This work was supported by funding from Optimeos Life Sciences, the National Science Foundation (CBET 1605816), the Bill and Melinda Gates Foundation (BMGF, OPP1150755), and the National Science Foundation Graduate Research Fellowship (DGE-1656466) awarded to K.D.R.

The authors have nothing to disclose.

The encapsulation of hydrophobic compounds such as vitamin E, as in step 1 of the Protocol, has been extensively described9,14,28. Relatively monodisperse particles are produced because the time scale for mixing is shorter than the time scale for the aggregation and growth of the particles. Specifically, the mixed solvent/antisolvent solution rapidly becomes homogeneous, which enables nucleation to occur uniformly. Assembly of the block copolymer to the particle surface then provides steric stabilization that halts particle growth5. Since mixing time in the chamber (turbulence) is a function of the inlet flow rates to the CIJ or the MIVM, there is an inlet rate, which occurs after the transition to turbulent mixing, where the particle size is essentially constant13. This provides additional robustness to the process as some batch-to-batch variation in inlet flowrate (i.e., syringe depression speed) can be tolerated without significant impact to the final NP size as seen from Figure 3. Slower or uneven inlet speeds can result in larger particles or more polydisperse distributions, as seen for the misfire example. FNP has also been extended to encapsulate hydrophilic compounds in nanoparticles by inverse Flash NanoPrecipitation. These inverted nanoparticles can then be used to create microparticles or be coated with PEG to create water-dispersible nanoparticles25. The underlying assembly principles remain the same, though there is the added complexity of crosslinking the particle core. This is necessary for stabilization of the particle in an aqueous environment. In general, a 1:1 charge ratio compared to the polyacid block is sufficient, though the ionic interactions can be promoted by pH adjustment through the addition of a base19. In this protocol, only the first process step to form inverted NPs has been described.
In addition to fast mixing, successful formulation by FNP or iFNP is limited to instances where several conditions can be met9,14. First, all stream inputs must be miscible. While emulsions have been used to produce NPs, FNP requires a uniform solution phase in the mixer. Second, the core component must be nearly insoluble at the solvent conditions in the mixer (for the CIJ, a 50/50 mixture by volume) to drive rapid nucleation. Otherwise, a significant portion will remain unencapsulated or will precipitate after further dilution with antisolvent. The MIVM can enable higher antisolvent content in the mixing chamber to address core material solubility limitations. It is often useful to generate supersaturation curves from solubility data as a function of solvent composition to guide process design9. Figure 6 shows representative curves for two compounds. Low supersaturation at the mixing chamber conditions merits operating at different compositions, typically using the MIVM. Higher supersaturation favors the nucleation of the core component over particle growth but a mismatch in assembly time of the core material and the stabilizer can result in large aggregates of the therapeutic. D&#39;Addio and Prud&#39;homme have reviewed the application of such supersaturation curves in detail9. Finally, the BCP must be molecularly dissolved in the solvent stream and the antisolvent stream must be selective for one block. The BCP must be sufficiently amphiphilic to provide both a solvophobic driving force from the collapsed block to anchor the stabilizer on the particle surface and for the solvated block to impart steric stability to the particle. Solvents other than those described in the protocol may be used as long as they meet these constraints.
Practice with manual syringe operation can improve the success rate during screening. As noted above, operation above the transition to homogeneous, turbulent mixing conditions means that small variations in flow rate are tolerated in the process28. Scale-up to pump-driven, computer-controlled flows results in even greater gains in consistency due to the reproducible inlet flow rates. At any point during post-processing of the particles, visual inspection or DLS analysis may indicate the presence of large aggregates which can be due to incidental dust or particle instability. When necessary, the stream can be filtered with an appropriate filter pore size. In the absence of aggregates, we have found that less than 5% mass is typically lost when filtering PEG-coated nanoparticles if the nominal filter size is larger than the particle size distribution. When filtering aggregates, experimental determination of mass lost during the process is necessary. Quantification of the mass loss can be carried out in one of two ways. The total solids mass in a given volume can be determined by thermogravimetric analysis before and after filtration to identify the extent of change (see Supplemental Information Section 2). Alternatively, the particles can be recovered (e.g.,&#160;by lyophilization) and dissolved in a good solvent. The concentration of the core material can then directly be measured by an appropriate technique such as ultraviolet-visible spectrophotometry or chromatography.
For FNP, the residual 10 vol% organic solvent (e.g., THF) must be removed from the aqueous dispersion. This can be done by evaporative distillation14,29, dialysis30, or tangential flow filtration31,32. Practical considerations for each processing step are described in the citations provided. For dialysis, typical membranes are 3.5 kDa or 6-8 kDa cutoffs, though larger options are available. This molecular weight cutoff is sufficient for solvent removal when dialyzed for 24 h using several bath changes. The use of tangential flow filtration entails some process development as care must be taken to avoid inducing aggregation due to concentration polarization at the membrane surface. We have found that reducing the organic solvent composition below a system-dependent value, usually 2-10 vol%, eliminates aggregation at the membrane surface. After processing, the concentration of nanoparticles is readily determined by thermogravimetric analysis (see Supplemental Information Section 2). It is often desirable to transport or store particles in a highly stable form. Aqueous dispersions can simply be frozen rapidly using a dry ice/acetone mixture and then stored at -80 &#176;C. Alternatively, dry powders can be obtained by lyophilization33,34 or spray drying24. Frequently, a cryoprotectant must be added to reduce nanoparticle aggregation during freezing or drying. Sugars (sucrose, trehalose, etc.), poly(ethylene glycol), or cyclodextrins can be screened for effectiveness over a range of concentrations by monitoring size by DLS35,36,37,38. Common NP stability problems during processing are often related to solubility or phase separation in the core resulting in rearrangement towards a lower energy state under conditions where mobility is increased. Use of co-core materials, alternative stabilizers, or modified external solution composition can help improve stability14,16,17,39,40,41.
As noted above, the MIVM enables higher antisolvent content in the mixing chamber when required to achieved high supersaturation. It can also allow for the physical segregation of species into more than two streams when reactivity or solubility constraints demand it. An example is the formation of zein protein-stabilized nanoparticles of the antibiotic clofazimine24. The hydrophobic clofazimine is introduced in an acetone stream; zein is introduced in a 60% ethanolic aqueous stream; casein, which complexes with zein, is brought in with an aqueous buffer stream, and the fourth stream is additional buffer to increase the ratio of water to acetone and ethanol. Two solvent streams are required since clofazimine and zein are not soluble in a common solvent. This process could not be accomplished in a two-jet CIJ mixer. This protein-stabilized formulation also demonstrates that FNP is not limited to BCP stabilizers. Janus particles have been produced without stabilizer42 and a range of low-cost stabilizers have been demonstrated for oral applications24. Notably, copolymers such as hydroxypropyl methylcellulose can be used in lieu of block copolymers24. Core materials can be made more hydrophobic by a number of techniques. Hydrophobic ion pairing has been applied to encapsulate a wide range of compounds that have intermediate solubility43,44,45. Extremely hydrophobic prodrugs have been generated and then encapsulated46. Nucleic acids have been encapsulated through complexation with cationic lipids47. Importantly, these studies have shown that FNP can produce a range of particle surface chemistries. Further, mixed stabilizers containing a fraction of BCP that has been modified with a targeting ligand on the chain end have been used. This enables precise control over ligand content on the surface since particle composition reflects the input stream composition23,48. Similarly, it is possible to incorporate multiple core components as well, including dyes and inorganic nanoparticles3,8.
Flash NanoPrecipitation is a scalable approach to polymeric nanoparticles comprised of either a hydrophobic or a hydrophilic core. If the criteria enumerated above are met, generally over 95% of the core material is encapsulated at high mass fraction in the particle. The three examples presented here were carried out at bench scale, requiring a few milligrams of material and about 0.5 mL in each inlet stream. This allows for rapid screening of particle conditions for formulation optimization. Scale-up of lead formulations to larger batch sizes is a matter of running the process for longer, which can readily be accomplished through the use of syringe pumps or flow controllers. By contrast, the scale-up of bulk addition nanoprecipitation faces well-documented challenges in maintaining sufficient micromixing at the point of addition and accounting for the effect of changing vessel geometry49. This is a major barrier, since it is crucial to manufacture particles in a consistent manner to meet FDA requirements50. Microfluidics techniques can also produce uniform, reproducible nanoparticles, but only enable production in the milligram range. For example, Karnik et al. reported production rates of 0.25 mg/min for a drug release study51. Further scale-up typically entails parallelization at high capital cost12. With FNP, it is straightforward to produce 1 gram of nanoparticles at 600 mg/min with a syringe pump and a few fittings to connect to the mixer inlets. Consequently, FNP represents both an accessible lab-scale screening tool as well as a scalable approach to NP production for translational work.

Since the late 1990s, there has been a steady increase in the number of clinical trials employing nanomaterials1,2. The rising interest reflects the promise of nanomaterials to improve the bioavailability of hydrophobic drugs and to enable preferential targeting within the body3. Polymeric nanoparticles (referred to as nanoparticles or NPs here) represent a growing proportion of this class of materials2. NPs have garnered interest because they have highly tunable properties such as size, composition, and surface functionalization4. When applied to the administration of poorly soluble drugs, NPs frequently have a core-shell structure where the therapeutic is encapsulated in the hydrophobic core and the shell consists of a hydrophilic polymer brush. A simple way to generate this structure employs an amphiphilic diblock copolymer (BCP) consisting of a degradable hydrophobic block, which forms part of the particle core, and a hydrophilic poly(ethylene glycol) (PEG) block, which forms the polymer brush and imparts steric stabilization4,5.
Nanoprecipitation is a common fabrication technique for polymeric nanoparticles because it is simple and not energy intensive6. In its simplest form, nanoprecipitation involves addition by pipette of NP components in an organic solvent like acetone to an excess volume of stirred water. The change in solvent to a dilute aqueous solution results in the precipitation of the insoluble core component. The stabilizer assembles on this growing particle surface, directed by adsorption of the collapsed hydrophobic block7,8,9,10. A uniform particle size distribution is obtained when the solvent and water rapidly mix to form a homogeneous solution. Mixing that is slower than the nucleation and assembly of the components results in a larger, more polydisperse particle population. Though readily accessible for a simple test, the stirred batch approach results in wide variability due to mixing inconsistency and is not amenable to scale-up6,11. Microfluidics have emerged as another avenue to NP production that can be run continuously. This means of production has been recently reviewed by Ding et al.11. A common approach uses laminar flow focusing to reduce the solvent length scale to sub-micron values. Mixing of the antisolvent occurs by diffusion, so small flow dimensions are crucial to ensure uniform particles11,12. Parallelization of multiple microfluidic chambers for scale-up is problematic for large production volumes.
The rapid mixing conditions that favor uniform nanoprecipitation in microfluidics alternately can be produced in confined, turbulent flows. Flash NanoPrecipitation (FNP) employs special mixing geometries to achieve these conditions under higher volumetric flowrates than possible with microfluidics. Inlet streams enter a mixing chamber under turbulent conditions that lead to the generation of vortices, so that solvent/anti-solvent lamellae form on the length scale of diffusion11,13. Thus, uniform mixing on a time scale shorter than nucleation and growth of the therapeutic is achieved. The confined geometry of the mixer does not permit stream bypassing of the region where turbulent energy dissipation occurs and the entire system experiences the same process history13. Nucleation occurs uniformly in the mixing chamber and particle growth proceeds until halted by the assembly of the BCP onto the surface9,14. The mixed stream containing stable particles may then be diluted with additional antisolvent to suppress Ostwald ripening of the particles15,16,17.
A confined impinging jet (CIJ) mixer is the simplest mixer design for FNP and permits mixing of two streams in a scalable and continuous fashion, as shown in Figure 1A13. A multi-inlet vortex mixer (MIVM) was developed to enable up to four different stream inputs while still achieving the rapid micromixing required for uniform particle formation, as shown in Figure 1B18. FNP enables simple formulation screening that can readily be translated to commercial-scale production. Due to the continuous nature of the process, larger batch sizes do not require new vessels but rather longer run times, enabling simple translation to kilogram-scale production in the same equipment train.
Hydrophilic compounds such as peptides and proteins (&#39;biologics&#39;) can also be encapsulated in a process termed inverse Flash NanoPrecipitation (iFNP). The technique requires an amphiphilic BCP where one block is hydrophobic and the other is a polyacid19. The initial step involves rapid mixing of a dimethyl sulfoxide (DMSO) stream containing the biologic and the BCP against a lipophilic solvent such as dichloromethane or chloroform. This results in the formation of particles stabilized with the hydrophobic block brush. Here, such an architecture will be termed an &#39;inverted&#39; NP. The core contains the polyacid, which is then ionically crosslinked using a multivalent cation. This stabilizes the particles for processing into an aqueous environment in the form of microparticles or PEG-coated nanoparticles by techniques that have been reported in the literature19,20,21.
This protocol can be employed for the lab-scale production of polymeric core-shell nanoparticles encapsulating either hydrophobic or hydrophilic compounds. The subsections of the protocol provide instructions on the use of both mixer classes - the CIJ and the MIVM. The reader should be able to adapt the protocol for novel core components and reproducibly generate nanoparticles of a desired size using the appropriate mixer for the stream inputs. Three example formulations using FNP and iFNP are presented below. Two employ the CIJ mixer and one requires the MIVM15,22. The first formulation demonstrates encapsulation of a model hydrophobic compound by FNP. The second formulation demonstrates encapsulation of a model hydrophilic compound by iFNP in a CIJ mixer. The final formulation provides an example of protein encapsulation by iFNP using a MIVM. The protocol for this third formulation describes the use of a small-scale, handheld MIVM termed the &#39;&#956;MIVM.&#39; The mixer design is smaller to allow for simplified formulation screening, but the scaling behavior is well understood and the mixer is not a microfluidic device22. The final section of the protocol includes some notes on scale-up of lead formulations identified in screening. These formulations are intended to provide access points for learning the process and consequently use non-degradable poly(styrene)-based polymers. Alternative stabilizers have been described in the literature, with a number of biocompatible commercial options available14,23,24.

<table>
	<tbody>
		<tr>
			<td>Confined Impinging Jets Mixer</td>
			<td>NA</td>
			<td>NA</td>
			<td>See supplemental information for engineering drawings. Review text for new mixer validation</td>
		</tr>
		<tr>
			<td>Luer fitting</td>
			<td>Idex Health &#38; Science</td>
			<td>P-604</td>
			<td>Assemble on CIJ or MIVM mixer inlet with corresponding threads</td>
		</tr>
		<tr>
			<td>Plug fitting</td>
			<td>Idex Health &#38; Science</td>
			<td>P-309</td>
			<td>Assemble on CIJ mixer sides (seal access point from drilling)</td>
		</tr>
		<tr>
			<td>Outlet fitting - CIJ</td>
			<td>Idex Health &#38; Science</td>
			<td>P-205</td>
			<td>Assemble with ferrule and tubing on CIJ chamber outlet</td>
		</tr>
		<tr>
			<td>Outlet ferrule - CIJ</td>
			<td>Idex Health &#38; Science</td>
			<td>P-200</td>
			<td>Assemble with outlet fitting (large end flush with tubing)</td>
		</tr>
		<tr>
			<td>Outlet tubing - CIJ</td>
			<td>Idex Health &#38; Science</td>
			<td>1517</td>
			<td>Use tubing cutter for clean ends. Ensure extra tubing doesn&#39;t protrodue into mixing chamber</td>
		</tr>
		<tr>
			<td>Tetrahydrofuran (THF)</td>
			<td>Fisher Scientific</td>
			<td>T425-4</td>
			<td>Use stabilizer-free THF to avoid solubility limits of BHT. Peroxides may interfere in some applications.</td>
		</tr>
		<tr>
			<td>Norm-ject syringe (3 ml)</td>
			<td>VWR</td>
			<td>53548-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin E (&#945;-tocopherol)</td>
			<td>Sigma-Aldrich</td>
			<td>90669-50G-F</td>
			<td>Store cold</td>
		</tr>
		<tr>
			<td>poly(styrene-b-ethylene glycol), PS1.6k-b-PEG5k</td>
			<td>Polymer Source</td>
			<td>P13141-SEO</td>
			<td>Other block sizes acceptable depending on application</td>
		</tr>
		<tr>
			<td>poly(styrene)1.8k</td>
			<td>Polymer Source</td>
			<td>P2275-S</td>
			<td>Example hydrophobic core material</td>
		</tr>
		<tr>
			<td>Scintillation vial</td>
			<td>DWK Lifesciences</td>
			<td>74504-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-slip plastic syringes, 1ml (100 pk)</td>
			<td>National</td>
			<td>S7510-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Maltodextrin DE 4-7</td>
			<td>Sigma-Aldrich</td>
			<td>419672-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(styrene-b-acrylic acid), PS5k-b-PAA4.8k</td>
			<td>Polymer Source</td>
			<td>P5917-SAA</td>
			<td>Other block sizes acceptable depending on application</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>D159-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihdyrate</td>
			<td>Sigma-Aldrich</td>
			<td>223506-25G</td>
			<td>Hygroscopic.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>AC423300250</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from chicken egg white (Ovalbumin, OVA)</td>
			<td>Sigma-Aldrich</td>
			<td>A5503-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Inlet Vortex Mixer</td>
			<td>NA</td>
			<td>NA</td>
			<td>See supplemental information for engineering drawings. Review text for new mixer validation</td>
		</tr>
		<tr>
			<td>Outlet fitting - MIVM</td>
			<td>Idex Health &#38; Science</td>
			<td>P-942</td>
			<td>Combination with ferrule</td>
		</tr>
		<tr>
			<td>Outlet tubing - MIVM</td>
			<td>NA</td>
			<td>NA</td>
			<td>Fit to ferrule ID.</td>
		</tr>
		<tr>
			<td>O-ring (MIVM)</td>
			<td>C.E. Conover</td>
			<td>MM1.5 35.50 V75</td>
			<td>Order bulk - consumable part. Ensure solvent compatibility if using an alternative source.</td>
		</tr>
		<tr>
			<td>Mixer stand</td>
			<td>NA</td>
			<td>NA</td>
			<td>See Markwalter &#38; Prud&#39;homme for design.17</td>
		</tr>
	</tbody>
</table>

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%.

Screening of NP formulations with FNP is rapid and requires small quantities of material (on the order of 1-10 mg). The FNP protocol to encapsulate hydrophobic compounds such as vitamin E (step 1) results in a stable, clear or lightly opalescent NP dispersion. Dynamic light scattering (DLS) provides a robust means to characterize the particle size. As shown in Figure 3, the process produces NPs with a low polydispersity in a reproducible fashion. The typical polydispersity index (PDI) is less than 0.20, indicating a relatively monodisperse population. The PDI is obtained from the autocorrelation function and is often implemented into instrument software. It is a ratio of the second to the first moment, where values of 0.1 are generally obtained for monodisperse particles26. For the four vitamin E/PS-b-PEG formulation replicates reported, the value was 0.12 &#177; 0.02 and the average diameter was 107 &#177; 7 nm. A typical &#34;misfire&#34; due to either uneven depression of the syringes or slower depression speed is also reported in Figure 3. The polydispersity was unaffected, but the size was slightly larger (135 nm). Including this sample, the new metrics for particle size are 113 &#177; 14 nm. A misfire results in periods of time where the chamber contains only a single stream type. It is important that the entire stream experiences the same process history and relative volumes of the organic and aqueous streams within the mixer. Without a stabilizer, an opaque solution with visible aggregates is produced. The DLS autocorrelation function for this sample is non-monotonic and does not decay smoothly, as seen in the Figure 3 inset.
Particle size control by FNP is demonstrated in Figure 4, where varying the relative amounts of core material &#8211; poly(styrene)1.8k in this case &#8211; and PS-b-PEG stabilizer resulted in particles sizes that ranged from 49-152 nm. These particle sizes were generated with THF streams containing a total mass concentration of core and stabilizer of 20 mg/mL, where 25%, 50%, or 75% of the mass was the poly(styrene) core material. The polydispersity of the nanoparticles was always less than 0.15. Extensive discussion of parameter effects on particle size produced by FNP may be found in the literature10. The loading can be tuned by holding the solvent volume constant and varying the relative volumes of the core and stabilizer stock solutions. Similarly, the total mass concentration can be varied by preparing stock solutions at values other than 10 mg/mL. Under certain conditions, it is possible to observe an empty micelle population by DLS27. This does not have any detrimental effect other than broadening the measured particle size distribution. When the sizes are similar, this may manifest as a single broad peak rather than two separate peaks.
The same CIJ mixer can also be used to encapsulate hydrophilic compounds by iFNP, as exemplified in step 2 of the Protocol. The particles produced in the reported formulation are around 65 nm with a low polydispersity of 0.08. The size distribution can be seen in Figure 5A (dashed lines). The effect of crosslinking the PAA carboxylic acid residues on particle stability is demonstrated by DLS analysis in a strong solvent such as DMSO, as shown in Figure 5B. The autocorrelation function for well-crosslinked particles should start near a value of 1 and drop off sharply to 0 at a characteristic time that is related to the particle size (solid line). Particles that swell extensively or dissolve are not crosslinked and show minimal autocorrelation signal (dotted line). For iFNP, failed trials manifest in similar ways as described for FNP above. Visible aggregates may be seen or poor DLS autocorrelation function shape may be observed. The MIVM can be used for FNP or iFNP when more than two inlet streams are required due to system constraints such as solubility or chemical incompatibility. A small-scale version of the MIVM (the &#956;MIVM) with its mixer stand is shown in Figure 2. As with the CIJ, this mixer can be used to encapsulate hydrophobic or hydrophilic compounds22. In step 3, a protocol for the encapsulation of a hydrophilic protein, OVA, by iFNP was described. The particle size distribution is shown in Figure 5A (solid line). The size is around 125 nm with a PDI of 0.16. A general protocol for syringe pump operation at larger scales is provided in step 4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58757/58757fig1.jpg" /> 
Figure 1: Mixer assembly and internal flow pattern schematics. (A) The confined impinging jets (CIJ) mixer with attached syringes is positioned above the quench bath. Not pictured are a stir bar in the quench bath vial and a stir plate. The mixing geometry is depicted in the expanded view showing the two stream inlets that impinge in the center of the chamber. (B) A multi-inlet vortex mixer (the &#956;MIVM) is shown with glass syringes and positioned in the stand above a quench bath. The mobile plate and the mechanical stops have been cropped from the picture. The expanded view shows the vortex chamber and the inlet channels schematically. (C) A schematic representation of core-shell NPs produced by FNP. Red spheres represent the therapeutic which, combined with the blue collapsed polymer block, comprise the NP core. The yellow polymer block forms the brush layer imparting steric stabilization to the NPs. <a href="https://www.jove.com/files/ftp_upload/58757/58757fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58757/58757fig2.jpg" /> 
Figure 2: &#956;MIVM terminology and components for assembly. The &#956;MIVM requires a mixer stand to enable uniform depression of the four syringes. In this case, the syringe plunger heights must all be uniform to ensure consistent mixing. It can alternatively be operated using syringe pumps. The mixer stand with labeled components is shown at left of the figure. On the right is the disassembled mixer with the O-ring in place on the mixing geometry disk. <a href="https://www.jove.com/files/ftp_upload/58757/58757fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58757/58757fig3.jpg" /> 
Figure 3: Particle size distribution of polymeric nanoparticles containing a core of vitamin E and stabilized by PS-b-PEG. Dynamic light scattering (DLS) provides intensity-weighted size distributions that indicate the NP diameter distribution. Curves are the average of triplicate analyses for each trial and have been rescaled to produce identical maximum peak heights. The four replicates (solid lines) indicate the high reproducibility of the method (standard deviation = 7 nm). Also included is a representative misfire (dashed line), such as slower syringe speed or uneven depression of the two syringes, which results in larger particle diameter. The standard deviation of the NP size including the misfire was 14 nm. (Inset) Without the PS-b-PEG stabilizer, large micron-scale aggregates (or droplets, in the case of an oil like vitamin E) are formed. The DLS autocorrelation function of a run without the stabilizer (dotted line) is shown along with a representative autocorrelation from a nanoparticle replicate (solid line). The autocorrelation function shows a number of characteristic timescales for the control sample, indicating a polydisperse population. <a href="https://www.jove.com/files/ftp_upload/58757/58757fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58757/58757fig4.jpg" /> 
Figure 4: Particle size control by FNP through varying relative ratios of core material to stabilizer. The intensity-weighted size distributions of three formulations with a poly(styrene) core stabilized by PS-b-PEG are depicted. The total mass concentration in THF was 20 mg/mL and the antisolvent was water. The formulations were prepared in a CIJ mixer. The fraction of the mass composed of the core material is listed in the legend. For example, the 25% core sample contained 5 mg/mL poly(styrene) and 15 mg/mL PS-b-PEG. The average sizes for the 25% (solid line), 50% (dashed line), and 75% (mixed dash line) core loadings were 49 nm, 96 nm, and 152 nm, respectively. All PDI values were less than 0.15. <a href="https://www.jove.com/files/ftp_upload/58757/58757fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58757/58757fig5.jpg" /> 
Figure 5: Characterization of inverted NPs made in a CIJ mixer or &#956;MIVM. (A) DLS curves are the average of triplicate analyses for each formulation. The dashed line indicates the size distribution of 3k MD particles made in the CIJ mixer while the solid line is the size distribution of OVA particles made in the &#956;MIVM. (B) The strength of crosslinking can be assessed by DLS using DMSO as the diluent. The DLS autocorrelation function indicates the strength of crosslinking through the initial autocorrelation value and the observation of a clean transition to a value of zero. The dashed line depicts the autocorrelation function for a particle with no crosslinker showing a weak initial signal and a broad decay time. The solid line depicts the autocorrelation after addition of a strong crosslinker (in this case, tetraethylenepentamine), which shows a strong initial signal and a defined decay timescale. <a href="https://www.jove.com/files/ftp_upload/58757/58757fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58757/58757fig6.jpg" /> 
Figure 6: Supersaturation, S, as a function of the relative mixing ratios of organic solvent to water. (A) Comparison of highest attainable supersaturation for (&#9675;) boscalid, a pesticide, and (&#9632;) peptide B, a seven-residue model peptide. The organic stream contains boscalid at a concentration of 230 mg/mL and peptide B at 200 mg/mL, their saturation concentrations. There is a maximum supersaturation that depends on each active pharmaceutical ingredient (API)/solvent system. (B) When the concentration of boscalid in the organic stream is decreased 20-fold, the conditions at which supersaturation and nanoprecipitation are achieved become limited. This figure is reprinted with permission from Elsevier9. <a href="https://www.jove.com/files/ftp_upload/58757/58757fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Files.&#160;<a href="https://www.jove.com/files/ftp_upload/58757/Supplemental_Files.zip" target="_blank">Please click here to download the files.</a>

Watch this Scientific Journal Video about Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles at JoVE.com

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

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 ...

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Bioengineering

21.8K Views.  Princeton University. 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.

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

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 ...

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...