Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

1. Particle master mold generation

NOTE: The 3D printing process can be performed with any 3D printer with sufficient spatial resolution; however, the current protocol describes the process for a multi-photon 3D printer.

1. Design microparticle structures using a computer-aided design (CAD) program.
   NOTE: The design specifications are as follows: 308 particles (diameter = 400 µm, height = 500 µm, and wall thickness = 100 µm) are arranged in a 22 by 14 array, with 600 µm of spacing between them. The design also contains a four-pointed star and a five-pointed star as fiducials immediately outside the array (Supplementary Figure 1).
2. Export the final design as an STL file (Supplementary Coding File 1) and upload the file to software capable of defining the print parameters as shown below. Then, save it as a file compatible with the 3D printer (see Table of Materials).
   NOTE: Slicing distance = 5 µm, hatching distance = 1 µm, shell contour = 50 µm, base slice count = 5 µm, scaffolding = hollow, scan mode = galvo, z-axis = microscope z-drive, scan speed = 100,000, power = 100.
3. Pre-treat a silicon printing substrate (see Table of Materials) using a plasma cleaning process (gas: O₂; power: 200 watts; temperature: 25 °C; flow: 20; time: 5 min), then immediately immerse the substrate into a solution of 30 mL of ethanol and 60 µl of 3-(trimethoxysilyl) propyl methacrylate in a glass Petri dish. Cover with aluminum foil and allow to incubate overnight.
   NOTE: This step increases print adhesion to the substrate, which is important during PDMS separation (step 2.6).
4. Upload the print file to the multi-photon 3D printer, apply the print resin to the treated substrate, load the 10x lens (see Table of Materials), and print the microstructures.
5. Once finished, submerge the print in propylene glycol methyl ether acetate (see Table of Materials) for 45 min to remove unexposed photoresist, then submerge the print in isopropyl alcohol for 5 min. Post-cure the master mold by exposing it to UV light at 254 nm for 120 min.
   ​NOTE: If available, UV light in the 405 to 365 nm range can be used for ~20 min to post-cure the printed structures.

2. Polydimethylsiloxane (PDMS) mold generation

1. Treat the surface of the 3D-printed master mold by placing it in a vacuum chamber containing a glass slide with 40 µL of Trichloro(1H,1H,2H,2H,-perfluorooctyl) (see Table of Materials) silane added to the surface. Pull the vacuum (relative pressure: -20 in.Hg) for 1 h.
   NOTE: This step ensures easy separation when demolding (step 2.6).
2. While the master mold surface is being treated, thoroughly mix the polydimethylsiloxane (PDMS) prepolymer base with the PDMS prepolymer curing agent in a 9:1 ratio by mass (at least 10 g of material is needed for each master mold). Once thoroughly mixed, transfer the uncured PDMS to a 50 mL centrifuge tube and centrifuge at 300 x g for 3 min at room temperature.
3. Once surface treatment is complete, place the master mold in an aluminum foil dish and pour the uncured PDMS on top of the mold, ensuring the features are entirely submerged. Place the aluminum foil dish in a vacuum chamber and pull the vacuum (relative pressure: -20 in.Hg) for 1 h to remove air bubbles.
4. Remove the aluminum foil dish from the vacuum chamber. Place 800 µm spacers at the ends of the master mold and overlay a clean glass slide onto the master mold, taking care to avoid introducing air bubbles. Use binder clips to clamp the mold and the slide together, localizing the clamping force over the spacers (Supplementary Figure 2).
5. Place the structure in an oven set to 120 °C for at least 4 h to cure the prepolymer into PDMS molds.
6. Remove the structure from the oven, carefully release the binder clips, and carefully separate the master mold from the cured PDMS mold using a razor blade.
   ​NOTE: The 3D-printed master mold can be reused to generate additional PDMS molds.

3. PLGA film fabrication

1. Weigh out 450 mg of PLGA (see Table of Materials) and place it on a 76 mm by 76 mm nonstick polymer sheet within a 250 µm thick ring shim, with an inner diameter of 50.8 mm. Place a second nonstick polymer sheet over the PLGA and compress the stack between two aluminum blocks using a 101.6 mm c-clamp until finger-tight.
   NOTE: 502H PLGA was used exclusively in this study; however, other types of PLGA are compatible with this process.
2. Place the c-clamped assembly in a vacuum oven set to 120 °C for 30 min under vacuum, with a relative pressure of -30 in.Hg. Then, remove the assembly and firmly tighten the clamp before placing it back in the vacuum oven for another 30 min.
   NOTE: The primary purpose of using a vacuum is to avoid PLGA degradation, which is accelerated at high temperatures.
3. Remove the assembly from the oven and allow it to cool for 4 h in a desiccator.
4. Once cool, loosen the clamp, remove the PLGA film from the nonstick polymer sheets, and place the film in a labeled Petri dish. Store the Petri dish inside a desiccator for later use.

4. PLGA particle generation

1. Treat the surface of the PDMS mold as described previously in step 2.1.
2. Using tweezers and/or a scalpel, cut out and place a portion of the 250 µm PLGA film, roughly the size of the array, on the treated PDMS mold.
3. Overlay a clean glass microscope slide on top of the PLGA film and PDMS mold and clamp the components together by placing a spring clamp directly over the array and PLGA film.
4. Place the clamped mold assembly in the vacuum oven set to 120 °C, and pull the vacuum with a relative pressure of -30 in.Hg for 1 h.
   NOTE: The time needed to form the particles depends on the PLGA, and can vary from 1-12 h.
5. Remove the clamped mold assembly from the oven and let it passively cool at room temperature for approximately 15 min, or until cool to the touch.
6. Using a razor blade, gently apply pressure between the PDMS mold and PLGA particle array to separate the two. Store the PLGA particles in a desiccator for future use.

5. Antigen concentration and purification

1. Thaw one aliquot of commercially available RABV antigen (see Table of Materials) at room temperature.
2. Assemble the filtration setup by placing two centrifugal spin filters with a 100 kDa molecular weight cutoff (MWCO; see Table of Materials) into two collection tubes.
3. Prewet the two centrifugal spin filters by adding 500 µL of UltraPure water.
4. Spin the filters at 2,400 x g for 1 min in a benchtop centrifuge at room temperature.
5. Remove the water from both the top and bottom compartments of the filtration setup using a 200 µL pipette.
   NOTE: Do not let the prewetted spin filter dry out.
6. Add 400 µL of distilled water to the top compartment of each spin filter, then add 100 µL of the thawed RABV antigen and mix with the pipette.
   NOTE: This study concentrated the antigen approximately fivefold and reduced the concentration of excipients <100 kDa by ~50-fold.
7. Load the two centrifugal spin filters into a benchtop centrifuge, ensuring that the filters face the center of the centrifuge. Mark which side of the filter faces toward the center of the centrifuge.
   NOTE: If oriented incorrectly, the solutions will not be properly filtered/concentrated.
8. Centrifuge the filters at 14,000 x g for 10 min at room temperature.
9. Retrieve the filters from the centrifuge. The collection tubes will contain the filtrate, while the filters will contain the concentrated sample (Supplementary Figure 3A).
10. Remove the filtrate in the collection tubes using a pipette and discard.
11. Add 450 µL of filtered distilled water to each filter, and mix the concentrated sample and water by pipetting up and down six times.
12. Centrifuge the two spin filters a second time at 14,000 x g for 10 min at room temperature. Ensure the filters face the center of the centrifuge in the same orientation as in step 5.7.
13. Retrieve the filter units from the centrifuge and, just as before, remove and discard the filtrate from each tube using a pipette.
14. Remove the spin filters from the collection tubes and cap the filter casings using the top of the collection tubes (Supplementary Figure 3B).
15. Place the bottom of the spin filter casings directly onto a vortex and vortex at 3,000 rpm for 30 s while holding the filter casings upright and at 45° angles (Supplementary Figure 3C).
    NOTE: This step is intended to resuspend any RABV antigen that formed a pellet during the centrifugation process.
16. Carefully remove the cap of the collection tubes from the spin filter casings. Place the filter casings back in the provided collection tubes and run a quick spin (2-3 s) to collect any volume that may have become trapped in the cap.
    NOTE: This quick spin must be performed in a benchtop microcentrifuge. The filters should be tangential to the centrifuge, opposite to the previous configuration, to ensure no solution is lost through the filters.
17. Invert each spin filter unit into a new collection tube provided with the spin filter kit (see Table of Materials), and centrifuge for 2 min at 1,000 x g at room temperature to collect the concentrated samples.
18. Consolidate the concentrated samples from the two collection tubes into one collection tube. Measure and record the resulting volume.
    NOTE: The resulting sample will have 5x the initial antigen concentration as the stock, have a small excipient (<100 kDa) concentration approximately 50-fold lower than the stock, and appear slightly milky white. The total volume should be approximately 44-48 µL.
19. Store the 5x concentrated, twice-washed antigen at 4 °C until the time of dispensing, but for no longer than 16 h. Prior to filling particles with this solution, centrifuge the tube at 1,000 x g for 1 min to remove any bubbles. The solution may be diluted using distilled water to attain the nominal target concentration for dispensing.

6. Particle filling

1. Vacuum filter 500 mL of deionized water through a 0.22 µm vacuum filter, then degas the solution by applying a vacuum (relative pressure: -20 in.Hg) while under sonication for 20 min.
2. During this time, attach the piezo dispense capillaries (PDCs; see Table of Materials) to the piezoelectric dispenser.
3. Prime the machine according to the manufacturer's instructions (see Table of Materials), and place the slide with the PLGA particles into the dispensing area.
4. Set up the "Find Target Reference Points", Run, and then set up the "Target Substrate", according to Supplementary Figure 4.
5. Load 25 µL of the concentrated antigen into the source plate and load it into the machine. Using the PDCs, aspirate 10 µL of concentrated antigen into the dispensing tip, wash the outside of the tip, then calibrate the dispensing alignment (Supplementary Figure 5).
6. Enter "Target Setup parameters" and click on the Run (Supplementary Figure 6).
7. Once complete, remove the filled particles and verify they are filled under a stereoscope.
8. Move directly to the next step in the protocol.

7. Particle sealing and harvesting

1. Place a stainless-steel block (see Table of Materials) on a hotplate. Place two microscope slides on the stainless-steel block so they are parallel. Ensure the stainless-steel block is level, then turn the hotplate on and set the temperature so that the surface temperature of the stainless steel is 200 °C. Verify the temperature of the hotplate before sealing.
2. Suspend the filled PLGA particles above the hotplate by placing them on the two glass slides, then immediately start a timer for 18 s.
   NOTE: The amount of time and heat particles need to seal will vary depending on the chemical properties of the PLGA used to make the particles. A custom 3D-printed slide holder can be used to handle the slides at a safe distance from the hotplate. The STL file is provided as Supplementary Coding File 2.
3. Once sealed, remove the particle array from the hotplate and suspend it over the lab bench by placing the particle array on two separate glass slides. Allow the particles to cool for 1 min.
4. Once cooled, the particles can be harvested using a scalpel while looking through a stereoscope. Hold the scalpel with the blade at a 45° angle to the slide and apply pressure to the base of the particle to separate it from the glass slide.
5. Once harvested, use the scalpel to transfer the particles into 0.5 mL low-protein binding tubes (see Table of Materials). Then, fill the tubes with 250 µL of a 1x phosphate buffer solution (PBS) containing 30 mg/mL bovine serum albumin (BSA) and 1 mg/mL glucose.
   ​NOTE: The 30 mg/mL BSA and 1 mg/mL glucose solution prepared in PBS will maintain the stability of the RABV antigen.
6. Crush the particles under a stereoscope using a pair of fine-tip tweezers. Ensure the RABV antigen in the core of the particle is available to be dissolved in the surrounding solution. Store the samples in a 4 °C refrigerator until antigen potency can be evaluated using an ELISA. Samples must be run on an ELSIA within 7 days of preparation.

8. Evaluating the antigen by ELISA

CAUTION: Do not let the microplates dry out at any point. Always stack plates, and always cover the upper plate with a plastic seal, an empty plate, or a lid to avoid drying out.

1. Prepare the buffers as instructed in Supplementary File 1.
2. Prepare 5 mL of coating solution (carbonate-bicarbonate buffer, pH 9.6) at a concentration of 2.5 µg/mL by adding 25 µL of the coating antibody to 4975 µL of coating buffer at pH 9.4-9.6.
   NOTE: 5 mL is needed to coat 96 wells with 50 µL each (with extra volume for pipetting loss). Increase the total volume by 5 mL for each additional ELISA plate needed.
3. Use a multichannel pipette to dispense 50 µL of the coating solution into each well of the microplate. The solution must be used for coating immediately after it is prepared.
4. Gently tap along the plate edges on a gloved palm to ensure the bottom of each well is evenly covered with liquid.
5. Seal the plate with adhesive film and place it at 37 °C for 1 h, then transfer to 4 °C overnight.
6. Retrieve the plates from cold storage and wash them three times with 200 µL of wash buffer in each well.
7. Remove the wash buffer and dispense 300 µL of blocking buffer into each well.
   CAUTION: When running multiple plates with the same samples repeated on different plates, it is important to proceed plate-by-plate (i.e., fill plate 1 with the material before proceeding to plate 2, and then to plate 3 and so on). Do not apply material X to all plates and then material Y, etc., as this would increase the risk of wells drying out. After the final wash step, the wash buffer should remain in the wells of the plate and only be discarded immediately before adding samples to the plate. A plastic 96-well plate lid needs to be used to cover all wells of the ELISA plate that are not being dispensed into straight away, and the lid sequentially moved to reveal additional wells to be filled with material.
8. Seal the plates with an adhesive plastic film (see Table of Materials) and place them in an incubator for 60 ± 5 min at 37 ± 2 °C.
9. Prepare the standard curve and test samples to be evaluated during this incubation.
   NOTE: The antigen being evaluated must be pre-diluted in diluent buffer at a recommended dilution of 0.125 IU/mL, with an excess volume of at least 40 µL to account for pipetting loss. All the samples are evaluated in duplicate (two technical replicates) on each ELISA plate. For the standard curve, a twofold dilution series for a total of eight points should be included in columns 2 and 3 of each ELISA plate. A dilution series may be performed for all other samples with an appropriate number of points based on the predicted amount of antigen being evaluated. Although less rigorous for higher throughput, a single sample dilution can be used as an alternative to the plating described above. However, the expected concentration of a single dilution must fall within the standard curve range.
10. In low-bind protein 96-well plates, or low-bind protein tubes (see Table of Materials), perform a twofold dilution series of each sample along the columns of the plate.
11. Load the start of the dilution series in row A for each respective sample at double the final volume required (per plate, 100 µL of sample is required per well, so all wells in A2-A11 should contain at least 240 µL of a sample, with an extra volume of 40 µL to account for pipetting loss).
12. Load 120 µL of diluent buffer into all other wells on the low-bind protein plate, excluding columns 1 and 12 (i.e., B2 through H11).
13. Using a multichannel pipette loaded with 10 tips, perform serial dilution by transferring 120 µL of sample from A2-A11 to B2-B11 and pipetting up and down several times to mix while avoiding splashing and bubbles. Repeat this process, moving down the plate along the columns until wells H2-H11. Discard the remaining 120 µL volume aspirated from the final row of wells.
    NOTE: The samples are now ready for analysis. If the blocking phase is not complete by this time, ensure the samples are kept on ice.
14. At the end of the incubation step, wash the plates three times with 200 µL of wash buffer.
15. Using a multichannel pipette, transfer 100 µL of diluent buffer into columns 1 and 12 as the "blanks".
16. Using a multichannel pipette, transfer 100 µL from each well of the low-bind protein 96-well plate containing sample dilutions to the washed ELISA plate. Repeat for all ELISA plates being run.
17. Seal the plates with an adhesive plastic film and place them in an incubator for 60 ± 5 min at 37 ± 2 °C.
18. Prepare the detection antibody solution (see Table of Materials) at a concentration of 0.2 µg/mL by adding 4.4 µL of the antibody solution to 10,995.6 µL of the diluent buffer and mix well by pipetting up and down.
    NOTE: A total of 11 mL is needed for 96 wells at 100 µL each (with extra volume for pipetting losses). Increase the total volume by 11 mL for each additional ELISA plate being evaluated. The solution must be used immediately following preparation.
19. At the end of the incubation step, wash the plates five times with 200 µL of wash buffer.
20. Aspirate the remaining wash buffer and dispense 100 µL of the detection antibody solution into each well on the microplate using a multichannel pipette.
21. Seal the plates with an adhesive plastic film and place them in an incubator for 60 ± 5 min at 37 ± 2 °C.
22. Prepare the conjugate toward the end of the detection antibody incubation step by adding 4.4 µL of streptavidin-peroxidase conjugate solution (see Table of Materials) to 10,995.6 µL of wash buffer.
    NOTE: A total of 11 mL is needed for 96 wells at 100 µL each (with extra volume for multichannel pipetting). Increase the total volume by 11 mL for each additional ELISA plate being evaluated.
23. Wash the plates five times with 200 µL of wash buffer.
24. Aspirate the remaining wash buffer and dispense 100 µL of the conjugate solution to each well.
25. Seal the plates with an adhesive plastic film and place them in a 37 °C incubator for 60 ± 5 min at 37 ± 2 °C.
26. Prepare the substrate during the final wash step according to the manufacturer's instructions (see Table of Materials). For each plate, dissolve one tablet of o-phenylenediamine dihydrochloride (OPD; see Table of Materials) in 9 mL of deionized water in the dark, then top up with 1 mL of 10x stable peroxide buffer. Adjust the number of tablets and total volume of solution (10 mL) according to the number of plates being evaluated.
27. Wash the plates five times with 200 µL of wash buffer.
28. Dispense 100 µL of the substrate to each well using a multichannel pipette.
29. Seal the plates with an adhesive plastic film and leave them on the bench for 10-20 min, protected from light, using aluminum foil.
    NOTE: Monitor color development visually to avoid saturation.
30. Add 50 µL of stop solution to each well and the read plate immediately for absorbance at 492 nm and a reference absorbance of 620 nm.
31. Analyze the data using the corrected absorbance reading (reading at 492 nm minus the reading at 620 nm) and subtract the average of the blank from all samples. Use a sigmoidal 4PL interpolation method to convert absorbance values to IU/mL. Finally, multiply by the 250 µL (total sample volume) to convert to IU.
    NOTE: This analysis can be done using data analysis software.