Novel retinoic acid nanoemulsion adjuvant for efficiently enhancing humoral immune response and mucosal immune response and preparation method and application of adjuvant
A technology of retinoic acid and nanoemulsion, which is applied in the field of biomedicine to achieve the effects of complete structure, enhanced immune response, and good uniformity
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Embodiment 1
[0045] Embodiment 1, formulation screening of retinoic acid nanoemulsion adjuvant (RA-NE)
[0046] Formula 1: Retinoic acid nanoemulsion adjuvant (100 g): 1 g of retinoic acid, 2 g of Tween-80, 2 g of propylene glycol, and 95 g of distilled water.
[0047] Recipe 2: Retinoic acid nanoemulsion adjuvant (100 g): 0.1 g of retinoic acid, 30 g of Tween-80, 10 g of glycerin, 10 g of glyceryl caprylate (GTCC), and 49.9 g of distilled water.
[0048] Formula 3: Retinoic acid nanoemulsion adjuvant (100 g): 2 g of retinoic acid, 5 g of Tween-20, 0.1 g of propylene glycol, 0.1 g of ethyl acetate, and 92.8 g of distilled water.
[0049] Formula 4: retinoic acid nanoemulsion adjuvant (100g): retinoic acid 5g, Tween-60 20g, propylene glycol 4g, medicinal paraffin oil 6g, distilled water 65g.
[0050] Formula 5: retinoic acid nanoemulsion adjuvant (100g): 2g retinoic acid, 30g Tween-85, 10g 1,3-butanediol, 15g GTCC, 43g distilled water.
[0051] Formula 6: retinoic acid nanoemulsion adjuva...
Embodiment 2
[0072] Example 2, Preparation and Characterization of Preferred atRA-NE
[0073] 1. Preparation of atRA-NE
[0074] Accurately weigh 10 mg of retinoic acid (atRA) in a 50 ml clean and dry beaker, drop the oil phase, surfactant and co-surfactant and stir until fully dissolved, then adopt the low-energy emulsification method, that is, slowly add sterilized pure Water until the volume of the system is 10mL, prepare retinoic acid nanoemulsion adjuvant (atRA-NE), and store at room temperature in the dark. Blank nanoemulsion adjuvant (BNE), there is no need to add retinoic acid in the prescription.
[0075] 2. Particle size and Zeta potential detection
[0076] The prepared nanoemulsion adjuvant was diluted 100 times with ultrapure water, and the particle size and Zeta potential were detected in a Nano ZA dynamic light scattering particle size potentiometer.
[0077] 3. Transmission electron microscope (TEM) detection
[0078] Dilute the prepared nanoemulsion adjuvant 100 times ...
Embodiment 3
[0088] Example 3, atRA-NE stimulates mouse lymphocyte maturation ability
[0089] 1. The ability of atRA-NE to promote the maturation of BMDC cells
[0090]Kill the mouse and take its femur, use a 1mL syringe to draw 1640 medium containing 10% FBS to wash the bone marrow 5 times, collect the bone marrow cell washing solution, centrifuge at 1500rpm for 15 minutes, and plant the collected cells in the cell culture plate, every other day Half the amount of medium was changed once, and the cells were collected by centrifugation on the 6th day to obtain mouse bone marrow-derived dendritic cells (BMDCs), and the cell concentration was adjusted to 5×10 5 pc / mL, add 2ml / well in a 6-well plate. Afterwards, 20 μL medium, 20 μL 1:100 diluted Blank-NE, 20 μg atRA or 20 μL 1:100 diluted atRA-NE were added to each well, mixed by blowing and placed in a cell culture incubator for 24 hours. Centrifuge and wash cells with PBS, dilute APC-labeled mouse CD11c antibody, PE-labeled mouse CD40 an...
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