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>
	<li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+health+impact+of+vaccination+against+ten+pathogens+in+98+low-income+and+middle-income+countries+from+2000+to+2030:+a+modelling+study.">Estimating the health impact of vaccination against ten pathogens in 98 low-income and middle-income countries from 2000 to 2030: a modelling study.</a> The Lancet. 397, 398-408 (2021).</li><li>Euliano, E. M., Sklavounos, A. A., Wheeler, A. R., McHugh, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+diagnostics+and+drug+delivery+technologies+to+low-resource+settings.">Translating diagnostics and drug delivery technologies to low-resource settings.</a> Science Translational Medicine. 14 (666), eabm1732 (2022).</li><li>Haider, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabies:+old+disease,+new+challenges.">Rabies: old disease, new challenges.</a> Canadian Medical Association Journal. 178 (5), 562-563 (2008).</li><li>Fisher, C. R., Streicker, D. G., Schnell, 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=The+spread+and+evolution+of+rabies+virus:+conquering+new+frontiers.">The spread and evolution of rabies virus: conquering new frontiers.</a> Nature Reviews Microbiology. 16 (4), 241-255 (2018).</li><li>Nagarajan, T., Rupprecht, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rabies and Rabies Vaccines. , (2020).</li><li>Shi, T., Dunham, E. F., Nyland, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabies+vaccination+compliance+and+reasons+for+incompletion.">Rabies vaccination compliance and reasons for incompletion.</a> The Western Journal of Emergency Medicine. 21 (4), 918-923 (2020).</li><li>Shankaraiah, R. H., Rajashekar, R. A., Veena, V., Hanumanthaiah, A. N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compliance+to+anti-rabies+vaccination+in+post-exposure+prophylaxis.">Compliance to anti-rabies vaccination in post-exposure prophylaxis.</a> Indian Journal of Public Health. 59 (1), 58-60 (2015).</li><li>Cleland, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-administration+vaccines:+controlled-release+technology+to+mimic+repeated+immunizations.">Single-administration vaccines: controlled-release technology to mimic repeated immunizations.</a> Trends in Biotechnology. 17 (1), 25-29 (1999).</li><li>Yeh, M. K., Coombes, A. G. A., Jenkins, P. G., Davis, S. 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P., Gander, B., Johansen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+divalent+vaccine+based+on+biodegradable+microspheres+induces+protective+immunity+against+tetanus+and+diphtheria.">An experimental divalent vaccine based on biodegradable microspheres induces protective immunity against tetanus and diphtheria.</a> Journal of Pharmaceutical Sciences. 92 (5), 957-966 (2003).</li><li>Mvan de Weert, M., Hennink, W. E., Jiskoot, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+instability+in+poly(lactic-co-glycolic+acid)+microparticles.">Protein instability in poly(lactic-co-glycolic acid) microparticles.</a> Pharmaceutical Research. 17 (10), 1159-1167 (2000).</li><li>Graf, T. 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A., Bronshtein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabies+vaccine+preserved+by+vaporization+is+thermostable+and+immunogenic.">Rabies vaccine preserved by vaporization is thermostable and immunogenic.</a> Vaccine. 33 (19), 2203-2206 (2015).</li><li>McHugh, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+fillable+microparticles+and+other+complex+3D+microstructures.">Fabrication of fillable microparticles and other complex 3D microstructures.</a> Science. 357 (6356), 1138-1142 (2017).</li><li>Sanchez, M. E. 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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

This method describes the encapsulation of the rabies antigen into biodegradable polymeric microparticles with structural and material properties that enable pulsatile release after a predetermined delay. Enzyme-linked immunosorbent assay (ELISA) assessment of the antigen retrieved from the particle core confirms the presence of intact trimeric rabies virus glycoprotein through particle fabrication.

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

fabrication-pulsatile-polymeric-microparticles-encapsulating-rabies

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JoVE Journal

Bioengineering

1.1K Views.  Rice University. This method describes the encapsulation of the rabies antigen into biodegradable polymeric microparticles with structural and material properties that enable pulsatile release after a predetermined delay. Enzyme-linked immunosorbent assay (ELISA) assessment of the antigen retrieved from the particle core confirms the presence of intact trimeric rabies virus glycoprotein through particle fabrication.

Video: Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.