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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.
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.
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, an....
The animal experiments were performed in accordance with the Guide to the Use and Care of Laboratory Animals and approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University.
1. Preparation of nanoemulsions (NEs)
In total, four nanoemulsion formulations were prepared and characterized by their particle size (Figure 1), their zeta potential and their encapsulation efficiency as presented in Table 2. The particle size was concentrated around 160-190nm and the addition of DOTAP reversed the Zeta potential of nanoemulsion. OVA-specific serum IgG and its subgroup antibody level in serum were detected 2 weeks post third immunization. The nanoemulsion adjuvant vaccine significantly increase.......
In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. Compared to traditional NE adjuvants, it has the following two advantages. First, in general, the surface of O/W NEs has a high negative charge, which makes it difficult to directly load antigens. Cationic NEs can effectively adsorb peptide or protein antigens and enhance the specific immunogenicity. Secondly, experience in traditional vaccine research has.......
This study was funded by Key Program of Chongqing Natural Science Foundation (No. cstc2020jcyj-zdxmX0027) and Chinese National Natural Science Foundation Project (No. 32270988).
....Name | Company | Catalog Number | Comments |
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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