This work was supported by Project supported by the National Key Research and Development Program of China (2021YFE020527, 2021YFC2302605, 2021YFC2300142), From 0 to 1 Original Innovation Project of Basic Frontier Scientific Research Program of Chinese Academy of Sciences (ZDBS-LY-SLH040), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant No. 21821005).

All methods described in this protocol have been approved by the Institute of Process Engineering, Chinese Academy of Sciences. All animal experiments were performed in strict accordance with the Regulations for the Care and Use of Laboratory Animals and Guideline for Ethical Review of Animal (China, GB/T35892-2018).
1. Preparation and characterization of PLGA nanoparticles
<ol>
	<li>Preparation of PLGA nanoparticles (PNPs)
	<ol>
		<li>Add 0.5 g of polyvinyl alcohol (PVA) to...

<ol>
	<li>Ma, G., Gu, Z., Wei, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+vaccine+delivery.">Advanced vaccine delivery.</a> Advanced Drug Delivery Reviews. 183, 114170 (2022).</li><li>Sharma, J., Carson, C. S., Douglas, T., Wilson, J. T., Joyce, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-particulate+platforms+for+vaccine+delivery+to+enhance+antigen-specific+cd8(+)+t-cell+response.">Nano-particulate platforms for vaccine delivery to enhance antigen-specific cd8(+) t-cell response.</a> Methods in Molecular Biology. 2412, 367-398 (2022).</li><li>Nguyen, T. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+immunogenicity+of+nanocovax,+a+sars-cov-2+recombinant+spike+protein+vaccine:+interim+results+of+a+double-blind,+randomised+controlled+phase+1+and+2+trial.">Safety and immunogenicity of nanocovax, a sars-cov-2 recombinant spike protein vaccine: interim results of a double-blind, randomised controlled phase 1 and 2 trial.</a> The Lancet Regional Health. Western Pacific. 24, 100474 (2022).</li><li>Coates, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+immunogenicity+of+a+trivalent+virus-like+particle+vaccine+against+western,+eastern,+and+venezuelan+equine+encephalitis+viruses:+a+phase+1,+open-label,+dose-escalation,+randomised+clinical+trial.">Safety and immunogenicity of a trivalent virus-like particle vaccine against western, eastern, and venezuelan equine encephalitis viruses: a phase 1, open-label, dose-escalation, randomised clinical trial.</a> The Lancet Infectious Diseases. 22 (8), 1210-1220 (2022).</li><li>Wei, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+a+nanoparticle+therapeutic+vaccine+in+patients+with+chronic+hepatitis+b:+a+randomized+clinical+trial.">Efficacy and safety of a nanoparticle therapeutic vaccine in patients with chronic hepatitis b: a randomized clinical trial.</a> Hepatology. 75 (1), 182-195 (2022).</li><li>Krishnan, R., Kim, J. 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We developed PLGA nanoparticle-stabilized oil/water emulsion as a delivery system for enhanced antigen internalization. The prepared PNPE possessed a densely packed surface to support the landing spot and unique softness and fluidity for potent cellular contact with the immune cell membrane. Furthermore, the oil/water interface offered high-content antigen loading, and amphiphilic PLGA conferred PNPE with high stability for the transportation of antigens to immune cells. The PNPE could rapidly adhere to the surface of th...

To combat epidemic, chronic, and infectious diseases, it is imperative to develop effective adjuvants for prophylactic and therapeutic vaccinations1,2. Ideally, the adjuvants should possess excellent safety and immune activation3,4,5. Effective uptake and process of antigens by antigen-presenting cells (APCs) are thought to be an essential stage in the downstream signaling cascades and initiation of the immune response6,7,8. Hence, gaining a clear understanding of the mechanism of interaction of immune cells with antigens and designing adjuvants to enhance internalization are efficient strategies to enhance the efficiency of vaccines.
Micro-/nanoparticles with unique properties have been previously investigated as antigen delivery systems to mediate the cellular uptake of antigens and the cellular interaction with pathogen-associated molecular patterns9,10. Upon contacting with cells, delivery systems begin to interact with the extracellular matrix and cell membrane, which led to internalization and subsequent cellular responses11,12. Previous studies have brought to light that the internalization of particles takes place through cell membrane-particle adhesion13, followed by flexible deformation of the cell membrane and diffusion of the receptor to the surface membrane14,15. Under these circumstances, the properties of the delivery system depend on the affinity to APCs, which subsequently affect the uptake quantity16,17.
To gain insights into the design of the delivery system for improved immune response, extensive efforts have been focused on the investigation of the relationship between the properties of particles and cellular uptake. The present study stemmed from the observation that solid micro-/nanoparticles with various charges, sizes, and shapes are often studied in this light, while the role of fluidity in antigen internalization is seldom investigated18,19. In fact, during adhesion, the soft particles demonstrated dynamic curvature changes and lateral diffusions to increase the contact area for multivalent interactions, which can hardly be replicated by the solid particles20,21. In addition, cell membranes are phospholipid bilayers (sphingolipids or cholesterol) at the site of uptake, and hydrophobic substances can alter the conformational entropy of lipids, reducing the amount of energy required for cellular uptake22,23. Thus, amplifying mobility and promoting hydrophobicity of the delivery system may be an effective strategy for strengthening antigen internalization to enhance immune response.
Pickering emulsion, stabilized by solid particles assembled at the interface between two immiscible liquids, have been widely used in the biological field24,25. In fact, the aggregating particles on the oil/water interface determine the formulation of multi-level structures, which promote multi-level delivery system-cellular interactions, and further induce multi-functional physiochemical properties in drug delivery. Because of their deformability and lateral mobility, Pickering emulsions were expected to enter in multi-valent cellular interaction with the immunocytes and be recognized by the membrane proteins26. In addition, as oily micelle cores in Pickering emulsions are not completely covered with solid particles, Pickering emulsions possess gaps of different sizes between particles on the oil/water interface, which cause higher hydrophobicity. Thus, it is crucial to explore the affinity of Pickering emulsions to APCs and elaborate on the subsequent internalization to develop efficient adjuvants.
Based on these considerations, we engineered a PLGA nanoparticle-stabilized Pickering emulsion (PNPE) as a fluidity vaccine delivery system that also helped to gain valuable insights in the affinity of the PNPE to BMDCs and cellular internalization. The real-time adhesion of bio- mimetic extracellular vesicles (bEVs; a replacement of BMDCs) to PNPE was monitored via a label-free method using a quartz crystal microbalance with dissipation monitoring (QCM-D). Following characterization of the affinity of PNPE to BMDCs, confocal laser scanning microscopy (CLSM) was used to determine the antigen uptake. The result indicated the higher affinity of PNPE to BMDCs, and the efficient internalization of the antigen. We anticipated that the PNPE would exhibit higher affinity to APCs, which may better stimulate the internalization of antigens to enhance immune responses.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AddVax</td>
			<td>InvivoGen</td>
			<td>Vac-adx-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Biosharp</td>
			<td>BS-70-CS</td>
			<td>70 &#956;m</td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope (CLSM)</td>
			<td>Nikon</td>
			<td></td>
			<td>A1</td>
		</tr>
		<tr>
			<td>Cy3 NHS Ester</td>
			<td>YEASEN</td>
			<td>40777ES03</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Staining Solution</td>
			<td>Beyotime</td>
			<td>C1005</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Phalloidin</td>
			<td>Solarbio</td>
			<td>CA1620</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastersizer 2000 Particle Size Analyzer</td>
			<td>Malvern</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA protein Assay Kit</td>
			<td>Thermo Science</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane emulsification equipment</td>
			<td>Zhongke Senhui Microsphere Technology</td>
			<td></td>
			<td>FM0201/500M</td>
		</tr>
		<tr>
			<td>Mini-Extruder</td>
			<td>Avanti Polar Lipids, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NANO ZS</td>
			<td>Malvern</td>
			<td></td>
			<td>JSM-6700F</td>
		</tr>
		<tr>
			<td>Polycarbonate membranes</td>
			<td>Avanti Polar Lipids, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly (lactic-co-glycolic acid) (PLGA)</td>
			<td>Sigma-Aldrich</td>
			<td>26780-50-7</td>
			<td>Mw 7,000-17,000</td>
		</tr>
		<tr>
			<td>Poly-L-lysine Solution</td>
			<td>Solarbio</td>
			<td>P2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly (vinyl alcohol) (PVA)</td>
			<td>Sigma-Aldrich</td>
			<td>9002-89-5</td>
			<td></td>
		</tr>
		<tr>
			<td>QSense Silicon dioxide sensor</td>
			<td>Biolin Scientific</td>
			<td>QSX 303</td>
			<td>Surface roughness &#60; 1 nm RMS</td>
		</tr>
		<tr>
			<td>Quartz Crystal Microbalance</td>
			<td>Biosharp</td>
			<td></td>
			<td>Q-SENSE E4</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640 basic</td>
			<td>Gibco</td>
			<td>C22400500BT</td>
			<td>L-Glutamine, 25 mM HEPES</td>
		</tr>
		<tr>
			<td>Scanning Electron Microscopy (SEM)</td>
			<td>JEOL</td>
			<td></td>
			<td>JSM-6700F</td>
		</tr>
		<tr>
			<td>Squalene</td>
			<td>Sigma-Aldrich</td>
			<td>111-02-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A simple one-step sonification was used to obtain PNPE. First, we prepared uniform PNPs for use as the solid stabilizer (Figure 1A). The morphology of PNPs were observed though SEM, showing that they are mostly uniform and spherical (Figure 1B). The hydrodynamic size and zeta potential of the formulations were detected via DLS. The diameter of the PNPs was 187.7 &#177; 3.5 nm and the zeta potential was -16.4 &#177; 0.4 mV (Figure 1C...

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Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

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 ...

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.

cellular-affinity-particle-stabilized-emulsion-to-boost-antigen

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JoVE Journal

Bioengineering

1.6K visualizações.  Tokyo University of Agriculture and Technology. Desenvolvemos a emulsão de Pickering estabilizada de nanopartículas PLGA. A emulsão de Pickering melhorou a afinidade celular com as células proteicas do antígeno, induzindo a internalização eficiente dos antígenos. As emulsões de Pickering estabilizadas com nanopartículas foram fáceis de preparar e fornecer antígeno de forma eficiente.Além disso, um método foi utilizado para monitorar a afinidade da emulsão à célula e elaborar a internalização. Este protocolo fornec...