乳液的Zeta电位。第三次免疫后 2 周检测 OVA 特异性血清 IgG 及其血清亚组抗体水平。纳米乳佐剂疫苗显着增加了血清中OVA特异性IgG，IgG1和IgG 2a抗体滴度（图2）。更重要的是，当OVA与CNE-RA佐剂时，阴?...

Watch this Scientific Journal Video about Author Spotlight: Development and Evaluation of a Cationic Nanoemulsion-Encapsulated Retinoic Acid System for Mucosal Vaccination at JoVE.com

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。

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen ...

我们正在研究一种粘膜疫苗，该疫苗可产生免疫反应作为感染组，具有预防感染和阻断传播的潜力。我们开发了一种紧张性纳米乳剂包封的视黄酸递送系统，通过肌内注射卵清蛋白进行免疫，可触发全身和粘膜免疫反应。在未来的研究中，肠道相关疾病的动物模型可用于进一步评估阳离子纳米乳包封的维甲酸在增强疫苗保护方面的效果和机制。

cationic-nanoemulsion-encapsulated-retinoic-acid-as-an-adjuvant-to

Research

JoVE Journal

Immunology and Infection

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA&#45;Specific Systemic and Mucosal Responses

415 次观看.  Third Military Medical University. 我们正在研究一种粘膜疫苗，该疫苗可产生免疫反应作为感染组，具有预防感染和阻断传播的潜力。我们开发了一种紧张性纳米乳剂包封的视黄酸递送系统，通过肌内注射卵清蛋白进行免疫，可触发全身和粘膜免疫反应。在未来的研究中，肠道相关疾病的动物模型可用于进一步评估阳离子纳米乳包封的维甲酸在增强疫苗保护方面的效果和机制。

视频: 作者聚焦：用于粘膜疫苗接种的阳离子纳米乳封装视黄酸系统的开发和评估

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid (RA) to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. By adding the FDA-approved RA to the nanoemulsion, antigen-specific sIgA was promoted in the vagina and small intestine after intramuscular injection of the nanoemulsion.