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

Abstract

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.

Introduction

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 antigens^7,8,9. The cationic surface charge of a variety of adjuvant systems is important for their adjuvant effects^10,11,12. 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.

Protocol

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)

1. For aqueous phase preparation, dissolve 0.15 g of polysorbate 80 in 28.2 mL of phosphate buffered saline (PBS) while stirring at 40 °C.
2. For oil phase preparation, use the oil phase formulation of the nanoemulsion shown in Table 1. Dissolve the sornitan trileate 85, DOTAP and RA in squalene while stirring at 40 °C.
3. For primary O/W emulsion preparation, stir the oil phase magnetically at 1400 rpm while adding the aqueous slowly and dropwise at a rate of 1 mL/min. Pre-emulsify the mixture using a high shear emulsifier at 30,000 rpm for 6 min.
4. For high pressure homogenization (HPH), treat the primary O/W emulsion prepared above with a high-pressure homogenizer for 12 cycles at 900 bar to obtain a homogeneous nano-emulsion in the 160-190 nm range.
5. At the end of the process, collect the emulsion in a 50 mL flask and filter-sterilize the emulsion through a 0.2 µm PES filter membrane.
6. Connect the flask to the Schlenk Line through the catheter, rotating the three-way stopcock to connect the system to the vacuum tube and turn on the vacuum pump to create vacuum in the flask. Then, rotating the three-way stopcock, connect the system to the Argon tube and fill it with Argon. Repeat this step 3x to ensure the flask is filled with Argon.
7. Replace the cork and seal with a paraffine film, wrap the flask in tin foil and store it at 4 °C.

2. Characterization of the nanoemulsions

1. Particle size and zeta potential detection
   1. Switch ON the instrument and wait 30 min for the laser light source to stabilize.
   2. Dilute the NE 50-fold with ultrapure water and add 1 mL of sample to the corresponding sample cell.
   3. To measure the particle size, set the temperature to 25 °C, select water as the dispersing medium, and select disposable plastic dishes as the measuring cell. Load the samples and measure automatically. Report the mean value of three repeated measurements for each sample as the final result.
   4. For measurement of zeta potential, set the temperature to 25 °C. Due to the choice of the aqueous phase as the dispersion medium, select the Smoluchowsi model, and the folded capillary cell as the measuring cell. Load the samples and measure automatically. Report the mean of three replicate measurements for each sample as the final result.
2. Determination of encapsulation rate and drug loading rate
   1. To determine the amount of RA loaded in the NE, use absolute ethanol to demulsify and extract the RA from the NE. To 5 µL of sample, add 995 µL of ethanol and determine the RA content of the solution using a spectrophotometer at 355 nm. Compare the absorbance to a standard curve. Use the following equation:
      Encapsulation = actual RA load/weight of drug added
      Drug loading rate =actual RA loading/ sample's quality

3. Immunization procedures and sample collection

1. Immunization procedure and grouping
   1. Group female Balb/C mice aged 6-8 weeks and weighing approximately 18-21 g in sets of 5 for immunization on day 0, day 14, day 28 according to the following experimental groups: (1) PBS group, (2) ovalbumin (OVA) group, (3) OVA+ NE-RA (OVA/ NE-RA) group, (4) OVA+CNE-RA(OVA/CNE-RA) group.
   2. Immunize all mice by intramuscular injection into the upper quadriceps muscles with 200 µL of different vaccine formulations: 100 µL of emulsion and 15 µg OVA absorbed in 100 µL of PBS, mixed before administration. Inject 100 µL/hind leg. For mice in the control group administer PBS alone.
   3. Euthanasia: Euthanize all mice by an intraperitoneal injection of 100 mg/kg of 1% sodium pentobarbital 2 weeks after completion of the three immunizations.
   4. Sample collection and processing: After euthanasia, use scissors to cut open the chest of mice and insert a syringe directly into the heart to aspirate approximately 0.5 mL of whole blood. Allow the blood to stand for 2 h at room temperature and then centrifuge at 1800 x g for 5 min at 4 °C. Collect serum, aliquot and store at -80 °C for further analysis.
   5. Collect whole blood, spleen, vaginal lavage fluid (VLF), bronchoalveolar lavage fluid (BALF), nasal lavage fluid (NLF), and small intestinal lavage fluid (SILF).
2. Isolation of splenic lymphocytes
   1. Soak mice in ethanol 75% (v/v) for a brief sterilization, transfer the mice to a clean bench. Cut the skin on the left abdomen with scissors, expose and remove the spleen.
   2. Place the spleen in a Petri dish containing 3 mL of PBS, grind and filter into a 15 mL centrifuge tube using a sieve (200 mesh, 70 µm) and grinding rod.
   3. Centrifuge the cells at 650 x g for 5 min at room temperature. Discard the supernatant and suspend the precipitate with 3 mL of red blood cell lysis solution, incubate at room temperature for 5 min.
   4. Add 10 mL of PBS to the tube and shake well to terminate the reaction, then centrifuge it at 650 x g for 5 min at room temperature.
   5. Discard the supernatant and resuspend the cells in 10 mL of PBS, add 20 µL of the cell dilution to the automated cell counter for cell counting.
   6. Centrifuge the cells at 650 x g for 5 min at room temperature. Discard the supernatant and dilute the cells to 1 x 10⁶ cells/mL with 1640 complete medium (1% penicillin-streptomycin, 10% fetal bovine serum).
3. VLF collection: Rinse the vagina 4x with 75 µL of 1%BSA/PBST, combine the lavage solution and collect in 1.5 mL centrifuge tubes, centrifuge at 5000 x g at 4 °C for 20 min and collect the supernatant with a pipette and store at -80 °C.
4. BALF and NLF collection
   1. Disinfect the mice after sacrifice with 75% (v/v) ethanol. Hold the mouse belly up and straighten the neck. Use scissors to make a longitudinal incision in the skin of the mouse neck and use forceps to separate the muscles and expose the trachea adequately.
   2. Cut the trachea through a small transverse opening and insert the hose toward the lungs, attach the syringe to the hose and inject 0.5 mL of PBS into the lungs and withdraw, repeat rinsing 3x and centrifuge at 650 x g at 4 °C for 5 min, store the supernatant (BALF) at -80 °C.
   3. Reverse the hose to the nasal cavity, attach the syringe to the hose and inject 0.5 mL of PBS, the liquid will flow through the nasal cavity into the 1.5 mL centrifuge tube. Aspirate the wash from the centrifuge tube and repeat the rinse 2x, then centrifuge at 650 x g at 4 °C for 5 min, store the supernatant (NLF) at -80 °C.
5. SILF collection
   1. Prepare PBS solution containing 0.1 mg/mL trypsin inhibitor, 50 mM/L EDTA-2Na, 1 mM/L phenylmethylsulfonyl fluoride (PMSF) as the small intestine lavage solution. Stir at room temperature to dissolve and store at 4 °C.
   2. Cut the small intestine open along the bowel tube and immerse in 3 mL of small intestinal lavage fluid. Mix by vertical shaking at 15 rpm for 30 min at 4 °C. Centrifuge at 1440 x g at 4 °C for 20 min and collect the supernatant with a pipette, then centrifuge at 5000 x g at 4 °C for 20 min. Store the supernatant (SILF) at -80 °C.

4. Evaluation of OVA-specific antibody response after intramuscular administration

1. Determine serum levels of IgG, subclasses of IgG1, IgG2a and the levels of specific sIgA in VLF, BALF, NLF and SILF by enzyme-linked immunosorbent assay (ELISA).
2. For antigen coating, dilute OVA to 5 µg/mL with coating buffer, and coat 96-well ELISA plates overnight at 4 °C with 100 µL of OVA solution per well.
3. Wash ELISA plates 5x with PBST and block by incubation with 250 µL of 1%BSA/PBST at 37 °C for 2 h.
4. Wash ELISA plates 5x with PBST, then add 100 µL of the sample diluted as described below to be tested and incubate at 37 °C for 1 h. Dilute serum, VLF, BALF, NLF with 0.5% BSA/PBST, dilute SILF with 20% fetal calf serum (FCS)/PBST.
   1. Start by diluting the serum 1000x using a two-step dilution procedure: dilute 20 µL of serum to 1 mL, then dilute 25 µL of this diluted serum to 500 µL. Use this dilution of serum for detection of IgG1, IgG2a.
5. Add 200 µL of the serum diluted at 1000x to the first row of the coated plate and add 100 µL of 0.5% BSA/PBST to all wells except the first row. Transfer 100 µL from the first row to the second row and mix 10x with the pipette. Repeat this step up to row 8 and discard 100 µL of liquid, the serum of different concentrations has been obtained for the detection of IgG.
6. Perform a 1:1 dilution of BALF, NLF and SILF to detect IgA. Use the same dilution procedure for VLF as that for serum.
7. Wash ELISA plates 5x with PBST. Add 100 µL of HRP-conjugated goat anti-mouse IgG/IgG1/IgG2a/IgA (diluted 1:10000 with PBST) to the appropriate wells and incubate at 37 °C for 40 min.
8. Wash ELISA plates 5x with PBST, add 100 µL of TMB ELISA substrate (highly sensitive), and transfer the plate to a place protected from light for 5 min. Immediately add 100 µL of 450 nm stop solution for TMB substrate to stop the reaction.
9. Measure the absorbance (OD) at 450 nm for each well and calculate the antibody titer by taking 2.1 times of the serum value of the blank group mouse as the positive response value.

5. ELISpot assay

1. Follow the guidelines for ELISpot Plus, use Mouse IFN-γ (ALP) from the kit to perform the assays.
2. Remove the plate from the sealed package and wash 4x with sterile PBS (200 µL/well). Condition the plate with 1640 complete medium (200 µL/well) and incubate for 30 min at room temperature.
3. Remove the medium and add the adjusted concentration of lymphocyte suspension prepared in step3.2.2 (100 µL/well) to the plate, then add 100 µL of the stimulus i.e., 1640 complete medium containing 20 µg/mL OVA257~264 or OVA323~339 or 1640 complete medium. Place the plate in a humidified incubator at 37 °C with 5% CO₂ for 48 h. Wrap the plate with aluminum foil to avoid evaporation.
4. Discard the cell suspension and wash the plate 5x with PBS (200 µL/well). Dilute the detection antibody (R4-6A2-biotin) to 1 µg/mL in PBS containing 0.5% fetal calf serum (PBS-0.5% FCS). Add 100 µL/well and incubate for 2 h at room temperature.
5. Wash plate as in step 5.2. Dilute the streptavidin-ALP (1:1000) in PBS-0.5%FCS and add 100 µL/well, incubate for 1 h at room temperature.
6. Wash plate as in step 5.2. Filter the ready-to-use substrate solution (BCIP/NBT-plus) through a 0.45 µm filter and add 100 µL/well. Develop until distinct spots emerge.
7. Stop color development by washing extensively in tap water, remove the soft plastic and leave the plate to dry.
8. Inspect and count spots in an ELISpot reader.

6. Statistical analysis

1. Analyze the differences among data of 4 groups by one-way ANOVA followed by Tukey multiple comparison post-test. Express all the results as mean ± SD. P value less than 0.05 was considered significant.

Representative Results

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.

Discussion

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 nanoemulsion^14,15,16, 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.

Materials

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