Name: Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation (Video) | JoVE Journal
Uploaded: 2018-08-11T14:00:00.000+00:00
Duration: 6 min 57 s
Description: Learn flash nanoprecipitation for scalable nanocarrier fabrication using confined impingement jets. This method enables researchers to produce sterile, low-polydispersity drug delivery systems loaded with bioactive proteins and small molecules.

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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.

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The authors declare that they have no competing financial interests.

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&quot; 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&quot; 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&quot; Bore A Diameter, 1/4&quot; Bore B Diameter, 1&quot; OD, 1-1/4&quot; 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&#039;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

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...

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 ...

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

Research

JoVE Journal

Bioengineering

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Learn flash nanoprecipitation for scalable nanocarrier fabrication using confined impingement jets. This method enables researchers to produce sterile, low-polydispersity drug delivery systems loaded with bioactive proteins and small molecules.

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation (Video) | JoVE Journal

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

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