Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

We developed the PLGA nanoparticles stabilized Pickering emulsion. The Pickering emulsion improved the cellular affinity to antigen protein cells, inducing efficient internalization of antigens. The nanoparticle stabilized Pickering emulsions were easy to prepare and deliver antigen efficiently.

Additionally, a method was used to monitor the affinity of emulsion to cell and elaborate on the internalization. This protocol provides insights for designing novel formulations with high cell efficiency and efficient antigen internalization, which provide a platform for the development of efficient vaccines. To begin, add 100 milligrams of polylactic-co-glycolic acid to 10 milliliters of acetone and ethanol mixture in a ratio of four to one to serve as the oil phase.

Place 20 milliliters of PVA aqueous solution under the fume hood and magnetically stir at 400 rotations per minute. Add five milliliters of the oil phase into the PVA solution drop-by-drop using a syringe pump. Then stir the mixture in the fume hood until the organic solvents completely evaporate.

After the volatilization of organic solvents, centrifuge the mixture at 15, 000 G.Wash three or more times until the final washing water is clear and transparent. Re-suspend the washed PLGA nanoparticles in two milliliters of deionized water and freeze the mixture at 80 degrees Celsius for 24 hours. To characterize the size and zeta potential of PLGA nanoparticles, add 10 microliters of PLGA nanoparticles into one milliliter of deionized water to obtain a diluent solution and transfer the diluent solution to a DTS1070 cell.

Switch on the computer and dynamic light scattering analyzer. Then place the DTS1070 cell in the DLS system. Click on the Zetasizer software and create a new measurement file to set up the determination procedure.

Then start the determination procedure to obtain the particle size and zeta potential distribution. To prepare PLGA nanoparticles stabilized Pickering emulsion, or PNPE, add freeze-dried PLGA nanoparticles to deionized water at a concentration of four milligrams per milliliter and then add squalane as the oil phase. Prepare PNPE via one-step sonication for five minutes at 100 watt in a water bath sonicator.

Dilute 20 microliters of PNPE and one milliliter of deionized water. Drop 20 microliters of the emulsion on the slide. Observe the morphology and homogeneity of the emulsion using optical microscopy at 40 times magnification and obtain photographs.

Turn on the spin coater by pressing the power button. Press the control button and set the coating time and speed. Connect the nitrogen pipe to the spin coater and adjust the fractionation of the gas cylinder to 0.4 kilopascals.

Place the clean chip on the suction cup of the spin coater. Add 100 microliters of poly-l-lysine dropwise to the center of the silicon dioxide sensor and close the top cover of the spin coater. Press the start button to start coating the sample and stop the machine when finished.

Turn off the vacuum pump, turn off the power and control buttons, and remove the poly-l-lysine modified silicon dioxide sensor. To determine the adhesion of biomimetic extracellular vesicles to PNPE, turn on the computer, the electronic unit, and the peristaltic pump, and activate the temperature control at the bottom right in the software to ensure that the set temperature is approximately one degree Celsius below room temperature. Place the poly-l-lysine modified silicon dioxide sensors in the flow cell according to the operating instructions and connect the measurement line between the flow cell and the flow pump.

Place the flow cell inside the flow module system. Click on the Acquisition toolbar and select Setup Measurement to search for 1, 3, 5, 7, 9, and 11 octaves of the chip in the channel used. To correct the baseline, click on Start Measurement to allow air to enter the flow module until the air baseline is smooth.

After flowing deionized water for 10 to 15 minutes to enable the solution baseline equilibrium again, pump the prepared PNPE solution into the flow module at a flow rate of 50 microliters per minute to achieve equilibrium adsorption on the silicon dioxide sensor. Pump the prepared biomimetic extracellular vesicle solution into the flow module at a flow rate of 50 microliters per minute to track the process of bEV adhesion to the PNPE surface. Incubate the bone marrow dendritic cells with 1640 complete media for seven days and then seed them into small confocal laser dishes at 1 times 10 to the 6 per well overnight at 37 degrees Celsius and a 5%carbon dioxide incubator.

Mix 0.5 milliliters of cyanine 5 labeled ovalbumin, with 0.5 milliliters of PNPE for one hour and remove the fluidic antigen by centrifugation at 5, 000 G for 20 minutes to develop vaccine formulation. After re-suspension with 200 microliters of deionized water, add 10 microliters of the formulation into the small confocal laser dishes under a super clean bench and co-culture with bone marrow dendritic cells for six hours. After removing the culture fluid from the cells, wash them twice with pre-warmed phosphate buffered saline.

Fix the cells with 4%formaldehyde solution and PBS for 10 minutes at room temperature. Wash the cells with PBS two or three times at room temperature for 10 minutes each. Permeabilize the cells with 100 microliters of 0.5%Triton X-100 solution for five minutes at room temperature.

After washing the cells again with PBS, cover the cells on the glass bottom dish of the small confocal laser dishes with 200 microliters of fluorescein isothiocyanate conjugated phalloidin working solution and incubate for 30 minutes at room temperature in the dark. After washing the cells with PBS three times for five minutes each, restain the nuclei with 200 microliters of DAPI solution for around 30 seconds. For image analysis, switch on the confocal microscope hardware in sequence including the laser, confocal, microscope and computer.

Click the software and select Nikon Confocal to enter the testing system. In the confocal microscope system, turn on the various units in the order noted. First, set up the FITC, DAPI and Cy5 channels and adjust the corresponding HV and offset.

Then after selecting the 100 times oil lens and put a drop of cedar oil on the top place the small confocal laser dishes containing stained bone marrow dendritic cells on the microscope stage. Click the scan button. Under fluorescence microscope, locate cells of interest by moving the x-axis, y-axis and z-axis.

Tune the laser intensity, image size and other parameters to enable scanning high quality confocal images. Finally, click Capture button and save the images. The adhesion of biomimetic extracellular vesicles to PNPE was tracked using QCM-D.

An immediate decrease in frequency, indicated a rapid adhesion of biomimetic extracellular vesicles to PNPE after the encounter. Moreover, delta F decreased with increasing biomimetic extracellular vesicles concentration, reflecting a concentration-dependent effect. PLGA microparticles and surfactant-stabilized nano emulsion were weakly bound to biomimetic extracellular vesicles, even at high concentration of 80 micrograms per milliliter, which probably resulted from a lack of contact sites with the immune cells.

The cyanine-5 ovalbumin fluorescent signal indicated that the total amount of antigen internalized into cells is significantly higher in the PNPE treated group compared to that in the PLGA microparticles and surfactant-stabilized nano emulsion treated group. The quantitative analysis of cellular uptake showed a significant higher relative intensity of fluorescence and bone marrow dendritic cells treated with PNPE than in those treated with PLGA microparticles and surfactant-stabilized nano emulsion. The obtained results demonstrated that the PNPE promoted the antigen internalization and effectively delivered the antigen intracellularly.

In order to obtain homogeneous emulsion, the mixture must be continuously shaken during sonication.