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 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.
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.
1. Synthesis of Poly(ethylene glycol)-block-poly(propylene sulfide)-Thiol
2. Assemble PEG-bl-PPS Nanocarriers via Hand-Powered Flash Nanoprecipitation
3. Characterize Nanocarrier Formulations
4. Fabrication of a high-speed syringe pump for FNP
5. Fabricate Polymersomes via FNP Using the Custom-Made High-Speed Syringe Pump
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 impingement of solutions in the CIJ mixer. Figure 1 provides an overview of the synthesis steps performed to produce PEG17–bl-PPS35-SH, the diblock copolymer used to self-assemble polymersome nanocarriers. An overview of the FNP protocol for assembling PEG-bl-PPS polymersomes loaded with therapeutics and/or imaging agents is diagramed in Figure 2. The polymer was impinged in a CIJ mixer (schematic shown in Figure 3a, originally described in 10) to form monodisperse polymersomes as the aggregate morphology, which can be validated by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryoTEM) (Figure 3b-3c). Polymersomes formed by FNP become smaller (Figure 3d) and more monodisperse (Figure 3e) with subsequent impingements, and may be loaded with hydrophilic and hydrophobic cargo (e.g., DiD lipophilic dye, small molecule therapeutics, protein etc.; Figure 4a). Nanocarriers formed under the sterile conditions described above are endotoxin free by both RAW Blue and LAL endotoxin assays and thus suitable for a wide range of in vitro and in vivo applications (Figure 4b, data not shown).
Lastly, we have designed and constructed an instrument to mechanically-control the flow rate and resulting impingement of solutions in the CIJ mixer (Figure 5). The creation of this instrument is essential, as commercially-available syringe pumps cannot achieve the flow rates needed for FNP. With the exception of custom modifications, commercially available syringe pumps have speed limitations imposed by their use of low-speed stepper motors, which are designed to reliably dispense fluid in a slow and steady fashion. In our instrument, reactant expulsion is controlled by a precision slide under the control of a 24 V brushed DC motor, which can achieve much greater speeds (4,252 rpm) than the slow stepper motors found in commercial syringe pumps. Custom software running on a single board computer is used to operate the instrument (Figure 6). 2D drawings have been provided in addition to 3D models of the parts. All drawings and models were created in FreeCAD (open-source parametric 3D CAD modeling software) to ensure that they are highly accessible to the research community. The software for operating the instrument was written in Python 2.7.12, allowing for the rapid development of custom FNP procedures to ensure the congruous production of nanocarriers (size, morphology, etc.). Software for operating the instrument will be made available upon request. Users should note that the software is not currently compatible with Python 3; however, this may change in future updates. By controlling reactant expulsion rate, this instrument eliminates the variable of human error from hand-operation.
Figure 1. Synthesis schema for the synthesis of PEG17-bl-PPS36-SH. Please click here to view a larger version of this figure.
Figure 2. Production of polymersomes via FNP in a hand-driven CIJ mixer. Diagram of the formation of polymersomes using FNP. The PEG-bl-PPS polymer is dissolved in organic solvent along with hydrophobic cargo, and is impinged against aqueous solvent with dissolved hydrophilic cargo. Rapid mixing occurs within the CIJ mixer, and efflux can be repeatedly impinged or allowed to complete the formation process through dilution in a reservoir of aqueous solvent. Please click here to view a larger version of this figure.
Figure 3. Characterization of polymersomes formed by FNP. (a) Design schematic of the CIJ mixer used in this study. All measurements are in millimeters. (b) Size distribution of polymersomes formed by FNP after 1 and 5 impingements, as measured by DLS. n = 6 formulations, mean of samples are graphed. (c) Example cryoTEM images of polymersomes formed after 1 and 5 impingements through the CIJ mixer, scale bar = 100 nm. Diameter (d) and polydispersity index (e) of polymersomes formed by FNP, measured by DLS. For comparison, polymersomes formed by thin film rehydration, with (TF-E) or without (TF-NE) subsequent extrusion, and formed by solvent dispersion (SD) were also measured, n=3, error bars represent standard deviation. Subfigures (c)–(e) taken with permission from Allen et al.11. Please click here to view a larger version of this figure.
Figure 4. Loading efficiency and endotoxin characterization. (a) Loading efficiency of small and macromolecules within polymersomes, n=3, error bars represent standard deviation. (b) RAW Blue LPS assay of polymersomes formed by sterile FNP, n=6, error bars represent standard deviation. Please click here to view a larger version of this figure.
Figure 5. Instrument for the mechanical control of solution impingement in the CIJ mixer. (a) 24 V brushed DC Motor. (b) Power supply (24 V, 2.5 A). (c) 4.5" stroke precision slide with 1.27 mm screw lead (connected to motor shaft by a screw beam coupling). (d) Expulsion platform constructed from rectangular metal plates and L-shaped corner braces. (e) CIJ mixer. (f) Expulsion carriage. (g) single board computer and 7" touchscreen. (h) mMotor control board encased in plastic housing (83 mm x 53 mm x 35 mm). (i) IR sensors (non-contact break-beam motion sensors). (j) Emergency stop button (NC). Please click here to view a larger version of this figure.
Figure 6. Core wiring diagram. The primary connections between the single board computer, motor controller, and IR sensors are displayed. The LCD touchscreen connections are not displayed here, as this component is non-essential (users may opt to use a standard computer monitor and mouse instead). Note that in the displayed configuration, the 24 V motor power supply and single board computer power supply are separate. Please click here to view a larger version of this figure.
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 need for additional extrusion steps during the fabrication of monodisperse polymersome nanocarriers. Polymersomes formed via FNP load both hydrophilic and hydrophobic cargo, and maintain the bioactivity of those molecules through the formulation process11. Additional protocols are described to ensure sterility when necessary, allowing the formation of polymersome formulations that are endotoxin-free and therefore suitable for biochemical and immunological assays as well as safe for administration in vivo. The hand-operated CIJ mixer is simple to set up and provides ease-of-use to the user, but introduces potential quality control issues due to user variability. To maintain flow consistency, we sought to create an instrument capable of achieving and reproducibly maintaining a comparable flow rate. Importantly, at the above specified channel dimensions, commercial syringe pumps cannot achieve sufficiently high flow rates (~1 mL/s) due to being equipped with low speed stepper motors. To combat this issue, and to afford greater control over the flow rate, fabrication of a high-speed syringe pump for FNP was described. Care was taken to utilize open source and easily customizable software for the system OS and code.
Control over alternative flow rates offers the potential to fine-tune the nanocarrier formulation and provides opportunities to further explore the assembly of diverse nanocarrier morphologies. The Reynolds number and corresponding mixing time was previously shown to impact the size of solid-core nanocarriers formed via FNP9, but it is not clear what impact it would have on the formation of polymersomes. This is a topic of current investigation, with the current recommended rate being 0.5 to 2 mL/s, with the representative results performed at approximately 1 mL/s. To increase control over flow rate even further, it may be necessary to replace the Linux-based OS with real-time control over the syringe pump motor.
Aside from adjusting the flow rate, there are a number of ways this FNP protocol can be modified to suite specific needs or applications. Smaller or larger amounts of polymer may be used. Concentrations as low as 1 mg/mL and as high as 100 mg/mL have been used to form stable nanocarriers. Larger volumes may be used for impingement, although consistent application of pressure during hand-driven FNP is more difficult at volumes greater than 1 mL per syringe. The volume of the reservoir may also be modified. Final organic:aqueous solvent ratios of greater than 1:3 may result in the incomplete formation of nanocarriers, and as such care should be taken to not decrease the volume of the reservoir without confirming the formation of nanocarriers. Aggregation may occur when attempting to load high concentrations of hydrophobic cargo, which can generally be alleviated by increasing the molar ratio of polymer:cargo.
An additional topic open for exploration is the further expansion of FNP polymersome formation to include other polymer systems beyond PEG-bl-PPS. Indeed, other systems have been used previously in the formation of micelles and solid-core drug nanocarriers16,17. However, it is not clear if there is a set of parameters that can lead to the formation of polymersomes via FNP using those other polymer systems. Given the number of potential variables to explore, it is entirely possible that other polymers can form polymersomes or other soft nanoarchitectures via FNP with adjusted experimental parameters, such as flow rate, temperature, solvent selection and polymer concentration.
As with all formulation techniques, there are limitations to FNP and restrictions that may make certain applications untenable. The rapid mixing process requires that the organic and aqueous solvents be miscible, which precludes the use of some common solvents used for the dissolution of many diblock copolymers, e.g., dichloromethane and chloroform. Some polymers may therefore be rendered incompatible with FNP if they are not able to be dissolved in a water-miscible organic solvent. The FNP protocol described here utilizes a 1:1 ratio of organic to aqueous solvent, which may decrease the activity of payloads sensitive to high concentrations of organic solvent, such as some bioactive proteins. It should be noted that influences on bioactivity will depend on the protein, as we have previously found minimal effects on the enzymatic activity of alkaline phosphatase following loading within polymersomes by FNP11. Multi-inlet vortex mixers18 are a more expensive but more customizable FNP platform that provides additional control over the ratio of organic to aqueous solvents, offering a versatile alternative to CIJ mixers for these contexts.
The authors have nothing to disclose.
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'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.
CanaKit Raspberry Pi 3 Ultimate Starter Kit – 32 GB Edition | CanaKit | UPC 682710991511 | |
Linear Bearing Platform (Small) – 8mm Diameter | Adafruit | 1179 | |
Linear Motion 8 mm Shaft, 330 mm Length, Chrome Plated, Case Hardened, Metric | VXB | kit11868 | |
Linear Rail Shaft Guide/Support – 8 mm Diameter | Adafruit | 1182 | |
Manual-Position Precision Slide 4.5" Stroke, 15 lb load capacity | McMaster-Carr | 5236A16 | |
MTPM-P10-1JK43 Iron Horse DC motor | Iron Horse | MTPM-P10-1JK43 | |
Official Raspberry Pi Foundation 7" Touchscreen LCD Display | Raspberry Pi | B0153R2A9I (ASIN) | |
PicoBorg Reverse – Advanced motor control for Raspberry Pi | PiBorg | BURN-0011 | |
Pololu Carrier with Sharp GP2Y0D810Z0F Digital Distance Sensor 10cm | Pololu | 1134 | |
Ruland PSR16-5-4-A Set Screw Beam Coupling, Polished Aluminum, Inch, 5/16" Bore A Diameter, 1/4" Bore B Diameter, 1" OD, 1-1/4" Length, 44 lb-in Nominal Torque | Ruland | PSR16-5-4-A | |
Polyethylene glycol monomethyl ether | Sigma Aldrich | 202495 | |
Methanesulfonyl chloride | Sigma Aldrich | 471259 | |
Toluene | Sigma Aldrich | 179418 | |
Toluene, Anhydrous | Sigma Aldrich | 244511 | |
Triethylamine | Sigma Aldrich | T0886 | |
Celite 545 (Diatomaceous Earth) | Sigma Aldrich | 419931 | |
Dichloromethane | Sigma Aldrich | 320269 | |
Diethyl ether | Sigma Aldrich | 296082 | |
N,N-Dimethylformamide, anhydrous | Sigma Aldrich | 227056 | |
Potassium carbonate | Sigma Aldrich | 791776 | |
Thioacetic acid | Sigma Aldrich | T30805 | |
Tetrahydrofuran | Sigma Aldrich | 360589 | |
Aluminum oxide, neutral, activated, Brockmann I | Sigma Aldrich | 199974 | |
Sodium methoxide solution, 0.5 M in methanol | Sigma Aldrich | 403067 | |
Propylene sulfide | Sigma Aldrich | P53209 | |
Acetic acid | Sigma Aldrich | A6283 | |
Methanol | Sigma Aldrich | 320390 | |
Sodium hydroxide solution 1.0 N | Sigma Aldrich | S2770 | |
Endotoxin-free water | GE Healthcare Life Sciences | SH30529.01 | |
Paper pH strips | Fisher Scientific | 13-640-508 | |
Endotoxin-free Dulbecco's PBS | Sigma Aldrich | TMS-012 | |
Borosilicate glass scintillation vials | Fisher Scientific | 03-337-4 | |
1 mL all-plastic syringe | Thermo Scientific | S75101 | |
Sepharose CL-6B | Sigma Aldrich | CL6B200 | |
Liquid chromatography column | Sigma Aldrich | C4169 | |
CIJ mixer, HDPE | Custom | ||
Triton X-100 | Sigma Aldrich | X100 | |
Hydrogen peroxide solution | Sigma Aldrich | 216763 | |
HEK-Blue hTLR4 | InvivoGen | hkb-htlr4 | |
RAW-Blue Cells | InvivoGen | raw-sp | |
QUANTI-Blue | InvivoGen | rep-qb1 | |
PYROGENT Gel Clot LAL Assays | Lonza | N183-125 |