Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

Summary

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.

Abstract

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 (ΔF) 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.

Introduction

To combat epidemic, chronic, and infectious diseases, it is imperative to develop effective adjuvants for prophylactic and therapeutic vaccinations^1,2. Ideally, the adjuvants should possess excellent safety and immune activation^3,4,5. 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 response^6,7,8. 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 patterns^9,10. Upon contacting with cells, delivery systems begin to interact with the extracellular matrix and cell membrane, which led to internalization and subsequent cellular responses^11,12. 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 membrane^14,15. Under these circumstances, the properties of the delivery system depend on the affinity to APCs, which subsequently affect the uptake quantity^16,17.

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 investigated^18,19. 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 particles^20,21. 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 uptake^22,23. 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 field^24,25. 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.

Protocol

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

1. Preparation of PLGA nanoparticles (PNPs)
   1. Add 0.5 g of polyvinyl alcohol (PVA) to 120 mL of deionized water at 90 °C and stir until completely dissolved to prepare the PVA solution. Store the solution in the refrigerator (4 °C) after cooling to room temperature.
   2. Add 100 mg of PLGA to 10 mL of acetone and ethanol mixture (ratio of 4:1) to serve as the oil phase.
   3. Place 20 mL of PVA aqueous solution under the fume hood and magnetically stir at 400 rpm/min. Add 5 mL 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.
      NOTE: With the increase in the concentration of the particles in the oil phase, the water phase solution gradually turns into a clear and transparent light bluish-white or milky white.
   4. After the volatilization of organic solvents (2-3 h), centrifuge the mixture at 15,000 x g. Wash three or more times until the final washing water is clear and transparent.
      ​NOTE: The purpose of this step is mainly to wash off the residual PVA on the particle surface to prevent it from affecting the Pickering emulsion preparation.
   5. Re-suspended the washed PNPs in 2 mL of deionized water and freeze the mixture at -80 °C for 24 h. Subsequently freeze-dry the particles in a lyophilizer for 48-72 h. The prepared PNPs are white flocky.
2. Characterization of PNPs
   1. To characterize the size and zeta potential of PNPs, add 10 µL of PNPs into 1 mL of deionized water to obtain diluent solution and transfer the diluent solution to a DTS1070 cell. Switch on the computer and dynamic light scattering analyzer (DLS), then place the DTS1070 cell in the DLS system.
   2. Click on the Zeta Size 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.
   3. To observe the PNPs morphology, evenly spread the 0.1 mL PNPs solution (diluted 40 times) on a 5 cm x 5 cm tin foil sheet and let the water evaporate naturally in a well-vented fume hood overnight.
   4. Cut out a small part of the tin foil and secure it on the sample table with conductive tape. Spray it with gold at a current of 10 mA for 120 s. Subsequently observe the surface morphology of the sample using scanning electron microscopy (SEM).

2. Preparation and characterization of PNPE

1. To prepare PNPE, add freeze-dried PNPs to deionized water at a concentration of 4 mg/mL (the aqueous phase) and then add squalene as the oil phase. Prepare PNPE via one-step sonication for 5 min at 100 W in a water bath sonicator. The oil-water phase ratio is 1:9.
2. Characterization of the prepared PNPE
   1. Open the Mastersizer 2000 software and the laser in sequence. Click on Measure to open the pre-set program and set the sample name. Click on Start to start measuring the background of the sample, then, using a dropper, add 1 mL of PNPE to the sample tank of the laser particle size analyzer to measure the particle size of the emulsion three times in parallel.
   2. Dilute 20 µL of PNPE in 1 mL of deionized water. Drop 20 µL of the emulsion on the slide. Observe the morphology and homogeneity of the emulsion using optical microscopy at 40x magnification and obtain photographs.
3. Antigen loading efficiency
   1. Dissolve 200 µg of ovalbumin (OVA) in 500 mL of deionized water. Then mix it with 500 mL of prepared PNPE and shake for 1 h at room temperature. Remove the fluidic antigen by centrifugation at 5000 x g for 20 min.
   2. Determine the fluidic antigen concentrations using bicinchoninic acid (BCA) assays²⁷. The formula for calculating the antigen loading efficiency is as follows:
      Antigen loading efficiency =
      ​Use the same method to determine the antigen loading efficiency of the SSE that is used as a control group.
4. Stability of PNPE
   1. Divide 6 mL of the prepared PNPE into six equal portions, and store three parts each at 4 °C and 25 °C, respectively. Dilute 20 µL of stored PNPE into 1 mL of deionized water (1:50) on day 0, 3, and 6. Then, measure the size and zeta potential of the PNPE stock solutions kept at different temperatures.
      NOTE: The different storage temperatures are set to compare the effect of different temperatures on the stability of PNPE, which can optimize the storage condition for high stability and long shelf life.
5. Preparation of PLGA microparticles (PMPs)
   1. Dissolve 500 mg of PLGA in 5 mL of dichloromethane as the oil phase, and then pour into 50 mL of external aqueous phase containing 1.5% PVA to obtain the oil/water mixture. Homogenize the mixture at 3000 rpm for 1 min using a homogenizer to obtain coarse-emulsions.
   2. Maintain the uniformity of PLGA particle size of microspheres (determined by coarse-emulsions) by membrane emulsification. Install a 5.2 µm membrane in the membrane tube. Adjust the pressure to 800 kPa and keep steady.
   3. Open the exhaust valve and feed valve, and after adding the coarse-emulsions to the sample tank, close the exhaust valve and feed valve, open the discharge valve and inlet valve, and take the post-film emulsion in a 200 mL beaker. Repeat the process three times to obtain pre-double emulsions.
   4. Stir the pre-double emulsions to evaporate the dichloromethane at room temperature to cure the microspheres. Wash the microspheres using deionized water at 7741 x g for 3 min five times. Pre-freeze in a freezer at -80 °C and finally freeze-dry to gain dried microspheres.
6. Characterization of PMPs
   1. Open the Mastersizer 2000 software and the laser in sequence. Click on Measure to open the pre-set program and set the sample name. Click on Start to start measuring the background of the sample. Then, add 1 mL of PMPs to the sample adding tank of the laser particle size analyzer with a dropper and measure the particle size of the microparticles three times in parallel.
7. Softness analysis
   NOTE: PNPE was coated onto the SiO₂ sensor to measure the softness.
   1. Clean the SiO₂ quartz sensor chip by UV-ozone treatment for 10 min, rinse twice with 5 mL of ethanol (75% v/v), and dry with N₂. Install the empty SiO₂ quartz sensor chip into the flow cell.
   2. Turn on the computer and activate the temperature control at the bottom right of the software to ensure that the set temperature is approximately 1 °C below room temperature.
   3. Click on the Acquisition button in the toolbar and select Setup Measurement to search for the 1, 3, 5, 7, 9, and 11 octaves of the chip in the channel used. Click on the Acquisition button in the toolbar and select Start Measurement.
   4. Correct the baseline with air, and when the baseline is balanced, select Stop and save the file as a blank.
   5. Clean the chip and spin coat 10 µg/mL of PNPE on the chip. Briefly, turn on the spin coater and set the operating parameter. Connect the N₂ pipe to the spin coater, adjust the fractionation of the gas cylinder to 0.4 kPa, and place the clean chip on the suction cup of the spin coater. Add 100 µL of PNPE dropwise to the center of the SiO₂ sensor and close the top cover of the spin coater to complete the coating.
   6. Install the PNPE coated chip into the flow cell. Repeat steps 2.7.3-2.7.4 and save the file as PNPE. Open both the blank and PNPE files and click on the Stitch button to obtain the related data of dissipation (ΔD).

3. Isolate and culture BMDCs ²⁸

NOTE: Make sure that all reagents and samples are placed on ice, as this has a positive effect on cell activity. To maintain sterility, perform all steps on an ultra-clean bench using sterile utensils.

1. Euthanize the C57BL/6 (female, 6-8 weeks old) mice via CO₂ inhalation and soak them in ethanol 70% (v/v) for a brief sterilization.
2. After soaking for 3-5 min, transfer the mice to a super clean bench. Remove leg muscles using stainless-steel scissors to expose the femur and tibia, and then separate the femur and tibia.
3. Fill a Petri dish with 70% (v/v) ethanol and soak the clean bones in it for 5-10 s to complete external sterilization. Clean all the bones and place them in a sterile tube with ice around them.
4. Trim the femur and tibia close to the joint using scissors. Insert the syringe needle into the bone to flush out the bone marrow into a centrifuge tube using pre-chilled RPMI medium (1640).
5. Rinse two or three times until the bone is completely white. Pipette the bone marrow several times to separate all clumps. Filter the cells into a 15 mL centrifuge tube using a 40 µm diameter sieve.
6. Centrifuge at 4 °C and 500 x g for 5 min. Discard the supernatant and add 2 mL of erythrocyte lysate to the sediment for 4 min.
7. After washing two or three times, re-suspend the cells in 2 mL of complete medium (1 % penicillin-streptomycin, 10 % fetal bovine serum, 20 ng/mL of IL-4, and 10 ng/mL of GM-CSF). Then, dilute 10 µL of cells into 1 mL of PBS and insert the chip of the handheld automated cell counter into the cell dilution for cell counting. Finally, seed cells at a density of 1 x 10⁶ cells/mL into a 10 mm culture dish with a complete medium.
8. Culture the cells in a cell incubator with 5% CO₂ at 37 °C. Change the medium every 2 days. On day 7, transfer the cells to 50 mL tube and centrifuge at 450 x g for 5 min to collect the cells.

4. Preparation of bio-mimetic extracellular vesicles (bEVs)

1. Assemble the mini-extruder in sequence as per the manufacturer's instructions. Make sure that the syringe housing is not cracked before using it. Ensure all O-rings are in good condition before every experiment and replace worn or damaged ones promptly. Worn or damaged O-rings can cause sudden pressure release when operating the extruder.
2. Aspirate the BMDCs repeatedly and transfer them to a centrifuge tube. Harvest the cells by centrifugation at 450 x g for 5 min at 4 °C. Pressurize the cell suspensions through 10 µm, 5 µm, and 1 µm polycarbonate membranes for 30 passes using the mini-extruder²⁹.
   NOTE: To enable bEV size uniformity, the BMDC suspensions were pushed through 10 µm, 5 µm, and 1 µm polycarbonate membranes for 30 passes. Additionally, during operation, make sure to apply force evenly and maintain the direction of force along the axis of the syringe.
3. Centrifuge the pooled supernatants for 5 min at 1000 x g and 4 °C to remove the cells. Collect the supernatant and centrifuge it at 3000 x g for 5 min at 4 °C to remove the remaining cells and cell debris.
4. Collect the supernatant and centrifuge it at 100,000 x g for 90 min at 4 °C. Discard supernatant and re-suspend the bEVs in 2 mL of HEPES-buffered saline (HBS). Purify the bEVs via filtration through a 0.45 µm membrane and store the purified bEVs at −80 °C.

5. bEVs adhere to the PNPE

NOTE: The SiO₂ sensor was modified via spin-coating method.

1. SiO₂ sensor modified with poly-L-lysine (PLL).
   1. Clean the SiO₂ quartz sensor chip using UV-ozone treatment for 10 min, rinse twice with ethanol, and dry with N₂.
   2. Turn on the spin coater by pressing the Power button, press the Control button, and set the coating time and speed. The initial rotation speed is 400 rpm, which is steadily increased to 5000 rpm.
   3. Connect the N₂ pipe to the spin coater and adjust the fractionation of the gas cylinder to 0.4 kPa. Place the clean chip on the suction cup of the spin coater. Add 100 µL of PLL dropwise to the center of the SiO₂ sensor and close the top cover of the spin coater.
   4. 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 PLL-modified SiO₂ sensor.
      ​NOTE: Before pressing Start, make sure that the coating time and speed have been set. Additionally, make sure that the SiO₂ sensor is placed in the center of the suction cup. Make sure that the lid is closed before running process to prevent accidents.
2. Determination of adhesion of bEVs to PNPE
   1. Turn on the computer, the electronic unit, the peristaltic pump, and activate the temperature control at the bottom right in the software to ensure that the set temperature is approximately 1 °C below room temperature.
   2. Place the PLL-modified SiO₂ sensors in the flow cell according to the operating instructions and connect the measurement line between the flow cell and flow pump. Place the flow cell inside the flow module system and rinse them with ultrapure water before beginning the experiments.
   3. 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. Subsequently, flow deionized water for 10-15 min to enable the solution baseline equilibrium again.
   4. Pump the prepared PNPE solution into the flow module at a flow rate of 50 µL/min to achieve equilibrium adsorption on the SiO₂ sensor.
      NOTE: The ΔF no longer changes when the PNPE is pumped into the flow module again, indicating that the surface of the SiO₂ sensors has been completely covered with PNPE.
   5. Pump the prepared bEVs solution into the flow module at a flow rate of 50 µL/min to track the process of bEV adhesion to the PNPE surface.

6. CLSM analysis of antigen uptake

1. Co-culture PNPE-OVA with BMDCs
   1. Incubate the BMDCs with 1640 complete media (1% penicillin-streptomycin, 10% fetal bovine serum, 20 ng/mL of IL-4, and 10 ng/mL of GM-CSF) for 7 days, and then seed them into small confocal laser dishes at 1 x 10⁶ per well overnight at 37 °C in a 5% CO₂ incubator.
   2. Mix 0.5 mL of Cy5-labeled OVA (400 µg/mL) with 0.5 mL of PNPE for 1 h and remove the fluidic antigen by centrifugation at 5000 x g for 20 min to develop vaccine formulation. After re-suspension with 200 µL of deionized water, add 10 µL of the formulation (10 µg/mL OVA) into the small confocal laser dishes under a super-clean bench and co-culture with BMDCs for 6 h.
2. Actin cytoskeleton stain
   1. Remove the culture fluid from the cells and wash them twice with pre-warmed phosphate buffered saline (PBS; pH 7.4).
   2. Fix the cells with 4% formaldehyde solution in PBS for 10 min at room temperature. During fixation, avoid fixatives containing methanol, which may destroy actin.
   3. Wash the cells with PBS two or three times at room temperature for 10 min each. Permeabilize the cells with 100 µL of 0.5% Triton X-100 solution for 5 min at room temperature. Wash the cells with PBS two or three times at room temperature for 10 min each again.
   4. Cover the cells on the glass-bottomed dish of the small confocal laser dishes with 200 µL of fluorescein isothiocyanate (FITC)-conjugated phalloidin working solution and incubate for 30 min at room temperature in the dark. To reduce background signal, add 1% bovine serum albumin to the FITC-conjugated phalloidin working solution. In addition, cover the lid of the small confocal laser dishes during incubation to prevent evaporation of the solution.
   5. Wash the cells with PBS thre times for 5 min each. Re-stain the nuclei with 200 µL of DAPI solution (concentration: 100 nM) for around 30 s.
3. Image analysis
   1. Switch on the confocal microscope hardware in sequence, including the laser, confocal, microscope, and computer. Click the NIS-Elements AR 5.20.00 software and select Nikon Confocal to enter the testing system.
   2. 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, select the 100x oil lens and put a drop of cedar oil on the top. Place the small confocal laser dishes containing stained BMDCs on the microscope stage.
   3. Click the Scan button. Under a 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.

Results

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 ± 3.5 nm and the zeta potential was -16.4 ± 0.4 mV (Figure 1C ...

Discussion

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...

Materials

List of materials used in this article

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