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

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.