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 120 mL of deionized water at 90 &#176;C and stir until completely dissolved to prepare the PVA solution. Store the solution in the refrigerator (4 &#176;C) after cooling to room temperature.</li>
		<li>Add 100 mg of PLGA to 10 mL of acetone and ethanol mixture (ratio of 4:1) to serve as the oil phase.</li>
		<li>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.</li>
		<li>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. 
		&#8203;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.</li>
		<li>Re-suspended the washed PNPs in 2 mL of deionized water and freeze the mixture at -80 &#176;C for 24 h. Subsequently freeze-dry the particles in a lyophilizer for 48-72 h. The prepared PNPs are white flocky.</li>
	</ol>
	</li>
	<li>Characterization of PNPs
	<ol>
		<li>To characterize the size and zeta potential of PNPs, add 10 &#181;L 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>
2. Preparation and characterization of PNPE
<ol>
	<li>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.</li>
	<li>Characterization of the prepared PNPE
	<ol>
		<li>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.</li>
		<li>Dilute 20 &#181;L of PNPE in 1 mL of deionized water. Drop 20 &#181;L of the emulsion on the slide. Observe the morphology and homogeneity of the emulsion using optical microscopy at 40x magnification and obtain photographs.</li>
	</ol>
	</li>
	<li>Antigen loading efficiency
	<ol>
		<li>Dissolve 200 &#181;g 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.</li>
		<li>Determine the fluidic antigen concentrations using bicinchoninic acid (BCA) assays27. The formula for calculating the antigen loading efficiency is as follows: 
		Antigen loading efficiency = <img alt="Equation 1" src="/files/ftp_upload/64406/64406eq01.jpg" style="margin: auto;" /> 
		&#8203;Use the same method to determine the antigen loading efficiency of the SSE that is used as a control group.</li>
	</ol>
	</li>
	<li>Stability of PNPE
	<ol>
		<li>Divide 6 mL of the prepared PNPE into six equal portions, and store three parts each at 4 &#176;C and 25 &#176;C, respectively. Dilute 20 &#181;L 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.</li>
	</ol>
	</li>
	<li>Preparation of PLGA microparticles (PMPs)
	<ol>
		<li>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.</li>
		<li>Maintain the uniformity of PLGA particle size of microspheres (determined by coarse-emulsions) by membrane emulsification. Install a 5.2 &#181;m membrane in the membrane tube. Adjust the pressure to 800 kPa and keep steady.</li>
		<li>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.</li>
		<li>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 &#176;C and finally freeze-dry to gain dried microspheres.</li>
	</ol>
	</li>
	<li>Characterization of PMPs
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Softness analysis 
	NOTE: PNPE was coated onto the SiO2 sensor to measure the softness.
	<ol>
		<li>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.</li>
		<li>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 &#176;C below room temperature.</li>
		<li>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.</li>
		<li>Correct the baseline with air, and when the baseline is balanced, select Stop and save the file as a blank.</li>
		<li>Clean the chip and spin coat 10 &#181;g/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 &#181;L of PNPE dropwise to the center of the SiO2 sensor and close the top cover of the spin coater to complete the coating.</li>
		<li>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 (&#916;D).</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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 &#181;m diameter sieve.</li>
	<li>Centrifuge at 4 &#176;C and 500 x g for 5 min. Discard the supernatant and add 2 mL of erythrocyte lysate to the sediment for 4 min.</li>
	<li>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 &#181;L 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.</li>
	<li>Culture the cells in a cell incubator with 5% CO2 at 37 &#176;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.</li>
</ol>
4. Preparation of bio-mimetic extracellular vesicles (bEVs)
<ol>
	<li>Assemble the mini-extruder in sequence as per the manufacturer&#39;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.</li>
	<li>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 &#176;C. Pressurize the cell suspensions through 10 &#181;m, 5 &#181;m, and 1 &#181;m polycarbonate membranes for 30 passes using the mini-extruder29. 
	NOTE: To enable bEV size uniformity, the BMDC suspensions were pushed through 10 &#181;m, 5 &#181;m, and 1 &#181;m 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.</li>
	<li>Centrifuge the pooled supernatants for 5 min at 1000 x g and 4 &#176;C to remove the cells. Collect the supernatant and centrifuge it at 3000 x g for 5 min at 4 &#176;C to remove the remaining cells and cell debris.</li>
	<li>Collect the supernatant and centrifuge it at 100,000 x g for 90 min at 4 &#176;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 &#181;m membrane and store the purified bEVs at &#8722;80 &#176;C.</li>
</ol>
5. bEVs adhere to the PNPE
NOTE: The SiO2 sensor was modified via spin-coating method.
<ol>
	<li>SiO2 sensor modified with poly-L-lysine (PLL).
	<ol>
		<li>Clean the SiO2 quartz sensor chip using UV-ozone treatment for 10 min, rinse twice with ethanol, and dry with N2.</li>
		<li>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.</li>
		<li>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 &#181;L of PLL dropwise to the center of the SiO2 sensor and close the top cover of the spin coater.</li>
		<li>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. 
		&#8203;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.</li>
	</ol>
	</li>
	<li>Determination of adhesion of bEVs to PNPE
	<ol>
		<li>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 &#176;C below room temperature.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Pump the prepared PNPE solution into the flow module at a flow rate of 50 &#181;L/min to achieve equilibrium adsorption on the SiO2 sensor. 
		NOTE: The &#916;F 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.</li>
		<li>Pump the prepared bEVs solution into the flow module at a flow rate of 50 &#181;L/min to track the process of bEV adhesion to the PNPE surface.</li>
	</ol>
	</li>
</ol>
6. CLSM analysis of antigen uptake
<ol>
	<li>Co-culture PNPE-OVA with BMDCs
	<ol>
		<li>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 &#176;C in a 5% CO2 incubator.</li>
		<li>Mix 0.5 mL of Cy5-labeled OVA (400 &#181;g/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 &#181;L of deionized water, add 10 &#181;L of the formulation (10 &#181;g/mL OVA) into the small confocal laser dishes under a super-clean bench and co-culture with BMDCs for 6 h.</li>
	</ol>
	</li>
	<li>Actin cytoskeleton stain
	<ol>
		<li>Remove the culture fluid from the cells and wash them twice with pre-warmed phosphate buffered saline (PBS; pH 7.4).</li>
		<li>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.</li>
		<li>Wash the cells with PBS two or three times at room temperature for 10 min each. Permeabilize the cells with 100 &#181;L 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.</li>
		<li>Cover the cells on the glass-bottomed dish of the small confocal laser dishes with 200 &#181;L 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.</li>
		<li>Wash the cells with PBS thre times for 5 min each. Re-stain the nuclei with 200 &#181;L of DAPI solution (concentration: 100 nM) for around 30 s.</li>
	</ol>
	</li>
	<li>Image analysis
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<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. O., Qadiri, S. S. N., Kim, J. O., Oh, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+viral+uptake+and+host-associated+immune+response+in+the+tissues+of+seven-band+grouper+following+a+bath+challenge+with+nervous+necrosis+virus.">Early viral uptake and host-associated immune response in the tissues of seven-band grouper following a bath challenge with nervous necrosis virus.</a> Fish &amp; Shellfish Immunology. 103, 454-463 (2020).</li><li>Mishra, D., Mishra, P. K., Dubey, V., Dabadghao, S., Jain, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+uptake+and+generation+of+immune+response+by+murine+dendritic+cells+pulsed+with+hepatitis+b+surface+antigen-loaded+elastic+liposomes.">Evaluation of uptake and generation of immune response by murine dendritic cells pulsed with hepatitis b surface antigen-loaded elastic liposomes.</a> Vaccine. 25 (39-40), 6939-6944 (2007).</li><li>Harwood, L. J., Gerber, H., Sobrino, F., Summerfield, A., Mccullough, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+internalization+of+foot-and-mouth+disease+virus:+influence+of+heparan+sulfate+binding+on+virus+uptake+and+induction+of+the+immune+response.">Dendritic cell internalization of foot-and-mouth disease virus: influence of heparan sulfate binding on virus uptake and induction of the immune response.</a> Journal of Virology. 82 (13), 6379-6394 (2008).</li><li>Jing, H., 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=Fluorescent+artificial+antigens+revealed+extended+membrane+networks+utilized+by+live+dendritic+cells+for+antigen+uptake.">Fluorescent artificial antigens revealed extended membrane networks utilized by live dendritic cells for antigen uptake.</a> Nano Letters. 22 (10), 4020-4027 (2022).</li><li>Meena, J., Goswami, D. G., Anish, C., Panda, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+uptake+of+polylactide+particles+induces+size+dependent+cytoskeletal+remodeling+in+antigen+presenting+cells.">Cellular uptake of polylactide particles induces size dependent cytoskeletal remodeling in antigen presenting cells.</a> Biomaterials Science. 9 (23), 7962-7976 (2021).</li><li>Yang, J., 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=Drug+delivery+via+cell+membrane+fusion+using+lipopeptide+modified+liposomes.">Drug delivery via cell membrane fusion using lipopeptide modified liposomes.</a> ACS Central Science. 2 (9), 621-630 (2016).</li><li>Rawle, R., Kasson, P., Boxer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disentangling+viral+membrane+fusion+from+receptor+binding+by+using+synthetic+dna-lipid+conjugates+totether+influenza+virus+to+model+lipid+membranes.">Disentangling viral membrane fusion from receptor binding by using synthetic dna-lipid conjugates totether influenza virus to model lipid membranes.</a> Biophysical Journal. 111 (1), 123-131 (2016).</li><li>Ha, H. K., Kim, J. W., Lee, M. R., Jun, W., Lee, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+uptake+and+cytotoxicity+of+&#946;-lactoglobulin+nanoparticles:+the+effects+of+particle+size+and+surface+charge.">Cellular uptake and cytotoxicity of &#946;-lactoglobulin nanoparticles: the effects of particle size and surface charge.</a> Asian-Australasian Journal of Animal Sciences. 28 (3), 420-427 (2015).</li><li>Malara, A., 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=Extracellular+matrix+structure+and+nano-mechanics+determine+megakaryocyte+function.">Extracellular matrix structure and nano-mechanics determine megakaryocyte function.</a> Blood. 118 (16), 4449-4453 (2011).</li><li>Dankovich, T. M., 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=Extracellular+matrix+remodeling+through+endocytosis+and+resurfacing+of+tenascin-r.">Extracellular matrix remodeling through endocytosis and resurfacing of tenascin-r.</a> Nature Communications. 12 (1), 7129 (2021).</li><li>Evans, E., Buxbaum, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+of+red-blood-cell+membrane+for+particle+surfaces+measured+by+the+extent+of+particle+encapsulation.">Affinity of red-blood-cell membrane for particle surfaces measured by the extent of particle encapsulation.</a> Biophysical Journal. 34 (1), 1-12 (1981).</li><li>Rohner, N. A., Purdue, L. N., Von Recum, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity-based+polymers+provide+long-term+immunotherapeutic+drug+delivery+across+particle+size+ranges+optimal+for+macrophage+targeting.">Affinity-based polymers provide long-term immunotherapeutic drug delivery across particle size ranges optimal for macrophage targeting.</a> Journal of Pharmaceutical Sciences. 110 (4), 1693-1700 (2021).</li><li>Zhou, X., Liu, Y., Wang, X. F., Li, X. M., Xiao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+particle+size+on+the+cellular+uptake+and+anti-inflammatory+activity+of+oral+nanotherapeutics.">Effect of particle size on the cellular uptake and anti-inflammatory activity of oral nanotherapeutics.</a> Colloids and Surfaces B-Biointerfaces. 187, 110880 (2020).</li><li>Zhang, D., 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=The+morphology+and+surface+charge-dependent+cellular+uptake+efficiency+of+upconversion+nanostructures+revealed+by+single-particle+optical+microscopy.">The morphology and surface charge-dependent cellular uptake efficiency of upconversion nanostructures revealed by single-particle optical microscopy.</a> Chemical Science. 13 (12), 3610 (2022).</li><li>Xi, Y. K., 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=Co2-responsive+pickering+emulsions+stabilized+by+soft+protein+particles+for+interfacial+biocatalysis.">Co2-responsive pickering emulsions stabilized by soft protein particles for interfacial biocatalysis.</a> Chemical Science. 13 (10), 2884-2890 (2022).</li><li>Trivedi, R. P., Klevets, I. I., Senyuk, B., Lee, T., Smalyukh, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconfigurable+interactions+and+three-dimensional+patterning+of+colloidal+particles+and+defects+in+lamellar+soft+media.">Reconfigurable interactions and three-dimensional patterning of colloidal particles and defects in lamellar soft media.</a> Proceedings of the National Academy of Sciences of United States of America. 109 (13), 4744-4749 (2012).</li><li>De Araujo, A. D., Hoang, H. N., Lim, J., Mak, J. Y. W., Fairlie, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+electrostatic+and+hydrophobic+surfaces+of+aromatic+rings+to+enhance+membrane+association+and+cell+uptake+of+peptides.">Tuning electrostatic and hydrophobic surfaces of aromatic rings to enhance membrane association and cell uptake of peptides.</a> Angewandte Chemie. 61 (29), 03995 (2022).</li><li>Waku, T., 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=Effect+of+the+hydrophilic-hydrophobic+balance+of+antigen-loaded+peptide+nanofibers+on+their+cellular+uptake,+cellular+toxicity,+and+immune+stimulatory+properties.">Effect of the hydrophilic-hydrophobic balance of antigen-loaded peptide nanofibers on their cellular uptake, cellular toxicity, and immune stimulatory properties.</a> International Journal of Molecular Sciences. 20 (15), 3781 (2019).</li><li>Meng, X., 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=A+soft+pickering+emulsifier+made+from+chitosan+and+peptides+endows+stimuli-responsiveness,+bioactivity+and+biocompatibility+to+emulsion.">A soft pickering emulsifier made from chitosan and peptides endows stimuli-responsiveness, bioactivity and biocompatibility to emulsion.</a> Carbohydrate Polymers. 277, 118768 (2022).</li><li>Wang, Z., 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=Fabrication+and+in+vitro/vivo+evaluation+of+drug+nanocrystals+self-stabilized+pickering+emulsion+for+oral+delivery+of+quercetin.">Fabrication and in vitro/vivo evaluation of drug nanocrystals self-stabilized pickering emulsion for oral delivery of quercetin.</a> Pharmaceutics. 14 (5), 897 (2022).</li><li>Ji, J., 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=Core-shell-structured+silica/polyacrylate+particles+prepared+by+pickering+emulsion:+influence+of+the+nucleation+model+on+particle+interfacial+organization+and+emulsion+stability.">Core-shell-structured silica/polyacrylate particles prepared by pickering emulsion: influence of the nucleation model on particle interfacial organization and emulsion stability.</a> Nanoscale Research Letters. 9 (1), 534 (2014).</li><li>Chen, 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=Quantitative+evaluation+of+proteins+with+bicinchoninic+acid+(bca):+resonance+raman+and+surface-enhanced+resonance+raman+scattering-based+methods.">Quantitative evaluation of proteins with bicinchoninic acid (bca): resonance raman and surface-enhanced resonance raman scattering-based methods.</a> Analyst. 137 (24), 5834-5838 (2012).</li><li>Colino, J., Shen, Y., Snapper, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+pulsed+with+intact+streptococcus+pneumoniae+elicit+both+protein-+and+polysaccharide-specific+immunoglobulin+isotype+responses+in+vivo+through+distinct+mechanisms.">Dendritic cells pulsed with intact streptococcus pneumoniae elicit both protein- and polysaccharide-specific immunoglobulin isotype responses in vivo through distinct mechanisms.</a> The Journal of Experimental Medicine. 195 (1), 1-13 (2002).</li><li>Zhang, Y., Wu, J., Zhang, H., Wei, J., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles-mimetic+encapsulation+improves+oncolytic+viro-immunotherapy+in+tumors+with+low+coxsackie+and+adenovirus+receptor.">Extracellular vesicles-mimetic encapsulation improves oncolytic viro-immunotherapy in tumors with low coxsackie and adenovirus receptor.</a> Frontiers in Bioengineering and Biotechnology. 8, 574007 (2020).</li><li>Cappellano, G., Abreu, H., Casale, C., Dianzani, U., Chiocchetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-microparticle+platforms+in+developing+next-generation+vaccines.">Nano-microparticle platforms in developing next-generation vaccines.</a> Vaccines. 9 (6), 606 (2021).</li><li>McClelland, R. D., Culp, T. N., Marchant, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+and+confocal+immunofluorescence+microscopy+of+virus-host+cell+interactions.">Imaging flow cytometry and confocal immunofluorescence microscopy of virus-host cell interactions.</a> Frontiers in Cellular Infection Microbiology. 11, 749039 (2021).</li><li>Konry, T., Sarkar, S., Sabhachandani, P., Cohen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+tools+and+technology+for+analysis+of+single+cells+and+cell-cell+interaction.">Innovative tools and technology for analysis of single cells and cell-cell interaction.</a> Annual Reviews of Biomedical Engineering. 18 (1), 259-284 (2016).</li><li>D'Aurelio, R., 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=A+comparison+of+EIS+and+QCM+nanoMIP-based+sensors+for+morphine.">A comparison of EIS and QCM nanoMIP-based sensors for morphine.</a> Nanomaterials. 11 (12), 3360 (2021).</li><li>Li, Y. J., 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=Artificial+exosomes+for+translational+nanomedicine.">Artificial exosomes for translational nanomedicine.</a> Journal of Nanobiotechnology. 19 (1), 242 (2021).</li><li>Rydell, G. E., Dahlin, A. B., Hook, F., Larson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QCM-D+studies+of+human+norovirus+VLPs+binding+to+glycosphingolipids+in+supported+lipid+bilayers+reveal+strain-specific+characteristics.">QCM-D studies of human norovirus VLPs binding to glycosphingolipids in supported lipid bilayers reveal strain-specific characteristics.</a> Glycobiology. 19 (11), 1176-1184 (2009).</li></ol>

The authors declare no conflicts of interest.

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...

Watch this Scientific Journal Video about Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization at JoVE.com

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 ...

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

Research

JoVE Journal

Bioengineering

1.7K Views.  Tokyo University of Agriculture and Technology. 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. 

Video: Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...