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

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.