Name: cationic nanoemulsion-encapsulated retinoic acid as an adjuvant to promote ova-specific systemic and mucosal responses
Uploaded: 2024-02-23T13:40:00.000+00:00
Duration: 6 min 2 s
Description: 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

This study was funded by Key Program of Chongqing Natural Science Foundation (No. cstc2020jcyj-zdxmX0027) and Chinese National Natural Science Foundation Project (No. 32270988).

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)
<ol>
	<li>For aqueous phase preparation, dissolve 0.15 g of polysorbate 80 in 28.2 mL of phosphate buffered saline (PBS) while stirring at 40 &#176;C.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>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.</li>
	<li>At the end of the process, collect the emulsion in a 50 mL flask and filter-sterilize the emulsion through a 0.2 &#181;m PES filter membrane.</li>
	<li>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.</li>
	<li>Replace the cork and seal with a paraffine film, wrap the flask in tin foil and store it at 4 &#176;C.</li>
</ol>
2. Characterization of the nanoemulsions
<ol>
	<li>Particle size and zeta potential detection
	<ol>
		<li>Switch ON the instrument and wait 30 min for the laser light source to stabilize.</li>
		<li>Dilute the NE 50-fold with ultrapure water and add 1 mL of sample to the corresponding sample cell.</li>
		<li>To measure the particle size, set the temperature to 25 &#176;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.</li>
		<li>For measurement of zeta potential, set the temperature to 25 &#176;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.</li>
	</ol>
	</li>
	<li>Determination of encapsulation rate and drug loading rate
	<ol>
		<li>To determine the amount of RA loaded in the NE, use absolute ethanol to demulsify and extract the RA from the NE. To 5 &#181;L of sample, add 995 &#181;L 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&#39;s quality</li>
	</ol>
	</li>
</ol>
3. Immunization procedures and sample collection
<ol>
	<li>Immunization procedure and grouping
	<ol>
		<li>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.</li>
		<li>Immunize all mice by intramuscular injection into the upper quadriceps muscles with 200 &#181;L of different vaccine formulations: 100 &#181;L of emulsion and 15 &#181;g OVA absorbed in 100 &#181;L of PBS, mixed before administration. Inject 100 &#181;L/hind leg. For mice in the control group administer PBS alone.</li>
		<li>Euthanasia: Euthanize all mice by an intraperitoneal injection of 100 mg/kg of 1% sodium pentobarbital 2 weeks after completion of the three immunizations.</li>
		<li>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 &#176;C. Collect serum, aliquot and store at -80 &#176;C for further analysis.</li>
		<li>Collect whole blood, spleen, vaginal lavage fluid (VLF), bronchoalveolar lavage fluid (BALF), nasal lavage fluid (NLF), and small intestinal lavage fluid (SILF).</li>
	</ol>
	</li>
	<li>Isolation of splenic lymphocytes
	<ol>
		<li>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.</li>
		<li>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 &#181;m) and grinding rod.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Discard the supernatant and resuspend the cells in 10 mL of PBS, add 20 &#181;L of the cell dilution to the automated cell counter for cell counting.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>VLF collection: Rinse the vagina 4x with 75 &#181;L of 1%BSA/PBST, combine the lavage solution and collect in 1.5 mL centrifuge tubes, centrifuge at 5000 x g at 4 &#176;C for 20 min and collect the supernatant with a pipette and store at -80 &#176;C.</li>
	<li>BALF and NLF collection
	<ol>
		<li>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.</li>
		<li>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 &#176;C for 5 min, store the supernatant (BALF) at -80 &#176;C.</li>
		<li>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 &#176;C for 5 min, store the supernatant (NLF) at -80 &#176;C.</li>
	</ol>
	</li>
	<li>SILF collection
	<ol>
		<li>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 &#176;C.</li>
		<li>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 &#176;C. Centrifuge at 1440 x g at 4 &#176;C for 20 min and collect the supernatant with a pipette, then centrifuge at 5000 x g at 4 &#176;C for 20 min. Store the supernatant (SILF) at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Evaluation of OVA-specific antibody response after intramuscular administration
<ol>
	<li>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).</li>
	<li>For antigen coating, dilute OVA to 5 &#181;g/mL with coating buffer, and coat 96-well ELISA plates overnight at 4 &#176;C with 100 &#181;L of OVA solution per well.</li>
	<li>Wash ELISA plates 5x with PBST and block by incubation with 250 &#181;L of 1%BSA/PBST at 37 &#176;C for 2 h.</li>
	<li>Wash ELISA plates 5x with PBST, then add 100 &#181;L of the sample diluted as described below to be tested and incubate at 37 &#176;C for 1 h. Dilute serum, VLF, BALF, NLF with 0.5% BSA/PBST, dilute SILF with 20% fetal calf serum (FCS)/PBST.
	<ol>
		<li>Start by diluting the serum 1000x using a two-step dilution procedure: dilute 20 &#181;L of serum to 1 mL, then dilute 25 &#181;L of this diluted serum to 500 &#181;L. Use this dilution of serum for detection of IgG1, IgG2a.</li>
	</ol>
	</li>
	<li>Add 200 &#181;L of the serum diluted at 1000x to the first row of the coated plate and add 100 &#181;L of 0.5% BSA/PBST to all wells except the first row. Transfer 100 &#181;L from the first row to the second row and mix 10x with the pipette. Repeat this step up to row 8 and discard 100 &#181;L of liquid, the serum of different concentrations has been obtained for the detection of IgG.</li>
	<li>Perform a 1:1 dilution of BALF, NLF and SILF to detect IgA. Use the same dilution procedure for VLF as that for serum.</li>
	<li>Wash ELISA plates 5x with PBST. Add 100 &#181;L of HRP-conjugated goat anti-mouse IgG/IgG1/IgG2a/IgA (diluted 1:10000 with PBST) to the appropriate wells and incubate at 37 &#176;C for 40 min.</li>
	<li>Wash ELISA plates 5x with PBST, add 100 &#181;L of TMB ELISA substrate (highly sensitive), and transfer the plate to a place protected from light for 5 min. Immediately add 100 &#181;L of 450 nm stop solution for TMB substrate to stop the reaction.</li>
	<li>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.</li>
</ol>
5. ELISpot assay
<ol>
	<li>Follow the guidelines for ELISpot Plus, use Mouse IFN-&#947; (ALP) from the kit to perform the assays.</li>
	<li>Remove the plate from the sealed package and wash 4x with sterile PBS (200 &#181;L/well). Condition the plate with 1640 complete medium (200 &#181;L/well) and incubate for 30 min at room temperature.</li>
	<li>Remove the medium and add the adjusted concentration of lymphocyte suspension prepared in step3.2.2 (100 &#181;L/well) to the plate, then add 100 &#181;L of the stimulus i.e., 1640 complete medium containing 20 &#181;g/mL OVA257~264 or OVA323~339 or 1640 complete medium. Place the plate in a humidified incubator at 37 &#176;C with 5% CO2 for 48 h. Wrap the plate with aluminum foil to avoid evaporation.</li>
	<li>Discard the cell suspension and wash the plate 5x with PBS (200 &#181;L/well). Dilute the detection antibody (R4-6A2-biotin) to 1 &#181;g/mL in PBS containing 0.5% fetal calf serum (PBS-0.5% FCS). Add 100 &#181;L/well and incubate for 2 h at room temperature.</li>
	<li>Wash plate as in step 5.2. Dilute the streptavidin-ALP (1:1000) in PBS-0.5%FCS and add 100 &#181;L/well, incubate for 1 h at room temperature.</li>
	<li>Wash plate as in step 5.2. Filter the ready-to-use substrate solution (BCIP/NBT-plus) through a 0.45 &#181;m filter and add 100 &#181;L/well. Develop until distinct spots emerge.</li>
	<li>Stop color development by washing extensively in tap water, remove the soft plastic and leave the plate to dry.</li>
	<li>Inspect and count spots in an ELISpot reader.</li>
</ol>
6. Statistical analysis
<ol>
	<li>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 &#177; SD. P value less than 0.05 was considered significant.</li>
</ol>

Evaluating Immunogenicity of Nanoemulsion&#45;Encapsulated Retinoic Acid in Mice

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The authors declare they have no conflicts of interest in this work.

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 (3&#946;- [N-(N&#8242;,N&#8242;-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.

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.

<table><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&amp;amp;L (HRP)</td><td>Abcam</td><td>Ab205720-500ug</td><td></td></tr><tr><td>Goat&amp;nbsp;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&amp;ndash;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

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 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66270/66270fig01.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66270/66270fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66270/66270fig02.jpg" /> 
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 &#177; SD (n=5), ***: P&#60;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&#60;0.05, ###p&#60;0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66270/66270fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66270/66270fig03.jpg" /> 
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 &#177; SD (n=5), ns: no significance, *p&#60;0.05; ***p&#60;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&#60;0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66270/66270fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Squalene</td>
			<td>Sorbitan trioleate 85</td>
			<td>DOTAP</td>
			<td>RA</td>
		</tr>
		<tr>
			<td>NE-RA</td>
			<td>1.5g</td>
			<td>0.15g</td>
			<td>0</td>
			<td>60mg</td>
		</tr>
		<tr>
			<td>CNE-RA</td>
			<td>1.5g</td>
			<td>0.15g</td>
			<td>45mg</td>
			<td>60mg</td>
		</tr>
	</tbody>
</table>
Table 1: The oil phase formulation of NEs.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Particle mean size (nm)</td>
			<td>Polydispersity index</td>
			<td>Zeta potential (mV)</td>
			<td>Encapsulation efficiency (%)</td>
			<td>Drug loading rate(mg/mL)</td>
		</tr>
		<tr>
			<td>NE-RA</td>
			<td>182.9&#177;3.4</td>
			<td>0.18&#177;0.02</td>
			<td>-23.0&#177;0.2</td>
			<td>40</td>
			<td>0.8</td>
		</tr>
		<tr>
			<td>CNE-RA</td>
			<td>162.7&#177;4.2</td>
			<td>0.16&#177;0.01</td>
			<td>42.2&#177;0.5</td>
			<td>40</td>
			<td>0.8</td>
		</tr>
	</tbody>
</table>
Table 2: Physicochemical characteristics of NEs.

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

Author Spotlight: Development and Evaluation of a Cationic Nanoemulsion&#45;Encapsulated Retinoic Acid System for Mucosal Vaccination

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

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

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

549 Views.  Third Military Medical University. We werken aan een mucosale vaccin dat een immuunrespons produceert als de set van infectie, die het potentieel heeft om infectie te voorkomen en de overdracht te onderbreken. We hebben een catatonisch nano-emulsie-ingekapseld retinoïnezuurafgiftesysteem ontwikkeld immunisatie met ovalbumine door intramusculaire injectie, die zowel systemische als mucosale immuunresponsen opwekt. In toekomstige studies kunnen diermodellen van darmgerelateerde ziekten worden gebruikt om het effect en het mecha...

Video: Auteur in de schijnwerpers: ontwikkeling en evaluatie van een kationisch nano-emulsie-ingekapseld retinoïnezuursysteem voor mucosale vaccinatie

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

Cancer Research

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

Medicine

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug delivery and immune response. The present study stemmed from the observation that the effects of charge, size, and shape of solid particles on cell affinity are usually considered, but we seldom realize the essential role of softness, dynamic restructuring phenomenon, and complex interface interaction in cellular affinity. Here, we developed poly-lactic-co-glycolic acid (PLGA) nanoparticle-stabilized Pickering emulsion (PNPE) that overcame the shortcomings of rigid forms and simulated the flexibility and fluidity of pathogens. A method was set up to test the affinity of PNPE to cell surfaces and elaborate on the subsequent internalization by immune cells. The affinity of PNPE to bio-mimetic extracellular vesicles (bEVs)-the replacement for bone marrow dendritic cells (BMDCs)-was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed real-time monitoring of cell-emulsion adhesion. Subsequently, the PNPE was used to deliver the antigen (ovalbumin, OVA) and the uptake of the antigens by BMDCs was observed using confocal laser scanning microscope (CLSM). Representative results showed that the PNPE immediately decreased frequency (&#916;F) when it encountered the bEVs, indicating rapid adhesion and high affinity of the PNPE to the BMDCs. PNPE showed significantly stronger binding to the cell membrane than PLGA microparticles (PMPs) and AddaVax adjuvant (denoted as surfactant-stabilized nano-emulsion [SSE]). Furthermore, owing to the enhanced cellular affinity to the immunocytes through dynamic curvature changes and lateral diffusions, antigen uptake was subsequently boosted compared with PMPs and SSE. This protocol provides insights for designing novel formulations with high cell affinity and efficient antigen internalization, providing a platform for the development of efficient vaccines.

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug ...

Bioengineering

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce validly immune responses. However, establishing a series of comprehensive protocols to evaluate the immune response of a novel nanoemulsion adjuvant vaccine is vital. Therefore, this article features a rigorous procedure to determine the physicochemical characteristics of a vaccine (by transmission electron microscopy [TEM], atomic force microscopy [AFM], and dynamic light scattering [DLS]), the stability of the vaccine antigen and system (by a high-speed centrifuge test, a thermodynamic stability test, SDS-PAGE, and western blot), and the specific immune response (IgG1, IgG2a, and IgG2b). Using this approach, researchers can evaluate accurately&#160;the protective effect of a novel nanoemulsion adjuvant vaccine in a lethal MRSA252 mouse model. With these protocols, the most promising nanoemulsion vaccine adjuvant in terms of effective adjuvant potential can be identified. In addition, the methods can help optimize novel vaccines for future development.

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus MRSA Infection

The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce ...

Preparation of a Nanoemulsion Adjuvant Vaccine

Source: Zhang, Z. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64299">Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates the production of nanoemulsion tumor vaccines. The stable oil reservoir depots carrying synthetic peptides and adjuvants are produced by low-energy nanoemulsion, offering significant promise for tumor immunotherapy.

1. Preparation of the I-OVA NE

	Admix 1 mg of monophosphoryl lipid A (MPLA) with 100 &#181;L of dimethyl sulfoxide (DMSO), vortex for 5 min, and allow it ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Immunogenics and Vaccine Development

Functionalized Nanoparticle Targeting of Ovarian Cancer: A Technique to Facilitate Folic Acid-conjugated Nanoparticle Contrast Agent Uptake by Ovarian Cancer Cells

First, mix the folic acid-capped copper sulfide nanoparticles at the proper concentration in fresh RPMI media. Add this nanoparticle solution to each well of the prepared 24-well plate that contains SKOV-3 cells at a concentration of 400 micrograms per milliliter. Incubate at 37 degrees Celsius with 5% carbon dioxide for two hours.
After this, trypsinize the cells with 0.5 milliliters of 0.25% trypsin and EDTA. To neutralize the trypsin, add at least 1 milliliter of folic acid-free RPMI 1640 complete growth media and centrifuge the cells at 123 x g for six minutes. To wash the cells, remove the supernatant.
Resuspend the cells in 2 milliliters of PBS and centrifuge at 123 x g for six minutes. Repeat this wash step twice to remove any unbound nanoparticles.
Then, resuspend the cells with 1 to 2 milliliters of a 2% Tween solution in PBS. Count the cells using a hemocytometer and Trypan Blue. Dilute the cells in a solution of 2% Tween and PBS to the chosen concentration for detection.

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

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