We would like to thank Dr. Bryan E. Tomlin affiliated with the elemental analysis lab within the department of chemistry at TAMU who assisted with the neutron activation analysis (NAA).

1. Polycaprolacyone triacrylate (PCLTA) synthesis
<ol>
	<li>Dry 3.2 mL of 400 Da polycaprolacyone (PCL) triol overnight at 50 &#176;C in an open 200 mL round bottom flask and K2CO3 in a glass vial at 90 &#176;C.</li>
	<li>Mix the triol with 6.4 mL of dichloromethane (DCM) and 4.246 g of potassium carbonate (K2CO3) under argon.</li>
	<li>Mix 2.72 mL of acryloyl chloride in 27.2 mL of DCM and add dropwise to the reaction mixture in the flask over 5 min.</li>
	<li>Cover the reaction mixture with aluminum foil and leave it undisturbed at room temperature for 24 h under argon.</li>
	<li>After 24 h, filter the reaction mixture using a filter paper on a Buchner funnel under vacuum to discard excess reagents.</li>
	<li>Precipitate filtrate from step 1.5 that contains the endcapped polymer in diethyl ether in a 1:3 (vol/vol) and rotovape at 30 &#176;C to remove the diethyl ether.</li>
</ol>
2. Formation of the polybubble
NOTE: Injecting polymer in the deionized (DI) water would cause the polybubbles to migrate to the bottom of the vial resulting in flattened bottom. Use 10% (wt/vol) carboxymethyl cellulose (CMC) fill the glass vial instead to avoid polybubble flattening.
<ol>
	<li>Prepare 10% (wt/vol) CMC solution in DI water.</li>
	<li>Fill a 0.92 mL glass vial with 0.8 mL of 10% CMC using a 1 mL transfer pipet.</li>
	<li>Mix 1000 mg/mL of 14 kDa PCL in DCM and synthesize PCLTA in a 1:3 (vol/vol) for a total volume of 200 &#181;L or prepare 200 &#181;L of 1000 mg/mL of 5 kDa poly (lactic-co-glycolic acid) diacrylate (PLGADA) in chloroform.</li>
	<li>Mix the 2-hydroxy-4&#8242;-(2-hydroxyethoxy)-2-methylpropiophenone (photoinitiator) with the polymer (PLGADA or PCL/PCLTA) mixture in 0.005:1 (vol/vol).</li>
	<li>Load 200 &#181;L of polymer mixture into a 1 mL glass syringe mounted on a syringe pump that is connected to a dispensing stainless-steel tube with an inner diameter of 0.016 inch.</li>
	<li>Use a micromotor to control the forward and backward motion of the polymer tube to inject polymer into the 10% CMC in the glass vial to form the polybubble.</li>
	<li>Cure the polybubbles under ultraviolet (UV) at 254 nm wavelength for 60 s at 2 W/cm2.</li>
	<li>Flash freeze the polybubbles in liquid nitrogen and lyophilize overnight at 0.010 mBar vacuum and at -85 &#176;C.</li>
	<li>Separate the polybubbles from the dried CMC using forceps and wash the polybubbles with DI water to remove any residual CMC. Note that other polymers can be used likely with modifications to alter the release kinetics.</li>
</ol>
3. Modulation of polybubble diameter
<ol>
	<li>Fill a 0.92 mL glass vial with 10% CMC using a 1 mL transfer pipet.</li>
	<li>Mix PCL/PCLTA in a 1:3 (vol/vol) with 1000mg/mL 14kDa PCL and synthesize PCLTA. Mix the photoinitiator with polymer mixture in a 0.005:1 (vol/vol).</li>
	<li>Load the polymer mixture into a 1 mL glass syringe mounted on a syringe pump that is connected to a dispensing stainless-steel tube with an inner diameter of 0.016 inch.</li>
	<li>Use a micromotor to control the forward and backward motion of the polymer tube to inject polymer into the 10% CMC in the glass vial to form the polybubble.</li>
	<li>To obtain polybubbles with various diameters, vary dispensing rate from 0.0005 to 1 &#181;L/s.</li>
	<li>Take images of the vial with the polybubbles with varying diameter.</li>
	<li>Use ImageJ to quantify the diameter of the polybubbles and use the size of the vial as scale.</li>
</ol>
4. Centering cargo within polybubble
<ol>
	<li>Modulation of PCL/PCLTA viscosity using K2CO3: 
	NOTE: Viscosity of PLGADA does not have to be modified using K2CO3 because the viscosity of 5 kDa PLAGDA at 1000 mg/mL is sufficient for centering the cargo.
	<ol>
		<li>Add K2CO3 (that was isolated after the PCLTA reaction) to the PCLTA at varying concentrations including 0 mg/mL, 10 mg/mL, 20 mg/mL, 40 mg/mL, and 60 mg/mL.</li>
		<li>Measure the dynamic viscosities of the solutions by changing the shear rate from 0 to 1000 1/s using rheometry.</li>
		<li>Manually inject the cargo in the middle (refer to step 4.2 to prepare the cargo mixture) of the polybubbles that were formed using the PCL/PCLTA solutions with different concentrations of K2CO3 (step 4.1.1). Determine the optimal concentration of K2CO3 by observing which solution from step 4.1.1 can result in retention of the cargo in the middle.</li>
	</ol>
	</li>
	<li>Centering of the cargo (already shown feasibility with small molecules) with CMC
	<ol>
		<li>Mix the cargo with 5% (wt/vol) CMC in a rotator overnight to increase the viscosity of the cargo.</li>
		<li>Manually inject 2 &#181;L of cargo mixture in the polybubble and proceed with UV curing at 254 nm wavelength for 60 s at 2 W/cm2.</li>
		<li>Flash freeze the polybubbles in liquid nitrogen for 30 s and lyophilize overnight at 0.010 mBar vacuum and at -85 &#176;C.</li>
		<li>Separate the polybubbles from the dried CMC using forceps and wash with DI water to remove any residual CMC.</li>
		<li>Cut the polybubble in half and image the halves using confocal microscopy to ensure that the cargo is centered (refer to step 6 for excitation and emission wavelengths used).</li>
	</ol>
	</li>
</ol>
5. Cargo Formulation
NOTE: Polybubble formulation can house various cargo types, including small molecules, proteins, and nucleic acids.
<ol>
	<li>Based on previous studies, in the case of protein cargo, use excipients including polyethylene glycol (PEG)6, polyvinylpyrrolidone (PVP), and glycopolymers6 to improve the stability of protein during polybubble formulation.</li>
	<li>Form polybubbles based on the protocol in step 2.</li>
	<li>Prepare the antigen solution by adding 17.11 g of trehalose to 625 &#181;L of HIV gp120/41 antigen.</li>
	<li>Manually inject 1 &#181;L of antigen solution in the middle of the polybubble.</li>
	<li>Open polybubbles on days 0, 7, 14, and 21, and record the fluorescence of antigen with excitation and emission wavelengths 497 nm and 520 nm, respectively.</li>
	<li>Determine the functionality of the antigen using enzyme-linked immunosorbent assay (ELISA) and use 5% nonfat milk as a blocking buffer.</li>
</ol>
6. Release of cargo
NOTE: Small molecule or antigen can be used as the cargo type
<ol>
	<li>Small molecule
	<ol>
		<li>Incubate polybubbles with centered acriflavine in 400 &#181;L of phosphate buffer saline (PBS) at 37 &#176;C, 50 &#176;C for PLGADA polybubbles and at 37 &#176;C, 50 &#176;C, 70 &#176;C for PCL/PCLTA polybubbles. 
		NOTE: The reason why we recommend testing above body temperatures is to a) simulate the temperature (50 &#176;C) at which the polybubble reaches while lasering the gold nanorods (AuNRs) within PCL and PLGA; and b) accelerate the degradation process of PCL (50 &#176;C, 70 &#176;C).</li>
		<li>At each time point, collect the supernatants and replace with 400 &#181;L of fresh PBS.</li>
		<li>Use a plate reader to quantify the fluorescence intensities in the collected supernatants. 
		NOTE: Use ex/em of 416 nm/514 nm for acriflavine.</li>
	</ol>
	</li>
	<li>Antigen
	<ol>
		<li>Incubate polybubbles with centered bovine albumin serum (BSA)-488 in 400 &#181;L of PBS at 37 &#176;C, 50 &#176;C for PLGADA polybubbles and at 37 &#176;C, 50 &#176;C for PCL/PCLTA polybubbles.</li>
		<li>At each time point, collect the supernatants and replace with 400 &#181;L fresh PBS.</li>
		<li>Use a plate reader to quantify the fluorescence intensities in the collected supernatants. Use ex/em of 497 nm/520 nm for BSA-488. 
		NOTE: Release study at 70 &#176;C for PCL/PCLTA polybubbles should not be conducted to avoid exposing the antigen to extreme temperature.</li>
	</ol>
	</li>
</ol>
7. Toxicity
<ol>
	<li>Quantifying chlorine content in polybubbles using neutron activation analysis (NAA)
	<ol>
		<li>Use polybubbles that were lyophilized for 2, 4, 6, 20, and 24 h for this study at 0.010 mBar vacuum and at -85 &#176;C.</li>
		<li>Measure 5-9 mg of polybubbles and place them on LDPE irradiation vials.</li>
		<li>Prepare 1000 g/mL of chlorine calibration solution from national institute of standards and technology (NIST)-traceable calibration solution.</li>
		<li>Use 1- megawatts Triga reactor to carry out neutron irradiations on each sample at neutron fluence rate of 9.1 &#215; 1012 /cm2&#183;s for 600 s.</li>
		<li>Transfer the polybubbles to unirradiated vials.</li>
		<li>Use HPGe detector to obtain gamma-ray spectra for 500 s after 360 s decay intervals.</li>
		<li>Use NAA software by canberra Industries to analyze the data.</li>
	</ol>
	</li>
	<li>Quantifying chlorine content released from polybubbles using NAA
	<ol>
		<li>Incubate polybubbles that were lyophilized overnight (at 0.010 mBar vacuum and at -85 &#176;C) in 400 &#181;L of PBS at 37 &#176;C.</li>
		<li>Collect the supernatants at weeks 1, 2, and 3 after incubation.</li>
		<li>Analyze the supernatants for chlorine content using NAA using the same method as described above in step 7.1.</li>
	</ol>
	</li>
</ol>
8. AuNR Synthesis by Kittler, S., et al.8
<ol>
	<li>Prepare AuNR seeding solution by mixing 250 &#181;L of 10 mM chloroauric acid (HAuCl4), 7.5 mL of 100 mM cetrimonium bromide (CTAB), and 600 &#181;L of 10 mM ice cold sodium borohydride (NaBH4).</li>
	<li>Prepare Growth solution by mixing 40 mL of 100 mM CTAB, 1.7 mL of 10 mM HAuCl4, 250 &#181;L of silver nitrate (AgNO3), and 270 &#181;L of 17.6 mg/mL ascorbic acid to a tube.</li>
	<li>Vigorously mix 420 &#181;L of seed solution with the growth solution at 1200 rpm for 1 min. Then leave the mixture undisturbed to react for 16 h.</li>
	<li>Remove the excess reagents from the mixture by centrifuging at 8000 &#215; g for 10 min and discard the supernatant.</li>
</ol>
9. Hydrophobicization of AuNRs by Soliman, M.G., et al.9
<ol>
	<li>Adjust pH of 1.5 mL of synthesized CTAB-stabilized AuNRs to 10 using 1 mM sodium hydroxide (NaOH).</li>
	<li>Stir the solution with 0.1 mL of 0.3 mM methylated PEG (mPEG) thiol at 400 rpm overnight.</li>
	<li>Mix PEGylated AuNRs with 0.4 M dodecylamine (DDA) in chloroform at 500 rpm for 4 days.</li>
	<li>Pipet out the top organic layer containing hydrophobicized AuNRs and store at 4 &#176;C until future use.</li>
</ol>
10. NIR-activation of polybubbles
<ol>
	<li>Mix the polymer (PLGADA or PCL/PCLTA) solution with hydrophobicized AuNRs in a 1:9 (vol/vol).</li>
	<li>Add photoinitiator to the polymer-AuNR mixture in a 0.005:1 (vol/vol).</li>
	<li>Form polybubbles by injecting the polymer-AuNR mixture into a 0.92 mL glass vial with 10% CMC (wt/vol) (refer to step 2).</li>
	<li>Cure the polybubbles at 254 nm wavelength for 60 s at 2 W/cm2.</li>
	<li>Flash freeze in liquid nitrogen for 30 s and lyophilize overnight at 0.010 mBar vacuum and at -85 &#176;C.</li>
	<li>Separate the dried polybubbles using forceps and wash with DI water to remove any residual CMC.</li>
	<li>Incubate the polybubbles in 400 &#181;L of PBS at 37 &#176;C.</li>
	<li>Activate the polybubbles using 801 nm NIR laser at 8A for 5 min every Monday, Wednesday, and Friday.</li>
	<li>Take forward-looking infrared (FLIR) images of the polybubble before and after laser activation to obtain temperature values.</li>
	<li>Calculate temperature differences between before and after laser activation based on the temperature values from the FLIR images.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+polymeric+nanoparticles+based+drug+delivery+systems.">Biodegradable polymeric nanoparticles based drug delivery systems.</a> Colloids and Surfaces B: Biointerfaces. 75 (1), 1-18 (2010).</li><li>Dai, C., Wang, B., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microencapsulation+peptide+and+protein+drugs+delivery+system.">Microencapsulation peptide and protein drugs delivery system.</a> Colloids and Surfaces B: Biointerfaces. 41 (2-3), 117-120 (2005).</li><li>Souery, W. 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G., Wolff, T., Eychm&#252;ller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy+and+Fast+Phase+Transfer+of+CTAB+Stabilised+Gold+Nanoparticles+from+Water+to+Organic+Phase.">Easy and Fast Phase Transfer of CTAB Stabilised Gold Nanoparticles from Water to Organic Phase.</a> Zeitschrift f&#252;r Physikalische Chemie. 229, 235 (2015).</li><li>Soliman, M. G., Pelaz, B., Parak, W. 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V., Heller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsatile+and+delayed+release+of+lysozyme+from+ointment-like+poly(ortho+esters).">Pulsatile and delayed release of lysozyme from ointment-like poly(ortho esters).</a> Journal of Controlled Release. 21 (1), 191-200 (1992).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instability,+stabilization,+and+formulation+of+liquid+protein+pharmaceuticals.">Instability, stabilization, and formulation of liquid protein pharmaceuticals.</a> International Journal of Pharmaceutics. 185 (2), 129-188 (1999).</li><li>Wong, D. Y., Hollister, S. J., Krebsbach, P. H., Nosrat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(epsilon-caprolactone)+and+poly+(L-lactic-co-glycolic+acid)+degradable+polymer+sponges+attenuate+astrocyte+response+and+lesion+growth+in+acute+traumatic+brain+injury.">Poly(epsilon-caprolactone) and poly (L-lactic-co-glycolic acid) degradable polymer sponges attenuate astrocyte response and lesion growth in acute traumatic brain injury.</a> Tissue Engineering. 13 (10), 2515-2523 (2007).</li><li>Davoodi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+systems+for+programmed+and+on-demand+release.">Drug delivery systems for programmed and on-demand release.</a> Advanced Drug Delivery Reviews. 132, 104-138 (2018).</li><li>Huang, Y. C., Lei, K. F., Liaw, J. W., Tsai, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+laser+intensity+activated+plasmonic+gold+nanoparticle-generated+photothermal+effects+on+cellular+morphology+and+viability:+a+real-time,+long-term+tracking+and+monitoring+system.">The influence of laser intensity activated plasmonic gold nanoparticle-generated photothermal effects on cellular morphology and viability: a real-time, long-term tracking and monitoring system.</a> Photochemical and Photobiological Sciences. 18 (6), 1419-1429 (2019).</li><li>Link, S., Burda, C., Nikoobakht, B., El-Sayed, M. 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Authors have nothing to disclose.

Current technologies and challenges 
Emulsion-based micro- and nanoparticles have been commonly used as drug delivery carriers. Although release kinetics of the cargo from these devices have been extensively studied, controlling burst release kinetics has been a major challenge11. Cargo versatility and functionality is also limited in emulsion-based systems owing to the exposure of cargo to excess aqueous and organic solvents. Protein-based cargo are often not compatible with...

Limited immunization coverage results in the death of 3 million people specifically caused by vaccine-preventable diseases1. Inadequate storage and transportation conditions lead to wastage of functional vaccines and thus contribute to reduced global immunization. In addition, incomplete vaccination due to not adhering to the required vaccine schedules also causes limited vaccine coverage, specifically in developing countries2. Multiple visits to medical personnel are required within the recommended period for receiving booster shots, thus limiting the percentage of population with complete vaccination. Hence, there is a need for developing novel strategies for controlled vaccine delivery to circumvent these challenges.
Current efforts towards developing vaccine delivery technologies include emulsion-based polymeric systems3,4. However, cargo is often exposed to greater quantity of organic solvent that can potentially cause aggregation and denaturation, specifically in the context of protein-based cargo5,6. We have developed a novel vaccine delivery platform, &#34;polybubbles&#34;, that can potentially house multiple cargo compartments while minimizing the volume of cargo that is exposed to solvent7. For example, in our polybubble core-shell platform, one cargo pocket of diameter 0.38 mm (SEM) is injected in the center of a 1 mm polybubble. In this case, surface area of cargo exposed to organic solvent would be approximately 0.453 mm2. After considering the packing density of spheres (microparticles) within a sphere (cargo depot), the actual volume of microparticles (10 &#181;m in diameter) that could fit in the depot is 0.17 mm3. The volume of one microparticle is 5.24x10-8 mm3 and thus number of particles microparticles that can fit the depot is ~3.2x106 particles. If each microparticle has 20 cargo pockets (as a result of double-emulsion) of 0.25 &#181;m diameter, then the surface area of cargo exposed to organic solvent is 1274 mm2. Cargo depot within the polybubble thus would have ~2800-fold less surface area exposed to organic solvent compared to that of organic solvent-exposed cargo in microparticles. Our polyester-based platform can thus potentially reduce the quantity of cargo exposed to organic solvent which can otherwise cause cargo aggregation and instability.
Polybubbles are formed based on phase-separation principle where the polyester in organic phase is injected into an aqueous solution resulting in a spherical bubble. Cargo in the aqueous phase can then be injected in the center of the polybubble. Another cargo compartment can potentially be achieved within the polybubble by mixing a different cargo with the polymer shell. The polybubble at this stage will be malleable and will then be cured to result in a solid polybubble structure with cargo in the middle. Spherical polybubbles were chosen over other geometrical shapes to increase the cargo capacity within the polybubble while minimizing the overall size of the polybubble. Polybubbles with cargo in the center were chosen to demonstrate delayed burst release. Polybubbles were also incorporated with near infrared (NIR)- sensitive (i.e., theranostic-enabling) agent, namely gold nanorods (AuNR), to cause increase in temperature of the polybubbles. This effect could potentially facilitate faster degradation and could be used for controlling kinetics in future applications. In this paper, we describe our approach to form and characterize polybubbles, to achieve delayed burst release from the polybubbles, and to incorporate AuNR within the polybubbles to cause NIR-activation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Step Ultra Tetramethylbezidine (TMB)-Enzyme-Linked Immunosorbent Assay (ELISA) Substrate Solution</td>
			<td>Thermo scientific</td>
			<td>34028</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Hydroxy-2-methylpropiophenone</td>
			<td>TCI AMERICA</td>
			<td>H0991</td>
			<td></td>
		</tr>
		<tr>
			<td>450 nm Stop Solution for TMB Substrate</td>
			<td>Abcam</td>
			<td>ab17152</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryloyl chloride</td>
			<td>Sigma Aldrich</td>
			<td>A24109-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Acriflavine</td>
			<td>Chem-Impex International</td>
			<td>22916</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethyl ether</td>
			<td>Fisher Chemical</td>
			<td>E138-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HIV1 gp120 antibody conjugated to horseradish peroxidase (HRP)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher BioReagents</td>
			<td>BP9700100</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-CF488 dye conjugates</td>
			<td>Invitrogen</td>
			<td>A13100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromosalicylic acid</td>
			<td>Acros Organics</td>
			<td>AC162142500</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxymethylcellulose (CMC)</td>
			<td>Millipore Sigma</td>
			<td>80502-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrimonium bromide (CTAB)</td>
			<td>MP Biomedicals</td>
			<td>ICN19400480</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Chemical</td>
			<td>C2984</td>
			<td></td>
		</tr>
		<tr>
			<td>Coating buffer</td>
			<td>Abcam</td>
			<td>ab210899</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma Aldrich</td>
			<td>270997-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Fisher Chemical</td>
			<td>E1384</td>
			<td></td>
		</tr>
		<tr>
			<td>Dodeacyl Amine</td>
			<td>Acros Organics</td>
			<td>AC117665000</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Fisher BioReagents</td>
			<td>BP251610</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Acros Organics</td>
			<td>A61 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Legato 100 Syringe Pump</td>
			<td>KD Scientific</td>
			<td>14 831 212</td>
			<td></td>
		</tr>
		<tr>
			<td>mPEG thiol</td>
			<td>Laysan Bio</td>
			<td>NC0702454</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonfat dry milk</td>
			<td>Andwin Scientific</td>
			<td>NC9022655</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium carbonate</td>
			<td>Acros Organics</td>
			<td>AC424081000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate saline buffer</td>
			<td>Fisher BioReagents</td>
			<td>BP3991</td>
			<td></td>
		</tr>
		<tr>
			<td>(Poly(caprolactone)</td>
			<td>Sigma Aldrich</td>
			<td>440744-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>(Poly(caprolactone) triol</td>
			<td>Acros Organics</td>
			<td>AC190730250</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly (lactic-co-glycolic acid) diacrylate</td>
			<td>CMTec</td>
			<td>280050</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium carbonate</td>
			<td>Acros Organics</td>
			<td>AC424081000</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant HIV1 gp120 + gp41 protein</td>
			<td>Abcam</td>
			<td>ab49054</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Acros Organics</td>
			<td>S181 25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Fisher Chemical</td>
			<td>S678 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrachloroauric acid</td>
			<td>Fisher Chemical</td>
			<td>G54 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trehalose</td>
			<td>Acros Organics</td>
			<td>NC9022655</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethyl amine</td>
			<td>Acros Organics</td>
			<td>AC157910010</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of near-infrared sensitive, core-shell vaccine delivery platform

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving immunization coverage worldwide. Here, a novel injectable, ultraviolet- curable and delayed burst release- enabling vaccine delivery platform called polybubbles is introduced. Cargo was injected into polyester-based polybubbles that were formed in 10% carboxymethycellulose -based aqueous solution. This paper includes protocols to maintain spherical shape of the polybubbles and optimize cargo placement and retention to maximize the amount of cargo within the polybubbles. To ensure safety, chlorinated solvent content within the polybubbles were analyzed using neutron activation analysis. Release studies were conducted with small molecules as cargo within the polybubble to confirm delayed burst release. To further show the potential for on-demand delivery of the cargo, gold nanorods were mixed within the polymer shell to enable near-infrared laser activation.

Polybubbles were extensively characterized using SEM and NAA. Cargo was successfully centered to result in a delayed burst release. Polybubbles were also successfully laser-activated because of the presence of AuNRs within the polybubbles.
Polybubble characterization 
Polybubbles injected in an aqueous solution without CMC resulted in a flattened polybubble due to their contact with the bottom of the glass ...

Watch this Scientific Journal Video about Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform at JoVE.com

Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

This article describes the protocols used to produce a novel vaccine delivery platform, &#34;polybubbles,&#34; to enable delayed burst release. Polyesters including poly(lactic-co-glycolic acid) and polycaprolactone were used to form the polybubbles and small molecules and antigen were used as cargo.

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving ...

production-near-infrared-sensitive-core-shell-vaccine-delivery

Research

JoVE Journal

Biology

5.1K Views.  Texas A&M University. This article describes the protocols used to produce a novel vaccine delivery platform, "polybubbles," to enable delayed burst release. Polyesters including poly(lactic-co-glycolic acid) and polycaprolactone were used to form the polybubbles and small molecules and antigen were used as cargo.

Video: Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...