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, 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.
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)
2. Characterization of the nanoemulsions
3. Immunization procedures and sample collection
4. Evaluation of OVA-specific antibody response after intramuscular administration
5. ELISpot assay
6. Statistical analysis
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 increased the OVA-specific IgG, IgG1, and IgG 2a antibody titers in serum (Figure 2). More importantly, the levels of specific sIgA in the vaginal lavage fluid and small intestine lavage fluid were significantly enhanced when OVA was adjuvanted with CNE-RA (Figure 3). In ELISpot assay, no significant spots were found.
Figure 1: Size diameter and distribution. (A) Size diameter and distribution of NE-RA. (B) Size diameter and distribution of CNE-RA. d.nm is the mean diameter of the particles in nm. Please click here to view a larger version of this figure.
Figure 2: OVA-specific serum IgG and its subgroup antibody levels in serum. OVA-specific serum IgG and its subgroup antibody levels in serum 2 weeks post the three immunizations were completed. (A) Log2 value of the IgG titers. (B) IgG1 optical density at 450 nm. (C) IgG2a optical density at 450 nm. Data are expressed as mean ± SD (n=5), ***: P<0.001. Comparisons of the differences between the groups and the PBS group are shown directly above the columns in the figure and are indicated as follows: ns: no significance, #p<0.05, ###p<0.001. Please click here to view a larger version of this figure.
Figure 3: OVA-specific sIgA. OVA-specific sIgA in vaginal lavage fluid, bronchoalveolar lavage fluid, nasal lavage fluid, small intestinal lavage fluid. (A) Vaginal lavage fluid, (B) bronchoalveolar lavage fluid, (C) Nasal lavage fluid, and (D) small intestinal lavage fluid. Data are expressed as mean ± SD (n=5), ns: no significance, *p<0.05; ***p<0.001. Comparisons of the differences between the groups and the PBS group are shown directly above the columns in the figure and are indicated as follows: ns: no significance, ###p<0.001. Please click here to view a larger version of this figure.
Sample | Squalene | Sorbitan trioleate 85 | DOTAP | RA |
NE-RA | 1.5g | 0.15g | 0 | 60mg |
CNE-RA | 1.5g | 0.15g | 45mg | 60mg |
Table 1: The oil phase formulation of NEs.
Sample | Particle mean size (nm) | Polydispersity index | Zeta potential (mV) | Encapsulation efficiency (%) | Drug loading rate(mg/mL) |
NE-RA | 182.9±3.4 | 0.18±0.02 | -23.0±0.2 | 40 | 0.8 |
CNE-RA | 162.7±4.2 | 0.16±0.01 | 42.2±0.5 | 40 | 0.8 |
Table 2: Physicochemical characteristics of NEs.
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 shown that it is difficult to stimulate the mucosal response by subcutaneous or intramuscular injection5. By adding the FDA-approved RA to the nanoemulsion14,15,16, OVA-specific sIgA was promoted in the vagina and small intestine by intramuscular injection. This protocol has not been validated in other antigens except for OVA. In future studies, animal models of intestinal-related diseases can be used to further evaluate the effect and mechanism of CNE-RA in enhancing vaccine protection.
High pressure homogenization, shear mixing and ultrasonication are the most common high-energy emulsification methods used to prepare NEs, with high pressure homogenization providing the best homogeneity17. However, high pressure homogenization methods often require expensive specialized equipment, with high preparation costs and non-negligible cooling problems18. In our previous experiments, it was found that the homogenization pressure and the number of cycles had an effect on the particle size of NEs, and within a certain range, the higher the homogenization pressure and the higher the number of cycles, the smaller the particle size tended to be. The preparation of oil-in-water emulsion colostrum effectively converts the oil and water phases into large droplets and reduces the number of homogenization cycles. If this step is omitted, increasing the number of homogenization cycles can eventually result in nano-emulsions with the same particle size and dispersion. Replacing PBS in the aqueous phase with 0.9% saline or sodium citrate buffer had no effect on the particle size and dispersion of the NEs.
Multiple cationic lipids are broadly applied in vaccine design19, DOTAP was chosen for that it has been approved for clinical use, shows good safety, and is well tolerated. Excessive positive charge on the NE surface may lead to cytotoxicity. For safety reasons, the type and amount of cationic lipids need to be taken into consideration when preparing cationic NEs. Other cationic lipids commonly used in vaccine studies are DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl4-(dimethylamino)butanoate)20, DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DOTMA (N,N,N-trimethyl-2,3-bis(octadec-9-en-1-yloxy)propan-1-aminium chloride)21, DC-Chol (3β- [N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride)22, and whether they can be used to prepare cationic NEs needs to be further investigated.
In recent years, RA has attracted attention due to its ability to induce immune cell homing to the intestinal mucosa through various pathways, however, RA is not only poorly water-soluble but also unstable to light and oxygen. During the preparation and storage of NEs, constant attention should be paid to the protection of RA from light and oxygen. RA will rapidly be oxidized and decomposed when exposed to light and oxygen, eventually losing its adjuvant effect.
The authors have nothing to disclose.
This study was funded by Key Program of Chongqing Natural Science Foundation (No. cstc2020jcyj-zdxmX0027) and Chinese National Natural Science Foundation Project (No. 32270988).
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 |