Name: fabrication of pulsatile polymeric microparticles encapsulating rabies antigen
Uploaded: 2023-05-12T13:30:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen at JoVE.com

We thank Chiron Behring and Bharat Biotech International for providing Particles for Humanity&#160;with the RABV antigen. We would also like to acknowledge Charles Rupprecht, VMD, MS, PhD., for his invaluable guidance and technical contributions. The authors would like to thank the generosity of Dr. Rebecca Richards-Kortum for allowing the use of her SciFLEXARRAYER S3 picoliter dispensing apparatus and Dr. Chelsey Smith&#39;s instruction on using the device. We also acknowledge the University of Massachusetts Chan Medical School for generating microscopy images of the rabies antigen.&#160;Finally, we thank Don Chickering and Erin Euliano for reviewing the document before submission. This work was supported by a grant (INV-004360) from the Bill and Melinda Gates Foundation.

<ol>
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Dr. McHugh has a patent filed based on the encompassed work and is currently a consultant for Particles for Humanity. Mr. Graf has a patent filed based on the encompassed work. Dr. Kadasia,&#160;Mr. Yang, and Ms. Brady are part of Particles for Humanity, a public benefit corporation that licensed a similar microparticle vaccine delivery technology from MIT during this work.

It is possible to alter particle geometry for specific needs; however, for cylindrical structures, the authors recommend maintaining a 5:4:1 ratio of the height:diameter:wall thickness described in the protocol. This aspect ratio ensures that enough PLGA material is present to seal the particles and remain mechanically robust enough for handling. Particle dimensions and shapes can easily be altered during the CAD process, enabling a myriad of geometries to be generated. Combining the flexibility of the CAD with 3D printing enables the rapid iteration of microparticle designs. Although this protocol uses a multi-photon 3D printer, any 3D printer with specifications capable of printing the microstructure dimensions in an appropriate material can be used to generate the initial master mold. Further, photolithography has previously been used to make similar structures in arrays much larger than those produced in this protocol; however, the labor, delay of ordering custom-made photomasks, and equipment accessibility would slow the iterative design process16. Finally, master mold generation can be outsourced to fee-for-service companies if in-house master mold fabrication is not feasible. Regardless of the 3D printer or method used to generate the master molds, the adhesion of the print to the substrate is critical for downstream steps. Specifically, if adhesion is inadequate during PDMS mold generation, printed particles will remain lodged in the PDMS mold, requiring manual removal of the printed particles and destruction of the master mold.
Particle filling is another critical aspect to consider. Microparticles have limited filling capacities, so filtration is used not only to concentrate the RABV antigen but also to remove stock excipients that would otherwise occupy a large portion of the microparticle core volume. However, given the large size of the RABV antigen (approximately 60 nm by 180 nm)17, it is possible to partially pellet out the antigen during the centrifugation steps. For this reason, it is important to resuspend the antigen by pipetting or vortexing after centrifugation to achieve a high recovery of the RABV antigen. A highly concentrated solution is ideal for dispensing, because it reduces the dispensing cycles and thereby limits antigen degradation during filling. However, viscosity is a major limitation of piezoelectric dispensing robots forming a stable drop, so dispensing a very high-concentration solution may not be possible or advisable. Diluting the filling solution is the easiest way to achieve a stable drop formation, but antigen stability over the additional filling cycles needed to achieve the desired loading and the longer amount of time required to fill particles should be considered.
Limitations 
This method requires highly specialized equipment to produce the initial molds and a specialized filling instrument for microparticle production. Although the need for a 3D printer with a printing resolution capable of generating the initial master molds can be subverted by a fee-for-service approach, accessibility to a piezoelectric dispensing robot is limiting. Procurement of a piezoelectric dispensing robot requires a significant initial upfront investment, often in the range of $80,000 to $200,000, depending on the brand, throughput, and capabilities. Although several other filling methods are potential alternatives, these methods have not been validated using the RABV antigen12.
Future applications 
A substantial proportion of encapsulated RABV antigen remained stable through the sealing process. In theory, by incorporating this antigen into particles composed of different types of PLGA that mimic the administration timeline of post-exposure prophylaxis treatment, all doses could be administered in a single injection. Eliminating the need for repeat hospital visits to administer additional doses will enhance patient compliance, resulting in better treatment outcomes. Further, having demonstrated the ability to retain the ELISA-reactivity of the highly complex inactivated rabies virus, it is likely that other antigens, including subunit vaccines, would be compatible with this encapsulation method. Using other prophylactic antigens with PULSED microparticles could save millions of lives in LMICs by increasing the vaccination rates of under-vaccinated populations. To accomplish this, however, vaccines must remain stable through not only encapsulation but also release, which may be challenging since the payload will be subjected to elevated temperatures and a potentially acidic microenvironment due to body heat and PLGA degradation products18. Future work will pursue stabilizing strategies of the antigen through release, which would open up the potential for a single-injection vaccination platform that is broadly applicable to prevent many infectious diseases.

Vaccination is an extremely effective healthcare tool, having prevented more than 37 million deaths between 2000 and 20191. Despite this effectiveness, vaccine-preventable diseases continue to pose a significant risk to global health, especially in low- and middle-income countries (LMICs) where high rates of un- and under-vaccination contribute to 1.5 million vaccine-preventable deaths annually2. Rabies is no exception to these disparities. Despite its status as the most deadly disease known to humankind, being almost universally fatal, rabies is fully treatable and is classified as eradicated in many high-income countries. Instead, the burden of rabies is disproportionately borne by people living in parts of Asia and Africa, where the disease has devastating outcomes on humans and livestock3,4.
Vaccination is critical to managing the global impact of rabies5. The cost of vaccination prohibits widespread implementation of pre-exposure prophylaxis (PrEP), considering the overall low incidence of the disease. Furthermore, in LMICs, the utility of post-exposure prophylaxis (PEP) is limited by socioeconomic pressures on patients seeking healthcare. Logistical factors, such as travel distance to healthcare access points, lost wages while obtaining treatment, the cost of treatment, appointments interfering with daily activities, and forgetfulness, result in PEP adherence rates as low as 60%6,7. This high patient attrition rate presents an opportunity for refining approaches to address gaps in rabies vaccination in order to combat the disease.
Single-injection (SI) vaccination systems that control the release of antigens have been explored as ways to obtain full immunization in one injection. Eliminating the need for multiple visits to a healthcare provider mitigates the burdens that prevent individuals from seeking adequate care. To achieve SI vaccination, an antigen is typically encapsulated within a biodegradable polymeric matrix that often takes the form of injectable microparticles. Once injected, the polymer degrades and releases the sequestered antigen. To date, two primary release strategies have been pursued to achieve SI vaccination. In one approach, the antigen is released continuously over an extended period of time. Although intended to enhance the immunogenicity of a single injection, it is unclear if this approach is sufficient to elicit a protective immune response against the rabies virus (RABV) in humans8. In the other, the antigen is released after a predetermined delay to mimic a conventional and proven prime-boost vaccine regimen. Spray drying and emulsion/solvent evaporation-based microparticle fabrication methods exhibit the former strategy, and have been used to successfully encapsulate both model vaccines9&#160;and highly stable antigens, such as tetanus toxoid10. However, these encapsulation methods involve stressors, including heat, solvent interaction, and physical forces, that can denature antigens11.
Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED) is a recently developed fabrication method that can be employed to encapsulate biologics in biodegradable microparticles. Micromolding is used to generate particles that are filled with a liquid payload and heated to allow the polymer to reflow and fully encapsulate the central depot of cargo within a contiguous layer of the biodegradable polymer. This microstructure results in the pulsatile release of the payload, after a duration that is dependent on the degradation rate of the polymeric shell12. This manuscript demonstrates the encapsulation of inactivated RABV within microparticles composed of poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer used in many FDA-approved formulations13, using the PULSED fabrication method to encapsulate stable RABV antigen as evaluated by an enzyme-linked immunosorbent assay (ELISA). By combining PLGA particles with different molecular weight and/or end groups, this approach has the potential to mimic the current rabies vaccination time course following a single injection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m PES filter</td>
			<td>Cole-Parmer+B4B2:B63</td>
			<td>04396-26</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 mm Shims</td>
			<td>McMaster Carr</td>
			<td>98090A935</td>
			<td></td>
		</tr>
		<tr>
			<td>0.75 inch Binder Clips</td>
			<td>Staples</td>
			<td>480114</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL&#160;Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe</td>
			<td>Fisherbrand</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>101.6 mm C-Clamp</td>
			<td>Amazon</td>
			<td>PT-SD-CP01A</td>
			<td>Black handle will eventually fall off. Use pliers to adjust once this happens.</td>
		</tr>
		<tr>
			<td>19 G needle</td>
			<td>EXCELINT</td>
			<td>26438</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL&#160;Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe</td>
			<td>Fisherbrand</td>
			<td>13-678-11</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(Trimethoxysilyl) Propyl Methacrylate&#160;</td>
			<td>Millipore Sigma</td>
			<td>M6514-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL&#160;Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tubes</td>
			<td>Corning&#160;</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL&#160;Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe</td>
			<td>Fisherbrand</td>
			<td>13-678-11F</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher</td>
			<td>AC268310010</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Block</td>
			<td>McMaster Carr</td>
			<td>9057K175</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Foil</td>
			<td>VWR</td>
			<td>89079-069</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 mL Centrifugal Filters, 100 kDa</td>
			<td>Millipore Sigma</td>
			<td>C82301</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rabies Virus Antibody, Serum Free Antibody, clone 1112-1, 100</td>
			<td>Fisherbrand</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rabies Virus Mouse Monoclonal Antibody, Clone D1-25, biotinylated</td>
			<td>Fisherbrand</td>
			<td>14-388-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxymethyl Cellulose</td>
			<td>Tokyo Chemical Industries</td>
			<td>C0045</td>
			<td></td>
		</tr>
		<tr>
			<td>ClipTip 300, Filter, Racked</td>
			<td>Fisherbrand</td>
			<td>13-678-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 0.65 mL Low Binding Snap Cap Microcentrifuge Tube</td>
			<td>Corning&#160;</td>
			<td>3206</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 1.7 mL Low Binding Snap Cap Microcentrifuge Tube</td>
			<td>Corning&#160;</td>
			<td>3207</td>
			<td></td>
		</tr>
		<tr>
			<td>Describe</td>
			<td>Nanoscribe</td>
			<td></td>
			<td>Software used to define the printing parameters for Nanoscribe 3D printer is step 1.2. 
			Software provided with the printer.</td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Fisher Scientific</td>
			<td>10529901</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Double-Sided Tape</td>
			<td>Staples</td>
			<td>649280</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (10x), No Calcium, No Magnesium</td>
			<td>Gibco</td>
			<td>14200075</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>89370-084</td>
			<td></td>
		</tr>
		<tr>
			<td>F1-ClipTip Multichannel Pipettes, 30 to 300 &#181;L</td>
			<td>Fisherbrand</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand SureOne Aerosol Barrier Pipette Tips, 0.1 &#8211; 10 &#181;L</td>
			<td>Fisherbrand</td>
			<td>13-678-11F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand SureOne Aerosol Barrier Pipette Tips, 100 &#8211; 1000 &#181;L</td>
			<td>Fisherbrand</td>
			<td>03-448-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand SureOne Aerosol Barrier Pipette Tips, 2 &#8211; 20 &#181;L</td>
			<td>Fisherbrand</td>
			<td>FB14955202</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand SureOne Aerosol Barrier Pipette Tips, 20 &#8211; 200 &#181;L</td>
			<td>Fisherbrand</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Elite Pipette Kit</td>
			<td>Fisherbrand</td>
			<td>05-408-137</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Pipet Controller&#160;</td>
			<td>Fisherbrand</td>
			<td>FB14955202</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri Dish, 90 mm</td>
			<td>VWR</td>
			<td>470313-346</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Slides</td>
			<td>Globe Scientific</td>
			<td>1380-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Helicon Focus 8</td>
			<td>HeliconSoft</td>
			<td></td>
			<td>Software used to focus stack images</td>
		</tr>
		<tr>
			<td>IP-Q Resin</td>
			<td>Nanoscribe</td>
			<td></td>
			<td>Printer resin is compatable with the 10x lens and is used for printing large microstructures on the Nanoscribe Photonic Professional GT2</td>
		</tr>
		<tr>
			<td>Lascar EL-USB-TC-LCD Thermocouple</td>
			<td>Amazon</td>
			<td>5053485896236</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Microscope Slide Box</td>
			<td>Millipore Sigma</td>
			<td>Z374385-1EA</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Nanoscribe Photonic Professional GT2 with 10X Objective</td>
			<td>Nanoscribe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoWrite</td>
			<td>Nanoscribe</td>
			<td></td>
			<td>Software used to interface with nanoscrive 3D printer. 
			Software provided with printer.</td>
		</tr>
		<tr>
			<td>Nunc MaxiSorp Flat-Bottom 96-well Plate</td>
			<td>Invitrogen</td>
			<td>44-2404-21</td>
			<td></td>
		</tr>
		<tr>
			<td>OPD Substrate Tablets (o-Phenylenediamine Dihydrochloride)</td>
			<td>Fisherbrand</td>
			<td>02-707-432</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M Wrapping Film, 4 in.</td>
			<td>Fisherbrand</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr>
			<td>PDC 60 with Type 3 Coating</td>
			<td>Scienion</td>
			<td>P-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS Particle Molds</td>
			<td>Rice University</td>
			<td>n/a</td>
			<td>N/A- Particles are 400 &#956;m in diameter with a wall thickness of 100 &#956;m, and a height of 500 &#956;m, resulting in an inner diameter of 200 &#956;m. The arrays are 14 x 22 particles spaced 600 &#956;m apart from each other. 4- and 5-point stars are used as fiducials, positioned 600 &#956;m to the right and left of the top right and top left particles on the array.</td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Fisher Scientific</td>
			<td>08-757-100D</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Stable Peroxide Substrate Buffer (10x)</td>
			<td>Fisherbrand</td>
			<td>02-707-430</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Cups</td>
			<td>Fisher Scientific</td>
			<td>S04170</td>
			<td></td>
		</tr>
		<tr>
			<td>PLGA Film, 502H</td>
			<td>Sigma</td>
			<td>502H: 719897-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene Glycol Monomethyl Ether Acetate</td>
			<td>Millipore Sigma</td>
			<td>484431</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabies Antigen</td>
			<td>Chiron Behring and Bharat Biotech International</td>
			<td></td>
			<td>Material was acquired by entering into a materials transfer agreement with the company.</td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>VWR</td>
			<td>21899-530 and 76457-512</td>
			<td></td>
		</tr>
		<tr>
			<td>SciFLEXARRAYER S3 with PCD 60</td>
			<td>Scienion</td>
			<td></td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Sealing Tape for 96-Well Plates</td>
			<td>Thermo Scientific</td>
			<td>15036</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>University Wafer</td>
			<td>1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Clamps</td>
			<td>IRWIN</td>
			<td>VGP58100</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Block</td>
			<td>McMaster Carr</td>
			<td>9083K12</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin&#8722;Peroxidase Polymer, Ultrasensitive</td>
			<td>Fisherbrand</td>
			<td>02-707-404</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>DOW</td>
			<td>2646340</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon Sheet</td>
			<td>McMaster Carr</td>
			<td>9266K12</td>
			<td>Used to make PLGA films. Must be cut into appropriately sized pieces.</td>
		</tr>
		<tr>
			<td>Teflon Sheet, 0.8 mm-thick</td>
			<td>McMaster Carr</td>
			<td>9266K81</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloro(1H, 1H, 2H, 2H-Perfluorooctyl) Silane</td>
			<td>Sigma</td>
			<td>448931-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers&#160;</td>
			<td>Pixnor</td>
			<td>ESD-16</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Fisher Scientific</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Oven, CL-1000S UV Crosslinker</td>
			<td>UVP</td>
			<td>95-0174-01</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>Bel-Art</td>
			<td>F420100000</td>
			<td>Note you will need two of these. One will be used exclusively to pre-treat samples with trichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane to prevent contamination.</td>
		</tr>
		<tr>
			<td>Vacuum Oven Capable of Reaching 120 &#176;C</td>
			<td>VWR</td>
			<td>97027-664</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Vacuum, CRVpro4&#160;</td>
			<td>Welch</td>
			<td>3041-01</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Wooden Tongue Depressors</td>
			<td>Electron Microscopy Sciences</td>
			<td>72320</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of pulsatile polymeric microparticles encapsulating rabies antigen

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately burdensome to those living in low- and middle-income countries (LMICs), where the majority of deadly exposures to rabies occur. Different drug delivery strategies have been explored to condense vaccine regimens to a single injection by encapsulating antigens into polymeric particles. However, harsh stressors during the encapsulation process can cause denaturation of the encapsulated antigen. This article describes a method for encapsulating the rabies virus (RABV) antigen into polymeric microparticles that exhibit tunable pulsatile release. This method, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED), generates microparticles using soft lithography to create inverse polydimethylsiloxane (PDMS) molds from a multi-photon, 3D-printed master mold. Poly(lactic-co-glycolic acid) (PLGA) films are then compression-molded into the PDMS molds to generate open-faced cylinders that are filled with concentrated RABV using a piezoelectric dispensing robot. These microstructures are then sealed by heating the top of the particles, allowing the material to flow and form a continuous, nonporous polymeric barrier. Post-fabrication, an enzyme-linked immunosorbent assay (ELISA) specific to the detection of intact trimeric rabies virus glycoprotein is used to confirm the high recovery of immunogenic antigen from the microparticles.

Particle sealing and filling are two of the most critical steps in this protocol. Particles were filled with fluorescein sodium salt to demonstrate ideal filling and some common errors. Fluorescein sodium salt was used in lieu of the RABV antigen for ease of visualization. During filling, it is important to dispense solution into the bottom of particle cores, then allow enough time for the solvent/water to evaporate. Once complete, a depot of the solute remains in the bottom of the particle core (Figure 1A). Once filled, it is critical to seal the particles correctly. Figure 1B demonstrates several outcomes (successful and unsuccessful) of the sealing process. After 12 s of sealing time, a distinct pathway remains from the particle center to the outside of the particle, demonstrating a particle that is not entirely sealed. Conversely, if left to seal for 36 s, the PLGA almost entirely melts, resulting in a microstructure with a shallow profile. The Ideal morphology can be visualized when particles are sealed for 18 and 24 s, as they contain cargo entirely encapsulated by the polymer while maintaining a particle structure. Figure 1C demonstrates several potential outcomes after filling and sealing. During dispensing, if the solvent does not reach the particle bottom, it leaves solute dried in the middle of the particle core (incorrectly filled); although these particles may still seal, the poor loading of cargo can limit loading efficiency. If particles are filled with too much cargo (overfilled), the sealing process is inhibited, as the cargo prevents PLGA from flowing over the opening. When correctly filled and sealed, particles of this geometry are small enough to fit easily inside a 19 G needle. Further, 10 particles consistently flowed through a 19 G needle (100% &#177; 0%) when injected with a viscous solution such as 2% carboxymethyl cellulose (Supplementary Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65147/65147fig01.jpg" /> 
Figure 1: Common problems with the filling and sealing process. (A) Images show the evaporation of solvent after one filling cycle, where particles are loaded with 6 nL of 100 mg/mL fluorescein sodium salt dissolved in water. (B) Representative images of 502H PLGA particles removed from the sealing process at 0, 12, 18, 24, and 36 s. (C) Different outcomes of the sealing process when particles are filled correctly, incorrectly, or overfilled. Images are generated by focus stacking multiple images and merging them using focus stacking software. Scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65147/65147fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Processing the antigen through a spin filter prior to loading it into the particles is important for two reasons. First, the centrifugation serves to remove stock excipients in the vaccine solution, that can limit particle loading capacity while retaining the RABV antigen. The current protocol purifies the antigen by approximately 50-fold. Second, the antigen is also concentrated during this process. Figure 2A shows a micrograph of intact RABV virions in the concentrated antigen sample. This antigen is approximately 4.4-fold more concentrated than the starting stock solution (Figure 2B). The amount of antigen initially loaded into the centrifugal spin filters can be altered to modulate the final fold concentration achieved. For example, loading 40 &#181;L of stock antigen results in an approximate 1.75-fold concentration. Supplementary Figure 8 demonstrates the importance of vortexing (step 5.15) in the concentration process. Neglecting to vortex or improperly vortexing the samples limits the concentration process.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65147/65147fig02.jpg" /> 
Figure 2: Antigen concentration. The concentration of antigen by centrifugal filtration shown by transmission electron microscopy (A) and confirmed by ELISA (B). Error bars indicate the standard deviation. Statistical analysis is done using Tukey&#39;s multiple comparison tests with one-way ANOVA. **p &#60; 0.01, ****p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/65147/65147fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3A depicts concentrated antigen filled into unsealed and sealed particles. Although a significant amount of antigen is loaded into the particles (0.0469 &#177; 0.0086 IU), this material comprises &#60;90% of the particle capacity, leaving ample space for the loading of additional antigen. Interestingly, unsealed particles only contain 0.0396 &#177; 0.0077 IU, comprising only 85% &#177; 16% of the total amount loaded. Although a statistically insignificant loss, some of the RABV antigen may have denatured during repeated rehydration and drying in the filling process. After sealing, 69% &#177; 5% of the antigen remains encapsulated in a bioactive form. Although this suggests significant loss occurs during the sealing process due to thermal stress, most of the inactivated viral antigen remains intact (Figure 3B). Co-encapsulation of stabilizing excipients along with the antigen is one possible strategy to further increase antigen stability throughout the fabrication process, and has previously been successful with other inactivated virus antigens14,15.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65147/65147fig03.jpg" /> 
Figure 3: Bioactive RABV antigen after particle fabrication. (A) Images show unsealed and sealed particles containing the RABV antigen. (B) Antigen stability through the particle fabrication process (n = 4). Loading control is generated by dispensing the antigen directly into the solution. Error bars indicate the standard deviation. Scale bar = 200 &#181;m. Statistical analysis is done using Tukey&#39;s multiple comparison tests with one-way ANOVA. *p &#60; 0.05. Images are generated by focus stacking multiple images and merging them using focus stacking software. <a href="https://www.jove.com/files/ftp_upload/65147/65147fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Particle, fiducial, and array dimensions. The figure shows the geometric properties of the four-pointed star fiducial (A), the cylindrical microparticle (B), the five-pointed star fiducial (C), and an array of particles with fiducials (D) displayed in the CAD software. <a href="https://www.jove.com/files/ftp_upload/65147/Figure S1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: This figure shows a cross-section of the structure placed into the oven to cure PDMS molds. Arrows indicate where binder clamps are applied. <a href="https://www.jove.com/files/ftp_upload/65147/Figure S2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Proper technique to efficiently recover concentrated antigen. At the end of the first spin, the concentrated sample circled in red (A) is retained in the filter, while the filtrate is collected in the bottom of the collection tube (larger outer tube). To resuspend the pelleted antigen, the centrifugal filter unit is capped using the collection tube (B) in preparation for vortexing. When vortexing, the tip of the tube is kept in contact with the vortex pad and the cap end rotated around while maintaining a 45&#176; angle with the vortex pad (C). <a href="https://www.jove.com/files/ftp_upload/65147/Figure S3.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4: Pre-run programming piezoelectric dispenser. (A) Set up the &#34;Find Target Reference Points&#34; by navigating from the &#34;Main tab&#34; to the Robot Setup &#62; Miscellaneous &#62;Div. Function &#62; Find Target Reference Points. Use the buttons highlighted in blue to set the fiduciary marks. First, select Learn Template and draw a box around the fiduciary mark of interest, the verify the template accuracy by clicking on Search Template, and save the template. Do this for the four- and five-pointed stars, and save the file names according to the instructions under Use Two Different Template Images. Next, ensure all the parameters in the black boxes match. Load the four-pointed star fiducial using the Load Template (green box). Save the &#34;Find Target Reference Points&#34; program by selecting Task List (orange box). (B) Set up the Run by navigating to the Robot Setup &#62; Tasks tab, then load the sequence of tasks shown in the blue box by adding tasks from the Task List (black box) using the Task in Run selections (green box). Finally, save the task (orange box). (C) Set up the &#34;Target Substrate&#34; by navigating to the Robot Setup &#62; Target Substrate, then add a target (blue box). (D) Enter the parameters shown here and select Save (blue box). <a href="https://www.jove.com/files/ftp_upload/65147/Figure S4.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 5: Loading antigen and calibrating dispending alignment. (A) Navigate to the Nozzle Setup &#62; Do Task tab and aspirate 10 &#181;L of the antigen into the dispensing tip by selecting TakeProbe10 uL (black box) and clicking on DO (black box). (B) This opens a separate window. Select the well the concentrated antigen was loaded into and select OK (blue box). (C) After the antigen has been aspirated, wash the tip by selecting the blue box, and repeat this wash two more times. Select the camera (black box) and determine the drop volume by selecting drop volume (green box). Ensure a stable drop is forming with a volume standard deviation (%) &#60;2 (orange box). (D) Following these steps will open up the Snap Drop Cam; select Image (blue box) in the drop-down menu and select Nozzle Head Camera Wizard. This opens a new window. Perform the next sequence of steps quickly. (E) Ensure the target made in Supplementary Figure 4 is loaded, then select Move To Target (blue box). Adjust the drops to 15 and select Spot (black box). Once spotted, immediately select Move (green box). Ensure Auto Find is selected and delete particle size = 12, then click on Start (orange boxes). If the auto-detection fails, repeat this process after moving to a different area on the slide (purple box). <a href="https://www.jove.com/files/ftp_upload/65147/Figure S5.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 6: Programing spotting array and beginning the run. (A) Navigate to the Target Setup &#62; Target, then fill in the parameters shown in the blue box. (B) Next, navigate to the &#34;Field Setup tab&#34; and enter 20 into the no. of drops field (blue box), select a well (black box), then select targets/particles to dispense into (green box). By selecting a different well and reselecting the targets/particles, additional filling cycles are performed during a single run. (C) On the main screen, ensure the run and target created in Supplementary Figure 5 are selected. (D) Navigate to the &#34;Run tab&#34; and select Start Run (blue box). <a href="https://www.jove.com/files/ftp_upload/65147/Figure S6.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 7: Microparticle injectability. (A-C) Focus-stacked stereoscope images of microparticles filled with fluorescein sodium salt and sealed in a 19 G needle. (D) A total of 10 particles were injected through a 19 G needle using a 2% carboxymethyl cellulose solution (n = 8). Scale bar = 1 mm. Error bars indicate standard deviation. <a href="https://www.jove.com/files/ftp_upload/65147/Figure S7.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 8: Potential issues with antigen concentration. The red line indicates the expected fold increase in concentration. Error bars indicate standard deviation. Statistical analysis was done using Tukey&#39;s multiple comparison tests with one-way ANOVA. *** p &#60; 0.001, ****p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/65147/Figure S8_Revised.pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Preparation of buffers and solutions for RABV ELISA. <a href="https://www.jove.com/files/ftp_upload/65147/Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 1: STL file containing particle array. <a href="https://www.jove.com/files/ftp_upload/65147/Supplemental File 1.STL" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 2: STL file containing geometry for the custom slide holder used for sealing particles. <a href="https://www.jove.com/files/ftp_upload/65147/Supplemental File 2.STL" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen at JoVE.com

Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

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The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately ...

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