Composite nanoparticles and methods of making using sequential nanoprecipitation

Abstract

A process for forming composite nanoparticles includes (a) mixing a first liquid with water, the first liquid comprising nanocrystal, hydrophobic organic molecule, or a combination thereof, and an organic solvent, forming particle core comprising the nanocrystal, hydrophobic organic molecule, or a combination thereof suspended in the water; and (b) mixing the particle core suspended in water with a second liquid, the second liquid comprising a stabilizer and the organic solvent, stabilizing the particle core comprising the nanocrystal, hydrophobic organic molecule, or a combination thereof with the stabilizer, and forming the composite nanoparticles. A time delay between steps (a) and (b) is about 2 millisecond (ms) to about 1 second.

Description

COMPOSITE NANOPARTICLES AND METHODS OF MAKING USING SEQUENTIAL NANOPRECIPITATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 712,295 filed October 25, 2024, which is incorporated by reference in its entirety for any and all purposes.FIELD

[0002] The present technology is generally related to methods of forming composite nanoparticles comprising nanocrystals, polymers, small molecules, peptides, or a combination of any two or more thereof.BACKGROUND

[0003] Drug delivery systems that use polymeric nanoparticles (NP) offer many advantages, such as sustained and / or triggerable drug release, improved drug bioavailability, and reduced off-target effects. Theranostic NPs, in which a single carrier can provide both diagnostic insight and deliver therapeutic payloads, have broad applications in the lab and the clinic. Bioimaging NPs, in which a single carrier provides contrast for bioimaging, has broad applications in the lab and clinic. Combining the versatility of polymeric NPs with potent bioimaging capabilities has rapidly advanced the level of precision in nanomedicine research.SUMMARY

[0004] Current manufacturing processes for the formation of inorganic-organic composite nanoparticles (CNPs) for drug delivery, theranostics, or bioimaging have significant limitations including a lack of scalability, lack of control over CNP properties, and the generation of poor quality heterogeneous CNPs that may include additional purification steps. These limitations can be overcome by using the sequential nanoprecipitation (SNaP) process. By controlling the order of addition of components via SNaP, remarkable improvements in CNP size control and uniformity may be achieved.

[0005] In an aspect, a method of forming composite nanoparticles comprises (a) mixing a first liquid with water, the first liquid comprising nanocrystal, hydrophobic organic molecule,or a combination thereof and an organic solvent, forming particle core comprising the nanocrystal, hydrophobic organic molecule, or a combination thereof suspended in the water; and (b) mixing the particle core suspended in water with a second liquid, the second liquid comprising a stabilizer and the organic solvent, stabilizing the particle core comprising the nanocrystal, hydrophobic organic molecule, or a combination thereof with the stabilizer, and forming the composite nanoparticles; wherein a time delay between steps (a) and (b) is about 2 millisecond (ms) to about 1 second.

[0006] The first liquid may further include a co-core excipient. The co-core excipient may include a hydrophobic polymer with a molecular weight of about 1 kDa to about 500 kDa. The core component may include an organic molecule having a molecule weight less than 1000 Daltons. The organic molecule may include a therapeutic molecule, a fluorescent molecule, an excipient, a prodrug, a small peptide, a counter-ion for a hydrophobic ion pairing formulation, or a combination of two or more thereof. The second liquid may include a cocore excipient. The co-core excipient may include a hydrophobic polymer with a molecular weight of about 1 kDa to about 500 kDa. The second liquid may include an organic molecule having a molecule weight less than 1000 Daltons.

[0007] The organic molecule may include a therapeutic molecule, a fluorescent molecule, an excipient, a prodrug, a small peptide, a counter-ion for a hydrophobic ion pairing formulation, or a combination of two or more thereof. The time delay between steps (a) and (b) may be about 2 ms to about 100 ms. The time delay between steps (a) and (b) may be about 2 ms to about 10 ms.

[0008] The nanocrystal may include gold, silver, copper, cobalt ferrite, manganese oxide, zinc oxide, cerium oxide, or a combination of any two or more thereof. The nanocrystal may include a first nanocrystal and a second nanocrystal different from the first nanocrystal, each of the first nanocrystal and the second nanocrystal comprising quantum dots, gold nanocrystals, iron oxide nanocrystals, or a combination of any two or more thereof.

[0009] The stabilizer may include an amphiphilic block copolymer, a surfactant, a lipid, or a combination of any two or more thereof. The composite nanoparticles may have a diameter of about 50 nm to about 15 pm. The composite nanoparticles may include about 5 wt.% to about 75 wt.% core component and the core component may include nanocrystals.

[0010] A combined concentration of solids in the first liquid and the second liquid may be about 5 mg / mL to about 200 mg / mL. The organic solvent may include tetrahydrofuran, dimethyl sulfoxide, 1,4-di oxane, ethanol, methanol, isopropanol, acetone, acetonitrile, dimethyl formamide, or a combination of two or more thereof. The core component may include quantum dots, and the method further comprising purifying the composite nanoparticles using serial fractionation via centrifugation.

[0011] In another aspect, a method of forming composite nanoparticles includes (a) introducing a first liquid into water via confined impinging jet, vortex mixing, or a combination thereof, the first liquid comprising iron oxide nanocrystals, poly(D,L-lactide), an organic molecule having a molecule weight less than 1000 Daltons, and a polar organic solvent, forming particle cores suspended in the water, the particle cores comprising nanocrystals, the poly(D,L-lactide), and the organic molecule; and (b) introducing a second liquid into the water comprising the particle cores via confined impinging jet, vortex mixing, or a combination thereof, the second liquid comprising polyethylene oxide)-block-poly(D,L lactide) and the polar organic solvent, stabilizing the particle core with poly(ethylene oxide)-block-poly(D,L lactide), and forming the composite nanoparticles. A time delay between steps (a) and (b) is about 2 ms to about 1000 ms.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1A is Schematic of SNaP IONC10-CNP formulation schemes: 1) PLA / R->NC+BCP, 2) NC^PLA / R+BCP, 3) NC+PLA / R^BCP. Schemes had the same formulation parameters: 15 mg / mL total solids content, 5% NC loading, and 2:1 ratio of stabilizer to core (33% core).

[0013] FIGS. 1B-1D are graphs of normalized, intensity-weighted size distributions of original, magnetic, and nonmagnetic fractions of each respective formulation, determined by DLS.

[0014] FIG. IE is a graph of peak emission intensity of rubrene in respective fractions, determined by fluorescence spectroscopy (lex = 492 nm).

[0015] FIG. IF is a graph of weight fraction of inorganic content of respective fractions, determined by TGA.

[0016] FIG. 2A is a schematic of IONCIO-CNPS with 5% loading synthesized using a 4-4 SNaP mixer with PLA in the first (scheme 3) or second (scheme 2) mixing stage compared with using a CI J (FNP).

[0017] FIG. 2B shows graphs of effects on CNP size of varying total solids content with constant 5% loading and 33% core. Arrows designate samples highlighted for further characterization.

[0018] FIG. 2C is a graph weight percent inorganic content of respective fractions determined by TGA. The attempted loading was 5 wt.%.

[0019] FIG. 2D is a graph of rubrene peak emission intensity of respective fractions determined by fluorescence spectroscopy, (kx = 492 nm).

[0020] FIGS. 3A-3D: Coloaded CNPs formulated with QD5 and IONC15 (m-qNPs). FIG.3 A is a schematic showing m-qNPs synthesized via SNaP. FIG. 3B is a graph of normalized, intensity-weighted size distributions of m-qNP fractions. FIG. 3C is a graph of relative emission intensity of QD5 in magnetic, nonmagnetic, and original fractions (kx = 405 nm). FIG. 3D shows representative negatively stained TEM images of individual m-qNPs.

[0021] FIG. 4A is a graph of modeled change in core component number density over time for standard CNP formulations with IONC5, IONC10, and IONC20 following classical diffusion-limited aggregation.

[0022] FIG. 4B is a graph of change in the growing core collision timescale (T) over time. The pink bar in both plots represents the estimated timescale in which the BCP micelle induction occurs. Tcross is the time at which the PLA trace intersects the other core component traces.

[0023] FIG. 5 is a schematic of the SNaP process.

[0024] FIGS. 6A-6C provide an image analysis method for characterizing MPs. FIG. 6A provides raw SEM images and zoomed views. FIG. 6B provides SEM images with detected circles outlined in green. FIG. 6C provides size distribution of pooled diameter data from several images of a MP batch.

[0025] FIGS. 7A-7F provide mixing geometry influences operating variable space. FIG.7A is a mixer setup schematics and mixing geometries for the CIJM-MIVM. FIGS. 7B and 7C are representative images of MPs synthesized at the labeled Ccore for the CIJM-MIVM. FIG. 7D is a mixer setup schematics and mixing geometries for the DIVM-MIVM. FIGS. 7E and 7F are representative images of MPs synthesized at the labeled Ccore for the DIVM-MIVM configurations.

[0026] FIGS. 8A-8C provide DIVM-MIVM setup schematics for Ta of 30, 60 and 90 ms, respectively. FIGS. 8D-8F provide respective photos of each setup, showing replaced delay tubing thickness and length.

[0027] FIGS. 9A-9G characterize the influence of Ta and Ccore on microparticle size. FIG.9A is a graph of particle diameter as a function of Ta for each Ccore. Influence of both Ta and Ccore on diameter were calculated to be statistically significant by two-way ANOVA test (a = 0.5, P <0.0001 for both parameters). FIG. 9B is a graph of linearized diameter vs Ta, plotted on a logarithmic scale. FIG. 9C is a graph of particle diameter as a function of the products of Ta and Ccore scaled to 1 / 3, with the linear relationship of Ccore 40, 60, and 80 mg / mL plotted. FIGS. 9D-9F are representative images of MP batches at labeled conditions. FIG. 9G provides a representative SEM image of SNaP MPs (100 mg / mL, 90 ms) indicating diameters of about 15 pm.

[0028] FIG. 10 is a SNaP process phase diagram for PLA microparticles. Summarized results of MP size tuning via Ccore and Ta. Relative particle sizes are illustrated with process instability zones shaded.

[0029] FIG. 11 provides SEM images and annotated sizes of PLA MPs stabilized by A. PVA B. PVP, and C. PEG-PLA. (N = 3)

[0030] FIG. 12A-12D characterize itraconazole-loaded MPs FIG. 12A is an illustration of itraconazole-loaded SNaP MP, FIG. 12B is a representative image and size distribution. FIG.12C is a graph of size and encapsulation efficiencies. FIG. 12D is a graph of cumulative itraconazole release from MPs (60 mg / mL, 90 ms) over 5 days in sink conditions at 37°C (N = 3, error bars smaller than symbol height).DETAILED DESCRIPTION

[0031] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s).

[0032] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

[0033] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments, and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential.

[0034] Colloidal nanocrystals (NC) are inorganic nanoparticles with distinct optical, electronic, and paramagnetic properties that are of particular interest in theranostics. The NCs may include an inorganic colloid stabilized with a hydrophobic ligand. Examples of the hydrophobic ligand may include, but are not limited to, oleic acid, oleylamine, octadectylamine, octanethiol, or a combination of any two or more thereof. Examples of the colloid may include, but are not limited to, gold, silver, copper, cobalt ferrite, manganese oxide, zinc oxide, cerium oxide, or a combination of any two or more thereof. For example, semiconducting quantum dots (QD) are promising bioimaging probes as they have high photostability, quantum yields, and tunable emission. Gold nanoparticles (AuNCs), due totheir surface plasmon resonance capabilities, have many uses in biomedicine, including as biosensors and imaging agents. Iron oxide nanocrystals (IONCS) can act as magnetic resonance imaging and magnetic particle imaging contrast agents. I0NC can also be locally manipulated in biological environments using a magnetic field, which is of interest in drug and gene delivery. Moreover, AuNC, I0NC, and other metallic NC, can act therapeutically through their ablative photothermal and hyperthermal capabilities.

[0035] NCs have distinct properties that rely on nanoscale-specific attributes such as size and aspect ratio. QD emission wavelength depends on size, IONC magnetic relaxivity scales with size, and AuNC and other plasmonic particles have distinct resonant profiles depending on size and morphology. Thus, it is useful to generate versatile theranostic nanoparticles (NPs), the ability to package NC into composite nanoparticles (CNP) independently of NC size and nondestructively of their original properties. Additionally, simultaneous efficient loading of small molecule therapeutics with variable chemical properties is useful for therapeutic versatility.

[0036] One advantage of polymeric NPs is the modularity of their physicochemical properties. NP attributes, including size and surface chemistry, directly inform their pharmacokinetic fate in terms of tissue retention and penetration, cell-specific uptake rates, and organ biodistribution. Thus, to optimize a theranostic CNP for a particular biomedical application, one may want to decouple the CNP properties from the cargo identity.

[0037] The Flash Nanoprecipitation (FNP) process is a scalable and tunable method for generating polymeric core-shell particles with high loading of bioactives. FNP is a one-step, controlled precipitation in which NP assembly is driven by hydrophobic interactions. Generally, the NP core consists of a hydrophobic homopolymer and small molecule therapeutics, while an amphiphilic block copolymer (BCP) comprises the shell, providing a dense hydrophilic polymer brush surface. In FNP, rapid and homogenous supersaturation of the hydrophobic components in an aqueous environment leads to diffusion-limited selfassembly and, thus, uniform NPs. Established micromixers such as the confined impingement jet (CIJ) mixer and multi -inlet vortex mixer (MIVM) provide this rapid solvent quality change, operating with Reynolds numbers above 2000, and thus, achieving mixing timescales on the order of a few milliseconds (ms).

[0038] FNP has been adopted to package therapeutics into monodisperse polymeric NPs, including hydrophobic small molecules, fluorophores, small biologies, and hydrophilic molecules through ion pairing.

[0039] The Sequential Nanoprecipitation (SNaP) process is a scalable approach for the controlled formation precipitation processes for the synthesis of polymeric particles. SNaP relies on a similar rapid solvent-switching and self-assembly principle as FNP using micromixing, but it involves a two-step assembly process in which the core formation is temporally separated from the particle stabilization. The particle core formation is initiated during a first micromixing step followed by particle stabilization through the addition of a stabilizer (e.g., BCP) in a second micromixing step. Forming composite nanoparticles with SNaP may include (a) mixing a water miscible organic stream against water, the first organic stream including nanocrystals and an organic solvent, forming particle cores comprising the nanocrystals suspended in the water; and (b) mixing the particle cores comprising the nanocrystals suspended in water against a second organic stream, the second organic stream comprising an amphiphilic block copolymer and the organic solvent, stabilizing the particles comprising the nanocrystals with the amphiphilic block copolymer, and forming the composite nanoparticles. The time between steps (a) and (b) is greater than 2 milliseconds and less than 1 second. Mixing steps are performed using a CIJ mixer, MIXVM, or another micromixer that can provide the rapid solvent quality change for formation of the particle.

[0040] Specifically, in the first step, the particle core is formed by rapid supersaturation of hydrophobic core components (e.g., one or more of a colloidal nanocrystal, a co-core excipient (e.g., a hydrophobic polymer), and an organic molecule) in an aqueous environment leading to diffusion-limited self-assembly of the particle core. In the second step, the particle core is stabilized by rapid supersaturation of a mixture including a stabilizer and any remaining core components not added in the first step in the aqueous environment. The modular SNaP mixer (e.g., a CIJ mixer, MIXVM, or another micromixer that can provide the rapid solvent quality change for formation of the particle) has a tunable inter-mixer length between spaced mixer containers to provide control over the delay time between the first and second mixing steps. This process provides the ability to generate particles from the nanometer to micron size range. Since SNaP allows for the sequential addition of components with millisecond resolution, larger core components, such as NCs, can be packaged in a more controlled and uniform manner by adjustment in the timing of core component aggregation.

[0041] Disclosed herein are methods of forming CNPs using sequential nanoprecipitation, and CNPs made using sequential nanoprecipitation. The CNPs made with SNaP include a hydrophobic core comprising one or more core components and a stabilizer forming a shell around the core. The core components may include colloidal nanocrystals, hydrophobic organic molecules, hydrophobic polymer co-core excipients, or a combination of any two or more thereof.

[0042] The hydrophobic organic molecules may be organic molecules with a molecular weight of 10 g / mol to about 5000 g / mol. The hydrophobic organic molecules may be insoluble or poorly soluble in water (e.g., solubility of less than 10 pg / mL, less than 1 pg / mL, or less than 0.1 pg / mL). The hydrophobic organic molecules may include therapeutic molecules (e.g., small molecule active pharmaceutical ingredients), fluorescent molecules, excipients, prodrugs, small peptides, counter-ions for hydrophobic ion pairing formulations, and any combination of two or more thereof. Nonlimiting examples of organic molecules include beta-carotene, alpha-tocopherol, curcumin, paclitaxel, cinnarizine, itraconazole, rubrene, and any combination of two or more thereof.

[0043] The colloidal nanocrystals may include an inorganic colloid stabilized with a hydrophobic ligand. Examples of the hydrophobic ligand may include, but are not limited to, oleic acid, oleylamine, octadectylamine, octanethiol, or a combination of any two or more thereof. Examples of the colloid may include, but are not limited to, gold, silver, copper, cobalt ferrite, manganese oxide, zinc oxide, cerium oxide, or a combination of any two or more thereof. For example, the colloidal nanocrystals may include quantum dots, gold nanocrystals, iron oxide nanocrystals, or a combination of any two or more thereof. In some embodiments, the nanocrystals include a first nanocrystal and a second nanocrystal different from the first nanocrystal, each of the first nanocrystal and the second nanocrystal comprising quantum dots, gold nanocrystals, iron oxide nanocrystals, or a combination of any two or more thereof. The nanocrystal may have a size of about 1 nm to about 50 nm, e.g., about 5 nm to about 25 nm, or about 3 nm to about 12 nm.

[0044] The stabilizer may include amphiphilic block copolymer, an amphiphilic polymer, a surfactant, a lipid, or a combination of any two or more thereof. The amphiphilic block copolymer may be a hydrophobic block copolymer with block sizes of about 1 kDa and about 100 kDa. Illustrative examples of amphiphilic block copolymers include polyethylene oxide) block copolymers (e.g., poly(ethylene oxide)-block-poly(D,L lactide), poly(ethylene oxide)-block-polystyrene, block-polylactic-co-glycolic acid, block-poly(caprolactone), block-poly(propylene oxide), or a combination of any two or more thereof), non- poly(ethylene oxide) block copolymers (e.g., poly-2-ethyl-2-oxazoline-b-poly(lactic acid), poly(ethylene oxide-co-glycidyl methyl ether)-b-poly(lactide), poly(hydroxyethyl methacrylate)-b-poly(lactose), or a combination of any two or more thereof), or a combination of any two or more thereof. The amphiphilic block copolymer may include a triblock copolymers (e.g., poly(ethylene oxide)-b-polypropylene oxide-b-PEO). For example, the amphiphilic block copolymer may include poly(ethylene oxide)-block-poly(D,L lactide). The poly(ethylene oxide)-block-poly(D,L lactide) having a molecular weight of about 5 kDa poly(ethylene oxide) and about 5 kDa poly(D,L lactide). Non block polymers may include hydrophobic polymers having a molecular weight of about 1 kDa and about 100 kDa. Illustrative examples include polyvinylpyrrolidone, polyvinyl alcohol, and combinations thereof. Surfactants may include water-soluble surfactants. Illustrative surfactants include D-a-tocopheryl polyethylene glycol succinate.

[0045] The hydrophobic polymer co-core excipients may include poly(D,L-lactide) or other hydrophobic polymers. The poly(D,L-lactide) may have a molecular weight of about 5 kDa to about 250 kDa, e.g., about 10 kDa to about 18 kDa. The second liquid may further include an organic molecule having a molecular weight less than 1000 Daltons.

[0046] The stabilizer may be a BCP, providing a dense hydrophilic polymer brush surface.

[0047] In some embodiments, the SNaP process is a three-step process. In a first step, the particle core is formed with the co-core excipient. In the second step, the nanocrystals are introduced into the core. In the third step, the stabilizer is introduced to form the shell around the particle core.

[0048] The method of forming composite nanoparticles includes mixing a first liquid with water, the first liquid comprising colloidal nanocrystals and an organic solvent, forming particle cores comprising the colloidal nanocrystals suspended in the water; and mixing the particles comprising the colloidal nanocrystals suspended in water with a second liquid, the second liquid comprising an amphiphilic block copolymer and the organic solvent, stabilizing the particles comprising the colloidal nanocrystals with the amphiphilic block copolymer, and forming the composite nanoparticles.

[0049] In some embodiments, the first liquid may further include a co-core excipient. The co-core excipient may include a hydrophobic polymer with a molecular weight of about 1 kDa to about 500 kDa. For example, the co-core excipient may include poly(D,L-lactide) or similar hydrophobic polymers. The first liquid may further include an organic molecule having a molecular weight less than 1000 Daltons. The organic molecule may be a hydrophobic organic molecule. The hydrophobic organic molecule may include therapeutic molecules (e.g., small molecule active pharmaceutical ingredients), fluorescent molecules, excipients, prodrugs, small peptides, counter-ions for hydrophobic ion pairing formulations, and any combination of two or more thereof. Examples of organic molecules include, but are not limited to, beta-carotene, alpha-tocopherol, curcumin, paclitaxel, cinnarizine, itraconazole, rubrene, and any combination of two or more thereof.

[0050] In some embodiments, the second liquid may further include a co-core excipient. The co-core excipient may include poly(D,L-lactide) or other hydrophobic polymers. The poly(D,L-lactide) may have a molecular weight of about 5 kDa to about 250 kDa, e.g., about 10 kDa to about 18 kDa. The second liquid may further include an organic molecule having a molecular weight less than 1000 Daltons. The organic molecule may be a hydrophobic organic molecule. The hydrophobic organic molecule may include therapeutic molecules (e.g., small molecule active pharmaceutical ingredients), fluorescent molecules, excipients, prodrugs, small peptides, counter-ions for hydrophobic ion pairing formulations, and any combination of two or more thereof. Examples of organic molecules include, but are not limited to, beta-carotene, alpha-tocopherol, curcumin, paclitaxel, cinnarizine, itraconazole, rubrene, and any combination of two or more thereof.

[0051] The method may include a time delay of a predetermined amount of time between the step of forming the particle cores comprising the colloidal nanocrystals suspended in the water and mixing in the amphiphilic block copolymer in organic solvent. The time delay may be about 2 millisecond (ms) to about 1 second, e.g., about 2 ms to about 100 ms, about 2 ms to about 10 ms, about 4 ms to about 6 ms, 10 ms to 1000 ms, or 100 ms to 1000 ms.

[0052] The colloidal nanocrystals may include an inorganic colloid stabilized with a hydrophobic ligand. Examples of the hydrophobic ligand may include, but are not limited to, oleic acid, oleylamine, octadectylamine, octanethiol, or a combination of any two or more thereof. Examples of the colloid may include, but are not limited to, gold, silver, copper, cobalt ferrite, manganese oxide, zinc oxide, cerium oxide, or a combination of any two ormore thereof. For example, the colloidal nanocrystals may include quantum dots, gold nanocrystals, iron oxide nanocrystals, or a combination of any two or more thereof. In some embodiments, the nanocrystals include a first nanocrystal and a second nanocrystal different from the first nanocrystal, each of the first nanocrystal and the second nanocrystal comprising quantum dots, gold nanocrystals, iron oxide nanocrystals, or a combination of any two or more thereof. The nanocrystal may have a size of about 1 nm to about 50 nm, e.g., about 5 nm to about 25 nm, or about 3 nm to about 12 nm.

[0053] The stabilizer may include amphiphilic block copolymer, an amphiphilic polymer, a surfactant, a lipid, or a combination of any two or more thereof. The amphiphilic block copolymer may be a hydrophobic block copolymer with block sizes of about 1 kDa and about 100 kDa. Illustrative examples of amphiphilic block copolymers include polyethylene oxide) block copolymers (e.g., poly(ethylene oxide)-block-poly(D,L lactide), poly(ethylene oxide)-block-polystyrene, block-polylactic-co-glycolic acid, block-poly(caprolactone), block-poly(propylene oxide), or a combination of any two or more thereof), non- poly(ethylene oxide) block copolymers (e.g., poly-2-ethyl-2-oxazoline-b-poly(lactic acid), poly(ethylene oxide-co-glycidyl methyl ether)-b-poly(lactide), poly(hydroxyethyl methacrylate)-b-poly(lactose), or a combination of any two or more thereof), or a combination of any two or more thereof. The amphiphilic block copolymer may include a triblock copolymers (e.g., poly(ethylene oxide)-b-polypropylene oxide-b-PEO). For example, the amphiphilic block copolymer may include poly(ethylene oxide)-block-poly(D,L lactide). The poly(ethylene oxide)-block-poly(D,L lactide) having a molecular weight of about 5 kDa poly(ethylene oxide) and about 5 kDa poly(D,L lactide). Non block polymers may include hydrophobic polymers having a molecular weight of about 1 kDa and about 100 kDa. Illustrative examples include polyvinylpyrrolidone, polyvinyl alcohol, and combinations thereof. Surfactants may include water-soluble surfactants. Illustrative surfactants include D-a-tocopheryl polyethylene glycol succinate.

[0054] The composite nanoparticles may have a core-shell structure with a diameter of about 80 nm to about 15 pm, e.g., about 100 nm to about 250 nm, about 90 nm to about 120 nm, about 100 nm to about 15 pm, about 500 nm to about 10 pm, or about 500 nm to about 1 pm. The composite nanoparticles may include about 5 wt.% to about 50 wt.% nanocrystals, e.g., about 5 wt.% to about 30 wt.%.

[0055] The diameter of the composite nanoparticles may increase with increasing the combined concentration of solids in the first liquid and the second liquid. A combined concentration of solids in the first liquid and the second liquid may be about 5 mg / mL to the polymer overlap concentration (e.g., about 80 mg / mL, 100 mg / mL, 200 mg / mL).

[0056] The organic solvent may be a water miscible polar organic solvent. Illustrative organic solvents include tetrahydrofuran, dimethyl sulfoxide, 1,4-di oxane, ethanol, methanol, isopropanol, acetone, acetonitrile, dimethyl formamide, or a combination of two or more thereof. For example, the solvent may include tetrahydrofuran.

[0057] Purification of the composite nanoparticles may alternatively or additionally include depletion column purification or, where the nanocrystals are magnetic, magnetic separators. In some embodiments, the nanocrystals include quantum dots, and the method further includes purifying the composite nanoparticles using serial fractionation via centrifugation.

[0058] The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.EXAMPLES

[0059] Disclosed herein are methods of encapsulation of IONCS of 5 nm to 25 nm in diameter and QDs of 3 nm to 12 nm within poly(lactic acid) (PLA)-based systems, using rubrene dye as a model hydrophobic small molecule. When the size of the NC was varied and the diffusivity of the NC diverged from the other core components, a disruption of CNP size control and homogeneity was observed. Thus, these examples indicate the use of SNaP to control CNP manufacturing. SNaP provides the ability to overcome mismatched component aggregation timescales through delaying stabilization, resulting in improved population uniformity, efficient co-encapsulation with rubrene dye, and restored size control independent of NC properties.EXAMPLE 1: SNaP Assembly Timescale in Synthesis

[0060] Materials. Poly(ethylene oxide)-block-poly(D,L lactide) (PEG-PLA) (5 kDa-5 kDa) was obtained from Evonik Industries. Oleic acid stabilized QDs of 540 nm emission (4-5 nm diameter) and 660 nm emission (10-12 nm diameter) suspended in toluene, and IONCsof 5 nm diameter, 10 nm diameter, 15 nm diameter, and 20 nm diameter, suspended in chloroform, were obtained from Ocean Nanotech. IONCS were characterized via transmission electron microscopy (TEM) before being used in synthesis. QDs and IONCs were solvent switched into tetrahydrofuran (THF) via rotary evaporation before use. Poly(D,L lactide) 10-18 kDa, rubrene, and THF were purchased from Sigma- Aldrich. Ultrapure deionized water was generating using Milli-Q Benchtop Purification System (Millipore- Sigma).

[0061] CNP Synthesis via SNaP. SNaP formulations were synthesized using a 3D-printed multistage vortex mixer with a 4-4 inlet configuration, as previously described. Flow rate was controlled using two synced PHD-Ultra Harvard Apparatus syringe pumps and gastight Hamilton syringes. Mixers were flushed in between formulations with 6 mL THF (36 ml / min) followed by 24 mL water (36 mL / min) and allowed to air dry for 30 min. Each mixing stage contained one organic stream of 2.5 mL volume, containing the chosen core or stabilizer components. Formulation parameters can be found in Table 1. The other 5 inlets contained equal volumes of water each. A flow rate of 36 mL / min was used, which corresponds to a calculated 5.8 ms delay time between stages. To ensure stable fluid flow, the initial and final 4 mL of the outgoing sample were discarded as the startup and leftover volumes. Sample volumes were collected in a quench bath of MilliQ water to bring the aqueous content to 90 vol%. The samples were then dialyzed and characterized via standard CNP characterization techniques.Table 1: CNP formulation parametersSNaP Formulations component(s) in mixing Mixer Total NC Core stage 1 -> components in mixing stage 2 Solids loading wt. % IONC10->PLA+BCP 2-4 15 5 33 IONC10+PLA->BCP 2-4 15 5 33 PLA->IONC10+BCP 2-4 15 5 33 IONC10+PLA->BCP 2-4 5 5 33 IONC10+PLA->BCP 2-4 30 5 33 PLA->IONC10+BCP 2-4 5 5 33 PLA->IONC10+BCP 2-4 30 5 33 IONC10->PLA+BCP 4-4 15 5 33 IONC10+PLA->BCP 4-4 15 5 33 PLA->IONC10+BCP 4-4 15 5 33 IONC10->PLA+BCP 4-4 5 5 33 IONC10->PLA+BCP 4-4 30 5 33 IONC10+PLA->BCP 4-4 5 5 33 IONC10+PLA->BCP 4-4 30 5 33 IONC10->PLA+BCP 4-4 15 15 33 IONC10->PLA+BCP 4-4 15 25 33 IONC10->PLA+BCP 4-4 15 50 33 IONC10->PLA+BCP 4-4 15 5 15 IONC10->PLA+BCP 4-4 15 5 50 IONC10->PLA+BCP 4-4 15 5 75IONC 15+QD5+PLA->BCP 4-4 5 5+5 33

[0062] CNP Purification. CNP formulations containing magnetic IONCS were purified using MidiMACS Separators and LD depletion columns (Miltenyi Biotec). Columns were first allowed to equilibrate (pre-wet) with 2 mL washes of MilliQ water. 2 mL of unpurified sample were added to the column and allowed to flow through. Equivalent volume washes with MilliQ water were also allowed to flow through. After the second wash, the column was removed from the magnet, 2 mL of MilliQ were added to the column and the sample was gently eluted using the plunger. QD-CNP formulations were purified using a serial fractionation via centrifugation. The original sample was centrifuged at low centrifugal force - 1000 g for 30 min, and the pellet and supernatant were separated. The resulting supernatant was then centrifuged at increasing centrifugal forces (starting at 2000 g and increasing to 25000 g) and the process was repeated until the pellet was too small to discern. The resulting pellets were resuspended in a small amount of water and for some analyses were pooled and analyzed together.

[0063] CNP Characterization. CNP size was determined using dynamic light scattering (DLS) with a Malvern ZetaSizer Nano ZS. Samples were diluted to approximately 0.05mg / mL concentration in MilliQ and measured using a detection angle of 173°. Reported sizes are intensity -weighted, average of 3 measurements of at least 10 runs as presented by the Malvern deconvolution software general purpose mode. Size distribution plots are reported as intensity -weighted traces, normalized to max intensity, to account for concentration and attenuator setting variability.

[0064] CNP total solids concentration was determined using thermogravimetric analysis (TGA), (Discovery 550 TGA, TA Instruments) as previously described. Briefly, aqueous samples were dispensed into tared platinum pans, then were heated under nitrogen from 25 °C to 105 °C at 10 °C / min and then held at 105 °C for 20 min. Sample concentration was then determined as the final weight divided by the initial sample volume. Sample composition, including NC loading, was determined using high temperature analysis to selectively burn off the polymer and retain the inorganic components.

[0065] CNP morphology was observed using TEM. Grids were prepared as follows: samples were drop-cast at about 0.3 mg / mL onto 300 mesh Au lacey carbon grids (TedPella) and allowed to dry. The grids were then negatively stained using 2 wt.% phosphotungstic acid (PTA) pH 6. The stain was deposited onto dry grids, allowed to sit for 10 min, then blotted. After, the grids were washed once with MilliQ before thoroughly drying them in air. Grids were imaged using a FEI Talos 120C electron microscope using a 120 kV beam with spot size 3. Image processing and analysis was done using ImageJ.

[0066] Absorbance and emission spectra of aqueous samples were generated using Duetta spectrophotometer-fluorimeter. (Horiba)

[0067] Delaying stabilization via SNaP overcomes assembly mismatches.

[0068] Example 2 results were conducted in accordance with the materials and methods in Example 1. Process parameters are defined according to Equations 1, 2, and 3.Total Solids = cBCP+ cPLA+ cNCEquation 1NC Loading = — — — x 100% Equation 2total solidsCore % =Cpla+Cncx 100% Equation 3total solidswhere each c refers to the components’ mass concentration in mg / mL in the solvent stream with the subscripts identifying the component.

[0069] To overcome the aggregation timescale mismatch and unify the competing assemblies seen in FNP, the emergent multistep synthesis method SNaP was used. In SNaP, an initial mixing stage is followed in series by a second mixing stage. This allows for a controlled delay between NP core aggregation and its stabilization; pre-assembling parts of the core before the shell is added. Adjusting the delay time between mixers can modulate the size of the resultant NP. Custom 3D printed mixer prototypes were used to achieve a range of short delay times between mixing chambers. In the present work, the delay time between mixing stages was kept constant at 5.8 ms to fully elucidate the other formulation parametric impacts.

[0070] To understand the importance of timing, the effects of changing the order of addition of the mismatched core components was investigated. FIG. 1A shows the scheme of these experiments: (1) PLA in the first mixing stage followed by NC and the stabilizing BCP in the second (PLA->NC+BCP), (2) NC in the first stage followed by PLA and BCP in the second (NC->PLA+BCP), and (3) both NC and PLA in the first stage followed by BCP (NC+PLA->BCP). In these experiments, IONCIOS were used with a maintained loading of 5 wt.%, total solids content of 15 mg / mL and 33 % core.

[0071] For these experiments, rubrene dye was added concurrently with the PLA to simulate the distribution of a small hydrophobic drug molecule inside the polymer matrix core. The distribution of the rubrene, easily quantified using fluorescence spectroscopy and qualitatively identifiable by a bright orange color, was used as a simple model for a multimodal theranostic CNP.

[0072] SNaP schemes 1, 2, and 3 yielded IONCIO-CNPS of 122 nm, 90 nm, and 164 nm, respectively (FIG. IB). Separating scheme 1 into magnetic and non-magnetic fractions revealed a larger NP size nonmagnetic fraction (142 nm diameter) than its magnetic counterpart (91 nm diameter), suggesting that the PLA continued to assemble more rapidly than the nanocrystal clusters. (FIG. IB). In comparison, Scheme 2 produced the smaller original CNP sample size, and separated into a magnetic fraction of the same size but reduced poly dispersity, with a nonmagnetic fraction around 38 nm consistent with PLA-PEG micelles.Scheme 3 yielded the larger CNP, and separated into a nearly identical trace around 200 nm, with a multimodal nonmagnetic fraction around 106 nm.

[0073] Next, the uniformity and co-encapsulation efficiency of the CNPs was characterized. Rubrene distribution within the original, magnetic, and nonmagnetic fractions was quantified using fluorimetry. It can be seen in FIG. IE that earlier addition of the PLA and rubrene with the NC (scheme 3) yields a magnetic fraction with the greater fluorescence intensity than when they are added with the stabilizer (FIG. IE). These emission intensity trends were consistent when the samples were diluted into an organic solvent, i.e., deformulated, thus indicating that the observed trends were not due to quenching of the rubrene within the CNP. To quantify the uniformity of the NC loading, thermal decomposition plots were generated via TGA to obtain the weight percent of inorganic content in the CNPs. Scheme 3 yielded a magnetic fraction with the closest inorganic content to the attempted loading of 5 wt.% (FIG. IF), followed closely by scheme 1.

[0074] Size and loading trends from this experiment revealed several insights into CNP assembly. Without being bound by any theory, a hypothesis was that pre-aggregating the PLA co-excipient in the first step before the addition of NCs in the second step (scheme 1) increases the PLA aggregate size and reduces its number density to match the NC aggregation behavior, leading to a more uniform sample. The alternate hypothesis was that the slower NC used extra time to aggregate to “catch up” with the fast components (scheme 2). It can be seen from both the size and loading data that adding the NC first resulted in the loaded CNP as the predominant species. Additionally, adding PLA and rubrene concurrently with the NC in the first step (scheme 3) afforded the higher co-encapsulation efficiency. These results indicated that solely delaying the stabilizer addition allowed for improved co-aggregation of mismatched components.

[0075] SNaP improves CNP size control and cargo uniformity. Next, SNaP was used to decouple CNP size from starting NC identity in a side-by-side comparison with FNP. Rubrene-loaded IONCIO-CNPS with PLA and PEG-PLA were synthesized utilizing schemes 2 and 3, i.e., NC in the first stage, and NC+PLA in the first stage (FIG. 2A). Scheme 3 provided a more pronounced size control; by varying the total solids content from 5 mg / mL to 30 mg / mL, sizes from 100 nm to 250 nm were achieved (FIG. 2B). Additionally, the original and magnetic fractions were nearly identical in size, indicating that the loaded CNP was the dominant species in the sample. Scheme 2 yielded only a slight size dependence,tuning from 90 nm to 120 nm. As expected, and in contrast to both SNaP samples, the FNP samples showed no change in size with increasing total solids content, remaining at around 75 nm for all samples. Also, as expected from the FNP library, the nonmagnetic fractions for the FNP samples showed a slight increase in size from 30 nm to 50 nm (FIG. 2B). In addition to total solids content, changing the core % was explored. The size dependence was less pronounced than with the total solids. Due to the delay of stabilizer addition, which quenches core growth in FNP, the parameter of core% is less impactful than the delay time between components in SNaP.

[0076] The 30 mg / ml samples were processed to highlight differences in drug and NC distribution. Quantitatively, both SNaP samples outperformed the FNP samples, with higher rubrene emission intensity (FIG. 2C) in the magnetic fraction than the analogous FNP sample, and a closer inorganic content to the attempted 5% loading (FIG. 2D), indicating a higher degree of co-encapsulation of the NC and fluorophore. Both SNaP schemes (3, 2) showed improvement in NC loading uniformity (about 3 -fold improvement) and rubrene coencapsulation (about 10-fold improvement) when compared to the analogous FNP sample.

[0077] The restoration of size control by temporally separating PLA and BCP addition is in agreement with previous SNaP studies, which show that size correlates with delay time. Additionally, the improved NC uniformity and co-encapsulation with rubrene underscores the importance of overcoming aggregation mismatch for improved CNP quality.

[0078] After observing that SNaP improved the uniformity of CNPs containing both NCs and small molecules and restored a method of IONC10-CNP size tuning, co-encapsulation of NCs of distinctly different sizes was accomplished. Multifunctional magnetic and fluorescent CNPs (referred to herein as “m-qNPs”) were synthesized containing larger 15 nm IONCS and smaller 5 nm QDs using SNaP scheme 3 (FIG. 3 A). m-qNPs of 190 nm were synthesized, which separated into a magnetic population of 210 nm and a nonmagnetic population of 36 nm (FIG. 3B). PL measurements of the m-qNPs and their fractions showed that the magnetic fraction of the m-qNPs was nearly equivalently emissive as the original, indicating achievement of uniform co-encapsulation of the two mismatched NCs (FIG. 3C). TEM images of m-qNPs indicated co-encapsulation of small and large objects (FIG. 3D). As 5 wt.% loading of both components was attempted, a 1:27 ratio of IONC15 to QD5 was expected if uniformity was achieved. While this ratio was not consistently observed in TEM, the higher number of co-encapsulated QD5 observed and the low number of QD-only CNPwas promising. The ability to robustly synthesize multifunctional CNPs with different-sized NCs, as well as potentially a small molecule drug, was an exciting application of SNaP due to the implications of countless NC-NC-drug combinations that are possible.

[0079] Predicting and modeling CNP assembly in SNaP. To explain the trends observed in SNaP, the experimental data was placed within the context of established diffusion-limited aggregation kinetics. Specifically, the aim was to understand how the combination of varying both aggregation timescales and number density affected CNP assembly in the milliseconds after mixing occurs.

[0080] Core components PLA and IONCS were considered as cores by themselves following diffusion-limited growth kinetics. To account for differential size and diffusivity of the aggregates, the time for two particles to collide was examined. The characteristic time for single particles to collide (t) in can be calculated as shown in Equation 4.- 3 it MW _t = - Equation 44NAkBTc1

[0081] Where . is the solvent viscosity (in this model, assumed the viscosity of water for simplicity), c is the particle mass concentration, NA is Avogadro’s number, kn is Boltzmann’s constant, T is the temperature in Kelvin, and MW is the molecular weight of individual particles. Examining t as a function of starting component, it is evident that these timescales scale with size due to number densities. However, t only represents the initial collision time. After the first collision, there is both a reduction in number density and an increase in size, which affect the collision time. To understand how both number density and collision time change, 3 standard formulations prepared in this study were considered - IONC5-5-33, IONC10-5-33, and IONC20-5-33. These formulations have the same initial number densities of PLA, and the same mass loading of NC (5 wt.%) but vary in the NC size and number density. Taking these initial number densities and their individual component characteristics, the change in aggregate number density was plotted overtime, assuming isolated aggregation. (FIG. 4 A) Change in number concentration (C) over time was calculated as:C = Equation 5where Cois the initial number density at the point of mixing. These number densities were used to calculate core collision time (T) relative to C for each component.31 T ,T = - Equation 64kBTC

[0082] The change in T was plotted over time. (FIG. 4B) It can be seen from these two plots that PLA number density drops rapidly, and subsequently, its aggregation rate starts high and quickly drops off. While the initial starting ratios of PLA:NC are high for the IONC5s and IONCIOS, within 2 ms there are fewer available PLA aggregates than NC, and they are slower to collide. However, for the IONC20s, this crossover point, noted as tcross , occurs later, at around 15 ms.

[0083] Building on this simple model of variable collision times, the timescale for the BCP stabilization was considered. Assigning a collision and growth model for BCP stabilization would not account for geometric aspects of the BCP molecule adhering to the surface of the growing core. Therefore, the timescale of BCP aggregation on the core surface was considered to be similar to its timescale of micellization. BCP self-assembly into spherical micelles has been modeled as a 2-step process, the first step as a rapid induction of unimers into overlapping brush aggregates and the second step as a slow rearrangement into micelles as equilibrium is achieved. For simplicity, this fast initial induction step for PEG-PLA was estimated to occur between 5-10 ms in accordance with previous studies.

[0084] Assigning a timescale of stabilization allowed to make inferences about CNP assembly regimes. At the estimated time of micelle induction for the model formulations, the component collision time for the growing IONC5 and IONC10 core is faster than that of the PLA core. The IONC20 core, however, still exhibits a much slower collision time at this point. Indeed, in this model formulation, even the initial collision time is slower than the estimates for BCP micellization.

[0085] Differential growth rates offer a rationale for why the IONC20 formulation seemed to exhibit a coating behavior rather than clustering - in the time it takes for the initial stabilization to occur, the polymer component is both aggregating faster than the NC and is available at a higher number concentration. Furthermore, this simple model can suggest how starting parameters may induce IONC20s into a clustering behavior. Increasing either the starting PLA number density or the IONC20 number density shifts the crossover point to the left; or, delaying stabilization by 5-10 ms via SNaP accommodates for a right-shifted crossover point. This model is thus consistent with the FNP and SNaP results discussed previously.

[0086] This crossover point model may also explain the divergent assembly in the IONC 10-CNPs as compared to IONC5-CNPs. For the IONC5 formulation, the crossover point occurs rapidly, under 0.5 ms. Thus, for this formulation, all core components sufficiently aggregate before stabilization. For IONC10, the crossover point is later, near 2 ms, which is approaching the micellization induction. While NC clustering was still observed, it is with these IONC 10 formulations that more pronounced divergent NP growth was observed, indicating that the BCPs are interfering with the aggregation.EXAMPLE 2: SNaP Microparticles

[0087] Polymeric microparticles (MPs) are useful drug delivery vehicles for extended-release applications, but current manufacturing techniques present significant challenges in balancing size control with scalability. This Example 2 provides an investigation of process parameters governing SNaP MP formation, using poly(lactic acid) (PLA) MPs stabilized with polyvinyl alcohol (PVA) as an example. Comparing vortex and impinging jet mixing geometries, indicated vortex mixing provides better core assembly, particularly at higher polymer concentrations. The influence of delay time (Ta) and core stream concentration (Ccore) on particle size were investigated, indicating that microparticle assembly follows Smoluchowski diffusion-limited growth kinetics within defined operational boundaries. Through this approach, precise control over microparticle size (e.g., 1.6 pm to 3.0 pm) with narrow poly dispersity was achieved. The versatility of SNaP was further demonstrated by successful formation of MPs with different stabilizers while maintaining consistent size control. Finally, the pharmaceutical relevance of SNaP was indicated by encapsulating itraconazole with high efficiency (e.g., 83%-85%) and characterizing its sustained release profile. These findings indicate SNaP as a robust, scalable platform for high-quality pharmaceutical microparticle production with superior control over quality attributes.

[0088] Synthesis and characterization of PLA. DL-lactide was added (12.6 g, 87.6 mmol) to a flame dried, silanized 200 mL round bottom flask equipped with a large stir bar before undergoing three vacuum / nitrogen purge cycles followed by overnight drying under high vacuum. In a glovebox, DBU (1.12 mmol) was added to a flame dried pear-shaped flask equipped with a stir bar. Anhydrous chloroform, extra dry over molecular sieves, was then used to dilute the monomer and catalyst with 82 mL and 18 mL, respectively. The monomer solution was then heated to 50 °C using a mineral oil bath. The initiator, HeBriB (1.19 mmol), was then added to the monomer solution and allowed to stir for 15 minutes. To initiate thereaction, the catalyst solution was transferred via cannula to the monomer solution for a final reaction molarity of 0.9 M. Following initiation, the heat was immediately turned off, and the reaction solution was allowed to remain in the cooling mineral oil under nitrogen. The reaction was terminated after 2 hours by extracting the catalyst with 1 M HCl(aq) followed by two brine washes. The organic phase was then concentrated and precipitated over 3 Ox excess ice-cold methanol to isolate the PLA. The product was transferred to salinized scintillation vials and dried overnight under high vacuum to yield a crystalline, white powder (12.2 g, 94.4% yield, D = 1.18).

[0089] Polymer characterization was performed byJH NMR and gel permeation chromatography (GPC).JH NMR spectra were acquired on a Bruker AVANCE NEO 500 MHz instrument with at least 32 scans and a 10-second relaxation delay time. 'H NMR (500 MHz, CDCh, 0.03 wt.% TMS) 85.38-4.99 (m, 155H), 4.50-4.27 (m, 5H), 1.92 (s, 6H), 1.82-1.34 (m, 499H).

[0090] Molecular weight distributions were analyzed on a Shimadzu GPC system equipped with a guard column (KD-G) and analytical column (KD-804) with a target molecular weight range of 2-200 kDa. The mobile phase consisted of HPLC-grade N,N’ -dimethylformamide containing 10 mM lithium bromide at a flow rate of 1 mL / min, with the column, UV, and RI detector temperatures maintained at 50°C. GPC samples were prepared as 2 mg / mL polymer solutions in mobile phase, sonicated for 10 minutes, and filtered through 0.45 pm PTFE syringe filters prior to injection (100 pL). Molecular weights were determined relative to narrow poly(methyl methacrylate) standards (Shodex M-75).

[0091] Assembly of Mixers. A 3D-printed dual-inlet vortex mixer (DIVM) was employed for the initial mixing step. The mixer was designed using Autodesk Fusion360 and fabricated on a Stratasys Objet30 Pro using Veroclear resin. Post-printing processing included the removal of support material under running water, installation of threaded heat inserts using an arbor press, and thorough compressed air cleaning to eliminate residual material. O-rings were positioned into the mixing stages, which were then assembled and secured using shoulder bolts, washers, and nuts.

[0092] For particle synthesis using the DIVM-MIVM configuration, the outlet of the 3D-printed mixer was connected to a stainless-steel multi-inlet vortex mixer (MIVM) via 0.04”or 0.06” inner diameter tubing. For the CIJM-MIVM configuration, a confined impinging jet mixer was used instead for the initial mixing step.

[0093] Microparticle Synthesis. MPs were synthesized via SNaP using the described mixer configurations. Before and after the MP synthesis, mixer streams were flushed with 3 mL of THF (60 mL / min) followed by 3 mL of water (60 mL / min).

[0094] FIG. 5 is a schematic of the SNaP process. An organic stream containing the microparticle core components was rapidly mixed against an antisolvent in the first micromixing step to initiate the core formation. The outlet was connected to a second micromixer, which introduced the stabilizer and arrested the microparticle growth. The delay time between the first and second mixing steps controlled the time of the core growth.

[0095] For the synthesis of MPs, a THF stream with dissolved PLA and rubrene (2 wt.%) was mixed against an equal volume stream of ultra-pure water in the first mixer, which was either a 3D-printed DIVM or a confined impinging jet mixer (CIJM). The output of this first mixing stage flowed into the inlet of the second mixer (MIVM), where it was mixed against three streams of 1 mg / mL PVA in ultra-pure water. For the synthesis of itraconazole loaded MPs, itraconazole was added to the THF stream at a 10 wt.% total solids concentration.

[0096] To achieve the desired Ta, the flow rate for each stream was at 60 mg / mL using a syringe pump (PHD Ultra Syringe pump, Harvard Apparatus) and the length / diameter of the tubing between mixing stages was varied.

[0097] To simulate a continuous flow system, MP samples were collected after the flow had fully developed. This solution was collected in an ultrapure water quenching bath to reduce the final organic concentration to 5 vol% THF. Startup and end volumes were not collected.

[0098] MPs were purified via dialysis to remove organic solvent and unencapsulated drug. Using regenerated cellulose dialysis tubing (12 kDa to 14 kDa MWCO, Repligen), particles were dialyzed against a large excess of ultrapure water, changing the water bath each hour for six hours.

[0099] Particle Characterization. MP morphology was evaluated using scanning electron microscopy (SEM). Samples were prepared by concentrating MPs via centrifugation (2,000ref, 5 minutes) followed by dropwise deposition onto silicon wafers. After ambient drying, samples were sputter-coated with a thin conductive platinum layer (3 nm) using an EMS 150R ES sputter coater and imaged using the SE2 detector on a Zeiss Merlin FE-SEM microscope operating at 1 kV EHT and 110 pA.

[0100] Particle size distributions were determined through image analysis of multiple SEM micrographs acquired at 1000* magnification. A custom MATLAB-based circular Hough transform algorithm was employed to detect particles and measure their diameters. For each formulation, at least 2,000 particles were analyzed across multiple fields of view to ensure statistical robustness.

[0101] Total solids concentration (Ctotai) in purified MP suspensions was determined using thermogravimetric analysis (TGA, Discovery 550 TGA, TA Instruments). Samples were heated under nitrogen from 25°C to 105°C at 10°C / min and maintained at 105°C for 20 minutes to ensure complete water evaporation. Total particle concentration was calculated by dividing the residual solid weight by the initial sample volume.

[0102] Itraconazole mass concentration (Cdrug) for purified MP samples was determined via High Performance Liquid Chromatography on a Thermofisher Vanquish Core HPLC equipped with an autosampler, quarternary pump, UV detector, and column (Hypersil Gold C18, 3 pm particle size, 175 A pore size, 4.6 mm x 150 mm, 30°C). HPLC samples were prepared by diluting NP solutions into acetonitrile to a final 50 vol% acetonitrile. The mobile phase was 50 vol% acetonitrile with 0.1 vol% trifluoracetic acid and 50 vol% water with 0.1 vol% trifluoracetic acid, with a total flow rate of 0.5 mL / min for 20 minutes. Detection was performed at 256 nm.

[0103] MP drug loading wt.% (DL) was calculated using the equation DL =Cdruax 100.ctotal Encapsulation efficiency (EE%), or the percent of drug that incorporated into MPs, was calculated using the equation EE% = — — — X 100 where DLtarget was 10% for all batches.DLtarget

[0104] To characterize the itraconazole release rate, MP dispersions were gently concentrated via centrifugation at 2000 ref for 5 minutes, followed by decanting off the supernatant. MPs were then redispersed at a 10-fold dilution into sink condition release media comprised of phosphate buffered saline and 10 v / v% Tween-80 surfactant. After initial sampling to determine the initial drug concentration (Cinitiai), the dispersion was agitated at 90rpm in a 37°C water bath. At predetermined time points, 1 mL aliquots were withdrawn and centrifuged (2000 ref, 5 minutes) to separate MPs from released drug. The supernatant was analyzed via HPLC for released itraconazole (Creieased), with the cumulative release calculated asCinitialx 100 for each time point.^released

[0105] Results and Discussion. In this study, SNaP process and formulation variables were identified and operating ranges for robust and tunable MP synthesis were determined. A quantitative size analysis methodology for MP size characterization was developed. The importance of mixing geometry was investigated by comparing impinging jet mixing and vortex mixing in the first micromixing step which initiated core assembly. The variable workspace of Ta and Ccore was investigated, including the influence of each parameter on particle size and identifying regions of predictable particle assembly. The versatility of SNaP was investigated by varying stabilizer composition and encapsulating a therapeutic agent.

[0106] Image Analysis Methodology for Microparticle Characterization. MP size is a quality attribute that influenced particle biodistribution, drug release rate, and efficacy. MPs synthesized in this study exhibited diameters exceeding 1 pm with sedimentation rates on the order of minutes, precluding reliable characterization via conventional dynamic light scattering (DLS) techniques. To address this limitation, scanning electron microscopy (SEM) was used to visualize the MPs. A high-throughput image analysis protocol was developed capable of providing both quantitative size distribution data and qualitative morphological insights that were useful for evaluating fabrication process.

[0107] SEM was selected as the primary imaging modality due to its superior resolution and magnification capabilities. Upon visual inspection, the SNaP-produced particles consistently exhibited high sphericity. Image analysis permitted accurate analysis of densely populated fields containing thousands of closely packed MPs (FIG. 6A-6B).

[0108] For statistical significance, multiple SEM micrographs were captured at 1000* magnification (1024 x 768 resolution) for each formulation. Particle diameters detected via the MATLAB-implemented algorithm were converted to micron scale and aggregated to generate comprehensive size distribution profiles (FIG. 6C). Each formulation was independently synthesized in triplicate (N=3) to evaluate process reproducibility, with at least 2,000 particles analyzed per formulation to ensure robust statistical representation. The microparticle poly dispersity index (PDI), the relative variance of particle size assuming aGaussian distribution, was calculated for each sample as — where D was mean particlediameter and o was distribution standard deviation.

[0109] Role of Mixing Geometry on SNaP Process. Micromixing efficiency can influence uniform particle assembly. The first micromixing step that initiated the assembly of the particle core influenced micromixing efficiency. Increasing polymer concentrations in the organic stream of the first mixing step (Ccore) may increase solution viscosity, potentially compromising mixing efficiency in less robust mixing configurations. Vortex mixing demonstrated higher efficiency compared to confined impinging jet mixing. SNaP MP assembly was compared between CIJM and DIVM geometries.

[0110] The CIJM-MIVM and DIVM-MIVM SNaP configurations (FIGS. 7A and 7D) at fixed Ta (30 ms) were investigated and the Ccore concentrations ,40 mg / mL and 100 mg / mL PLA, were explored, which represented lower and higher values in the concentration range. MPs were stabilized by 0.1 wt.% PVA in all three water streams in the second mixing step. The organic stream dynamic viscosities were calculated for Ccore of 40 mg / mL and 100 mg / mL using the Mark-Houwink-Sakurada equation for intrinsic viscosity [q] (Equation 7) and a modified Huggins equation for solution viscosity q (Equation 8):[q] = KMa(Equation 7)q= qs(l + [q]c + kH[t]]2c2) (Equation s)where K and a represent Mark-Houwink parameters for PLA in THF (0.0174 mL / g and 0.736, respectively), M is the polymer molecular weight (17.5 kDa), qsis THF viscosity (0.46 mPa s), and kn is the Huggins coefficient (0.3 for good solvents). This analysis revealed a greater than two-fold increase in solution viscosity between 40 mg / mL (0.99 mPa*s) and 100 mg / mL (2.21 mPa*s) formulations.[OHl] In the CIJM-MIVM setup, uniform particle assembly was observed at 40 mg / mL, producing monodisperse 1.9 pm particles (FIG. 7B). However, at 100 mg / mL, severe poly dispersity and irregular morphologies emerged, including pear and disc-shaped aggregates, which, without being bound by any theory may indicate insufficient mixing during core assembly (FIG. 7C). In contrast, the DIVM-MIVM configuration maintained uniform assembly at both concentrations, producing spherical populations with diameters of 1.6 pm and 2.2 pm, respectively (FIGS. 7E-7F). These observations were consistent at longerdelay times (Td = 90 ms), where the CIJM-MIVM configuration produced 3.0 pm particles at 40 mg / mL, and aggregates at 100 mg / mL, and the DIVIM-MIVM configuration produced 2.2 pm and 3.0 pm particles at the same respective Ccore values.

[0112] Without being bound by any theory, the performance of the vortex mixing geometry may stem from its enhanced tolerance to increased stream viscosity, effectively expanding the operational parameter space for Ccore conditions. Vortex mixing accommodated asymmetric flowrates, providing greater flexibility in anti sol vent / sol vent ratios and supersaturation states. Based on these advantages, the DIVM-MIVM configuration was selected for subsequent investigations.

[0113] Controlling Microparticle Sizes via Delay Time and Core Concentration. Having established the mixing configuration, parameters of MP formation during SNaP were investigated. Ta, the residence time between core nucleation in the first mixer and stabilization in the second mixer, was a determinant of MP size. Size-tunable assembly of MPs from 0.7 pm to 1.2 pm using delayed times of 7-23 ms, and the present study extended this investigation to longer delay times to form larger MPs.

[0114] To achieve the extended delay times, two millifluidic mixers were connected in series with interchangeable tubing with variable dimensions. The system consisted of a DIVM for the imitation of core formation followed by an MIVM for stabilization. The delay time between mixing steps was controlled by adjusting the thickness and length of the connecting tubing between micromixers. At constant flow rate Q, the delay time was calculated using equations 9 and 10:V = (V' + a * L) (Equation 9)Td= (Equation 10)where V represents the total delay channel volume, a and L denote the cross-sectional area and length of the interchangeable tubing, and V’ accounts for the volume contribution of fixed delay channel segments. By maintaining a constant flow rate of 60 mL / min in each inlet while varying the tubing dimensions, delay times of 30, 60, and 90 ms were established (FIGS. 8A-8C).

[0115] The influence of Ccore on MP characteristics was investigated. FNP increased the total solids concentration resulting in larger nanoparticles for systems involving core material aggregation. Similar trends occurred with Ccore concentrations in the SNaP process, providing tuning of MP size. Ccore values were evaluated ranging from 40 mg / mL to 100 mg / mL PL A, with the upper bound approaching the solubility limit of PLA (18 kDa) in THF, which was approximately 120 mg / mL.

[0116] Within these parameter ranges, the operational space for SNaP was mapped by determining achievable MP sizes (FIG. 9A). As expected, increasing Ta led to larger MP sizes across core concentrations, though the effect was less pronounced at the 100 mg / mL PLA Ccore. Without being bound by any theory, this is consistent with core growth continuing during the Ta between two mixers. Longer core assembly times resulted in larger particles. Similarly, increasing Ccore produced larger particles, providing an additional parameter for controlling MP size. Without being bound by any theory, this effect may stem from an increase in the core growth rate relative to the core nucleation rate. Within this variable space, MPs were produced ranging from 1.59 ± 0.01 pm (40 mg / mL PLA, 30 ms Ta) to 2.98 ± 0.35 pm (100 mg / mL PLA, 90 ms Ta) with narrow size distributions. The MP PDIs increased at the higher 80 mg / mL and 100 mg / mL PLA Ccore concentrations. Example SEM images of the spherical MPs are shown in FIGS. 9D-9F. The data can be found in Table 2. FIG. 9G provides a representative SEM image of SNaP MPs (100 mg / mL, 90 ms) indicating diameters of about 15 pm.

[0117] For delay times less than 23 ms, the particle radius, R, scaled with Ta as described by Smoluchowski’s model of diffusion limited growth shown in Equation 11 :1R=rtkBTccore\3 (Equation 11)V ^VPcore 'where t is growth time (in our case Ta), T is temperature, rj is solution dynamic viscosity, p is core material density, Ccore is the core concentration, and kB is the Boltzmann constant. Without being bound by any theory, the SNaP particle formation may proceed via diffusionlimited aggregation of hydrophobic species in the delay channel. This analysis extended to longer delay times up to 90 ms. When linearizing microparticle size data (FIG. 9B), two distinct trends were observed depending on core concentration. For Ccore values of 40 mg / mL, 60 mg / mL, and 80 mg / mL, the data followed diffusion-limited growth kinetics with slopevalues of approximately 1 / 3, consistent with Smoluchowski’s model. However, at 100 mg / mL Ccore, this relationship broke down, and particle size became largely independent of delay time, suggesting a shift in the underlying assembly mechanism.

[0118] Without being bound by any theory, at 100 mg / mL PLA, the overlap concentration (C*) may approach where PLA chains begin to entangle in solution and thus no longer precipitate as individual globules prior to assembly. To test this hypothesis, we calculated C* for our linear PLA (17.5 kDa) by first determining the radius of gyration (Rg) using De Gennes’s scaling law (Equation 12) and then calculating the C* using coil overlap concentration equation (Equation 13):n Nb^Rg = (Equation 12)Q / VfC* — - =■ (Equation 13)where N is the number of Kuhn segments (121), b is the Kuhn length (8.81 A), M is molecular weight (17.5 kDa), and NA is Avogadro’s number. The calculated Rgvalue of 39.6 A yielded a C* of 112 mg / mL, indicating that the 100 mg / mL formulation approached the threshold for chain entanglement. Without being bound by any theory, this may explain the deviation from Smoluchowski kinetics at this concentration, as polymer chain entanglements can lead to network formation during precipitation, altering the assembly mechanism. Additional experiments at a Ccore of 120 mg / mL (above C*) resulted in visible PLA macroprecipitates immediately after synthesis, further indicating that polymer chain entanglements in the organic stream lead to uncontrolled aggregation. These findings establish a process constraint: polymeric materials in SNaP may remain below their overlap concentration in the solvent stream to ensure controlled assembly.

[0119] Next, using the Smoluchowski scaling law, MP size data was collapsed from formulations with 40 mg / mL, 60 mg / mL, and 80 mg / mL PLA Ccore’ s onto a single predictive curve (FIG. 9C). Without being bound by any theory, this finding indicated that particle size in SNaP processes followed diffusion-limited aggregation principles, providing precise prediction of microparticle dimensions based on Ccore and Ta. Such predictive capability represents an advancement in controlled microparticle manufacturing.

[0120] Based on the analysis, a process phase diagram was developed for 17.5 kDa PLA microparticles (FIG. 10) that delineated regions of controlled, diffusion-limited growth from zones of process instability. This diagram serves as a practical guide for formulation scientists, providing rational selection of formulation (Ccore) and process parameters (Ta) to achieve target particle specifications. Within the stable operating region, reproducible size control was demonstrated from 1.8 pm to 2.9 pm by systematically varying core concentration and delay time.

[0121] The process may be used for work with small batches for formulation development and for scale up under continuous flow. For a single batch, accounting for start-up volume and flow stabilization, 120 mg to 300 mg of PLA was used. A SNaP mixer set-up operated under continuous flow yielded 144 g to 360 g of microparticles hourly (18 L / hr). Furthermore, the vortex mixing geometries employed in the SNaP mixers were amenable to scale-up.Table 2. Average MP size and PDI from organic stream concentration and delay times tested. (N=3).Ccore (mg / mL) Td (ms) Diameter (yim) PDI40 30 1.59± 0.01 0.28 ± 0.0240 60 1.86 ± 0.07 0.12 ± 0.0140 90 2.24 ± 0.05 0.15 ± 0.0560 30 1.92 ± 0.09 0.11 ± 0.0360 60 2.24 ± 0.09 0.17 ± 0.0460 90 2.57 ± 0.15 0.12 ± 0.0580 30 2.01 ± 0.20 0.29 ± 0.1580 60 2.36 ± 0.17 0.27 ± 0.1080 90 2.87 ± 0.12 0.34 ± 0.09100 30 2.86 ± 0.11 0.17 ± 0.03100 60 2.88 ± 0.33 0.24 ± 0.11100 90 2.98 ± 0.35 0.39 ± 0.15*Dissolved solids for all formulations contained 2 wt.% rubrene, e.g., core concentration 40 mg / mL corresponded to 39.2 mg / mL PLA and 0.8 mg / mL rubrene. (N = 3).

[0122] Variation of Stabilizing Polymers. The surface properties of drug delivery vehicles influenced their biological behavior. To demonstrate the versatility of SNaP for creating MPswith diverse surface chemistries, PLA particles were synthesized using three different stabilizing polymers: polyvinyl alcohol (PVA, 18 kDa), polyvinyl pyrrolidone (PVP, 40 kDa), and the amphiphilic block copolymer polyethylene glycol-polylactic acid (PEG-PLA, 5kDa-5kDa). Formulations used identical core streams (Ccore = 40 mg / mL PLA) and delay times (Ta = 13.5 ms).

[0123] While hydrophilic stabilizers (PVA and PVP) were introduced in the aqueous streams of the second mixing step, the amphiphilic PEG-PLA was incorporated in a second organic stream that replaced one of the water streams. As shown in FIG. 11, all three formulations produced MPs with virtually identical morphologies and mean diameters. PVP-stabilized MPs exhibited slightly higher poly dispersity, likely attributable to the substantially higher molecular weight of PVP (40 kDa) compared to PVA (18 kDa) and PEG-PLA (10 kDa), resulting in slower diffusion kinetics during the stabilization phase.

[0124] Without being bound by any theory, the consistency in particle size across diverse stabilizers with fundamentally different stabilization mechanisms - adsorption of water-soluble polymers versus hydrophobic anchoring of amphiphilic copolymers - provided evidence that MP size was determined by core growth during the intermixer delay time rather than by stabilizer characteristics.

[0125] Loading Small Molecule Therapeutics into Microparticles via SNaP. To evaluate the pharmaceutical relevance of SNaP-produced MPs, the encapsulation of itraconazole was investigated, where itraconazole is a weakly hydrophobic antifungal agent (FIG. 12A). Itraconazole was incorporated into the core organic stream at a 10 wt.% target loading while maintaining Ccore at 60 mg / mL. Delay times were varied from 30 ms to 90 ms to assess the influence of particle size on drug loading efficiency.

[0126] Itraconazole loaded MPs were synthesized at all three delay times. A representative SEM image of the spherical MPs is shown in FIG. 12B. Shown in FIG. 12C, the drug-loaded MPs exhibited slightly smaller diameters than their non-drug-loaded counterparts, attributable to the higher density of itraconazole (1.60 g / mL) compared to PLA (1.25 g / mL). Encapsulation efficiency remained consistent across all delay times, with values of 83%, 84%, and 85% for 30 ms, 60 ms, and 90 ms, respectively. Moreover, the consistent drug loading efficiency across varying delay times indicated that MP size can be independentlytuned without compromising therapeutic incorporation, highlighting the orthogonal control of quality attributes afforded by the SNaP process.

[0127] Release kinetics studies revealed a biphasic pattern: an initial burst release of approximately 23% upon dispersion into sink conditions, followed by a slow sustained release over the five days tested (FIG. 12D).

[0128] While certain embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.

[0129] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.

[0130] The present disclosure is not to be limited in terms of the particular embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and compositions within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can of course vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0131] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0132] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0133] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0134] Other embodiments are set forth in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of forming composite nanoparticles comprising:(a) mixing a first liquid with water, the first liquid comprising a core component and an organic solvent, forming a particle core comprising the core component suspended in the water, the core component comprising a nanocrystal, hydrophobic organic molecule, or a combination thereof; and(b) mixing the particle core suspended in water with a second liquid, the second liquid comprising a stabilizer and the organic solvent, stabilizing the particle core comprising the core component with the stabilizer, and forming the composite nanoparticles;wherein a time delay between steps (a) and (b) is about 2 millisecond (ms) to about 1 second.

2. The method of claim 1, wherein the first liquid further comprises a co-core excipient.

3. The method of claim 2, wherein the co-core excipient comprises a hydrophobic polymer with a molecular weight of about 1 kDa to about 500 kDa.

4. The method of claim 1, wherein the core component comprises an organic molecule having a molecule weight less than 1000 Daltons.

5. The method of claim 4, wherein the organic molecule comprises a therapeutic molecule, a fluorescent molecule, an excipient, a prodrug, a small peptide, a counter-ion for a hydrophobic ion pairing formulation, or a combination of two or more thereof.

6. The method of claim 1, wherein the second liquid further comprises a co-core excipient.

7. The method of claim 6, wherein the co-core excipient comprises a hydrophobic polymer with a molecular weight of about 1 kDa to about 500 kDa.

8. The method of claim 1, wherein the second liquid further comprises an organic molecule having a molecule weight less than 1000 Daltons.

9. The method of claim 8, wherein the organic molecule comprises a therapeutic molecule, a fluorescent molecule, an excipient, a prodrug, a small peptide, a counter-ion for a hydrophobic ion pairing formulation, or a combination of two or more thereof.

10. The method of claim 1, wherein the time delay between steps (a) and (b) is about 2 ms to about 100 ms.

11. The method of claim 1, wherein the time delay between steps (a) and (b) is about 2 ms to about 10 ms.

12. The method of claim 1, wherein the nanocrystal comprises gold, silver, copper, cobalt ferrite, manganese oxide, zinc oxide, cerium oxide, or a combination of any two or more thereof.

13. The method of claim 12, wherein the nanocrystal comprises a first nanocrystal and a second nanocrystal different from the first nanocrystal, each of the first nanocrystal and the second nanocrystal comprising quantum dots, gold nanocrystals, iron oxide nanocrystals, or a combination of any two or more thereof.

14. The method of claim 1, wherein the stabilizer comprises an amphiphilic block copolymer, a surfactant, a lipid, or a combination of any two or more thereof.

15. The method of claim 1, wherein the composite nanoparticles have a diameter of about 50 nm to about 15 pm.

16. The method of claim 1, wherein the composite nanoparticles comprise about 5 wt.% to about 75 wt.% core component and the core component comprises nanocrystals.

17. The method of claim 1, wherein a combined concentration of solids in the first liquid and the second liquid is about 5 mg / mL to about 200 mg / mL.

18. The method of claim 1, wherein the organic solvent comprises tetrahydrofuran, dimethyl sulfoxide, 1,4-di oxane, ethanol, methanol, isopropanol, acetone, acetonitrile, dimethyl formamide, or a combination of two or more thereof.

19. The method of claim 1, wherein the core component comprises quantum dots, and the method further comprising purifying the composite nanoparticles using serial fractionation via centrifugation.

20. A method of forming composite nanoparticles comprising:(a) introducing a first liquid into water via confined impinging jet, vortex mixing, or a combination thereof, the first liquid comprising iron oxide nanocrystals, poly(D,L-lactide), an organic molecule having a molecule weight less than 1000 Daltons, and a polar organic solvent, forming particle cores suspended in the water, the particle cores comprising nanocrystals, the poly(D,L-lactide), and the organic molecule; and(b) introducing a second liquid into the water comprising the particle cores via confined impinging jet, vortex mixing, or a combination thereof, the second liquid comprising poly(ethylene oxide)-block-poly(D,L lactide) and the polar organic solvent, stabilizing the particle cores with poly(ethylene oxide)-block-poly(D,L lactide), and forming the composite nanoparticles;wherein a time delay between steps (a) and (b) is about 2 ms to about 1 second.

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