Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

The significance of this approach is that it could be used to administer multiple dose vaccines in a single injection, potentially saving millions of lives, particularly in low-and middle-income countries where newborns are not immunized due to access challenges. This fabrication method allows rabies vaccine to retain its native immunity conferring form when encapsulated in microparticles whose release mimics current vaccination schedules with only a single injection. Rabies is a fatal disease, requiring up to five repeated vaccine injections in some cases to prevent death.

However, administering the complete vaccine regimen in one visit can improve adherence and save lives. This technique represents a platform technology capable of delivering diverse therapeutics or prophylactics for various diseases. To begin, treat the 3D-printed master mold surface by placing it in a vacuum chamber containing a glass slide with 40 microliters of trichlorosilane on the surface.

Start the vacuum for one hour. Mix the PDMS prepolymer base and curing agent at a mass ratio of 9:1 into a plastic cup. Then, transfer the solution into a 50-milliliter tube and centrifuge 300 G for three minutes at room temperature.

After centrifugation, make sure the solution is clear. Once surface treatment is complete, place the master mold in an aluminum foil dish and pour the uncured PDMS onto the mold, ensuring the features are entirely submerged. Place the aluminum foil dish in a vacuum chamber and pull the vacuum for one hour to remove any air bubbles.

After removing the aluminum foil dish, place 800-micrometer spacers at each end of the master mold and overlay a clean glass slide onto the master mold, avoiding bubbles. Use binder clips to clamp the mold over the 800-micron spacers. Cure the prepolymer in the oven at 120 degrees Celsius for four hours.

Once it is cured, carefully release the binder clips. Use a razor blade to separate the master mold from the cured PDMS mold. For preparing the PLGA film, place 450 milligrams of PLGA on a non-stick polymer sheet within a ring shim.

Then, overlay a second non-stick polymer sheet on the PLGA and use a 101.6-millimeter C-clamp to compress the stack between two aluminum blocks until finger-tight. Place the C-clamped assembly in a vacuum oven. After 30 minutes in the oven, tighten the clamp and place it back for another 30 minutes.

Then, transfer the assembly into a desiccator for four hours to cool. Once cooled, loosen the clamp, remove the PLGA film from the non-stick polymer sheets, and place it in a labeled Petri dish. Store the Petri dish inside a desiccator for later use.

To generate PLGA particles, treat the PDMS mold surface as demonstrated previously. Using tweezers or a scalpel, cut the 250-micron PLGA film to be approximately the size of the particle array and place it on the treated PDMS mold. Overlay a clean glass microscope slide on the PLGA film atop the PDMS mold and secure them together by placing a spring clamp over the array and PLGA film.

Keep the clamped mold assembly in the vacuum oven for one hour, then cool it at room temperature for 15 minutes. Once cooled, gently apply the pressure using a razor blade to separate the PDMS mold from the PLGA particle array and store the PLGA particles in a desiccator for further use. To fill the particles with concentrated rabies vaccine antigen, prepare the piezoelectric dispenser and place the slide containing the PLGA particles into the dispensing area.

Load the concentrated antigen on the plate, then enter the target setup parameters and click on Run. The piezoelectric robot will then fill the particles. Once completed, use a stereoscope to verify that the particles have been filled.

For sealing the filled particle, place a stainless steel block on a hot plate and two parallel microscope slides on the stainless steel block. After leveling the stainless steel block, turn on the hot plate and set the temperature at 205 degrees Celsius. Once the desired surface temperature of the stainless steel is reached, suspend the filled PLGA particles on the two glass slides and immediately start a timer for 18 seconds.

After removing the sealed particle array from the hot plate, suspend it on two glass slides on the lab bench and cool it for one minute. To harvest sealed particles, hold the scalpel blade at a 45-degree angle to the slide and apply pressure to the base of the particles to separate them from the slide. Using a scalpel, move the harvested particles into a 0.5-milliliter low-protein-binding tube.

Add 250 microliters of PBS containing bovine serum albumin and glucose to maintain rabies vaccine stability Place the sample under the stereoscope and crush the particles using one prong on a pair of fine-tip tweezers. During particle filling, sufficient drying time is required for complete solvent or water evaporation. After drying, a solute depot remains at the bottom of the particle core.

The ideal morphology of particles was observed between ceiling times of 18 and 24 seconds for this PLGA, as particles containing cargo were completely sealed at those times without losing particle structure. Particles were small enough to fit easily in a 19-gauge needle when properly packed and sealed. Furthermore, 10 particles flowed consistently through a 19-gauge needle when injecting them in a viscous solution, such as 2%carboxymethyl cellulose.

The transmission electron micrograph indicated intact rabies variants in the concentrated antigen sample, and these were approximately 4.4-fold more concentrated than the starting stock solution. After sealing, approximately 69%of the antigen remains encapsulated in its bioactive form, suggesting that heat stress causes significant loss during sealing, but most of the viral antigen remains intact. Microparticle filling is one of the most critical steps.

If particles are not filled correctly, it is unlikely the particles will seal correctly. To understand stability, additional formulation development, including excipients and other biologically relevant materials in the particle core, would be necessary.