The flash nanoprecipitation, or FNP techniques demonstrated here offer a scalable and straightforward platform for encapsulating hydrophobic or hydrophilic compounds inside polymeric nanoparticles. For clarity, the technique used to encapsulate biologics is called inverse flash nanoprecipitation, or IFNP. The underlying principles are unchanged, but the resulting nanoparticle structure is different.
Generally, new users need practice to consistently operate the inlet syringes when manually making small nanoparticle batches. Larger volumes can be produced with computer controlled syringe pumps. Our protocol also provides details regarding post processing of nanoparticle solutions, which is often critical to successful application of a formulation.
Before starting the process, check the CIJ mixer fittings, and ensure that the outlet tubing is not crimped. Then, fill two five milliliter polypropylene rubber free Luer lock syringes with two to three milliliters of acetone, or another cleaning solvent. Lock the syringes into the inlet adapters, and set the assembly over a waste container.
Steadily depress the plungers to send the solvent through the mixing chamber over the course of a few seconds. Then, remove the syringes, and dry the mixture with a stream of nitrogen gas. Next, to begin preparing the solvent input stream, pipette 0.25 milliliters of a 10 milligrams per milliliter solution of vitamin E, in stabilizer free tetrahydrofuran, into a 1.5 milliliter microcentrifuge tube.
Then, pipette 0.25 milliliters of a 10 milligrams per milliliter solution of a block copolymer stabilizer in THF into the same tube. Vortex the mixture for five to 10 seconds, and then centrifuge it at one thousand G for five to 10 seconds to recover liquid adhering to the cap. Prepare a 1.5 milliliter centrifuge tube containing 0.525 milliliters of deionized water as the anti-solvent.
Then, pipette four milliliters of deionized water into a 20 milliliter scintillation vial to make the quench bath. Place a small stir bar in the vial. Place the clean CIJ mixer over the quench bath in a rack or test tube block on a stir plate.
Start stirring the quench bath at about 75%of the maximum possible speed. Then, fit a blunt tipped needle to a one milliliter polypropylene rubber free syringe, and draw up the anti-solvent. Carefully expel air bubbles from the syringe, and then remove and dispose of the needle.
Adjust the plunger so that the liquid comes just to the end of the syringe. Then, attach the syringe to one of the CIJ inlets. Draw the solvent mixture into a second syringe in the same way, and attach it to the other inlet.
To form the nanoparticles, simultaneously depress both plungers with a smooth uniform motion in less than 0.5 seconds. It is critical to depress the syringes rapidly, evenly, and smoothly. You should not suddenly strike the syringes, but should begin the motion already in contact with the tops of the syringes.
Afterwards, set the CIJ mixer over the waste container, without removing the syringes to ensure that the holdup volume does not drain into the dispersion. Remove the stir bar from the scintillation vial, and cap it. Then, remove and discard the solvent and anti-solvent syringes.
Clean the mixer before the next flash nanoprecipitation trial. To prepare a sample for dynamic light scattering analysis, pipette 100 microliters of the dispersion into a cuvette. Add 900 microliters of quench bath solvent, and mix well by pipetting before starting the sample analysis.
To begin assembling the micro MIVM, place the O-ring in the mixing geometry disc. Align the holes in the mixing disc with the pegs on the top disc, and fit them together, being careful not to displace the O-ring. Loosen the outlet tubing fitting in the bottom receiver, and then screw the connected discs into the receiver.
Fit a spanner wrench to the pegs of the top disc, and tighten the assembly. Tighten the outlet tubing fitting so that it sits firmly against the bottom face of the mixing geometry disc. Ensure that the syringe fittings on the top disc are snug.
Raise the mobile plate on the mixer stand to keep it out of the way, then place the assembled mixer on the stand, with the outlet tubing threaded through the support plate. Next, place a 15 milliliter centrifuge tube containing 5.25 milliliters of chloroform as the quench bath under the outlet tubing. Then, draw 0.75 milliliters of solution A, which is a five milligram per milliliter solution of ovalbumin in dimethyl sulfoxide in 10%water by volume, into a one milliliter gas tight Luer lock syringe with a blunt tipped needle.
Carefully expel air bubbles, remove the needle, and prime the solution to the end of the Luer fitting. Connect the syringe to a mixer inlet. Repeat this process with 0.75 milliliters each of solution B, which is six milligrams per milliliter of block copolymer stabilizer in DMSO, and solution C, which is THF.
Connect the syringes to the mixer inlets clockwise, in alphabetical order. Prepare a 2.5 milliliter gas tight syringe containing 1.85 milliliters of solution D, which is chloroform, and connect it to the fourth inlet. Confirm that there is no significant difference in the syringe heights.
Then, carefully grip the bearing housing on each side of the mobile plate, and slowly lower the plate until it is just barely resting evenly on top of the syringes. To generate the nanoparticles, steadily and smoothly depress the plate in about 0.5 to one second. Then, remove and cap the quench bath tube.
Disassemble and clean the mixer when finished. To work with larger volumes, load the solutions into gas tight syringes, and connect polytetrafluoroethylene tubing with Luer fittings to the syringes. Prime the solutions to the ends of the tubing.
Clamp the syringes into syringe pumps, and attach the tubing to the appropriate mixer inlets. Place a small waste vial under the outlet tubing for collecting the startup volume. Prepare the quench bath and keep it nearby.
Simultaneously, start the pumps, and let about five milliliters of effluent flow into the waste vial. Then, start collecting the nanoparticles in the quench bath as usual. A stir plate may be used to mix the quench bath if desired.
Four replicates of FNP of polymeric nanoparticles with a hydrophobic core, prepared in the CIJ mixer, showed high replicability, and were relatively monodisperse, with an average diameter of 107 nanometers. A representative misfire from slow or uneven syringe depression, produced slightly larger particles, while polydispersity was unaffected in this example, misfires can result in more polydisperse distributions. The DLS auto-correlation function decayed smoothly for a representative polymeric nanoparticle sample.
This smooth decay was not observed when formulation was attempted without the block copolymer stabilizer, which produced micron scale oil droplets instead. The relative amount of core material to stabilizer controlled the particle size, as shown here, in nanoparticles with a polystyrene core. The PDI was below 0.15 in each formulation.
Nanoparticles with hydrophilic cores where produced by IFNP. Particles with a maltodextrin core, prepared in the CIJ mixer, were about 65 nanometers in diameter, and had a PDI of 0.08. Particles with an ovalbumin core prepared in the micro MIVM were about 125 nanometers in diameter, and had a PDI of 0.16.
FNP and IFNP are powerful tools for processing molecules and the nanoparticles. Before performing either technique, be sure to consider the solubilities of each component in all solvents, or solvent mixtures, to ensure high super saturation during mixing. The basic technique is straightforward, but mastering the syringe depression steps takes some practice.
If you have trouble with this, consider using a syringe pump setup like the one shown in this video to improve consistency. For charged hydrophilic molecules, or molecules with intermediate solubility, FNP may be combined with hydrophobic ion pairing to enable efficient encapsulation. FNP can also be used to encapsulate multiple compounds in the same nanoparticle core.
Consult the text and other literature resources to learn how to best remove organic solvents from the nanoparticle dispersions in your desired application. There are also a range of techniques for stabilizing nanoparticles during storage.
