Name: rapid, scalable assembly and loading of bioactive proteins and immunostimulants into diverse synthetic nanocarriers via flash nanoprecipitation
Uploaded: 2018-08-11T14:00:00
Description: Watch this Scientific Journal Video about Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation at JoVE.com

We acknowledge staff and instrumentation support from the Structural Biology Facility at Northwestern University. The support from the R.H. Lurie Comprehensive Cancer Center of Northwestern University and the Northwestern University Structural Biology Facilities is acknowledged. The Gatan K2 direct electron detector was purchased with funds provided by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust. We also thank the following facilities at Northwestern University: the Keck Interdisciplinary Surface Science Facility, the Structural Biology Facility, the Biological Imaging Facility, the Center for Advanced Molecular Imaging, and the Analytical Bionanotechnology Equipment Core. This research was supported by the National Science Foundation grant 1453576, the National Institutes of Health Director&#39;s New Innovator Award 1DP2HL132390-01, the Center for Regenerative Nanomedicine Catalyst Award and the 2014 McCormick Catalyst Award. SDA was supported in part by NIH predoctoral Biotechnology Training Grant T32GM008449.

1. Synthesis of Poly(ethylene glycol)-block-poly(propylene sulfide)-Thiol
<ol>
	<li>Synthesize methoxy-poly(ethylene glycol) mesylate (Mn: 750) (MeO-PEG17-Ms, I).
	<ol>
		<li>Dissolve 10 g of MeO-PEG17-OH in 200 mL of 100% toluene within a 3-neck round bottom flask (RBF) under magnetic stirring at 600 rpm.</li>
		<li>Connect the 3-neck RBF to a Dean-Stark apparatus, itself attached to a condenser, keep the entire system under inert gas, either nitrogen or argon.</li>
		<l...

<ol>
	<li>Stano, A., Scott, E. A., Dane, K. Y., Swartz, M. A., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+T+cell+immunity+towards+a+protein+antigen+using+polymersomes+vs.+solid-core+nanoparticles.">Tunable T cell immunity towards a protein antigen using polymersomes vs. solid-core nanoparticles.</a> Biomaterials. 34 (17), 4339-4346 (2013).</li><li>Discher, B. M., 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=Polymersomes:+tough+vesicles+made+from+diblock+copolymers.">Polymersomes: tough vesicles made from diblock copolymers.</a> Science. 284 (5417), 1143-1146 (1999).</li><li>Vasdekis, A. E., Scott, E. A., O'Neil, C. P., Psaltis, D., Hubbell, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+fabrication+of+monodisperse+biocompatible+and+biodegradable+polymersomes+with+controlled+permeability.">Microfluidic fabrication of monodisperse biocompatible and biodegradable polymersomes with controlled permeability.</a> Journal of the American Chemical Society. 130 (29), 9543-9549 (2008).</li><li>Pessi, J., 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=Microfluidics-assisted+engineering+of+polymeric+microcapsules+with+high+encapsulation+efficiency+for+protein+drug+delivery.">Microfluidics-assisted engineering of polymeric microcapsules with high encapsulation efficiency for protein drug delivery.</a> International Journal of Pharmaceutics. 472 (1-2), 82-87 (2014).</li><li>O'Neil, C. P., Suzuki, T., Demurtas, D., Finka, A., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+encapsulation+of+biomolecules+into+polymersomes+via+direct+hydration.">A novel method for the encapsulation of biomolecules into polymersomes via direct hydration.</a> Langmuir. 25 (16), 9025-9029 (2009).</li><li>Saad, W. S., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+nanoparticle+formation+by+flash+nanoprecipitation.">Principles of nanoparticle formation by flash nanoprecipitation.</a> Nano Today. 11 (2), 212-227 (2016).</li><li>Johnson, B. K., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+for+rapid+self-assembly+of+block+copolymer+nanoparticles.">Mechanism for rapid self-assembly of block copolymer nanoparticles.</a> Physical Review Letters. 91 (11), 118302 (2003).</li><li>Han, J., 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=A+simple+confined+impingement+jets+mixer+for+flash+nanoprecipitation.">A simple confined impingement jets mixer for flash nanoprecipitation.</a> Journal of Pharmaceutical Sciences. 101 (10), 4018-4023 (2012).</li><li>Allen, S., Osorio, O., Liu, Y. G., Scott, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+assembly+and+loading+of+theranostic+polymersomes+via+multi-impingement+flash+nanoprecipitation.">Facile assembly and loading of theranostic polymersomes via multi-impingement flash nanoprecipitation.</a> Journal of Controlled Release. 262, 91-103 (2017).</li><li>Bobbala, S., Allen, S. D., Scott, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+nanoprecipitation+permits+versatile+assembly+and+loading+of+polymeric+bicontinuous+cubic+nanospheres.">Flash nanoprecipitation permits versatile assembly and loading of polymeric bicontinuous cubic nanospheres.</a> Nanoscale. 10 (11), 5078-5088 (2018).</li><li>Allen, S. D., 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=Polymersomes+scalably+fabricated+via+flash+nanoprecipitation+are+non-toxic+in+non-human+primates+and+associate+with+leukocytes+in+the+spleen+and+kidney+following+intravenous+administration.">Polymersomes scalably fabricated via flash nanoprecipitation are non-toxic in non-human primates and associate with leukocytes in the spleen and kidney following intravenous administration.</a> Nano Research. , (2018).</li><li>Karabin, N. B., 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=Sustained+micellar+delivery+via+inducible+transitions+in+nanostructure+morphology.">Sustained micellar delivery via inducible transitions in nanostructure morphology.</a> Nature Communications. 9 (1), 624 (2018).</li><li>Mascoli, C. C., Weary, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limulus+amebocyte+lysate+(LAL)+test+for+detecting+pyrogens+in+parenteral+injectable+products+and+medical+devices:+advantages+to+manufacturers+and+regulatory+officials.">Limulus amebocyte lysate (LAL) test for detecting pyrogens in parenteral injectable products and medical devices: advantages to manufacturers and regulatory officials.</a> Journal of the Parenteral Drug Association. 33 (2), 81-95 (1979).</li><li>Pustulka, K. M., 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=Flash+nanoprecipitation:+particle+structure+and+stability.">Flash nanoprecipitation: particle structure and stability.</a> Molecular Pharmaceutics. 10 (11), 4367-4377 (2013).</li><li>Tang, C., Amin, D., Messersmith, P. B., Anthony, J. E., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+directed+self-assembly+of+pH-responsive+antioxidant+nanoparticles.">Polymer directed self-assembly of pH-responsive antioxidant nanoparticles.</a> Langmuir. 31 (12), 3612-3620 (2015).</li><li>Gindy, M. E., Panagiotopoulos, A. Z., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+block+copolymer+stabilized+nanoparticles:+simultaneous+encapsulation+of+organic+actives+and+inorganic+nanostructures.">Composite block copolymer stabilized nanoparticles: simultaneous encapsulation of organic actives and inorganic nanostructures.</a> Langmuir. 24 (1), 83-90 (2008).</li></ol>

We have provided detailed instructions for the rapid fabrication of polymersomes using PEG17-bl-PPS35-SH as the diblock copolymer. Vesicular polymersomes are the primary aggregate morphology assembled at this ratio of hydrophilic PEG and hydrophobic PPS block molecular weight. When impinged multiple times, they have a diameter and polydispersity that matches polymersomes extruded through a 200 nm membrane after being formed via thin film hydration. This protocol thus eliminates the...

Nanocarriers allow for the controlled delivery of small and macromolecular cargo, including active entities that, if not encapsulated, would be either highly degradable and/or too hydrophobic for administration in vivo. Of the nanocarrier morphologies regularly fabricated, polymeric vesicles analogous to liposomes (also called polymersomes) offer the ability to simultaneously load hydrophilic and hydrophobic cargo1,2. Despite their promising advantages, polymersomes are still rare in clinical applications due, in part, to several key challenges in their manufacturing. For clinical use, polymersome formulations need to be made in large-scale, sterile, and consistent batches.
A number of techniques can be used to form polymersomes from a diblock copolymer, such as poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-bl-PPS), that include solvent dispersion3, thin film rehydration1,4, microfluidics 5,6, and direct hydration7. Solvent dispersion involves long incubation times in the presence of organic solvents, which may denature some bioactive payloads, like proteins. Thin film rehydration does not offer control over the polydispersity of the formed polymersomes, often requiring expensive and time-consuming extrusion techniques to achieve acceptable monodispersity. Furthermore, both microfluids and direct hydration are difficult to scale up for larger production volumes. Of the different nanocarrier fabrication methods, flash nanoprecipitation (FNP) offers the ability to make large-scale and reproducible formulations8,9,10. While FNP was previously reserved for the formulation of solid-core nanoparticles, our lab has recently expanded the use of FNP to include the consistent formation of diverse PEG-bl-PPS nanostructure morphologies11,12, including polymersomes11 and bicontinuous nanospheres12. We found that FNP was capable of forming monodisperse formulations of polymersomes without the need for extrusion, resulting in superior polydispersity index values compared to non-extruded polymersomes formed by thin film rehydration and solvent dispersion11. Bicontinuous nanospheres, with their large hydrophobic domains, were not able to be formed by thin film rehydration, despite forming under a number of solvent conditions with FNP12.
Here, we provide a detailed description for the synthesis of the PEG-bl-PPS diblock copolymer used in polymersome formation, the confined impingement jets (CIJ) mixer used for FNP, the FNP protocol itself, and the implementation of an automated system to reduce user variability. Included is information on how to sterilize the system sufficiently to produce endotoxin-free formulations for use in vivo, and representative data concerning the characterization of polymersomes formed by FNP. With this information, readers with interest in utilizing polymersomes for in vitro and in vivo work will be able to fabricate their own sterile, monodisperse formulations. Readers with experience in nanocarrier formulations and with polymer synthesis expertise will be able to rapidly test their own polymer systems using FNP as a potential alternative to their current formulation techniques. Additionally, the protocols described herein may be used as educational tools for the formulation of nanocarriers in nanotechnology laboratory courses.

<table>
	<tbody>
		<tr>
			<td>CanaKit Raspberry Pi 3 Ultimate Starter Kit - 32 GB Edition</td>
			<td>CanaKit</td>
			<td>UPC 682710991511</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Bearing Platform (Small) - 8mm Diameter</td>
			<td>Adafruit</td>
			<td>1179</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Motion 8 mm Shaft, 330 mm Length, Chrome Plated, Case Hardened, Metric</td>
			<td>VXB</td>
			<td>kit11868</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Rail Shaft Guide/Support - 8 mm Diameter</td>
			<td>Adafruit</td>
			<td>1182</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual-Position Precision Slide 4.5&#34; Stroke, 15 lb load capacity</td>
			<td>McMaster-Carr</td>
			<td>5236A16</td>
			<td></td>
		</tr>
		<tr>
			<td>MTPM-P10-1JK43 Iron Horse DC motor</td>
			<td>Iron Horse</td>
			<td>MTPM-P10-1JK43</td>
			<td></td>
		</tr>
		<tr>
			<td>Official Raspberry Pi Foundation 7&#34; Touchscreen LCD Display</td>
			<td>Raspberry Pi</td>
			<td>B0153R2A9I (ASIN)</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoBorg Reverse - Advanced motor control for Raspberry Pi</td>
			<td>PiBorg</td>
			<td>BURN-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Pololu Carrier with Sharp GP2Y0D810Z0F Digital Distance Sensor 10cm</td>
			<td>Pololu</td>
			<td>1134</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruland PSR16-5-4-A Set Screw Beam Coupling, Polished Aluminum, Inch, 5/16&#34; Bore A Diameter, 1/4&#34; Bore B Diameter, 1&#34; OD, 1-1/4&#34; Length, 44 lb-in Nominal Torque</td>
			<td>Ruland</td>
			<td>PSR16-5-4-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol monomethyl ether</td>
			<td>Sigma Aldrich</td>
			<td>202495</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanesulfonyl chloride</td>
			<td>Sigma Aldrich</td>
			<td>471259</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Sigma Aldrich</td>
			<td>179418</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene, Anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>244511</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Celite 545 (Diatomaceous Earth)</td>
			<td>Sigma Aldrich</td>
			<td>419931</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>320269</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma Aldrich</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide, anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium carbonate</td>
			<td>Sigma Aldrich</td>
			<td>791776</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T30805</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran</td>
			<td>Sigma Aldrich</td>
			<td>360589</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum oxide, neutral, activated, Brockmann I</td>
			<td>Sigma Aldrich</td>
			<td>199974</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium methoxide solution, 0.5 M in methanol</td>
			<td>Sigma Aldrich</td>
			<td>403067</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene sulfide</td>
			<td>Sigma Aldrich</td>
			<td>P53209</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>320390</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution 1.0 N</td>
			<td>Sigma Aldrich</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free water</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30529.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper pH strips</td>
			<td>Fisher Scientific</td>
			<td>13-640-508</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free Dulbecco&#39;s PBS</td>
			<td>Sigma Aldrich</td>
			<td>TMS-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>03-337-4</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL all-plastic syringe</td>
			<td>Thermo Scientific</td>
			<td>S75101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sepharose CL-6B</td>
			<td>Sigma Aldrich</td>
			<td>CL6B200</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid chromatography column</td>
			<td>Sigma Aldrich</td>
			<td>C4169</td>
			<td></td>
		</tr>
		<tr>
			<td>CIJ mixer, HDPE</td>
			<td>Custom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma Aldrich</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-Blue hTLR4</td>
			<td>InvivoGen</td>
			<td>hkb-htlr4</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW-Blue Cells</td>
			<td>InvivoGen</td>
			<td>raw-sp</td>
			<td></td>
		</tr>
		<tr>
			<td>QUANTI-Blue</td>
			<td>InvivoGen</td>
			<td>rep-qb1</td>
			<td></td>
		</tr>
		<tr>
			<td>PYROGENT Gel Clot LAL Assays</td>
			<td>Lonza</td>
			<td>N183-125</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid, scalable assembly and loading of bioactive proteins and immunostimulants into diverse synthetic nanocarriers via flash nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Here, we have presented a simple protocol for the formulation of nanocarriers capable of loading hydrophilic and hydrophobic cargo that are safe for in vivo mouse and non-human primate administration11,13. We have also included a detailed protocol for the synthesis of the polymer used in our representative results, along with a description for the fabrication of a custom instrument for the mechanically-controlled impingem...

Watch this Scientific Journal Video about Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation at JoVE.com

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

We believe that the use of flash nanoprecipitation to assemble complex nanostructures will allow researchers who do not have extensive experience in chemistry, engineering or nanomaterial science to fabricate advanced drug delivery systems independently for their own specific applications and needs. The main advantage of this technique is the generation of large volumes of diverse nanostructures at high concentration, low polydispersity, and loaded with bioactive molecules including small molecule drugs and proteins all formed under sterile conditions. Generally, individuals new to polymer synthesis may struggle because the polypropylene sulfide component is highly sensitive to oxidation by exposure to air.Visual demonstration of this method is critical as the multiple impingement steps are difficult to learn because they need to be performed very quickly in succession. To sterilize the confined impingement jets, or CIJ mixer, work within a biological safety cabinet to submerge the mixer with all parts disassembled into zero point one molar sodium hydroxide overnight. Reassemble the CIJ mixer and flow endotoxin-free water through the mixer using luerlock syringes, test the ph of the water, and continue to flow water through until the ph registers as neutral.To prepare impingement solution one, first weigh 20 milligrams of the synthesized polymer into a one point five milliliter tube. Then, add hydrophobic dyes, drugs, or other cargo. Finally, add 500 microliters of 100%tetrahydrofuran to the polymer and cargo, vortex vigorously to dissolve.To prepare impingement solution two, dissolve the hydrophilic cargo to be loaded within the polymer vesicles in 500 microliters of an aqueous buffer. Add 2.5 milliliters of an aqueous buffer of choice to a suitably sized reservoir. Now, place the reservoir under the CIJ mixer such that the outflow from the mixer directly enters the reservoir.Load the impingement solutions into separate one milliliter plastic disposable syringes, impinge the solutions against each other to simultaneously form nanostructures by inserting syringes into luerlock adapters at the top of the CIJ mixer. In a single, smooth, and rapid motion, depress both syringes simultaneously and with equal force. If multiple impingements are going to be performed, it is critical that they are done as quickly as possible to reduce the loss of valtower organic solvent.To perform multiple impingements, split the nascent nanostructure solution between two syringes and repeat this step up to four more times. Collect the outflow in the aqueous buffer filled reservoir and gently stir to ensure mixing. There are several options to remove the unloaded cargo and organic solvent.The nanocarrier formulation can be removed through excise exclusion or desalting column, through a tangential flow filtration system, through vacuum desiccation, or through dialysis as demonstrated here. Dialyze the nanocarrier formulation in the same aqueous buffer and reservoir used for impingement. Use tubing of an appropriate molecular weight cutoff for at least 24 hours, with at least two buffer changes.Once the unloaded cargo has been removed from the organic solvent, concentrate the nanocarrier formulation using a spin concentrator system following the manufacturer's instructions. Nanocarriers can be stored at four degrees celsius for weeks to months. Prior to use after storage, nanocarrier formulations should be briefly vortexed.Perform fabrication of a high-speed syringe pump as described in the text protocol. To use autorun mode, select autorun from the main menu. The system will prompt users to allow the motor to automatically position the syringe expulsion platform to the beginning of the precision slide.Ensure that the path in front of and behind the metal plate is clear prior to proceeding. Load the CIJ mixer into the rectangular opening of the rear expulsion carriage. Then, load one milliliter plastic syringes as before and mount syringes onto the female lure connectors of the CIJ mixer.Prior to running the instrument, set the desired motor speed and check that the system is clear as described in the text protocol. To expel reactants from the syringes and into the CIJ mixer, press the run button in the software interface. Representative cryogenic transmission electron micrographs demonstrate smaller and more mono-dispersed polymersomes after repeated impingements.Dynamic light scattering analysis confirmed a leftward shift of the size distribution of the polymersomes after repeated impingements. After five repeated impingements, the average diameter and polydispersity of the polymersomes match that of the extruded samples formed by thin film rehydration. Flash nanoprecipitation can be used to load a variety of hydrophobic and hydrophilic small molecules and macromolecules in polymersomes.Though this method can provide insight into how immunotherapies and vaccines function, it can also be applied to the study of how polymers self assemble into intricate nanostructures. Following this procedure, other methods like intravenous or subcutaneous administration to animal models can be performed in order to determine which cell subsets internalize the nanoparticles or what the route of clearance is for different nanostructures. The implications of this technique extend toward therapy of heart diseases and cancer as well as the development of vaccines for infectious diseases because these nanocarriers can target multiple drugs to very specific immune cells.

rapid-scalable-assembly-loading-bioactive-proteins-immunostimulants

Research

JoVE Journal

Bioengineering

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

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 ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...