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WO2025207378A1 - Scalable and high throughput assembly of layer-by-layer nanoparticles - Google Patents

Scalable and high throughput assembly of layer-by-layer nanoparticles

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Publication number
WO2025207378A1
WO2025207378A1 PCT/US2025/020523 US2025020523W WO2025207378A1 WO 2025207378 A1 WO2025207378 A1 WO 2025207378A1 US 2025020523 W US2025020523 W US 2025020523W WO 2025207378 A1 WO2025207378 A1 WO 2025207378A1
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Prior art keywords
polyelectrolyte
nanoparticle
solution
poly
relation
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French (fr)
Inventor
Darrell J. Irvine
Paula T. Hammond-Cunningham
Ivan Susin PIRES
Ezra Zevulun Reingold GORDON
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Massachusetts Institute of Technology
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Massachusetts Institute of Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures

Definitions

  • Nanoparticles have been extensively studied as drug carriers to alter the pharmacokinetics and pharmacodynamics of therapeutics, with engineering of NP surface chemistry representing an important strategy to optimize delivery of drugs to disease sites and/or avoid off-target tissue uptake.
  • One approach towards to engineer surface chemistry is by layer-by-layer (LbL) assembly of polymer coatings on nanoparticles.
  • LbL technique consists of the deposition of nanoscale polymer films on surfaces through alternating adsorption of molecular layers of complementary polymers, most typically polycations and polyanions.
  • the resulting multilayer assemblies which can be several nanometers in thickness, can enable controlled drug release as well as modulation of surface properties and the resulting interactions with biological interfaces.
  • LbL-NPs for targeted drug delivery and controlled drug release has been successfully demonstrated in many applications. Assembly of polymer films on NPs can yield increased NP plasma half-life and stability, tumor targeting, and control over the subcellular localization of NPs. Moreover, LbL-NPs can be used to effectively deliver small-molecule therapeutics and nucleic acids which can be loaded into the polymer film in the NP. The high versatility of this platform approach enables the facile assembly of combination therapies within a single particle to target diverse diseases. [0004] A challenge of LbL-NP synthesis is the serial nature of the layer deposition process.
  • a method of making a composition comprising a layered nanoparticle comprising: a first step of microfluidic mixing of a solution comprising a nanoparticle with a solution comprising a first polyelectrolyte to form a solution comprising a first-layered nanoparticle.
  • kits comprising: a solution comprising a nanoparticle; a solution comprising a first polyelectrolyte; a solution comprising a second polyelectrolyte; one or more microfluidic mixing chips; and instructions for its use.
  • a solution comprising a nanoparticle comprising a solution comprising a first polyelectrolyte; a solution comprising a second polyelectrolyte; one or more microfluidic mixing chips; and instructions for its use.
  • FIGs. 1A-1D show schematics of nanoparticle assembly and the characterization of nanoparticle surface charge conversion upon polyelectrolyte deposition.
  • FIG. 1A-1D show schematics of nanoparticle assembly and the characterization of nanoparticle surface charge conversion upon polyelectrolyte deposition.
  • FIG. 1A shows a schematic for small-scale deposition of polymers onto interleukin-12 (IL-12) loaded NPs.
  • FIG. 1B shows intensity-weighted particle size (Z-avg), number-averaged (#-avg) size, polydispersity index (PDI), and zeta potential of the resulting polymer-nanoparticle mixtures at increasing PLR weight equivalents to bare anionic IL-12-NP.
  • FIG. 1C shows Z-avg, #- avg, PDI, and zeta potential of the resulting polymer-nanoparticle mixtures from increasing weight equivalent PLE to PLR-coated IL-12 NPs.
  • FIG. 1D a schematic representing the different possible resulting assemblies upon increasing polymer weight equivalents.
  • FIG. 2E shows Z-avg size, PDI and zeta potential of the resulting PLR/PLE-IL-12-NPs generated via different layering strategies on TFF purified PLR-IL-12-NPs. (mean ⁇ s.d.).
  • FIGs. 3A-3I show microfluidic fluid mixing chips enable homogeneous LbL-NP assembly without the need of purification steps.
  • FIG. 3A shows Z-avg size and PDI of target PLE/PLR-IL-12-NPs compared to small scale bath sonication mixing of IL-12 NPs with titrated polymer-to-NP wt. eq. of PLR and PLE.
  • FIG. 3F shows Z-avg size, PDI, and zeta potential of the resulting PLR-IL-12-NPs generated using titrated PLR-to-NP wt.eq. via microfluidic (MCF) mixing compared to target NP measurements.
  • FIG.3G shows Z-avg size, PDI, and zeta potential of the resulting PLE/PLR-IL-12-NPs generated using titrated PLR-to- NP wt.eq. then titrated PLE-to-NP wt. eq. via MCF compared to target NP measurements.
  • FIG. 3H shows fraction of fluorescently labeled PLE polymer associated with NPs after optimized TFF-based layering before and after TFF purification compared to titrated polymer layering using MCF.
  • FIG. 3I shows negative stain transmission electron microscopy (TEM) of unlayered (UL) IL-12 NPs or PLE/PLR-IL-12-NPs assembled via either the optimized TFF- based protocol or the titrated polymer-to-NP wt. eq. MCF protocol. Scale bars represent 100 nm (mean ⁇ s.d.).
  • FIGs. 4A-4E show that microfluidic fluid mixing chips enable homogeneous LbL-NP assembly in optimized buffer solutions.
  • FIG. 4A-4E show that microfluidic fluid mixing chips enable homogeneous LbL-NP assembly in optimized buffer solutions.
  • FIG. 4A shows Z-avg size and PDI of NPs layered with increasing PLR-to-NP wt. eq. in 25 mM HEPES and 20 mM NaCl.
  • FIG. 4B shows Zeta potential of NPs layered with increasing PLR-to-NP wt. eq. in 25 mM HEPES and 20 mM NaCl.
  • FIG. 4C shows a schematic, Z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using small-scale bath sonication mixing of polymers and NPs.
  • FIG. 4A shows Z-avg size and PDI of NPs layered with increasing PLR-to-NP wt. eq. in 25 mM HEPES and 20 mM NaCl.
  • FIG. 4C shows a schematic, Z-avg size, PDI, and zeta potential of NPs layered with P
  • FIG. 4D shows a schematic, Z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using large- scale bath sonication mixing of polymers and NPs.
  • FIG. 4E shows a schematic, Z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using MCF mixing of polymers and NPs.
  • FIGs. 5A-5D shows MCF-LbL-NPs maintain the desired in vitro characteristics of PLE/PLR LbL-NPs.
  • FIG. 5A shows the fluorescence ratio of NPs associated with HM-1 cells relative to NPs remaining in the supernatant at 4 and 24 hours after dosing (median).
  • FIG.6A shows the HEK-Blue IL-12 reporter cell line response curves for free IL-12 or IL-12 loaded onto LbL-NPs generated via either TFF-based or MCF-based LbL assembly.
  • FIG. 6B shows the calculated EC 50 of curves in FIG.6A.
  • FIG. 6C shows in vivo treatment scheme of metastatic ovarian cancer. IL- 12 doses of 20 ⁇ g were given on days 7 and 14 for each construct
  • FIG.6D shows whole animal bioluminescence reading via IVIS of luciferase expressing tumors cells from mice treated as in FIG.
  • FIG. 6C shows survival curves of mice treated as in FIG. 6C.
  • Statistical comparisons between survival curves were performed using a log-rank (Mantel–Cox) test.
  • asterisks denote p-values: ****p ⁇ 0.0001, ***p ⁇ 0.001, **p ⁇ 0.01, *p ⁇ 0.05.
  • FIG. 7 shows MCF LbL-NPs, TFF-based LbL-NPs (STD), and unlayered NPs (UL) colloidal stability data in NaCl solution. Colloidal stability is measured by dynamic light scattering (DLS).
  • FIG. 8 shows MCF LbL-NPs, TFF-based LbL-NPs (STD), and unlayered NPs (UL) colloidal stability data in physiological saline buffered solution supplemented with serum.
  • FIGs.9A-9G show MCF-LbL-NPs can be assembled with varying polymer chemistries and NP cores.
  • FIG. 9A shows zeta potential of PLR-IL-12-NPs mixed with varying wt. eq.
  • FIG. 9B shows Z-avg size and PDI of final HA, PLD, or PAA-coated IL-12 NPs after MCF assembly.
  • FIG. 9C shows Zeta potential of particles from FIG. 9B.
  • FIG. 9C shows zeta potential of CML NPs of varying sizes mixed with increasing wt. eq. of PLR. Zeta potential values at 0.01 wt. eq are from UL CML.
  • FIG. 9E shows CML NPs of varying sizes and their final MCF-assembled LbL-NPs with PLR/PLE films.
  • FIG. 9F shows flow cytometry median fluorescence intensity (MFI) of HM-1 cells incubated with CML NPs at 10 ⁇ g/mL for 4 hours.
  • FIG. 9G shows correlation between the log-fold change in CML NP MFI of LbL-NPs relative to UL-NPs to log of the estimated total available NP surface area (SA) in solution
  • FIG. 10 shows TEM micrographs of unlayered IL-12 NPs and LbL NPs assembled via either TFF or MCF.
  • FIG. 11 shows Comparison of dynamic light scattering (DLS) electrophoretic mobility results from independent IL-12-LbL-NP batches generated via either optimized TFF-based (with excess polymers) or MCF-based (using POP wt.
  • DLS dynamic light scattering
  • FIG. 12 shows TEM micrographs of carboxy-modified latex (CML) beads unlayered or layered with PLR/PLE.
  • FIGs. 13A-13E show microfluidic fluid mixing chips enable homogeneous LbL-NP assembly in optimized buffer solutions.
  • FIG.13A shows the z-avg size and PDI of NPs layered with increasing PLR-to-NP wt. eq. in 25 mM HEPES and 20 mM NaCl (mean ⁇ s.d.).
  • FIG. 13B shows the zeta potential of NPs layered with increasing PLR-to-NP wt. eq.
  • FIG.13C shows the z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using small-scale bath sonication mixing of polymers and NPs (mean ⁇ s.d.).
  • FIG.13D shows the z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using large-scale bath sonication mixing of polymers and NPs (mean ⁇ s.d.).
  • FIG.13C shows the z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using small-scale bath sonication mixing of polymers and NPs (mean ⁇ s.d.).
  • FIG. 14 shows a flow chart comparing previous methods of assembling LbL-NPs to the MCF method provided herein.
  • the term “microfluidic mixing” encompasses methods which enable rapid and efficient mixing through the manipulation of fluids in channels or layers of small dimensions.
  • the microfluidic mixing can utilize micro-scale channels.
  • the microfluidic mixing can utilize milli-scale channels.
  • the microfluidic mixing can utilize micro-scale layers.
  • particles include polymeric particles, single-emulsion particles, double-emulsion particles, coacervates, liposomes, microparticles, nanoparticles, macroscopic particles, pellets, crystals, aggregates, composites, pulverized, milled or otherwise disrupted matrices, and cross-linked protein or polysaccharide particles, each of which have an average characteristic dimension of about less than about 1 mm and at least 1 nm, where the characteristic dimension, or “critical dimension,” of the particle is the smallest cross-sectional dimension of the particle.
  • a particle may be composed of a single substance or multiple substances.
  • the particle is not a viral particle.
  • the particle is not a liposome.
  • the particle is not a micelle. In certain embodiments, the particle is substantially solid throughout. In certain embodiments, the particle is a nanoparticle. In certain embodiments, the particle is a microparticle. [0024]
  • the term “nanoparticle” refers to a particle having an average (e.g., mean) dimension (e.g., diameter) of between about 1 nanometer (nm) and about 1 micrometer ( ⁇ m) (e.g., between about 1 nm and about 400 nm, about 1 nm and about 300 nm, between about 1 nm and about 100 nm, between about 1 nm and about 30 nm, between about 1 nm and about 10 nm, or between about 1 nm and about 3 nm), inclusive.
  • polyelectrolyte refers to a polymer which under a particular set of conditions (e.g., physiological conditions) has a net positive or negative charge.
  • the polyelectrolyte is or comprises a polycation.
  • a polyelectrolyte is or comprises a polyanion. Polycations have a net positive charge and polyanions have a net negative change. The net charge of a given polyelectrolyte may depend on the surrounding chemical conditions (e.g., pH).
  • Exemplary polyelectrolytes for use in polymeric coatings in the method of this disclosure include but are not limited to: poly(L-arginine) (PLR), poly-L-glutamic acid (PLE), polyarginine, polyglutamic acid, polylysine, hyaluronic acid (HA), poly-L-aspartate, polyaspartic acid, polyaminoacid composites, block co-polymers of poly amino acids and poly-ethylene glycol, polyacrylic acid, dextran sulfate, heparin folate, heparin sulfate, D-amino acid polymers or L/D amino acid mixtures, nucleic acids, fucoidan, sulfated- ⁇ -cyclodextran, polyglutamic acid-block- polyethylene glycol, polystyrene sulfonate (SPS), linear poly(ethylene imine), poly(diallyldimethyl ammonium chloride).
  • PLR poly(L-arginine)
  • polystyrene sulfonate alginate, and chondroitin sulfate.
  • substantially free refers to a solution with reduced amounts of free, or unbound, polyelectrolyte. In some embodiments, substantially free means the solution does not need to undergo purification to remove free or unbound polyelectrolyte.
  • substantially free indicates less than about 5%, 4%, 3,%, 2%, or 1% of free polyelectrolyte in solution. In some embodiments, substantially free indicates less than about 5% free polyelectrolyte in solution. In some embodiments, substantially free indicates an undetectable amount of free polyelectrolyte in solution.
  • agent e.g., molecule
  • pharmaceutical agent e.g., pharmaceutically acceptable agent
  • a compound may be any agent that can be represented by a chemical formula, chemical structure, or sequence.
  • an agent may be provided as a salt, ester, hydrate, or solvate.
  • an agent is cell- permeable, e.g., within the range of typical agents that are taken up by cells and acts intracellularly, e.g., within mammalian cells, to produce a biological effect. Certain compounds may exist in particular geometric or stereoisomeric forms.
  • Such compounds including cis- and trans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)-isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated.
  • Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates [e.g., with water (i.e. hydrates) or common solvents] and/or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms.
  • a “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds.
  • the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long.
  • a protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed.
  • amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification.
  • a protein may also be a single molecule or may be a multi-molecular complex.
  • a protein may be a fragment of a naturally occurring protein or peptide.
  • a protein may be naturally occurring, recombinant, synthetic, or any combination of these.
  • a protein can be tethered to a particle.
  • Exemplary proteins include but are not limited to interleukin-12, interleukin-15 super agonist, interleukin 18, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , interleukin-2, anti-PD1 antibodies, anti-PDL1 antibodies, anti-CTLA4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti- NKG2A antibodies, anti-CD73 antibodies, anti-A2aR antibodies, anti-B7-H3 antibodies, anti- B7-H4 antibodies, single-chain interleukin-12, tumor necrosis factor alpha, interleukin-10, interleukin-8, TNF-related apoptosis-inducing ligand (TRAIL), FMS-like tyrosine kinase 3 ligand (FLT3LG).
  • TRAIL TNF-related apoptosis-inducing ligand
  • FLT3LG FMS-like tyrosine kinase 3 ligand
  • a “polymer” refers to a compound comprised of at least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeating covalently bound structural units.
  • a polymer is naturally occurring.
  • a polymer is synthetic (i.e., not naturally occurring).
  • a “small molecule” refers to an organic molecule with a molecular weight of less than 800 g/mol (e.g., less than 700 g/mol, less than 600 g/mol, less than 500 g/mol, less than 400 g/mol, less than 300 g/mol, less than 200 g/mol, less than 100 g/mol, between 50 to 800 g/mol, inclusive, between 100 to 800 g/mol, inclusive, or between 100 to 500 g/mol, inclusive).
  • nucleic acid or “nucleic acid sequence,” “nucleic acid molecule,” “nucleic acid fragment” or “polynucleotide” may be used interchangeably with “gene,” “mRNA encoded by a gene,” and “cDNA”.
  • plateau point As used herein the terms “plateau point,” “plateau onset point” or “POP” are used interchangeably and refer to the point at which complete conversion of surface charge of the layered nanoparticle is achieved (FIG. 1C-1D). In some embodiments, “titrated amount,” or “POP amount” refers to the amount of polyelectrolyte needed to achieve charge conversion and/or plateau.
  • This value can be determined by titration experiments, such as those described herein, or calculated as described herein (Example 9), to identify the minimum amount of polyelectrolyte needed to achieve reach the POP (i.e., to complete conversion of surface charge of the layered nanoparticles). Charge conversion is indicated by the complete reversal of surface charge (e.g., negative to positive or positive to negative, measured by zeta potential).
  • the term “excess” polyelectrolyte refers to the amount of polyelectrolyte, in weight equivalents relative to the nanoparticle, which surpasses the titration amount or plateau point of polyelectrolyte.
  • excess is at least 0.3 wt. eq. of polyarginine. In some embodiments, excess polyelectrolyte is greater than 0.2 wt. eq. of polyarginine. In some embodiments, excess is at least 1 wt. eq. of polyglutamate. In some embodiments, excess polyelectrolyte is greater than 0.6 wt. eq. of polyglutamate.
  • asserts The terms “assess,” “determine,” “evaluate,” and “assay” are used interchangeably herein to refer to any form of detection or measurement, and include determining whether a substance, signal, disease, condition, etc., is present or not.
  • the result of an assessment may be expressed in qualitative and/or quantitative terms. Assessing may be relative or absolute. “Assessing the presence of” includes determining the amount of something that is present or determining whether it is present or absent. [0035] Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, or more typically, within 5%, 4%, 3%, 2%, or 1% of a given value or range of values.
  • the dispersity of a polymer can be modified, for example, using polymer fractionation (e.g., preparative SEC, Baker-Williams fractionation, continuous spin fractionation), or modifying the work-up procedure (e.g., by partially dissolving a polymer, an insoluble high molar mass fraction may be filtered off resulting in a large reduction in Mw and a small reduction in Mn, thus reducing polydispersity).
  • polymer fractionation e.g., preparative SEC, Baker-Williams fractionation, continuous spin fractionation
  • modifying the work-up procedure e.g., by partially dissolving a polymer, an insoluble high molar mass fraction may be filtered off resulting in a large reduction in Mw and a small reduction in Mn, thus reducing polydispersity.
  • salts or “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1–19.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2–hydroxy–ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2– naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pec
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C1–4alkyl)4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.
  • composition and “formulation” are used interchangeably.
  • D ETAILED D ESCRIPTION OF C ERTAIN E MBODIMENTS [0041] The present disclosure demonstrates a novel approach for multilayer assembly onto NPs using microfluidic (MCF) mixing technology. LbL-NPs have been used in preclinical models for controlled drug release, tumor and immune cell targeting, to attempt to improve pharmacokinetics and biodistribution, and to attempt to control cellular trafficking and uptake mechanisms.
  • MCF microfluidic
  • MCF is scalable, continuous, and readily implemented under cGMP conditions for clinical-grade NP production.
  • MCF allows for LbL electrostatic assembly using titrated polymer-to-NP weight equivalent ratios where no excess polymer is required to achieve a given LbL layering. Under such conditions, no time- consuming purification is needed, increasing LbL-NP throughput and avoiding the loss of NPs during purification.
  • interleukin-12 interleukin-12
  • IL-12 interleukin-12
  • poly(amino acid) polymers which have shown promising therapeutic efficacy in preclinical mouse models of metastatic ovarian cancer.
  • the inventors demonstrate herein equivalent efficacy in vitro and in vivo to LbL-NPs generated via traditional lab-scale batch-wise polymer adsorption and tangential flow filtration purification.
  • the inventors further demonstrate herein that, by rational selection of polymer-to-NP ratios for surface charge conversion without the addition of excess polymers, this approach enables LbL films to be constructed without the need for time-consuming purification steps, greatly simplifying LbL-NP preparation.
  • MCF can assemble LbL films of various chemistries and on various NP core substrates.
  • a method of making a composition comprising a layered nanoparticle comprising: a first step of microfluidic mixing of a solution comprising a nanoparticle with a solution comprising a first polyelectrolyte to form a solution comprising a first-layered nanoparticle.
  • the method further comprises a second step of microfluidic mixing of the solution comprising the first-layered nanoparticle with a solution comprising a second polyelectrolyte to form a solution comprising a second-layered nanoparticle.
  • the first step results in charge conversion from the nanoparticle to the first-layered nanoparticle.
  • the second step results in charge conversion from the nanoparticle to the second-layered nanoparticle.
  • a method of making a composition comprising a layered nanoparticle comprising: microfluidic mixing of a solution comprising a nanoparticle with a solution comprising a first polyelectrolyte to form a solution comprising a first-layered nanoparticle; and microfluidic mixing of the solution comprising the first-layered nanoparticle, with a solution comprising a second polyelectrolyte to form a solution comprising a second-layered nanoparticle.
  • the first polyelectrolyte comprises hyaluronic acid, poly- glutamate, poly-aspartate, poly-amino acid composite, block co-polymers of poly amino acids and poly-ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, nucleic acids, or a fluorescently labeled derivative thereof.
  • the second polyelectrolyte comprises hyaluronic acid, poly- glutamate, poly-aspartate, poly-amino acid composite, block co-polymers of poly amino acids and poly-ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, nucleic acids, or a fluorescently labeled derivative thereof.
  • the first polyelectrolyte is poly-amino acid composite. In some embodiments, the first polyelectrolyte comprises block co-polymers of poly amino acids and poly-ethylene glycol. In some embodiments, the first polyelectrolyte comprises poly-arginine. In some embodiments, the first polyelectrolyte comprises poly-lysine. In some embodiments, the first polyelectrolyte comprises poly-acrylic acid. In some embodiments, the first polyelectrolyte comprises dextran sulfate. In some embodiments, the first polyelectrolyte comprises heparin sulfate. In some embodiments, the first polyelectrolyte comprises heparin folate.
  • the first polyelectrolyte comprises nucleic acids.
  • the second polyelectrolyte is hyaluronic acid, poly-glutamate, poly-aspartate, poly-amino acid composite, block co-polymers of poly amino acids and poly-ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, or nucleic acids.
  • the second polyelectrolyte comprises hyaluronic acid.
  • the second polyelectrolyte comprises poly-glutamate.
  • the second polyelectrolyte comprises heparin folate. In some embodiments, the second polyelectrolyte comprises nucleic acids. In some embodiments, the second polyelectrolyte is hyaluronic acid, poly-glutamate, poly- aspartate, poly-amino acid composite, block co-polymers of poly amino acids and poly- ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, or nucleic acids. [0055] In certain embodiments, the method further comprises determining an amount of polyelectrolyte needed to achieve a plateau onset point (POP).
  • POP plateau onset point
  • the POP amount is determined using the procedures and/or calculations described herein. In some embodiments, the POP amount is determined using methods known in the art. [0056] In some embodiments, the solution comprising the first polyelectrolyte comprises at least 80% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises at least 90% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises at least 95% of the amount of polyelectrolyte needed to achieve the POP.
  • the solution comprising the first polyelectrolyte comprises at least 99% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises about 100% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises at least 105% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises at least 110% of the amount of polyelectrolyte needed to achieve the POP.
  • the solution comprising the first polyelectrolyte comprises between 80% and 100% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 80% and 85% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 85% and 90% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 90% and 95% of the amount of polyelectrolyte needed to achieve the POP.
  • the solution comprising the first polyelectrolyte comprises between 95% and 100% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 95% and 105% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 90% and 110% of the amount of polyelectrolyte needed to achieve the POP. [0058] In some embodiments, the solution comprising the first polyelectrolyte comprises 100% of the amount of polyelectrolyte needed to achieve the POP.
  • the solution comprising the first polyelectrolyte comprises about 100% of the amount of polyelectrolyte needed to achieve the POP.
  • the solution comprising the second polyelectrolyte comprises at least 80% of the amount of polyelectrolyte needed to achieve the POP.
  • the solution comprising the second polyelectrolyte comprises at least 90% of the amount of polyelectrolyte needed to achieve the POP.
  • the solution comprising the second polyelectrolyte comprises at least 95% of the amount of polyelectrolyte needed to achieve the POP.
  • the solution comprising the second polyelectrolyte comprises at least 99% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the second polyelectrolyte comprises about 100% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the second polyelectrolyte comprises at least 105% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the second polyelectrolyte comprises at least 110% of the amount of polyelectrolyte needed to achieve the POP.
  • the solution comprising the second polyelectrolyte comprises between 80% and 100% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the second polyelectrolyte comprises between 80% and 85% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the second polyelectrolyte comprises between 85% and 90% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the second polyelectrolyte comprises between 90% and 95% of the amount of polyelectrolyte needed to achieve the POP.
  • the solution comprising the second polyelectrolyte comprises about 100% of the amount of polyelectrolyte needed to achieve the POP.
  • the POP amount is an amount of polyelectrolyte polymer added to the NP such that the particle net charge is neutral. In some embodiments, the POP amount is about 1.5 to about 3 times the amount of polyelectrolyte required to achieve neutrality. In some embodiments, the POP amount is about 1.5 times the amount of polyelectrolyte required to achieve neutrality. In some embodiments, the POP amount is about 2 times the amount of polyelectrolyte required to achieve neutrality.
  • the POP amount is about 3 times the amount of polyelectrolyte required to achieve neutrality.
  • the solution comprising the first-layered nanoparticle is substantially free of unbound first polyelectrolyte after mixing.
  • the solution comprising the second-layered nanoparticle is substantially free of unbound second polyelectrolyte after mixing.
  • the solution comprising the first-layered nanoparticle does not comprise an excess of the first polyelectrolyte in relation to the nanoparticle.
  • the solution comprising the second-layered nanoparticle does not comprise an excess of the second polyelectrolyte in relation to the first-layered nanoparticle.
  • the second polyelectrolyte has a net positive surface change. In some embodiments, the nanoparticle and the second polyelectrolyte have net positive surface charges and the first polyelectrolyte has a net negative surface charge. In some embodiments, the nanoparticle and the second polyelectrolyte have net negative surface charges and the first polyelectrolyte has a net positive surface charge. In certain embodiments, the nanoparticle and second polyelectrolyte have the same surface charge. In certain embodiments, the nanoparticle and second polyelectrolyte have opposite surface charges.
  • the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.7 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.05 to 0.7 weight equivalents of poly-arginine in relation to the nanoparticle. [0069] In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.1 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.1 to 0.15 weight equivalents of the first polyelectrolyte in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises between about 0.35 to 0.4 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.4 to 0.45 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.45 to 0.5 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.5 to 0.55 weight equivalents of the first polyelectrolyte in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises between about 0.55 to 0.6 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.6 to 0.65 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.65 to 0.7 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. [0070] In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.05 to 0.1 weight equivalents of the first polyelectrolyte in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises between 0.1 to 0.15 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.15 to 0.2 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.2 to 0.25 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.25 to 0.3 weight equivalents of the first polyelectrolyte in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises between 0.3 to 0.35 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.35 to 0.4 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.4 to 0.45 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.45 to 0.5 weight equivalents of the first polyelectrolyte in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises between 0.5 to 0.55 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.55 to 0.6 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.6 to 0.65 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.65 to 0.7 weight equivalents of the first polyelectrolyte in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises about 0.1 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.1 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.2 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.2 weight equivalents of the first polyelectrolyte in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises about 0.3 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.3 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.4 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.4 weight equivalents of the first polyelectrolyte in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises about 0.5 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.5 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.6 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.6 weight equivalents of the first polyelectrolyte in relation to the nanoparticle.
  • the first polyelectrolyte is a positively charged polyelectrolyte selected form the group consisting of poly-amino acid composite, block co-polymers of poly amino acids and poly-ethylene glycol, poly-arginine, poly-lysine, and a fluorescently labeled derivative thereof. [0073] In some embodiments, the first polyelectrolyte comprises a negative charge. In some embodiments, the first polyelectrolyte is negatively charged.
  • the first polyelectrolyte is a negatively charged polyelectrolyte selected form the group consisting of hyaluronic acid, poly-glutamate, polyaspartic acid, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, nucleic acids, and a fluorescently labeled derivative thereof.
  • first polyelectrolyte is poly-arginine. In some embodiments, first polyelectrolyte comprises poly-arginine.
  • the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.05 to 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. [0076] In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.1 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.1 to 0.15 weight equivalents of poly-arginine in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises between about 0.15 to 0.2 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.2 to 0.2.5 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.25 to 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises between about 0.1 and about 0.3 weight equivalents of poly-arginine in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises between 0.05 to 0.1 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.1 to 0.15 weight equivalents of poly-arginine in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises between 0.1 and 0.2 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.15 to 0.2 weight equivalents of poly-arginine in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises between 0.2 to 0.2.5 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.25 to 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. [0078] In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.3 weight equivalents of the first poly electrolyte in relation to the nanoparticle. [0079] In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.1 weight equivalents of poly-arginine in relation to the nanoparticle.
  • the solution comprising the first polyelectrolyte comprises 0.1 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises less than 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. [0080] In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.05 to 0.8 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.05 to 0.8 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle.
  • the solution comprising the second polyelectrolyte comprises between about 0.05 to 0.1 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.1 to 0.15 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.15 to 0.2 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle.
  • the solution comprising the second polyelectrolyte comprises between about 0.35 to 0.4 weight equivalents of the second polyelectrolyte in relation to the first- layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.4 to 0.45 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.45 to 0.5 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle.
  • the solution comprising the second polyelectrolyte comprises between 0.05 to 0.1 weight equivalents of the second polyelectrolyte in relation to the first- layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.1 to 0.15 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.15 to 0.2 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle.
  • the second polyelectrolyte is a positively charged polyelectrolyte selected form the group consisting of poly-amino acid composite, block co-polymers of poly amino acids and poly- ethylene glycol, poly-arginine, poly-lysine, and a fluorescently labeled derivative thereof. [0084] In some embodiments, the second polyelectrolyte comprises a negative charge. In some embodiments, the second polyelectrolyte is negatively charged.
  • the solution comprising the second polyelectrolyte comprises about 0.2 weight equivalents of the second polyelectrolyte in relation to the first- layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.3 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.4 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.5 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle.
  • the solution comprising the second polyelectrolyte comprises about 0.6 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.7 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.8 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. [0086] In some embodiments, the solution comprising the second polyelectrolyte comprises 0.1 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle.
  • the solution comprising the second polyelectrolyte comprises 0.6 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises 0.7 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises 0.8 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. [0087] In certain embodiments, the second polyelectrolyte is selected from the group consisting of poly-glutamate, hyaluronic acid, polyaspartic acid, and polyacrylic acid.
  • the solution comprising the second polyelectrolyte comprises 0.35 to 0.4 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte about 0.4 to 0.5 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.45 to 0.5 weight equivalents of hyaluronic acid in relation to the nanoparticle. [0093] In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.4 weight equivalents of hyaluronic acid in relation to the nanoparticle.
  • the solution comprising the second polyelectrolyte comprises 0.4 weight equivalents of hyaluronic acid in relation to the nanoparticle.
  • the second polyelectrolyte is polyaspartic acid.
  • the solution comprising the second polyelectrolyte comprises about 0.2 to about 0.4 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.2 to about 0.25 weight equivalents of polyaspartic acid in relation to the nanoparticle.
  • the solution comprising the second polyelectrolyte comprises about 0.25 to about 0.3 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.3 to about 0.35 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.35 to about 0.4 weight equivalents of polyaspartic acid in relation to the nanoparticle. [0096] In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.2 to about 0.4 weight equivalents of polyaspartic acid in relation to the nanoparticle.
  • the solution comprising the second polyelectrolyte comprises 0.1 to 0.15 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.15 to 0.2 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.2 to 0.25 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.25 to 0.3 weight equivalents of polyacrylic acid in relation to the nanoparticle.
  • the metallic nanoparticle is gold. .In some embodiments, the nanoparticle comprises an empty core. In some embodiments, the nanoparticle comprises a liquid core. In some embodiments, the nanoparticle comprises a polymeric core. In certain embodiments, the nanoparticle comprises a polymeric micelle. In some embodiments, the nanoparticle comprises a polymersome. In some embodiments, the nanoparticle comprises a dendrimer core. In some embodiments, the nanoparticle comprises a lipid carrier. In some embodiments, the nanoparticle comprises a silver nanoparticle. In some embodiments, the nanoparticle comprises an iron oxide core. In some embodiments, the nanoparticle comprises a quantum dot. In some embodiments, the nanoparticle is mesoporous.
  • the nanoparticle is a polymeric nanoparticle. In some embodiments, the nanoparticle is a nanotube. In some embodiments, the nanoparticle is a quantum dot. In some embodiments, the nanoparticle is a micelle. In some embodiments, the nanoparticle is a lipid nano disc. In some embodiments, the nanoparticle is a liposome. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the nanoparticle is a solid nanoparticle. In some embodiments, the nanoparticle is a latex bead. In certain embodiments, the latex bead is carboxy-modified. In certain embodiments, the nanoparticle comprises a negative charge.
  • the at least one additional pharmaceutical agent comprises a protein or small molecule.
  • the protein is interleukin-12, interleukin-15 super agonist, interleukin 18, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , interleukin-2, anti-PD1 antibodies, anti-PDL1 antibodies, anti-CTLA4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-NKG2A antibodies, anti-CD73 antibodies, anti-A2aR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, single-chain interleukin-12, tumor necrosis factor alpha, interleukin- 10, interleukin-8, TNF-related apoptosis-inducing ligand (TRAIL), or FMS-like tyrosine kinase 3 ligand (FLT3LG).
  • TRAIL TNF-related apoptosis-inducing ligand
  • FLT3LG FMS-like tyrosine kinas
  • the protein is a cytokine.
  • the at least one additional pharmaceutical agent comprises a cytokine.
  • the at least one additional pharmaceutical agent comprises IL-12.
  • the additional pharmaceutical agent is a small molecule.
  • the small molecule is Oxaliplatin, Doxorubicin, Paclitaxel, Lurbinectedi, Mitomycin, Trabectedin, Lobenguane, Lutetium, Radium, Cisplatin, or Sorafenib.
  • the layered nanoparticle comprises a targeting moiety.
  • the method does not comprise purification by ultrafiltration.
  • the method does not comprise purification by tangential flow filtration, size exclusion chromatography, or dialysis.
  • NPs are mixed with polyelectrolytes using commercially available bifurcating mixer MCF cartridges.
  • said microfluidic mixing comprises the use of at least one microfluidic chip.
  • the at least one microfluidic mixing chip comprises a bifurcating channel.
  • said microfluidic mixing comprises the use of at least two microfluidic chips comprising a bifurcating channel.
  • said microfluidic mixing comprises the use of at least three microfluidic chips comprising a bifurcating channel.
  • the at least one, two, or three microfluidic chip does not comprise a bifurcating channel.
  • the at least one microfluidic chip comprises a T-shaped channel.
  • the at least one microfluidic chip comprises a Y-shaped channel.
  • said microfluidic mixing comprises the use of at least two microfluidic chips comprising a herring bone-shaped channel.
  • said microfluidic mixing comprises the use of at least three microfluidic chips comprising a herring bone-shaped channel.
  • the at least one, two, or three microfluidic chip does not comprise a herring-bone shaped channel.
  • each microfluidic mixing step increases the nanoparticle size by at least 1nm. In certain embodiments, each microfluidic mixing step increases the nanoparticle in size by less than 50nm.
  • the layered nanoparticle has a polydispersity index of about 0.05 to about 0.15. In some embodiments, the layered nanoparticle has a polydispersity index of about 0.05 to about 0.1. In some embodiments, the layered nanoparticle has a polydispersity index of about 0.1 to about 0.15. In some embodiments, the layered nanoparticle has a polydispersity index of 0.05 to 0.15.
  • the layered nanoparticle has a PDI of between 0 and 0.2. In some embodiments, the layered nanoparticle has a PDI of 0.2. In some embodiments, the layered nanoparticle has a PDI of 0.1. [00114] In certain embodiments, the layered nanoparticle has a polydispersity index of less than 0.25. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of between 0 and 0.3. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of between 0 and 0.1. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of between 0 and 0.2.
  • the wherein the layered nanoparticle has a polydispersity index of between 0.2 and 0.3. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of 0.1. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of 0.2. [00115] In certain embodiments, the layered nanoparticle is smaller than 400 nm in size. In certain embodiments, the layered nanoparticle is smaller than 300 nm in size. In certain embodiments, the layered nanoparticle is smaller than 200 nm in size. In certain embodiments, the layered nanoparticle is between 110-200 nm in size.
  • the solution comprising the nanoparticle has a concentration of 1 mg/mL. In some embodiments, the solution comprising the nanoparticle has a concentration of 5 mg/mL. In some embodiments, the solution comprising the nanoparticle has a concentration of 6 mg/mL. [00119] In certain embodiments, the solution comprising the nanoparticle has a concentration of at about 0.5 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of at about 1 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of at about 5 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 0.5 mg/mL and 5 mg/mL.
  • the solution comprising the nanoparticle has a concentration of between about 0.5 mg/mL and 1 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 1 mg/mL and 2 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 2 mg/mL and 3 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 3 mg/mL and 4 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 4 mg/mL and 5 mg/mL.
  • LbL assembly has previously been performed by alternating sequential incubations of the liposomal core particle with excess PLR and PLE in a low ionic strength buffer. Under these self-assembly conditions, each step of polyelectrolyte adsorption is accompanied by extensive washing/purification to remove excess unbound polymer from the coated particles before the next round of adsorption. [00125] To understand and optimize LbL assembly, the polymer:particle weight ratio variation during nanolayer deposition was assessed. Unlayered anionic IL-12 NPs were incubated with increasing PLR weight equivalents (wt.
  • PLR/NPs purified PLR-coated cationic IL- 12-NPs
  • TFF-LbL-NP MCF-LbL-NP (IL-12 PLR/PLE) (IL-12 PLR/PLE) # -avg (nm) 76.2 9.24 (12) 68.6 7.34 (11) Zeta Potential (mV) 8.38 (17) 4.07 (8.2) [00139] As some LbL-NP assemblies benefit from solution conditions during layering beyond deionized water (DI) such as in PLE/PLR-IL-12-NPs, the MCF strategy was employed in buffer conditions. Firstly, the varying PLR wt. eq.
  • MCF LbL-NPs maintain desired particle properties in vitro and maintain IL-12- LbL-NP efficacy in vivo in a metastatic ovarian cancer mouse model.
  • Benefits of PLE/PLR coating on NPs is increased ovarian cancer cell affinity and prevention of NP endocytosis leading to high retention of the LbL-NPs on the cancer cell membrane.
  • PLE/PLR-IL-12-NPs generated via the titrated polymer-to-NP wt. eq. had the same desired traits in vitro.
  • Fluorescent NPs were administered to a murine metastatic ovarian cancer cell line, OV2944-HM1 (HM-1), and the amount of NPs associated with the cells after 4 and 24 hours were quantified.
  • HM-1 murine metastatic ovarian cancer cell line
  • MCF LbL-NPs and TFF-based LbL- NPs had significantly increased HM-1 cell association compared to unlayered (UL) NPs (FIG. 5A).
  • MCF LbL-NPs have increased stability in serum-supplemented buffer.
  • serum components can impact stability LbL-NPs from aggregating.
  • particles were incubated in HEPES saline buffer (15 mM HEPES, 150 mM NaCl) supplemented with 10% fetal bovine serum (FBS). As shown in FIG.
  • CML beads were layered with varying core sizes with PLR and PLE.
  • Smaller CML particles required larger wt. eq. to reach the POP given their higher surface area to mass ratios (FIG. 9D).
  • FIG. 9E PLR/PLE coated CML beads of varying sizes were readily assembled.
  • TEM confirmed that particles maintained their homogeneity (FIG. 12).
  • Example 8 [00146] The experiments above carried out layering in deionized water, but LbL assembly is also sometimes carried out in the presence of salts to create LbL films with thicker layers. Thus, it was assessed whether LbL-NPs could be generated in the presence of salt-containing buffers.
  • anionic liposomes devoid of IL-12 with a composition optimized for siRNA loading and delivery from LbL-NPs were used.
  • First varying PLR:liposome mass ratios were examined to find the POP of PLR required for LbL assembly in 25 mM HEPES and 20 mM sodium chloride (NaCl), a buffer composition previously found optimal for loading of siRNA into LbL films. Around 0.15 wt. eq.
  • the LbL platform is a technique to modulate the surface properties of NPs for the development of therapeutic drug carriers.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • POPG 1-palmitoyl-2- oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt)
  • POPG 1,2-distearoyl-sn- glycero-3-phospho-(1'-rac-glycerol) (sodium salt)
  • DSPG 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (sodium salt) (18:1 MPB-PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (DOPE-DBCO), and cholesterol were purchased from Avanti Polar Lipids.
  • DOPE 1,2-distearoyl-sn-glycero-3-
  • Hyaluronic acid (HA, 20 kDa) was purchased from Lifecore Biomedical.
  • Poly-L-acrylic acid (PAA, 15 kDa) was purchased from Sigma Aldrich.
  • Yellow fluorescent carboxy-modified latex (CML) beads (Fluorospheres) with diameters of 20 nm, 40 nm, 100 nm and 200 nm were purchased from ThermoFisher Scientific.
  • Recombinant single-chain IL-12 production Single-chain IL-12 sequence was synthesized as a genomic block (Integrated DNA Technologies) and cloned into a gWIZ expression vector (Genlantis). Plasmids were transiently transfected into Expi293 cells (ThermoFisher Scientific).
  • IL-12 conjugated liposome synthesis Lipid solutions composed of 65 mol% DSPC, 24 mol% Cholesterol, 6 mol% POPG and 5 mol% of MPB-PE were made in chloroform and then dried into a thin film using a rotovap.
  • IL-12 free liposomes were made with a 33 mol% DSPC, 33 mol% DSPG, and 33 mol% cholesterol and hydrated and extruded as described previously.
  • Microfluidics LbL assembly Microfluidic mixing was performed on NanoAssemblr IgniteTMNxGenTM cartridge (NIN0061) that uses the NxGenTM mixing (Precision Nanosystems) chip. Luer lock syringes were separately filled with either polymer solution or the liposomes and attached to the microfluidics chip. A syringe pump (Pump 11 Elite, Harvard Apparatus) was then used to control the fluid flow rate into the mixing chip. With the exception of the flow rate studies, assembly was performed with flow rates of 9 mL/min (per channel).
  • PLR-TR was purified via Reverse-Phase High Pressure Liquid Chromatography (RP- HPLC) on a Jupiter C4 column (5 ⁇ m particles, 300 ⁇ – Phenomenex) using a water:acetonitrile gradient which started at 20% acetonitrile for 5 minutes, then increased to 35% in a linear gradient until 10 minutes. Isocratic elution at 35% was performed for 30 minutes then the elution buffer was increased to 95% to clean out the column for 10 minutes then dropped back to 20% acetonitrile to re-equilibrate the column for 5 minutes. After HPLC purification, the polymer-dye conjugated was then lyophilized and stored at -20 °C.
  • RP- HPLC Reverse-Phase High Pressure Liquid Chromatography
  • PLE was reacted with 5 molar excess of sulfo-cy3-NHS-ester (Lumiprobe) in pH 9 PBS (adjusted by adding 0.1 M sodium bicarbonate) for 3 hours at room temperature then left overnight at 4 °C. Excess dye was removed via extensive dialysis (3 kDa, Spectrum) against 0.9% NaCl then dialyzed against DI to remove salts. The resulting PLE-cy3 polymer was lyophilized and stored at -20 °C.
  • IL-12 NPs were generated via either the standard TFF- based LbL protocol or the MCF protocol, but with the PLE layering solution having 50% PLE- cy3. Free polymers were separated from NPs on a 300 kDa centrifugal filter (Vivaspin500, Sartorius) at 30 xg for 20 min. Polymer fluorescence on the permeate fluid was then compared to that of the initial sample to determine the fraction of polymer bound to NPs.
  • HM-1 cells were plated on a tissue-culture 96-well plate at a density of 50k cells per well. The next day, wells were dosed with NPs to 0.05 mg/mL and left for the target incubation time (4 hrs or 24 hrs). For analysis of association, the supernatant was removed from the well and diluted 10X with DMSO. Cells were then washed three times with PBS then dissolved with DMSO. Fluorescence of NPs associated with cells was then normalized to supernatant fluorescence. The relative fluorescence of each formulation was then compared to an unlayered liposome control containing the same fluorophore as described previously.
  • HM-1 cells were plated onto wells at a density of 10k/well and left to adhere overnight prior to NP treatment. After the desired incubation time with NPs, cells were washed 3x with PBS. After washing, cells were fixed in 4% paraformaldehyde for 10 minutes then washed (3x with PBS) and stained with wheat germ agglutinin (WGA) conjugated to Alexa Fluor488 (Invitrogen) and Hoechst 33342 (Thermo Scientific) following manufacturer instructions. Images were analyzed using ImageJ. Slides were imaged on an Olympus FV1200 Laser Scanning Confocal Microscope. [00172] Mice: B6C3F1 mice were purchased from Jackson Laboratories.
  • mice were randomized into treatment groups. At days 7 and 14 post- inoculation, mice were treated intraperitoneally with vehicle (5% dextrose) or 20 ⁇ g of IL-12 either as a free cytokine, or conjugated to unlayered (UL) NPs, or LbL-NPs. Mice weights were tracked daily after treatments for signs of toxicity. Bioluminescence was tracked for 30 days after tumor inoculation or as needed to evaluate tumor burden. [00174] Statistical Analysis: GraphPad PRISM 9 was used to perform statistical analyses. Comparisons between two groups was performed via unpaired t-tests.
  • NP colloidal stability under increasing ionic strength Particles were diluted to 0.1 mg/mL in water buffered with 10 mM HEPES (pH 7.3) and assessed for their size via DLS. A 1 M NaCl stock solution was then used to adjust the salt concentration in the sample. After salt addition, samples were left to incubate for 10 minutes before size measurement on a DLS. After size measurement, the amount of NaCl was further increased and the sample again allowed to incubate for 10 minutes. This process was repeated until reaching super physiological levels of NaCl (i.e., > 200 mM NaCl).
  • NP colloidal stability in serum supplemented buffer Particles were diluted to 0.1 mg/mL in a solution containing 15 mM HEPES (pH 7.3), 150 mM NaCl, and 10% FBS. The overall size of the sample was evaluated via DLS.
  • Recombinant single-chain IL-12 production Single-chain IL-12 sequence was synthesized as a genomic block (Integrated DNA Technologies®) and cloned into a gWIZ expression vector (Genlantis®). Plasmids were transiently transfected into Expi293TM cells (ThermoFisher Scientific).
  • a method of making a composition comprising a layered nanoparticle comprising: microfluidic mixing of a solution comprising a nanoparticle with a solution comprising a first polyelectrolyte to form a solution comprising a first-layered nanoparticle.
  • Embodiment 2 The method of embodiment 1, further comprising: microfluidic mixing of the solution comprising the first-layered nanoparticle with a solution comprising a second polyelectrolyte to form a solution comprising a second-layered nanoparticle.
  • the first polyelectrolyte is poly-arginine.
  • Embodiment 11 The method of embodiment 10, wherein the solution comprising the first polyelectrolyte comprises less than 0.3 weight equivalents of poly-arginine in relation to the nanoparticle.
  • Embodiment 12. The method of embodiment 11, wherein the solution comprising the first polyelectrolyte comprises between 0.1 and 0.2 weight equivalents of poly-arginine in relation to the nanoparticle.
  • Embodiment 13 The method of embodiment 11, wherein the solution comprising the first polyelectrolyte comprises 0.1 weight equivalents of poly-arginine in relation to the nanoparticle.
  • Embodiment 15 The method of embodiment 14, wherein the solution comprising the second polyelectrolyte comprises less than 0.6 weight equivalents of poly-glutamate in relation to the nanoparticle.
  • Embodiment 16 The method of embodiment 15, wherein the solution comprising the second polyelectrolyte comprises between 0.6 and 0.4 weight equivalents of poly-glutamate in relation to the nanoparticle.
  • Embodiment 17. The method of embodiment 16, wherein the solution comprising the second polyelectrolyte comprises 0.5 weight equivalents of poly-glutamate in relation to the nanoparticle.
  • the composition further comprises at least one additional pharmaceutical agent.
  • Embodiment 20 The method of embodiment 19, wherein the at least one additional pharmaceutical agent comprises a protein, or fragment thereof, a small molecule, or a nucleotide.
  • Embodiment 21 The method of embodiment 19 or 20, wherein the at least one additional pharmaceutical agent comprises a protein.
  • Embodiment 22 The method of any one of embodiments 19-21, wherein the at least one additional pharmaceutical agent comprises a cytokine.
  • Embodiment 23 The method of any one of embodiments 19-22, wherein the at least one additional pharmaceutical agent comprises IL-12.
  • Embodiment 24 The method of any one of embodiments 1-23, wherein the method does not comprise purification by ultrafiltration.
  • Embodiment 25. The method of any one of embodiments 1-24, wherein said microfluidic mixing comprises the use of at least one microfluidic chip.
  • Embodiment 26. The method of embodiment 25, wherein the at least one microfluidic chip comprises a herring bone-shaped channel.
  • Embodiment 27. The method of any one of embodiments 2-26, wherein said microfluidic mixing comprises the use of at least two microfluidic chips comprising a herring bone-shaped channel.
  • Embodiment 28. The method of embodiment 27, wherein the at least two microfluidic chips are connected in series.
  • each microfluidic mixing step independently comprises a flow rate of at least 1 mL/min.
  • Embodiment 30 The method of any one of embodiments 1-29, wherein each microfluidic mixing step independently comprises a flow rate of at least 3 mL/min.
  • Embodiment 31 The method of any one of embodiments 1-30, wherein the method does not comprise sonication or agitation.
  • Embodiment 32 The method of any one of embodiments 1-31, wherein the layered nanoparticle has a polydispersity index of less than 0.25.
  • Embodiment 33 The method of any one of embodiments 1-32, wherein the layered nanoparticle is smaller than 200 nm in size.
  • Embodiment 34 The method of any one of embodiments 1-328, wherein each microfluidic mixing step independently comprises a flow rate of at least 1 mL/min.

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Abstract

Surface modification of nanoparticles (NPs) via the layer-by-layer (LbL) technique is a approach to generate targeted drug delivery vehicles. A simple and scalable synthesis method for LbL-NPs that can be adapted for clinical translation is of great interest. Presented herein is a robust and scalable method of polymer deposition onto nanoparticles.

Description

SCALABLE AND HIGH THROUGHPUT ASSEMBLY OF LAYER-BY- LAYER NANOPARTICLES RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application 63/569,409 filed March 25, 2024, the entirety of which is incorporated herein by reference. GOVERNMENT SUPPORT [0002] This invention was made with government support under CA235375, CA274651, AI161297, AI161818, and AI048240 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND [0003] Spatiotemporal control of drug delivery has been a major objective of new therapeutic platforms. Nanoparticles (NPs) have been extensively studied as drug carriers to alter the pharmacokinetics and pharmacodynamics of therapeutics, with engineering of NP surface chemistry representing an important strategy to optimize delivery of drugs to disease sites and/or avoid off-target tissue uptake. One approach towards to engineer surface chemistry is by layer-by-layer (LbL) assembly of polymer coatings on nanoparticles. The LbL technique consists of the deposition of nanoscale polymer films on surfaces through alternating adsorption of molecular layers of complementary polymers, most typically polycations and polyanions. The resulting multilayer assemblies, which can be several nanometers in thickness, can enable controlled drug release as well as modulation of surface properties and the resulting interactions with biological interfaces. Based on the versatility of this approach, the utility of LbL-NPs for targeted drug delivery and controlled drug release has been successfully demonstrated in many applications. Assembly of polymer films on NPs can yield increased NP plasma half-life and stability, tumor targeting, and control over the subcellular localization of NPs. Moreover, LbL-NPs can be used to effectively deliver small-molecule therapeutics and nucleic acids which can be loaded into the polymer film in the NP. The high versatility of this platform approach enables the facile assembly of combination therapies within a single particle to target diverse diseases. [0004] A challenge of LbL-NP synthesis is the serial nature of the layer deposition process. While improvements have been made in LbL-NP assembly methods at the lab scale, these approaches have still required multiple purification steps and/or hard-to-scale methods of mixing polymers and NPs. This includes the use of sonication or vortex mixers coupled with centrifugation of membrane-based purification approaches that suffer from potential interactions of NPs with the membrane. In recent years, there have been advancements in implementing streamlined and scalable LbL assembly via fluidics due to its high degree of control and reproducibility. However, the methods used have been highly scale-dependent, such as size exclusion of particles from channels, magnetic diversion of particles, or fine-tuned microparticle retention in fluidized beds. This scale requirement has precluded many of these LbL techniques from being extended to NPs. Some newer approaches have been applied to nanomaterials but require heating which alters the polymer structure and potentially damages the NP. To this end, the present disclosure provides a scalable and high throughput method to the assembly of LbL NPs. SUMMARY OF THE INVENTION [0005] In one aspect, provided herein is a method of making a composition comprising a layered nanoparticle, comprising: a first step of microfluidic mixing of a solution comprising a nanoparticle with a solution comprising a first polyelectrolyte to form a solution comprising a first-layered nanoparticle. [0006] In another aspect, provided herein is a kit comprising: a solution comprising a nanoparticle; a solution comprising a first polyelectrolyte; a solution comprising a second polyelectrolyte; one or more microfluidic mixing chips; and instructions for its use. BRIEF DESCRIPTION OF THE DRAWINGS [0007] The accompanying drawings, which constitute a part of this specification, illustrate several embodiments of the invention and together with the description, provide non-limiting examples of the invention. [0008] FIGs. 1A-1D show schematics of nanoparticle assembly and the characterization of nanoparticle surface charge conversion upon polyelectrolyte deposition. FIG. 1A shows a schematic for small-scale deposition of polymers onto interleukin-12 (IL-12) loaded NPs. FIG. 1B shows intensity-weighted particle size (Z-avg), number-averaged (#-avg) size, polydispersity index (PDI), and zeta potential of the resulting polymer-nanoparticle mixtures at increasing PLR weight equivalents to bare anionic IL-12-NP. FIG. 1C shows Z-avg, #- avg, PDI, and zeta potential of the resulting polymer-nanoparticle mixtures from increasing weight equivalent PLE to PLR-coated IL-12 NPs. FIG. 1D a schematic representing the different possible resulting assemblies upon increasing polymer weight equivalents. [0009] FIGs. 2A-2E show that excess polymers prevent NP aggregation during layering via optimized bath sonication protocol. FIG. 2A shows a schematic for optimized LbL synthesis protocol using bath sonication and excess polymers followed by TFF. FIG. 2B shows Z-avg size, PDI and zeta potential of IL-12 NPs during LbL assembly. FIG. 2C shows polymer-to- NP wt. eq. retained with the NPs after TFF purification. FIG. 2D shows Z-avg size, PDI and zeta potential of the resulting PLR-IL-12-NPs generated via different layering strategies. FIG. 2E shows Z-avg size, PDI and zeta potential of the resulting PLR/PLE-IL-12-NPs generated via different layering strategies on TFF purified PLR-IL-12-NPs. (mean ± s.d.). [0010] FIGs. 3A-3I show microfluidic fluid mixing chips enable homogeneous LbL-NP assembly without the need of purification steps. FIG. 3A shows Z-avg size and PDI of target PLE/PLR-IL-12-NPs compared to small scale bath sonication mixing of IL-12 NPs with titrated polymer-to-NP wt. eq. of PLR and PLE. FIG. 3B shows a schematic for polymer deposition onto NPs via microfluidics chip. FIG.3C shows Z-avg size and PDI, of NPs layered with plateau onset point (POP) PLR using a microfluidics chip with increasing channel fluid flow rates (both polymer and NP channels had equal flow rates). Shaded regions indicate target NP measurements from optimal layering conditions for the current NP formulation. FIG. 3D shows Zeta potential measurements of particle mixtures from FIG. 3C. FIG. 3E shows a schematic for LbL-NP assembly using titrated polymer-to-NP wt. eq. without the need of purification steps with a 30 min incubation step. FIG. 3F shows Z-avg size, PDI, and zeta potential of the resulting PLR-IL-12-NPs generated using titrated PLR-to-NP wt.eq. via microfluidic (MCF) mixing compared to target NP measurements. FIG.3G shows Z-avg size, PDI, and zeta potential of the resulting PLE/PLR-IL-12-NPs generated using titrated PLR-to- NP wt.eq. then titrated PLE-to-NP wt. eq. via MCF compared to target NP measurements. FIG. 3H shows fraction of fluorescently labeled PLE polymer associated with NPs after optimized TFF-based layering before and after TFF purification compared to titrated polymer layering using MCF. FIG. 3I shows negative stain transmission electron microscopy (TEM) of unlayered (UL) IL-12 NPs or PLE/PLR-IL-12-NPs assembled via either the optimized TFF- based protocol or the titrated polymer-to-NP wt. eq. MCF protocol. Scale bars represent 100 nm (mean ± s.d.). [0011] FIGs. 4A-4E show that microfluidic fluid mixing chips enable homogeneous LbL-NP assembly in optimized buffer solutions. FIG. 4A shows Z-avg size and PDI of NPs layered with increasing PLR-to-NP wt. eq. in 25 mM HEPES and 20 mM NaCl. FIG. 4B shows Zeta potential of NPs layered with increasing PLR-to-NP wt. eq. in 25 mM HEPES and 20 mM NaCl. FIG. 4C shows a schematic, Z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using small-scale bath sonication mixing of polymers and NPs. FIG. 4D shows a schematic, Z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using large- scale bath sonication mixing of polymers and NPs. FIG. 4E shows a schematic, Z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using MCF mixing of polymers and NPs. [0012] FIGs. 5A-5D shows MCF-LbL-NPs maintain the desired in vitro characteristics of PLE/PLR LbL-NPs. FIG. 5A shows the fluorescence ratio of NPs associated with HM-1 cells relative to NPs remaining in the supernatant at 4 and 24 hours after dosing (median). FIG.5B. shows representative confocal microscopy of HM-1 cells dosed with UL NPs for 24 hours. FIG.5C shows representative confocal microscopy of HM-1 cells dosed with TFF-based LbL- NPs for 24 hours. FIG. 5D shows representative confocal microscopy of HM-1 cells dosed with MCF-based LbL-NPs for 24 hours. Group statistical comparisons was performed using two-way ANOVA with Tukey’s multiple-comparisons test. Asterisks denote p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. [0013] FIGs. 6A-6E show MCF-LbL-NPs maintain the bioactivity of IL-12 in vitro and in vivo effectiveness in a metastatic mice model of ovarian cancer. FIG.6A shows the HEK-Blue IL-12 reporter cell line response curves for free IL-12 or IL-12 loaded onto LbL-NPs generated via either TFF-based or MCF-based LbL assembly. FIG. 6B shows the calculated EC50 of curves in FIG.6A. FIG. 6C shows in vivo treatment scheme of metastatic ovarian cancer. IL- 12 doses of 20 µg were given on days 7 and 14 for each construct FIG.6D shows whole animal bioluminescence reading via IVIS of luciferase expressing tumors cells from mice treated as in FIG. 6C. (mean ± s.e.m.; n = 5 for Dextrose, IL-12 and UL and n = 8 for TFF-LbL and MCF- LbL). FIG. 6E shows survival curves of mice treated as in FIG. 6C. Statistical comparisons between survival curves were performed using a log-rank (Mantel–Cox) test. (mean ± s.d.) asterisks denote p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. [0014] FIG. 7 shows MCF LbL-NPs, TFF-based LbL-NPs (STD), and unlayered NPs (UL) colloidal stability data in NaCl solution. Colloidal stability is measured by dynamic light scattering (DLS). [0015] FIG. 8 shows MCF LbL-NPs, TFF-based LbL-NPs (STD), and unlayered NPs (UL) colloidal stability data in physiological saline buffered solution supplemented with serum. [0016] FIGs.9A-9G show MCF-LbL-NPs can be assembled with varying polymer chemistries and NP cores. FIG. 9A shows zeta potential of PLR-IL-12-NPs mixed with varying wt. eq. of HA, PLD, or PAA polymers. Arrows indicate POP wt. eq. FIG.9B shows Z-avg size and PDI of final HA, PLD, or PAA-coated IL-12 NPs after MCF assembly. FIG. 9C shows Zeta potential of particles from FIG. 9B. FIG. 9C shows zeta potential of CML NPs of varying sizes mixed with increasing wt. eq. of PLR. Zeta potential values at 0.01 wt. eq are from UL CML. FIG. 9E shows CML NPs of varying sizes and their final MCF-assembled LbL-NPs with PLR/PLE films. FIG. 9F shows flow cytometry median fluorescence intensity (MFI) of HM-1 cells incubated with CML NPs at 10 µg/mL for 4 hours. FIG. 9G shows correlation between the log-fold change in CML NP MFI of LbL-NPs relative to UL-NPs to log of the estimated total available NP surface area (SA) in solution [0017] FIG. 10 shows TEM micrographs of unlayered IL-12 NPs and LbL NPs assembled via either TFF or MCF. [0018] FIG. 11 shows Comparison of dynamic light scattering (DLS) electrophoretic mobility results from independent IL-12-LbL-NP batches generated via either optimized TFF-based (with excess polymers) or MCF-based (using POP wt. eq.). [0019] FIG. 12 shows TEM micrographs of carboxy-modified latex (CML) beads unlayered or layered with PLR/PLE. [0020] FIGs. 13A-13E show microfluidic fluid mixing chips enable homogeneous LbL-NP assembly in optimized buffer solutions. FIG.13A shows the z-avg size and PDI of NPs layered with increasing PLR-to-NP wt. eq. in 25 mM HEPES and 20 mM NaCl (mean ± s.d.). FIG. 13B shows the zeta potential of NPs layered with increasing PLR-to-NP wt. eq. in 25 mM HEPES and 20 mM NaCl (mean ± s.d.). FIG.13C shows the z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using small-scale bath sonication mixing of polymers and NPs (mean ± s.d.). FIG.13D shows the z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using large-scale bath sonication mixing of polymers and NPs (mean ± s.d.). FIG. 13E shows the z-avg size, PDI, and zeta potential of NPs layered with PLR and PLE using MCF mixing of polymers and NPs (mean ± s.d.). [0021] FIG. 14 shows a flow chart comparing previous methods of assembling LbL-NPs to the MCF method provided herein. DEFINITIONS [0022] As used herein, the term “microfluidic mixing” encompasses methods which enable rapid and efficient mixing through the manipulation of fluids in channels or layers of small dimensions. In some embodiments, the microfluidic mixing can utilize micro-scale channels. In certain embodiments, the microfluidic mixing can utilize milli-scale channels. In some embodiments, the microfluidic mixing can utilize micro-scale layers. In certain embodiments, the microfluidic mixing can utilize milli-scale layers. In some embodiments the microfluidic mixing occurs in a bifurcated mixer, herringbone mixer, T-mixer, or jet mixers (i.e., coaxial or impinging). In certain embodiments, microscale mixing can be scaled up according to production needs. [0023] The term “particle” refers to a small object, fragment, or piece of a substance that may be a single element, inorganic material, organic material, or mixture thereof. Examples of particles include polymeric particles, single-emulsion particles, double-emulsion particles, coacervates, liposomes, microparticles, nanoparticles, macroscopic particles, pellets, crystals, aggregates, composites, pulverized, milled or otherwise disrupted matrices, and cross-linked protein or polysaccharide particles, each of which have an average characteristic dimension of about less than about 1 mm and at least 1 nm, where the characteristic dimension, or “critical dimension,” of the particle is the smallest cross-sectional dimension of the particle. A particle may be composed of a single substance or multiple substances. In certain embodiments, the particle is not a viral particle. In other embodiments, the particle is not a liposome. In certain embodiments, the particle is not a micelle. In certain embodiments, the particle is substantially solid throughout. In certain embodiments, the particle is a nanoparticle. In certain embodiments, the particle is a microparticle. [0024] The term “nanoparticle” refers to a particle having an average (e.g., mean) dimension (e.g., diameter) of between about 1 nanometer (nm) and about 1 micrometer (µm) (e.g., between about 1 nm and about 400 nm, about 1 nm and about 300 nm, between about 1 nm and about 100 nm, between about 1 nm and about 30 nm, between about 1 nm and about 10 nm, or between about 1 nm and about 3 nm), inclusive. Nanoparticles described herein were constructed using methods previously described by Correa, S., et al. ACS Nano 13, 5623– 5634 (2019). [0025] The term “polyelectrolyte” as used herein, refers to a polymer which under a particular set of conditions (e.g., physiological conditions) has a net positive or negative charge. In some embodiments, the polyelectrolyte is or comprises a polycation. In some embodiments, a polyelectrolyte is or comprises a polyanion. Polycations have a net positive charge and polyanions have a net negative change. The net charge of a given polyelectrolyte may depend on the surrounding chemical conditions (e.g., pH). Exemplary polyelectrolytes for use in polymeric coatings in the method of this disclosure include but are not limited to: poly(L-arginine) (PLR), poly-L-glutamic acid (PLE), polyarginine, polyglutamic acid, polylysine, hyaluronic acid (HA), poly-L-aspartate, polyaspartic acid, polyaminoacid composites, block co-polymers of poly amino acids and poly-ethylene glycol, polyacrylic acid, dextran sulfate, heparin folate, heparin sulfate, D-amino acid polymers or L/D amino acid mixtures, nucleic acids, fucoidan, sulfated-β-cyclodextran, polyglutamic acid-block- polyethylene glycol, polystyrene sulfonate (SPS), linear poly(ethylene imine), poly(diallyldimethyl ammonium chloride). Polyallylamine hydrochloride, poly(L-lactide-co- L-lysine, polyserine ester, poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid], sodium polystyrene sulfonate, alginate, and chondroitin sulfate. [0026] As used herein, the phrase “substantially free” refers to a solution with reduced amounts of free, or unbound, polyelectrolyte. In some embodiments, substantially free means the solution does not need to undergo purification to remove free or unbound polyelectrolyte. In some embodiments, substantially free indicates less than about 5%, 4%, 3,%, 2%, or 1% of free polyelectrolyte in solution. In some embodiments, substantially free indicates less than about 5% free polyelectrolyte in solution. In some embodiments, substantially free indicates an undetectable amount of free polyelectrolyte in solution. [0027] The terms “agent,” “therapeutic agent,” or “pharmaceutical agent” are used herein to refer to any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound may be any agent that can be represented by a chemical formula, chemical structure, or sequence. Example of agents, include, e.g., small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids, polysaccharides, etc. In general, agents may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the agent. An agent may be at least partly purified. In some embodiments, an agent may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the agent, in various embodiments. In some embodiments, an agent may be provided as a salt, ester, hydrate, or solvate. In some embodiments, an agent is cell- permeable, e.g., within the range of typical agents that are taken up by cells and acts intracellularly, e.g., within mammalian cells, to produce a biological effect. Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)-isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated. Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates [e.g., with water (i.e. hydrates) or common solvents] and/or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable. [0028] A “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds. The term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, synthetic, or any combination of these. In some embodiments, a protein can be tethered to a particle. Exemplary proteins include but are not limited to interleukin-12, interleukin-15 super agonist, interleukin 18, interferon-α, interferon-β, interferon-ϒ, interleukin-2, anti-PD1 antibodies, anti-PDL1 antibodies, anti-CTLA4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti- NKG2A antibodies, anti-CD73 antibodies, anti-A2aR antibodies, anti-B7-H3 antibodies, anti- B7-H4 antibodies, single-chain interleukin-12, tumor necrosis factor alpha, interleukin-10, interleukin-8, TNF-related apoptosis-inducing ligand (TRAIL), FMS-like tyrosine kinase 3 ligand (FLT3LG). [0029] As used herein, a “polymer” refers to a compound comprised of at least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeating covalently bound structural units. In certain embodiments, a polymer is naturally occurring. In certain embodiments, a polymer is synthetic (i.e., not naturally occurring). In certain embodiments, the term “polymer” is used interchangeably with “polyelectrolyte.” [0030] As used herein, a “small molecule” refers to an organic molecule with a molecular weight of less than 800 g/mol (e.g., less than 700 g/mol, less than 600 g/mol, less than 500 g/mol, less than 400 g/mol, less than 300 g/mol, less than 200 g/mol, less than 100 g/mol, between 50 to 800 g/mol, inclusive, between 100 to 800 g/mol, inclusive, or between 100 to 500 g/mol, inclusive). In certain embodiments, the small organic molecule is a therapeutically active agent such as a drug (e.g., a small organic molecule approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)). The small organic molecule may also comprise a metal. In some embodiments, small molecules can be tethered to a particle. Exemplary small molecules which can be tethered to a particle include but are not limited to Oxaliplatin, Doxorubicin, Paclitaxel, Lurbinectedi, Mitomycin, Trabectedin, Lobenguane, Lutetium, Radium, Cisplatin, or Sorafenib. [0031] The terms “nucleic acid” or “nucleic acid sequence,” “nucleic acid molecule,” “nucleic acid fragment” or “polynucleotide” may be used interchangeably with “gene,” “mRNA encoded by a gene,” and “cDNA”. [0032] As used herein the terms “plateau point,” “plateau onset point” or “POP” are used interchangeably and refer to the point at which complete conversion of surface charge of the layered nanoparticle is achieved (FIG. 1C-1D). In some embodiments, “titrated amount,” or “POP amount” refers to the amount of polyelectrolyte needed to achieve charge conversion and/or plateau. This value can be determined by titration experiments, such as those described herein, or calculated as described herein (Example 9), to identify the minimum amount of polyelectrolyte needed to achieve reach the POP (i.e., to complete conversion of surface charge of the layered nanoparticles). Charge conversion is indicated by the complete reversal of surface charge (e.g., negative to positive or positive to negative, measured by zeta potential). [0033] As utilized herein, the term “excess” polyelectrolyte refers to the amount of polyelectrolyte, in weight equivalents relative to the nanoparticle, which surpasses the titration amount or plateau point of polyelectrolyte. In some embodiments, excess is at least 0.3 wt. eq. of polyarginine. In some embodiments, excess polyelectrolyte is greater than 0.2 wt. eq. of polyarginine. In some embodiments, excess is at least 1 wt. eq. of polyglutamate. In some embodiments, excess polyelectrolyte is greater than 0.6 wt. eq. of polyglutamate. [0034] The terms “assess,” “determine,” “evaluate,” and “assay” are used interchangeably herein to refer to any form of detection or measurement, and include determining whether a substance, signal, disease, condition, etc., is present or not. The result of an assessment may be expressed in qualitative and/or quantitative terms. Assessing may be relative or absolute. “Assessing the presence of” includes determining the amount of something that is present or determining whether it is present or absent. [0035] Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, or more typically, within 5%, 4%, 3%, 2%, or 1% of a given value or range of values. [0036] As used herein “targeting moiety’” refers to a small molecule, protein, or fragment thereof which allows for or enhances binding to a target. [0037] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular. [0038] “Dispersity” (D) as used herein is a measure of the distribution of molecular mass in a given polymer sample and is calculated by dividing the weight average molar mass (Mw) by the number average molar mass (Mn). The dispersity of a given sample can have a value equal to or greater than 1. As the polymer chains approach uniform chain length, the dispersity approaches unity (1). The dispersity of a polymer can be modified, for example, using polymer fractionation (e.g., preparative SEC, Baker-Williams fractionation, continuous spin fractionation), or modifying the work-up procedure (e.g., by partially dissolving a polymer, an insoluble high molar mass fraction may be filtered off resulting in a large reduction in Mw and a small reduction in Mn, thus reducing polydispersity). [0039] As used herein, the term “salt” or “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1–19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2–hydroxy–ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2– naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3–phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p–toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1–4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt. [0040] The terms “composition” and “formulation” are used interchangeably. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS [0041] The present disclosure demonstrates a novel approach for multilayer assembly onto NPs using microfluidic (MCF) mixing technology. LbL-NPs have been used in preclinical models for controlled drug release, tumor and immune cell targeting, to attempt to improve pharmacokinetics and biodistribution, and to attempt to control cellular trafficking and uptake mechanisms. Provided herein is a method for polymer deposition onto NPs enabled through MCF mixing. MCF is scalable, continuous, and readily implemented under cGMP conditions for clinical-grade NP production. In addition to increased process robustness, MCF allows for LbL electrostatic assembly using titrated polymer-to-NP weight equivalent ratios where no excess polymer is required to achieve a given LbL layering. Under such conditions, no time- consuming purification is needed, increasing LbL-NP throughput and avoiding the loss of NPs during purification. The utility of this system was demonstrated using interleukin-12 (IL-12)- loaded liposomal NPs layered with poly(amino acid) polymers which have shown promising therapeutic efficacy in preclinical mouse models of metastatic ovarian cancer. [0042] Using the provided methods, the inventors demonstrate herein equivalent efficacy in vitro and in vivo to LbL-NPs generated via traditional lab-scale batch-wise polymer adsorption and tangential flow filtration purification. The inventors further demonstrate herein that, by rational selection of polymer-to-NP ratios for surface charge conversion without the addition of excess polymers, this approach enables LbL films to be constructed without the need for time-consuming purification steps, greatly simplifying LbL-NP preparation. Moreover, it was shown that MCF can assemble LbL films of various chemistries and on various NP core substrates. [0043] The details of certain embodiments, of the invention are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the invention will be apparent from the Definitions, Examples, Figures, and Claims. It should be understood that the aspects described herein are not limited to specific embodiments, methods, or configurations, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting. [0044] In one aspect, provided herein is a method of making a composition comprising a layered nanoparticle, comprising: a first step of microfluidic mixing of a solution comprising a nanoparticle with a solution comprising a first polyelectrolyte to form a solution comprising a first-layered nanoparticle. [0045] In some embodiments, the method further comprises a second step of microfluidic mixing of the solution comprising the first-layered nanoparticle with a solution comprising a second polyelectrolyte to form a solution comprising a second-layered nanoparticle. [0046] In some embodiments, the first step results in charge conversion from the nanoparticle to the first-layered nanoparticle. [0047] In certain embodiments, the second step results in charge conversion from the nanoparticle to the second-layered nanoparticle. [0048] In another aspect, provided herein is a method of making a composition comprising a layered nanoparticle, comprising: microfluidic mixing of a solution comprising a nanoparticle with a solution comprising a first polyelectrolyte to form a solution comprising a first-layered nanoparticle; and microfluidic mixing of the solution comprising the first-layered nanoparticle, with a solution comprising a second polyelectrolyte to form a solution comprising a second-layered nanoparticle. [0049] In some embodiments, the first and second polyelectrolytes independently comprise one or more of hyaluronic acid, poly-glutamate, polyaspartic acid, poly-amino acid composite, block co-polymers of poly amino acids and poly-ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, nucleic acids, or a fluorescently labeled derivative thereof. In some embodiments, the first and second polyelectrolytes are different. In some embodiments, the first and second polyelectrolytes comprise opposite charges. [0050] In some embodiments, the first polyelectrolyte comprises hyaluronic acid, poly- glutamate, poly-aspartate, poly-amino acid composite, block co-polymers of poly amino acids and poly-ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, nucleic acids, or a fluorescently labeled derivative thereof. [0051] In some embodiments, the second polyelectrolyte comprises hyaluronic acid, poly- glutamate, poly-aspartate, poly-amino acid composite, block co-polymers of poly amino acids and poly-ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, nucleic acids, or a fluorescently labeled derivative thereof. [0052] In some embodiments, the first polyelectrolyte is hyaluronic acid, poly-glutamate, poly-aspartate, poly-amino acid composite, block co-polymers of poly amino acids and poly- ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, or nucleic acids. [0053] In some embodiments, the first polyelectrolyte comprises hyaluronic acid. In some embodiments, the first polyelectrolyte comprises poly-glutamate. In some embodiments, the first polyelectrolyte comprises poly-aspartic acid. In some embodiments, the first polyelectrolyte is poly-amino acid composite. In some embodiments, the first polyelectrolyte comprises block co-polymers of poly amino acids and poly-ethylene glycol. In some embodiments, the first polyelectrolyte comprises poly-arginine. In some embodiments, the first polyelectrolyte comprises poly-lysine. In some embodiments, the first polyelectrolyte comprises poly-acrylic acid. In some embodiments, the first polyelectrolyte comprises dextran sulfate. In some embodiments, the first polyelectrolyte comprises heparin sulfate. In some embodiments, the first polyelectrolyte comprises heparin folate. In some embodiments, the first polyelectrolyte comprises nucleic acids. In some embodiments, the second polyelectrolyte is hyaluronic acid, poly-glutamate, poly-aspartate, poly-amino acid composite, block co-polymers of poly amino acids and poly-ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, or nucleic acids. [0054] In some embodiments, the second polyelectrolyte comprises hyaluronic acid. In some embodiments, the second polyelectrolyte comprises poly-glutamate. In some embodiments, the second polyelectrolyte comprises poly-aspartate. In some embodiments, the second polyelectrolyte is poly-amino acid composite. In some embodiments, the second polyelectrolyte comprises block co-polymers of poly amino acids and poly-ethylene glycol. In some embodiments, the second polyelectrolyte comprises poly-arginine. In some embodiments, the second polyelectrolyte comprises poly-lysine. In some embodiments, the second polyelectrolyte comprises poly-acrylic acid. In some embodiments, the second polyelectrolyte comprises dextran sulfate. In some embodiments, the second polyelectrolyte comprises heparin sulfate. In some embodiments, the second polyelectrolyte comprises heparin folate. In some embodiments, the second polyelectrolyte comprises nucleic acids. In some embodiments, the second polyelectrolyte is hyaluronic acid, poly-glutamate, poly- aspartate, poly-amino acid composite, block co-polymers of poly amino acids and poly- ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, or nucleic acids. [0055] In certain embodiments, the method further comprises determining an amount of polyelectrolyte needed to achieve a plateau onset point (POP). In some embodiments, the POP amount is determined using the procedures and/or calculations described herein. In some embodiments, the POP amount is determined using methods known in the art. [0056] In some embodiments, the solution comprising the first polyelectrolyte comprises at least 80% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises at least 90% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises at least 95% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises at least 99% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises about 100% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises at least 105% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the first polyelectrolyte comprises at least 110% of the amount of polyelectrolyte needed to achieve the POP. [0057] In some embodiments, the solution comprising the first polyelectrolyte comprises between 80% and 100% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 80% and 85% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 85% and 90% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 90% and 95% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 95% and 100% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 95% and 105% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises between 90% and 110% of the amount of polyelectrolyte needed to achieve the POP. [0058] In some embodiments, the solution comprising the first polyelectrolyte comprises 100% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the first polyelectrolyte comprises about 100% of the amount of polyelectrolyte needed to achieve the POP. [0059] In some embodiments, the solution comprising the second polyelectrolyte comprises at least 80% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the second polyelectrolyte comprises at least 90% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the second polyelectrolyte comprises at least 95% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the second polyelectrolyte comprises at least 99% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the second polyelectrolyte comprises about 100% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the second polyelectrolyte comprises at least 105% of the amount of polyelectrolyte needed to achieve the POP. In certain embodiments, the solution comprising the second polyelectrolyte comprises at least 110% of the amount of polyelectrolyte needed to achieve the POP. [0060] In some embodiments, the solution comprising the second polyelectrolyte comprises between 80% and 100% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the second polyelectrolyte comprises between 80% and 85% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the second polyelectrolyte comprises between 85% and 90% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the second polyelectrolyte comprises between 90% and 95% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the second polyelectrolyte comprises between 95% and 100% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the second polyelectrolyte comprises between 95% and 105% of the amount of polyelectrolyte needed to achieve the POP. In some embodiments, the solution comprising the second polyelectrolyte comprises between 90% and 110% of the amount of polyelectrolyte needed to achieve the POP. [0061] In certain embodiments, the solution comprising the second polyelectrolyte comprises 100% of the amount of polyelectrolyte needed to achieve the POP. [0062] In certain embodiments, the solution comprising the second polyelectrolyte comprises about 100% of the amount of polyelectrolyte needed to achieve the POP. [0063] In some embodiments, the POP amount is an amount of polyelectrolyte polymer added to the NP such that the particle net charge is neutral. In some embodiments, the POP amount is about 1.5 to about 3 times the amount of polyelectrolyte required to achieve neutrality. In some embodiments, the POP amount is about 1.5 times the amount of polyelectrolyte required to achieve neutrality. In some embodiments, the POP amount is about 2 times the amount of polyelectrolyte required to achieve neutrality. In some embodiments, the POP amount is about 3 times the amount of polyelectrolyte required to achieve neutrality. [0064] In certain embodiments, the solution comprising the first-layered nanoparticle is substantially free of unbound first polyelectrolyte after mixing. In some embodiments, the solution comprising the second-layered nanoparticle is substantially free of unbound second polyelectrolyte after mixing. [0065] In some embodiments, the solution comprising the first-layered nanoparticle does not comprise an excess of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the second-layered nanoparticle does not comprise an excess of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the first-layered nanoparticle is substantially free of unbound first polyelectrolyte after mixing and the solution comprising the second-layered nanoparticle is substantially free of unbound second polyelectrolyte after mixing. In some embodiments, the solution comprising the first-layered nanoparticle does not comprise an excess of the first polyelectrolyte in relation to the nanoparticle and the solution comprising the second-layered nanoparticle does not comprise an excess of the second polyelectrolyte in relation to the first-layered nanoparticle. [0066] In some embodiments, the nanoparticle comprises one layer. In some embodiments, the nanoparticle comprises more than one layer. In some embodiments, the nanoparticle comprises more than two layers. In some embodiments, the nanoparticle comprises more than three layers. [0067] In certain embodiments, the nanoparticle has a surface charge that is opposite the charge of the first polyelectrolyte. In some embodiments, the first polyelectrolyte and second polyelectrolyte have opposite charges. In some embodiments, the nanoparticle has a net positive surface charge. In some embodiments, the nanoparticle has a net negative surface change. In some embodiments, the first polyelectrolyte has a net negative surface change. In some embodiments, the first polyelectrolyte has a net positive surface change. In some embodiments, the second polyelectrolyte has a net negative surface change. In some embodiments, the second polyelectrolyte has a net positive surface change. In some embodiments, the nanoparticle and the second polyelectrolyte have net positive surface charges and the first polyelectrolyte has a net negative surface charge. In some embodiments, the nanoparticle and the second polyelectrolyte have net negative surface charges and the first polyelectrolyte has a net positive surface charge. In certain embodiments, the nanoparticle and second polyelectrolyte have the same surface charge. In certain embodiments, the nanoparticle and second polyelectrolyte have opposite surface charges. [0068] In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.7 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.05 to 0.7 weight equivalents of poly-arginine in relation to the nanoparticle. [0069] In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.1 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.1 to 0.15 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.15 to 0.2 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.2 to 0.25 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.25 to 0.3 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.3 to 0.35 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.35 to 0.4 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.4 to 0.45 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.45 to 0.5 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.5 to 0.55 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.55 to 0.6 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.6 to 0.65 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.65 to 0.7 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. [0070] In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.05 to 0.1 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.1 to 0.15 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.15 to 0.2 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.2 to 0.25 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.25 to 0.3 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.3 to 0.35 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.35 to 0.4 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.4 to 0.45 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.45 to 0.5 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.5 to 0.55 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.55 to 0.6 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.6 to 0.65 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.65 to 0.7 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. [0071] In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.1 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.1 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.2 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.2 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.3 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.3 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.4 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.4 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.5 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.5 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.6 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.6 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.7 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.7 weight equivalents of the first polyelectrolyte in relation to the nanoparticle. [0072] In some embodiments, the first polyelectrolyte comprises a positive charge. In some embodiments, the first polyelectrolyte is positively charged. In some embodiments, the first polyelectrolyte is a positively charged polyelectrolyte selected form the group consisting of poly-amino acid composite, block co-polymers of poly amino acids and poly-ethylene glycol, poly-arginine, poly-lysine, and a fluorescently labeled derivative thereof. [0073] In some embodiments, the first polyelectrolyte comprises a negative charge. In some embodiments, the first polyelectrolyte is negatively charged. In some embodiments, the first polyelectrolyte is a negatively charged polyelectrolyte selected form the group consisting of hyaluronic acid, poly-glutamate, polyaspartic acid, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, nucleic acids, and a fluorescently labeled derivative thereof. [0074] In some embodiments, first polyelectrolyte is poly-arginine. In some embodiments, first polyelectrolyte comprises poly-arginine. [0075] In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.05 to 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. [0076] In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.1 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.1 to 0.15 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.15 to 0.2 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.2 to 0.2.5 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.25 to 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises between about 0.1 and about 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. [0077] In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.05 to 0.1 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.1 to 0.15 weight equivalents of poly-arginine in relation to the nanoparticle. In some embodiments, the solution comprising the first polyelectrolyte comprises between 0.1 and 0.2 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.15 to 0.2 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.2 to 0.2.5 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises between 0.25 to 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. [0078] In certain embodiments, the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.3 weight equivalents of the first poly electrolyte in relation to the nanoparticle. [0079] In some embodiments, the solution comprising the first polyelectrolyte comprises about 0.1 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises 0.1 weight equivalents of poly-arginine in relation to the nanoparticle. In certain embodiments, the solution comprising the first polyelectrolyte comprises less than 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. [0080] In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.05 to 0.8 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.05 to 0.8 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. [0081] In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.05 to 0.1 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.1 to 0.15 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.15 to 0.2 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.2 to 0.25 weight equivalents of the second polyelectrolyte in relation to the first- layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.35 to 0.3 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.3 to 0.35 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.35 to 0.4 weight equivalents of the second polyelectrolyte in relation to the first- layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.4 to 0.45 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.45 to 0.5 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.5 to 0.55 weight equivalents of the second polyelectrolyte in relation to the first- layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.55 to 0.6 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.6 to 0.65 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.65 to 0.7 weight equivalents of the second polyelectrolyte in relation to the first- layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.7 to 0.75 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.75 to 0.8 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. [0082] In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.05 to 0.1 weight equivalents of the second polyelectrolyte in relation to the first- layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.1 to 0.15 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.15 to 0.2 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.2 to 0.25 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.35 to 0.3 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.3 to 0.35 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.35 to 0.4 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.4 to 0.45 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.45 to 0.5 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.5 to 0.55 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.55 to 0.6 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.6 to 0.65 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.65 to 0.7 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.7 to 0.75 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises between 0.75 to 0.8 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. [0083] In some embodiments, the second polyelectrolyte comprises a positive charge. In some embodiments, the second polyelectrolyte is positively charged. In some embodiments, the second polyelectrolyte is a positively charged polyelectrolyte selected form the group consisting of poly-amino acid composite, block co-polymers of poly amino acids and poly- ethylene glycol, poly-arginine, poly-lysine, and a fluorescently labeled derivative thereof. [0084] In some embodiments, the second polyelectrolyte comprises a negative charge. In some embodiments, the second polyelectrolyte is negatively charged. In some embodiments, the second polyelectrolyte is a negatively charged polyelectrolyte selected form the group consisting of hyaluronic acid, poly-glutamate, polyaspartic acid, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, nucleic acids, and a fluorescently labeled derivative thereof. [0085] In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.1 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.2 weight equivalents of the second polyelectrolyte in relation to the first- layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.3 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.4 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.5 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.6 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.7 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises about 0.8 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. [0086] In some embodiments, the solution comprising the second polyelectrolyte comprises 0.1 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises 0.2 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises 0.3 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises 0.4 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises 0.5 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises 0.6 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises 0.7 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises 0.8 weight equivalents of the second polyelectrolyte in relation to the first-layered nanoparticle. [0087] In certain embodiments, the second polyelectrolyte is selected from the group consisting of poly-glutamate, hyaluronic acid, polyaspartic acid, and polyacrylic acid. In some embodiments, the second polyelectrolyte is poly-glutamate. In some embodiments, the solution comprising the second polyelectrolyte comprises between about 0.25 to about 0.8 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between about 0.25 to about 0.35 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between about 0.35 to about 0.45 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between about 0.45 to about 0.55 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between about 0.55 to about 0.65 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between about 0.65 to about 0.75 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between about 0.7 to about 0.8 weight equivalents of poly-glutamate in relation to the nanoparticle. [0088] In some embodiments, the solution comprising the second polyelectrolyte comprises between 0.25 to 0.8 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between 0.25 to 0.35 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between 0.35 to 0.45 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between 0.45 to 0.55 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between 0.55 to 0.65 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between 0.65 to 0.75 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between 0.7 to 0.8 weight equivalents of poly-glutamate in relation to the nanoparticle. [0089] In certain embodiments, the solution comprising the second polyelectrolyte comprises between about 0.4 and about 0.6 weight equivalents of poly-glutamate in relation to the nanoparticle. [0090] In certain embodiments, the solution comprising the second polyelectrolyte comprises less than 0.6 weight equivalents of poly-glutamate in relation to the nanoparticle. In some embodiments, the solution comprising the second polyelectrolyte comprises between 0.4 and 0.6 weight equivalents of poly-glutamate in relation to the nanoparticle. In certain embodiments, the solution comprising the second polyelectrolyte comprises 0.5 weight equivalents of poly-glutamate in relation to the nanoparticle. [0091] In some embodiments, the second polyelectrolyte is hyaluronic acid. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.3 to about 0.5 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.3 to about 0.35 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.35 to about 0.4 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.4 to about 0.5 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.45 to about 0.5 weight equivalents of hyaluronic acid in relation to the nanoparticle. [0092] In some embodiments, the second polyelectrolyte is hyaluronic acid. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.3 to 0.5 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.3 to 0.35 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.35 to 0.4 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte about 0.4 to 0.5 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.45 to 0.5 weight equivalents of hyaluronic acid in relation to the nanoparticle. [0093] In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.4 weight equivalents of hyaluronic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.4 weight equivalents of hyaluronic acid in relation to the nanoparticle. [0094] In certain embodiments the second polyelectrolyte is polyaspartic acid. [0095] In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.2 to about 0.4 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.2 to about 0.25 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.25 to about 0.3 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.3 to about 0.35 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.35 to about 0.4 weight equivalents of polyaspartic acid in relation to the nanoparticle. [0096] In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.2 to about 0.4 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.2 to 0.25 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.25 to 0.3 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.3 to 0.35 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.35 to 0.4 weight equivalents of polyaspartic acid in relation to the nanoparticle. [0097] In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.3 weight equivalents of polyaspartic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.3 weight equivalents of polyaspartic acid in relation to the nanoparticle. [0098] In certain embodiments the second polyelectrolyte is polyacrylic acid. [0099] In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.1 to about 0.3 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.1 to about 0.15 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.15 to 0.2 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.2 to about 0.25 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises about 0.25 to about 0.3 weight equivalents of polyacrylic acid in relation to the nanoparticle. [00100] In certain embodiments the solution comprising the second polyelectrolyte comprises 0.1 to 0.3 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.1 to 0.15 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.15 to 0.2 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.2 to 0.25 weight equivalents of polyacrylic acid in relation to the nanoparticle. In certain embodiments the solution comprising the second polyelectrolyte comprises 0.25 to 0.3 weight equivalents of polyacrylic acid in relation to the nanoparticle. [00101] In some embodiments the solution comprising the second polyelectrolyte comprises about 0.15 to 0.8 weight equivalents of second polyelectrolyte. [00102] In certain embodiments, the nanoparticle is selected from the group consisting of lipid nanoparticles, liposomes, solid nanoparticles, metallic nanoparticles, polymeric nanoparticles, nanotubes, quantum dots, micelles, and lipid nano discs. [00103] In some embodiments, the nanoparticle comprises a liposome. In some embodiments, the nanoparticle comprises a lipid nanoparticle. In some embodiments, the nanoparticle comprises a solid core. In some embodiments, the nanoparticle comprises a metallic nanoparticle. In some embodiments, the metallic nanoparticle is gold. .In some embodiments, the nanoparticle comprises an empty core. In some embodiments, the nanoparticle comprises a liquid core. In some embodiments, the nanoparticle comprises a polymeric core. In certain embodiments, the nanoparticle comprises a polymeric micelle. In some embodiments, the nanoparticle comprises a polymersome. In some embodiments, the nanoparticle comprises a dendrimer core. In some embodiments, the nanoparticle comprises a lipid carrier. In some embodiments, the nanoparticle comprises a silver nanoparticle. In some embodiments, the nanoparticle comprises an iron oxide core. In some embodiments, the nanoparticle comprises a quantum dot. In some embodiments, the nanoparticle is mesoporous. [00104] In some embodiments, the nanoparticle is a polymeric nanoparticle. In some embodiments, the nanoparticle is a nanotube. In some embodiments, the nanoparticle is a quantum dot. In some embodiments, the nanoparticle is a micelle. In some embodiments, the nanoparticle is a lipid nano disc. In some embodiments, the nanoparticle is a liposome. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the nanoparticle is a solid nanoparticle. In some embodiments, the nanoparticle is a latex bead. In certain embodiments, the latex bead is carboxy-modified. In certain embodiments, the nanoparticle comprises a negative charge. [00105] In certain embodiments, the nanoparticle comprises at least one additional pharmaceutical agent. In certain embodiments, the composition comprises at least one additional pharmaceutical agent. In some embodiments, the at least one additional pharmaceutical agent comprises a protein, or fragment thereof, a small molecule, or a nucleotide. In certain embodiments, the at least one additional pharmaceutical agent comprises a protein. In certain embodiments, the at least one additional pharmaceutical agent comprises an antibody or fragment thereof. In some embodiments, the additional pharmaceutical agent is a small molecule. In some embodiments, the additional pharmaceutical agent is a polynucleotide. In certain embodiments, the at least one additional pharmaceutical agent comprises at least one oligosaccharide. In certain embodiments, the at least one additional pharmaceutical agent comprises at least one monosaccharide. In some embodiments, the at least one additional pharmaceutical agent comprises a protein or small molecule. In some embodiments the protein is interleukin-12, interleukin-15 super agonist, interleukin 18, interferon-α, interferon-β, interferon-ϒ, interleukin-2, anti-PD1 antibodies, anti-PDL1 antibodies, anti-CTLA4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-NKG2A antibodies, anti-CD73 antibodies, anti-A2aR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, single-chain interleukin-12, tumor necrosis factor alpha, interleukin- 10, interleukin-8, TNF-related apoptosis-inducing ligand (TRAIL), or FMS-like tyrosine kinase 3 ligand (FLT3LG). In some embodiments the protein is a cytokine. In some embodiments, the at least one additional pharmaceutical agent comprises a cytokine. In certain embodiments, the at least one additional pharmaceutical agent comprises IL-12. In some embodiments, the additional pharmaceutical agent is a small molecule. In certain embodiments, the small molecule is Oxaliplatin, Doxorubicin, Paclitaxel, Lurbinectedi, Mitomycin, Trabectedin, Lobenguane, Lutetium, Radium, Cisplatin, or Sorafenib. In some embodiments, the layered nanoparticle comprises a targeting moiety. [00106] In some embodiments, the method does not comprise purification by ultrafiltration. In some embodiments, the method does not comprise purification by tangential flow filtration, size exclusion chromatography, or dialysis. [00107] In some embodiment, NPs are mixed with polyelectrolytes using commercially available bifurcating mixer MCF cartridges. In certain embodiments, said microfluidic mixing comprises the use of at least one microfluidic chip. In some embodiments, the at least one microfluidic mixing chip comprises a bifurcating channel. In some embodiments, said microfluidic mixing comprises the use of at least two microfluidic chips comprising a bifurcating channel. In some embodiments, said microfluidic mixing comprises the use of at least three microfluidic chips comprising a bifurcating channel. In some embodiments, the at least one, two, or three microfluidic chip does not comprise a bifurcating channel. In some embodiments, the at least one microfluidic chip comprises a T-shaped channel. In some embodiments, the at least one microfluidic chip comprises a Y-shaped channel. In some embodiments, said microfluidic mixing comprises the use of at least two microfluidic chips comprising a herring bone-shaped channel. In some embodiments, said microfluidic mixing comprises the use of at least three microfluidic chips comprising a herring bone-shaped channel. In some embodiments, the at least one, two, or three microfluidic chip does not comprise a herring-bone shaped channel. [00108] In some embodiments, said microfluidic mixing comprises the use of at least two microfluidic chips comprising a bifurcating channel. In some embodiments, said microfluidic mixing comprises the use of at least three microfluidic chips comprising a bifurcating channel. [00109] In some embodiments, the method is a continuous process. In certain embodiments, the at least two microfluidic chips are connected in series. [00110] In some embodiments, each microfluidic mixing step independently comprises a flow rate of at least 1 mL/min. In some embodiments, each microfluidic mixing step independently comprises a flow rate of at least 3 mL/min. In some embodiments, each microfluidic mixing step independently comprises a flow rate greater than 1 mL/min. In some embodiments, each microfluidic mixing step independently comprises a flow rate greater than 3 mL/min. In some embodiments, each microfluidic mixing step independently comprises a flow rate of between 1 mL/min and 3 mL/min. In some embodiments, each microfluidic mixing step independently comprises a flow rate of between 3 mL/min and 10 mL/min. In some embodiments, each microfluidic mixing step independently comprises a flow rate of between 3 mL/min and 5 mL/min. In some embodiments, the flow rate is about 1 mL/min. In some embodiments, the flow rate is about 2 mL/min. In some embodiments, the flow rate is about 3 mL/min. In some embodiments, the flow rate is about 4 mL/min. In some embodiments, the flow rate is about 5 mL/min. In some embodiments, the flow rate is about 6 mL/min. In some embodiments, the flow rate is about 7 mL/min. In some embodiments, the flow rate is about 8 mL/min. In some embodiments, the flow rate is about 9 mL/min. In some embodiments, the flow rate is about 10 mL/min. [00111] In certain embodiments, the method does not comprise sonication or agitation. In certain embodiments, the method does not comprise sonication. In certain embodiments, the method does not comprise agitation. [00112] In some embodiments, each microfluidic mixing step increases the nanoparticle size by at least 1nm. In certain embodiments, each microfluidic mixing step increases the nanoparticle in size by less than 50nm. [00113] In some embodiments, the layered nanoparticle has a polydispersity index of about 0.05 to about 0.15. In some embodiments, the layered nanoparticle has a polydispersity index of about 0.05 to about 0.1. In some embodiments, the layered nanoparticle has a polydispersity index of about 0.1 to about 0.15. In some embodiments, the layered nanoparticle has a polydispersity index of 0.05 to 0.15. In some embodiments, the layered nanoparticle has a polydispersity index of 0.05 to 0.1. In some embodiments, the layered nanoparticle has a polydispersity index of 0.1 to 0.15. In some embodiments, the layered nanoparticle has a polydispersity index (PDI) of less than 0.25. In some embodiments, the layered nanoparticle has a PDI of less than 0.2. In some embodiments, the layered nanoparticle has a PDI of greater than 0.2. In some embodiments, the layered nanoparticle has a PDI of less than 0.1. In some embodiments, the layered nanoparticle has a PDI of between 0 and 0.3. In some embodiments, the layered nanoparticle has a PDI of between 0 and 0.2. In some embodiments, the layered nanoparticle has a PDI of 0.2. In some embodiments, the layered nanoparticle has a PDI of 0.1. [00114] In certain embodiments, the layered nanoparticle has a polydispersity index of less than 0.25. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of between 0 and 0.3. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of between 0 and 0.1. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of between 0 and 0.2. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of between 0.2 and 0.3. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of 0.1. In some embodiments, the wherein the layered nanoparticle has a polydispersity index of 0.2. [00115] In certain embodiments, the layered nanoparticle is smaller than 400 nm in size. In certain embodiments, the layered nanoparticle is smaller than 300 nm in size. In certain embodiments, the layered nanoparticle is smaller than 200 nm in size. In certain embodiments, the layered nanoparticle is between 110-200 nm in size. In some embodiments, the layered nanoparticle is between 100-140 nm in size. In some embodiments, the layered nanoparticle is about 110nm in size. In some embodiments, the layered nanoparticle is about 120nm in size. [00116] It should be known to those skilled in the art that the nanoparticle sizes described herein refer to the Z-average size. [00117] In certain embodiments, the layered nanoparticle is between 1 and 50 nm in size. In certain embodiments, the layered nanoparticle is between 50 nm and 100 nm in size. In certain embodiments, the layered nanoparticle is between 1 nm and 100 nm in size. In certain embodiments, the layered nanoparticle is between 1 nm and 200 nm in size. In certain embodiments, the layered nanoparticle is between 100 nm and 200 nm in size. In certain embodiments, the layered nanoparticle is between 150 nm and 200 nm in size. In certain embodiments, the layered nanoparticle is between 400 nm and 999 nm in size. In certain embodiments, the layered nanoparticle is between 200 nm and 500 nm in size. In certain embodiments, the layered nanoparticle is between 200 and 400 nm in size. In some embodiments, the layered nanoparticle is between 500 nm and 999 nm in size. [00118] In certain embodiments, the solution comprising the nanoparticle has a concentration of at least 0.5 mg/mL. In some embodiments, the solution comprising the nanoparticle has a concentration of 1 mg/mL. In some embodiments, the solution comprising the nanoparticle has a concentration of 5 mg/mL. In some embodiments, the solution comprising the nanoparticle has a concentration of 6 mg/mL. [00119] In certain embodiments, the solution comprising the nanoparticle has a concentration of at about 0.5 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of at about 1 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of at about 5 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 0.5 mg/mL and 5 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 0.5 mg/mL and 1 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 1 mg/mL and 2 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 2 mg/mL and 3 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 3 mg/mL and 4 mg/mL. In certain embodiments, the solution comprising the nanoparticle has a concentration of between about 4 mg/mL and 5 mg/mL. [00120] In one aspect, provided herein a kit comprising: the solution comprising the nanoparticle, the solution comprising the first polyelectrolyte, and the solution comprising the second polyelectrolyte provided herein; the microfluidic chips of the present disclosure; and instructions for its use. [00121] In another aspect, provided is a kit comprising: a solution comprising a nanoparticle; a solution comprising a first polyelectrolyte; a solution comprising a second polyelectrolyte; one or more microfluidic mixing chips; and instructions for its use. [00122] In some embodiments, the kit further comprises packaging to contain the contents. In some embodiments, the kit further comprises a label. EXAMPLES [00123] In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting in their scope. Example 1. Excess polymer is required for LbL assembly without particle aggregation during batchwise polyelectrolyte adsorption [00124] As a model system for targeted immunotherapy delivery to cancer cells, anionic liposomes that were surface-conjugated with the potent cytokine IL-12 (IL-12 NPs), and subsequently coated by alternating layers of poly(L-arginine) (PLR) and poly(L-glutamate) (PLE) were prepared. LbL assembly has previously been performed by alternating sequential incubations of the liposomal core particle with excess PLR and PLE in a low ionic strength buffer. Under these self-assembly conditions, each step of polyelectrolyte adsorption is accompanied by extensive washing/purification to remove excess unbound polymer from the coated particles before the next round of adsorption. [00125] To understand and optimize LbL assembly, the polymer:particle weight ratio variation during nanolayer deposition was assessed. Unlayered anionic IL-12 NPs were incubated with increasing PLR weight equivalents (wt. eq.), or alternatively, purified PLR-coated cationic IL- 12-NPs (PLR/NPs) were incubated with increasing PLE wt. eq relative to the liposome lipid mass (FIG. 1A). After polymer and NP mixing, the resulting unpurified polymer-NP assemblies' size and charge were characterized via dynamic light scattering (DLS) and electrophoretic mobility, respectively. Starting from very low polymer concentrations, increased polymer-to-NP wt. eq. led to increasing NP charge up to a charge neutralization point (isoelectric point), at which particles rapidly aggregated due to a lack of charge-charge repulsion (FIGs. 1B-1C). [00126] Further increases in polymer-to-NP wt. eq. led to reductions in particle size as the overall NP charge inverted, and the zeta potential neared a plateau onset point (POP), where complete charge reversal was achieved. Given the known composition of the liposomes used here, the charge neutralization and POP wt. eq. for PLR onto the IL-12 liposomes was estimated based on charge balance and was found to have values of ~0.05 and 0.1, respectively, which were similar to our experimental results (see below derivation). Analogously, charge neutralization and POP wt.eq. for PLE of ~0.2 and ~0.45 were estimated and found to be consistent with empirical observations. Yet, further increasing the polymer-to-NP wt. eq beyond the zeta potential plateau onset point (POP) was required to form polymer-coated NPs with the lowest overall size and polydispersity index (PDI). Due to this reduced size and increased homogeneity, the polymer wt. eq. beyond the POP are used for LbL-NP synthesis. [00127] These results suggest that excess polymers present during the layering may prevent bridging of NPs by the polymer to avoid aggregate formation (FIG.1D). It has been previously shown that polymer deposition onto NP surfaces is a kinetically controlled reaction and is expected to be quantitative until the POP. [00128] Indeed, increasing the volume of NP preparations can yield lower quality LbL-NPs due to reduced mixing efficiency even in the presence of bath sonication such that low NP concentration and high polymer wt. eq. need to be used practically. Nonetheless, to remove excess polymers, NP centrifugation or ultrafiltration techniques such as tangential flow filtration (TFF) were employed. Example 2. Increasing production scale requires excess polymer to yield homogenous LbL- NPs. [00129] To validate that further increasing the polymer wt. eq. beyond the POP did not increase the amount of adsorbed polymer on the NPs, the optimized protocol of LbL-NP synthesis for lab-scale production (0.3 and 1 wt. eq. of PLR and PLE, respectively, with NPs at 0.5 mg/mL) was performed using fluorescently tagged PLR and PLE polymers. This process consisted of the addition of NPs to a solution of polymers under bath sonication (FIG.2A). An Erlenmeyer flask with <30% of its rated volume was used to facilitate homogeneous NP distribution over the polymer solution and minimize the chance of inhomogeneities in the sample (PLR wt. eq. of 0.3 and PLE wt. eq. of 1). The excess polymer was then removed via TFF after each polymer layer deposition. The production of LbL-NPs via this protocol had the characteristic reversion of surface charge and minor increases in overall size while maintaining overall low PDI (FIG. 2B). Quantification of the amount of polymer remaining attached to the NPs after purification demonstrated that only ~0.1 wt. eq. of PLR and ~0.5-0.6 wt. eq. of PLE were used for surface adsorption (FIG. 2C). These results confirmed that the polymer wt. eq. beyond the POP observed in FIG. 1 did not increase the number of polymers in the LbL films. [00130] Based on these results, it was theorized that the assembly of LbL-NPs at the POP could allow the omission of purification steps, as no excess polymers should be present in the solution. To better understand LbL-NP assembly at the POP polymer-to-NP wt. eq., particles generated with POP wt. eq. of PLR or PLE were compared to the standard layering with excess polymers under bath sonication followed by TFF. Reasonably sized LbL-NPs with POP wt. eq. of PLR were generated in small-scale test batches (<50 µL and < 1 mg/mL lipids), however, a noticeable increase in NP size was observed when layering with a larger batch size of NPs (~5 mg) was attempted (FIG.2D). Omitting sonication during the layering led to a further increase in the resulting NP size compared to the standard approach. Similarly, PLE layering with POP wt. eq. (0.5 wt.eq. PLE-to-lipids) on purified PLR-NPs resulted in NP aggregation during layering (FIG. 2E). Thus, large-scale LbL assembly in static solution even in the presence of sonication could only successfully be performed under conditions of excess polymer, requiring subsequent purification steps. Example 3. Microfluidics-enabled mixing generates homogeneous LbL-NPs without intermediate purification steps. [00131] As polymer wt. eq. beyond the plateau point only increased the number of free polymers in the solution without affecting the LbL film, the inventors posited that mixing limitations during the standard bath sonication protocol may lead to the observed NP aggregate formation. The ability of titrated polymer wt. eq. in the assemble LbL-NPs without purification steps was accordingly evaluated. Sequential small-scale mixing of titrated PLR and PLE wt. eq. with sonication was first assessed. This method yielded ~200 nm PLR/PLE IL-12 NPs (FIG. 3A). The increased NP size (~190 nm compared to ~120 nm target) and increased PDI (~0.2 compared to target ~0.1) could limit the application of these NPs. Further, as shown in FIG. 2, standard LbL-NP assembly with titrated polymer wt. eq. had limited process robustness, especially at larger scales. [00132] To overcome these limitations and manufacture large-scale LbL-NPs without the need for purification, a commercial MCF cartridge with a bifurcating mixer was utilized (FIG.3B). This microfluidic chip platform has been developed for lab and clinical-scale cGMP manufacturing of NPs, making it an ideal candidate for clinical-scale manufacturing of LbL- NPs. For mixing, solutions of liposomes and polyelectrolytes were each introduced into one of two entry flow ports (FIG.3B); the two solution streams converged at the mixer and the mixed sample was collected from the outlet port. [00133] The effect of flow rate through the channels on the resulting polymer-coated NP was examined. The effect of flow rate on the adsorption of PLR to anionic IL-12 liposomes was first evaluated, combining PLR and liposomes at the POP. The target was to achieve NPs similar in size to that of the standard optimized bath sonication protocol. Increasing the flow rate led to reduced PLR-NP Z-avg size and PDI while maintaining charge conversion (FIGs. 3C, 3D), demonstrating improved sample homogeneity without impacting polymer adsorption. [00134] Importantly, flow rates of >3 mL/min, resulted in NPs of target size for the PLR-NPs even at its POP wt. eq. Higher flow rates than 10 mL were unachievable due to flow rate limitations of the MCF chip. Nonetheless, the MCF mixing enabled large-scale polymer deposition at the POP wt. eq. [00135] Based on these findings, a two-stage microfluidic mixing process was designed to allow two rounds of polyelectrolyte adsorption at POP wt. eq., in order to generate LbL-NPs without purification steps (FIG.3E). IL-12-liposomes and PLR were combined in a first-stage MCF mixer followed by a 30 min incubation step to ensure polymer adsorption and perform quality tests. The output of the first stage and a PLE solution were used as the input in a second- stage mixer. When POP wt. eq. PLR was deposition onto IL-12-NPs at increased scales (>1 mL and >5 mg) using MCF, the target PLR-NP sizes of the standard bath sonication production protocol across multiple batches was readily achieved (FIG. 3F). In addition to reduced processing time and complexity, such an approach can be scaled up under cGMP conditions. Indeed, using titrated PLR wt. eq. deposition onto IL-12-NPs at increased scales (>1 mL) using MCF readily yielded the target PLR-NP sizes from the optimized TFF production protocol. Similarly, depositing PLE at its POP wt. eq. onto PLR-IL-12 NPs from MCF-generated PLR- IL-12 NPs yielded desired PLR/PLE IL-12 NPs with reproducibility (FIG. 3G). To validate that all polymers were adsorbed on NPs using the MCF protocol, fluorescently tagged PLE polymers were used to assemble PLE/PLR IL-12 NPs with both the standard optimized TFF- based protocol and the new MCF LbL-NPs. Any free polymers in the LbL-NP solution were separated using centrifugal filters and, as expected from the POP of ~ 0.5 wt. eq. for PLE, only ~50% of PLE was bound to TFF-LbL-NPs prior to purification due to the excess polymers used during assembly with 1 wt. eq. with PLE. [00136] On the other hand, both purified TFF-LbL-NPs and MCF LbL-NPs both had >95% of PLE bound to NPs (FIG. 3H), confirming the lack of free polymers. Analysis of sample morphology via negative stain transmission electron microscopy (TEM) confirmed that both TFF-based and the MCF-based PLE/PLR-IL-12 NPs had a polymer film on the NP based on the reduced staining around the liposome (FIG. 3I). Further, no signs of significant polyplex formation in either LbL-NP formulation on the TEM micrographs were observed (FIG. 10). [00137] To compare the reproducibility between LbL assembly on IL-12 liposomes via either the standard TFF-based approach or MCF, the data from independent batches of TFF-LbL (n = 10) and MCF-LbL (n = 8) over the course of over six months was compiled. Both methods yielded overall similar NPs with Z-avg 110-150 nm, zeta potential of approximately -50 mV, and PDI ≤ 0.2 (FIG.11, Table 1). However, MCF-LbL NPs yielded smaller size and PDI with reduced standard deviations, demonstrating increased sample homogeneity and reduction in amount of partially aggregated LbL-NPs. [00138] Table 1. Comparison of dynamic light scattering (DLS) electrophoretic mobility results from independent IL-12-LbL-NP batches generated via either optimized TFF-based (with excess polymers) or MCF-based (using POP wt. eq.). TFF-LbL-NP MCF-LbL-NP (IL-12 PLR/PLE) (IL-12 PLR/PLE) #-avg (nm) 76.2 9.24 (12) 68.6 7.34 (11) Zeta Potential (mV) 8.38 (17) 4.07 (8.2) [00139] As some LbL-NP assemblies benefit from solution conditions during layering beyond deionized water (DI) such as in PLE/PLR-IL-12-NPs, the MCF strategy was employed in buffer conditions. Firstly, the varying PLR wt. eq. were tested to find the titrated amount of PLR required for LbL assembly in 25 mM HEPES and 20 mM sodium chloride (NaCl) which is a buffer composition found to optimize the loading of siRNA onto LbL films.25 Around 0.15 wt. eq. of PLR was required to reach the plateau point (FIGs. 4A-4B). Next, the size and zeta potential of LbL-NP synthesized from small-scale tests were compared between those synthesized using bath sonication and TFF to MCF LbL-NPs. Similar to IL-12-LbL-NPs, it was determined that assembly with MCF enabled more homogenous particle formulations (FIGs.4C-4E), confirming the benefit of NP layering via MCF mixing and the generalizability of this strategy for LbL-NP formulations. Example 4. MCF LbL-NPs maintain desired particle properties in vitro and maintain IL-12- LbL-NP efficacy in vivo in a metastatic ovarian cancer mouse model. [00140] Benefits of PLE/PLR coating on NPs is increased ovarian cancer cell affinity and prevention of NP endocytosis leading to high retention of the LbL-NPs on the cancer cell membrane. PLE/PLR-IL-12-NPs generated via the titrated polymer-to-NP wt. eq. had the same desired traits in vitro. Fluorescent NPs were administered to a murine metastatic ovarian cancer cell line, OV2944-HM1 (HM-1), and the amount of NPs associated with the cells after 4 and 24 hours were quantified. At 4 and 24 hours after dosing, MCF LbL-NPs and TFF-based LbL- NPs had significantly increased HM-1 cell association compared to unlayered (UL) NPs (FIG. 5A). Moreover, MCF-LbL-NPs had equal or better HM-1 association compared to TFF LbL NPs, confirming that MCF layering maintained and potentially improved the desired high ovarian cancer cell affinity. However, an important characteristic of PLE/PLR LbL-NPs is its cell-membrane retention which enables for high intratumoral extracellular residence time of cytokines such as IL-12. Next evaluated was the subcellular localization of NPs 24 hours after by confocal microscopy. While UL particles were all endocytosed (FIG. 5B), both TFF-LbL NPs (FIG. 5C) and MCF-LbL NPs (FIG. 5D) presented with NPs on the cancer cell membrane, confirming that PLE/PLR-IL-12-NPs retained their cell surface retention properties when prepared via MCF mixing. [00141] Having validated that LbL-NP assembly via MCF maintained the desired cancer cell association properties, the bioactivity of loaded IL-12 NPs was assessed. HEK-Blue IL-12 reporter cell lines were dosed with either free IL-12 or IL-12 bound to PLE/PLR-IL-12-NPs generated via the standard TFF-based protocol or MCF. The IL-12 on MCF-LbL NPs maintained its activity like that of TFF-LbL NPs (FIGs. 6A-6B). To evaluate the particle performance in vivo in mice bearing peritoneally disseminated metastatic HM-1 tumors, the same dosing paradigm shown previously to extend survival in this mice model with IL-12 loaded PLE/PLR-LbL-NPs was used (FIG. 6C) (PCT/US2023/076753). Both TFF-based and MCF-based LbL-NPs were found to have better control of tumor growth based on whole- mouse tumor bioluminescence readings (FIG. 6D), significantly extending survival of mice compared to either free IL-12 or UL IL-12 NPs (FIG. 6E). Example 5. MCF LbL-NPs have increased stability under increasing ionic strength. [00142] To observe differences in polyelectrolyte configuration on the NP surfaces, particles generated via the standard TFF or the MCF LbL assembly methods were exposed to increasing sodium chloride (NaCl) concentrations. Increasing NaCl levels increases the solution ionic strength which reduces the charge-based NP colloidal stability (due to charge-screening effects). As shown in FIG. 7 below, it was determined that LbL-NPs assembled via the new MCF approach had substantially increased stability to increasing ionic strength, having minimal aggregation at even physiological levels of NaCl (~150 mM). As the benefits of NP delivery may rely on their small size, preventing NP aggregation when exposed to physiological levels of ionic strength may allow for improved benefits of LbL-NPs for in vivo applications. Example 6. MCF LbL-NPs have increased stability in serum-supplemented buffer. [00143] It is known that serum components can impact stability LbL-NPs from aggregating. To evaluate if the MCF LbL-NPs had improved colloidal stability in a serum-containing solution, particles were incubated in HEPES saline buffer (15 mM HEPES, 150 mM NaCl) supplemented with 10% fetal bovine serum (FBS). As shown in FIG. 8 ̧while standard TFF- based LbL-NPs rapidly coalesced into large particles, LbL-NPs assembled via MCF had minimal size expansion and were found to be stable for over one hour. Example 7. MCF LbL-NPs can be readily assembled with varied polymer chemistries and NP core sizes for screening of LbL-NPs interactions [00144] The LbL platform is a modular approach for tuning NP surface chemistries with a wide range of polymer chemistries showing promising results in preclinical models. To validate that MCF assembly of LbL films was amenable to other polymer chemistries, titration experiments of IL-12 loaded PLR-NPs with varying wt. eq. of hyaluronic acid (HA), poly-L- aspartic acid (PLD), or polyacrylic acid (PAA) were performed. Each polymer chemistry showed a distinct POP wt. eq. which followed the trend of mass charge density (PAA>PLD>HA) for each of the polymers tested (FIG. 9A). Further, these POP wt. eq. were employed for each of these varying polymer chemistries to generate monodisperse LbL-NPs using MCF mixing (FIGs. 9B-9C). [00145] In addition to varying chemistries, LbL assembly may be performed on differing NP cores. However, little has been explored on the effect of NP size on LbL-NP properties. Thus, carboxy-modified latex (CML) beads were layered with varying core sizes with PLR and PLE. Smaller CML particles required larger wt. eq. to reach the POP given their higher surface area to mass ratios (FIG. 9D). However, no discernable trend for the plateau zeta potential values could be determined, without wishing to be bound by theory, it is hypothesized that it is likely due to the effect of NP size and roughness on the apparent zeta potential. Nonetheless, PLR/PLE coated CML beads of varying sizes were readily assembled (FIG. 9E). TEM confirmed that particles maintained their homogeneity (FIG. 12). When evaluated for their binding towards HM-1 cells via flow cytometry, all LbL-NPs showed an increase in median fluorescence intensity (MFI) compared to UL NPs (FIG.9F). It was determined that increasing UL NP size increased NP association whereas smaller LbL-NPs showed higher association. Without wishing to be bound by theory, it is hypothesized that this effect on small NPs was due to their increased surface area which should allow for higher LbL-NP film interaction with cancer cells. Indeed, when the correlation was assessed between the log-fold change in LbL- NP MFI relative to UL-NP MFI and the available NP surface area, it was determined that the data showed a correlation (FIG. 9G). Example 8 [00146] The experiments above carried out layering in deionized water, but LbL assembly is also sometimes carried out in the presence of salts to create LbL films with thicker layers. Thus, it was assessed whether LbL-NPs could be generated in the presence of salt-containing buffers. Here anionic liposomes devoid of IL-12 with a composition optimized for siRNA loading and delivery from LbL-NPs were used. First varying PLR:liposome mass ratios were examined to find the POP of PLR required for LbL assembly in 25 mM HEPES and 20 mM sodium chloride (NaCl), a buffer composition previously found optimal for loading of siRNA into LbL films. Around 0.15 wt. eq. of PLR was preferred to reach the POP (FIGs. 13A-13B). Next, the size and zeta potential of LbL-NP synthesized from small-scale tests under bath sonication was compared to increased bath sonication and TFF was compared to that of MCF LbL-NPs. Similar to IL-12-LbL-NPs, it was determined that assembly with MCF enabled more homogenous particle formulations (FIGs. 13C-13E). [00147] The LbL platform is a technique to modulate the surface properties of NPs for the development of therapeutic drug carriers. The present disclosure demonstrates that microfluidic mixing is highly effective for combining polyelectrolytes and NPs, achieving LbL assembly under conditions amenable to scalable, continuous synthesis for clinical-scale manufacturing, including under cGMP conditions. Moreover, it was determined that the MCF mixing supported LbL assembly at polymer-to-NP weight equivalents that were devoid of excess polymers, allowing for the production LbL-NPs without time-consuming purification steps and loss of NP yields. Thus this approach readily increases yield, reduces waste polymers, increases throughput, and requires less expensive equipment compared to the prior optimized LbL protocol for NPs based on TFF (summary of comparison between TFF and MCF assembly of LbL-NPs provided in Table 2). These process modifications do not alter the properties of LbL-NPs in vitro and in vivo based on the known properties of IL-12 loaded PLE/PLR-IL-12- NPs. Moreover, it was shown that this approach can be implemented for various LbL-NP film chemistries and employed in NPs of differing core compositions and sizes. With a library of PLR/PLE CML NPs of varying sizes it was shown that the increase in LbL-NP association relative to its UL-NP was directly correlated to the total available NP surface area. [00148] Taken together, this work provides a method to assemble LbL-NPs at various scales and at increased throughput. The MCF-LbL technique presented here may also be applied to larger particles. This approach should facilitate both clinical development of LbL-NPs as well as ease the production of libraries of LbL-NPs which can be implemented to screen for desired traits. [00149] Table 2. Comparison of LbL-NP assembly via TFF or MCF. MCF LbL-NPs TFF LbL-NPs / Example 9. Derivation of polymer-to-nanoparticles weight equivalent required for charge neutralization and plateau onset point (POP) [00150] For a given liposome, the molar averaged MW of the components (^^^^^) and average charge per component are given by: [00151] Where ^^^%^, ^^^ and ^^ are the molar percentage of component a, the molecular weight of component a, and the charge magnitude of component a in the liposome, respectively. For the formulation in this study, ^^^^^= 706 Da and ^^^^ = 0.17 (POPG net charge of -1 and MPB-PE net charge of -2 after ring opening reaction). The polymer 1-to-lipid weight ratio (wt. eq.) needed to achieve charge neutralization (^^^^^^^^^) can be calculated with the following equation: [00152] Where ^^^^^^^^^^ is the first polymer molecular weight and ^^^^^^^^^is the charge of the first polymer. For PLR (length 50), ^^^^^^^^^ = 0.046. Here, it was assumed that all lipids in the liposome contributed to the polymer deposition given that the Debye screening length in deionized water (~1 µm) is significantly larger than the bilayer thickness (~ 5 nm) such that the inner core lipids contribute to the net surface charge of the liposomes. Moreover, PLR was assumed to be fully charged given its high pKa. [00153] For the wt. eq. required for first plateau onset point (POP, ^^^^^^^^), it was assumed that the polymer weight and zeta potential (^ ) were linearly related between ^^^^ = 0 and ^^^^ = ^^^^^^^^.The equation below describes this relationship in terms of ^^^^^^^^^, the zeta potential of unlayered NPs (^ !), and the plateau zeta potential with excess PLR (^^"#^$$^). [00154] For PLR onto liposomes with ^ ! = -40 mV and ^^"#^$$^ = +50 mV, this yielded ^^^^^^^^ =0.10. For the second layer, the wt. eq. for charge neutralization (^^^^^^^^') can be estimated via the ratios of the polymer charge to molecular weights assuming that NP is covered with the first polymer. Note the subtracted term accounts for the component of polymer 1 cancels (-) core charge. [00155] Here, ^^^^^^^^^' was the second polymer molecular weight and ^^^^^^^^'was the magnitude of the second polymer’s charge. For PLE (length 100) deposition onto PLR-NPs, ^^^^^^^^' = 0.21. Here PLE was assumed to be ~22% charged, given PLE’s estimated pKa of 6.05 and the pH of deionized water of ~5.5. Lastly, the mass of PLE for the second POP (^^^^^^^') was estimated, using an equivalent equation to the one above: ^^^^^^^' = ^^^^^^^^' [00156] For PLE onto PLR-NP with ,^ = +50 this yielded ^^^^^^^' = 0.44 Experimental Methods. [00157] Materials: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2- oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt) (POPG), 1,2-distearoyl-sn- glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DSPG), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (sodium salt) (18:1 MPB-PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (DOPE-DBCO), and cholesterol were purchased from Avanti Polar Lipids. Poly-L-arginine (PLR) with a molecular weight (MW) of 9.6 kDa and poly-L-glutamic acid (PLE) with a MW of 15 kDa, and poly-L- aspartic acid (PLD) with a MW of 14 kDa were purchased from Alamanda Polymers. Borondipyrromethene tetramethylrhodamine (BDP TMR) azide (Lumiprobe) and BDP 630/650 azide (Lumiprobe) were conjugated to DOPE-DBCO in chloroform to generate DOPE-TMR and DOPE-630/650. Successful conjugation was validated via thin-layer chromatography which indicated <1% free dye. Hyaluronic acid (HA, 20 kDa) was purchased from Lifecore Biomedical. Poly-L-acrylic acid (PAA, 15 kDa) was purchased from Sigma Aldrich. Yellow fluorescent carboxy-modified latex (CML) beads (Fluorospheres) with diameters of 20 nm, 40 nm, 100 nm and 200 nm were purchased from ThermoFisher Scientific. [00158] Recombinant single-chain IL-12 production: Single-chain IL-12 sequence was synthesized as a genomic block (Integrated DNA Technologies) and cloned into a gWIZ expression vector (Genlantis). Plasmids were transiently transfected into Expi293 cells (ThermoFisher Scientific). After 5 days, cell culture supernatants were collected and protein was purified in an ÄKTA pure chromatography system using HiTrap HP Niquel sepharose affinity column, followed by size exclusion using Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences). Endotoxin levels in purified protein was measured using Endosafe Nexgen-PTS system (Charles River) and assured to be <5 EU/mg protein. [00159] IL-12 conjugated liposome synthesis: Lipid solutions composed of 65 mol% DSPC, 24 mol% Cholesterol, 6 mol% POPG and 5 mol% of MPB-PE were made in chloroform and then dried into a thin film using a rotovap. Films were allowed to further dry overnight in a desiccator. Lipid films were hydrated at 0.5-1 mg/mL using deionized water at a pH of 5 and sonicated for 3-5 minutes at 65 °C then extruded (Avestin Liposofast-50) at 65 °C once against a 100 nm membrane then thrice against 50 nm membranes. Extruded liposomes were added to an ice bath then the pH was adjusted to pH 7.0 with 10 mM HEPES buffer prior to addition of scIL-12 containing a terminal cysteine residue at 0.17 wt. eq. with liposomes at 0.33 mg/mL. After overnight incubation with IL-12 at 4 °C, any remaining maleimides were quenched with 100-fold molar excess of L-cysteine (Sigma) for 1.5 hrs on ice. For fluorescence labeling of liposomes, 0.2 mol% of DSPC content was replaced by either DOPE-TMR or DOPE-630/650. IL-12 concentration was measured via enzyme-linked immunoassay (ELISA) (Peprotech) and lipid content was quantified via fluorescence. IL-12 free liposomes were made with a 33 mol% DSPC, 33 mol% DSPG, and 33 mol% cholesterol and hydrated and extruded as described previously. [00160] Small-scale layer-by-layer (LbL) assembly: For small-scale (~50 µL) polymer adsorption onto NP surfaces, polymer solution was added to a 1.7 mL tube. The container was then placed under bath sonication (Branson 2510) and the NPs were quickly (<1 s) added to the polymer solution. Samples were allowed to incubate for 5 minutes analysis. Any subsequent layers were deposited without any purification unless otherwise indicated. For all polymer-to- NP wt. eq. values, only the NP core mass is considered. [00161] Large-scale LbL assembly and purification via tangential flow filtration (TFF): Assembly of polyelectrolyte layers at larger scale (>1 mL) was performed as described previously. Briefly, unlayered liposomes were added to a solution with 0.3-0.4 wt. eq. of PLR relative to lipid in a glass Erlenmeyer flask under sonication. After 30-minutes incubation on ice, excess PLR polymer was removed by tangential-flow filtration (TFF) through a 100 kDa mPES membrane (Repligen) pre-treated with 10 mg/mL solution of free PLR. For the terminal PLE layer, purified particles coated with PLR were added to a solution with PLE in a glass flask under sonication at 1 wt. eq. of polymer to lipid. Particles coated with both PLR and PLE were the purified by TFF on a separate 100 kDa mPES membrane (Repligen) to remove any excess PLE. For layering, a glass Erlenmeyer flask with <30% of its rated volume was used to facilitate homogeneous NP distribution over the polymer solution and minimize the chance of inhomogeneities in the sample. [00162] Microfluidics LbL assembly: Microfluidic mixing was performed on NanoAssemblr IgniteTMNxGenTM cartridge (NIN0061) that uses the NxGenTM mixing (Precision Nanosystems) chip. Luer lock syringes were separately filled with either polymer solution or the liposomes and attached to the microfluidics chip. A syringe pump (Pump 11 Elite, Harvard Apparatus) was then used to control the fluid flow rate into the mixing chip. With the exception of the flow rate studies, assembly was performed with flow rates of 9 mL/min (per channel). [00163] Characterization of particle preparations: Dynamic light scattering (DLS) and zeta potential measurements were made on a Zetasizer Nano ZSP (Malvern). Nanoparticle micrographs were acquired using Transmission Electron Microscopy (TEM) on a JEOL 2100F microscope (200 kV) with a magnification range of 10,000-60,000X. [00164] Fluorescent polymer synthesis. PLR was reacted with 2 molar equivalents of BDP- TR-NHS-ester (Lumiprobe) in DMSO catalyzed with one molar equivalent of triethylamine (TEA). PLR-TR was purified via Reverse-Phase High Pressure Liquid Chromatography (RP- HPLC) on a Jupiter C4 column (5 µm particles, 300 Å – Phenomenex) using a water:acetonitrile gradient which started at 20% acetonitrile for 5 minutes, then increased to 35% in a linear gradient until 10 minutes. Isocratic elution at 35% was performed for 30 minutes then the elution buffer was increased to 95% to clean out the column for 10 minutes then dropped back to 20% acetonitrile to re-equilibrate the column for 5 minutes. After HPLC purification, the polymer-dye conjugated was then lyophilized and stored at -20 °C. PLE was reacted with 5 molar excess of sulfo-cy3-NHS-ester (Lumiprobe) in pH 9 PBS (adjusted by adding 0.1 M sodium bicarbonate) for 3 hours at room temperature then left overnight at 4 °C. Excess dye was removed via extensive dialysis (3 kDa, Spectrum) against 0.9% NaCl then dialyzed against DI to remove salts. The resulting PLE-cy3 polymer was lyophilized and stored at -20 °C. [00165] Polymer retention quantification: To assess the amount of polymer retained on LbL- NPs after TFF processing, IL-12 NPs with 0.2 mol% of DOPE-630/650 was layered with 0.3 wt. eq. of a PLR solution composed of 50% PLR and 50% PLR-TR. After purification of excess PLR/PLR-TR via TFF, particles were layered with 1 wt. eq. of a PLE solution composed of 50% of PLE and 50% PLE-cy3. After purification of excess PLE/PLE-cy3 via TFF, the sample was diluted 10x into dimethyl sulfoxide (DMSO) to disrupt NPs. The fluorescence of lipids and polymers were separately quantified on a plate reader (TECAN) compared to standard curves to determine the polymer-to-NP wt. eq. of the purified NPs. [00166] Excess polymer quantification: IL-12 NPs were generated via either the standard TFF- based LbL protocol or the MCF protocol, but with the PLE layering solution having 50% PLE- cy3. Free polymers were separated from NPs on a 300 kDa centrifugal filter (Vivaspin500, Sartorius) at 30 xg for 20 min. Polymer fluorescence on the permeate fluid was then compared to that of the initial sample to determine the fraction of polymer bound to NPs. Permeate DOPE-630/650 fluorescence was measured to confirm complete particle separation. [00167] Assembly of LbL-NPs with various outer layer polymers. PLR-coated IL-12-NPs were first titrated with increasing concentrations of either HA, PLD, or PAA to determine the POP wt. eq. Then the solution of NP at 1-2 mg/mL was mixed the polymer at the POP wt. eq. as described herein. [00168] Assembly of LbL films on CML beads. CML beads of varying sizes were titrated with varying concentrations of PLR to determine the POP wt. eq. Particles were then mixed with the determined PLR wt. eq. as described in Microfluidics LbL assembly. The same process was repeated with PLE to generated LbL-CML NPs. [00169] Cell Culture: OV2944-HM-1 cells were acquired through Riken BRC and were cultured in α-MEM while MC38 were cultured in DMEM. Cell media was also supplemented with 10% FBS and penicillin/streptomycin with cells incubated in a 5% CO2 humidified atmosphere at 37 °C. All cell lines were murine pathogen tested and confirmed mycoplasma negative by Lonza MycoAlert™ Mycoplasma Detection Kit. [00170] In vitro cellular association: The day before dosing, HM-1 cells were plated on a tissue-culture 96-well plate at a density of 50k cells per well. The next day, wells were dosed with NPs to 0.05 mg/mL and left for the target incubation time (4 hrs or 24 hrs). For analysis of association, the supernatant was removed from the well and diluted 10X with DMSO. Cells were then washed three times with PBS then dissolved with DMSO. Fluorescence of NPs associated with cells was then normalized to supernatant fluorescence. The relative fluorescence of each formulation was then compared to an unlayered liposome control containing the same fluorophore as described previously. [00171] For analysis via flow cytometry of CML beads, NPs were dosed at the indicated concentrations and allowed to incubate with cells at 37°C for 4 hours. Cells were washed with PBS then detached from the plates using 0.25% trypsin and stained with DAPI (15 min incubation) for viability assessment and fixed with 2% paraformaldehyde (30 min incubation) until analysis by flow cytometry using an LSR Fortessa (BD Biosciences). For confocal imaging, 8-well chambered coverglass (Nunc Lab-Tek II, Thermo Scientific) were treated with rat tail collagen type I (Sigma-Aldrich) per manufacturer’s instructions. HM-1 cells were plated onto wells at a density of 10k/well and left to adhere overnight prior to NP treatment. After the desired incubation time with NPs, cells were washed 3x with PBS. After washing, cells were fixed in 4% paraformaldehyde for 10 minutes then washed (3x with PBS) and stained with wheat germ agglutinin (WGA) conjugated to Alexa Fluor488 (Invitrogen) and Hoechst 33342 (Thermo Scientific) following manufacturer instructions. Images were analyzed using ImageJ. Slides were imaged on an Olympus FV1200 Laser Scanning Confocal Microscope. [00172] Mice: B6C3F1 mice were purchased from Jackson Laboratories. Female mice were used between 8-12 weeks of age unless otherwise noted with weights of 20-25 g. All animal work was conducted under the approval of the Massachusetts Institute of Technology Division of Comparative Medicine in accordance with federal, state, and local guidelines. Before mouse treatments or imaging, mice were anesthetized with 2-3% isoflurane. [00173] Efficacy studies with metastatic ovarian cancer model: B6C3F1 mice were inoculated intraperitoneally with 106 cells of HM-1-luc in PBS. Five days after inoculation, bioluminescence was measured on an In Vivo Imaging System Spectrum CT (IVIS, Perkin Elmer) 10 minutes after i.p. injection of 3 µg of D-luciferin sodium salt (GoldBio) to confirm tumor engraftment and mice were randomized into treatment groups. At days 7 and 14 post- inoculation, mice were treated intraperitoneally with vehicle (5% dextrose) or 20 µg of IL-12 either as a free cytokine, or conjugated to unlayered (UL) NPs, or LbL-NPs. Mice weights were tracked daily after treatments for signs of toxicity. Bioluminescence was tracked for 30 days after tumor inoculation or as needed to evaluate tumor burden. [00174] Statistical Analysis: GraphPad PRISM 9 was used to perform statistical analyses. Comparisons between two groups was performed via unpaired t-tests. For multiple groups or multiple variable analysis, one-way, or two-way ANOVAs were used with Tukey’s posthoc correction. [00175] Assessment of NP colloidal stability under increasing ionic strength. Particles were diluted to 0.1 mg/mL in water buffered with 10 mM HEPES (pH 7.3) and assessed for their size via DLS. A 1 M NaCl stock solution was then used to adjust the salt concentration in the sample. After salt addition, samples were left to incubate for 10 minutes before size measurement on a DLS. After size measurement, the amount of NaCl was further increased and the sample again allowed to incubate for 10 minutes. This process was repeated until reaching super physiological levels of NaCl (i.e., > 200 mM NaCl). [00176] Assessment of NP colloidal stability in serum supplemented buffer. Particles were diluted to 0.1 mg/mL in a solution containing 15 mM HEPES (pH 7.3), 150 mM NaCl, and 10% FBS. The overall size of the sample was evaluated via DLS. [00177] Recombinant single-chain IL-12 production. Single-chain IL-12 sequence was synthesized as a genomic block (Integrated DNA Technologies®) and cloned into a gWIZ expression vector (Genlantis®). Plasmids were transiently transfected into Expi293™ cells (ThermoFisher Scientific). After 5 days, cell culture supernatants were collected and protein was purified in an ÄKTA pure chromatography system using HiTrap HP Niquel sepharose affinity column, followed by size exclusion using Superdex® 200 Increase 10/300 GL column (GE Healthcare Life Sciences). Endotoxin levels in purified protein was measured using Endosafe® Nexgen-PTS™ system (Charles River®) and assured to be <5 EU/mg protein. [00178] Assembly of LbL-NPs with various outer layer polymers. PLR-coated IL-12-NPs were first titrated with increasing concentrations of either HA, PLD, or PAA to determine the POP wt. eq. Then the solution of NP at 1-2 mg/mL was mixed the polymer at the POP wt. eq. as described in Microfluidics LbL assembly. [00179] Assembly of LbL films on CML beads. CML beads of varying sizes were titrated with varying concentrations of PLR to determine the POP wt. eq. Particles were then mixed with the determined PLR wt. eq. as described in Microfluidics LbL assembly. The same process was repeated with PLE to generated LbL-CML NPs. EMBODIMENTS Embodiment 1. A method of making a composition comprising a layered nanoparticle, comprising: microfluidic mixing of a solution comprising a nanoparticle with a solution comprising a first polyelectrolyte to form a solution comprising a first-layered nanoparticle. Embodiment 2. The method of embodiment 1, further comprising: microfluidic mixing of the solution comprising the first-layered nanoparticle with a solution comprising a second polyelectrolyte to form a solution comprising a second-layered nanoparticle. Embodiment 3. The method of embodiment 2, further comprising: microfluidic mixing of the solution comprising the nanoparticle with the solution comprising the first polyelectrolyte to form the solution comprising the first-layered nanoparticle; and microfluidic mixing of the solution comprising the first-layered nanoparticle, with the solution comprising the second polyelectrolyte to form the solution comprising the second- layered nanoparticle. Embodiment 4. The method of any one of embodiments 1-3, wherein the first and second polyelectrolytes independently comprise one or more of hyaluronic acid, poly- glutamate, poly-aspartate, poly-amino acid composite, block co-polymers of poly amino acids and poly-ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, and nucleic acids. Embodiment 5. The method of any one of embodiments 1-4, wherein the solution comprising the first-layered nanoparticle is substantially free of unbound first polyelectrolyte. Embodiment 6. The method of any one of embodiments 2-5, wherein the solution comprising the second-layered nanoparticle is substantially free of unbound second polyelectrolyte. Embodiment 7. The method of any one of embodiments 2-6, wherein each solution comprising the first and second polyelectrolytes does not comprise an excess of polyelectrolyte in relation to the nanoparticle. Embodiment 8. The method of any one of embodiments 1-7, wherein the nanoparticle comprises a surface charge that is opposite the charge of the first polyelectrolyte. Embodiment 9. The method of any one of embodiments 2-8, wherein the first polyelectrolyte and second polyelectrolyte comprise opposite charges. Embodiment 10. The method of any one of embodiments 1-9, wherein the first polyelectrolyte is poly-arginine. Embodiment 11. The method of embodiment 10, wherein the solution comprising the first polyelectrolyte comprises less than 0.3 weight equivalents of poly-arginine in relation to the nanoparticle. Embodiment 12. The method of embodiment 11, wherein the solution comprising the first polyelectrolyte comprises between 0.1 and 0.2 weight equivalents of poly-arginine in relation to the nanoparticle. Embodiment 13. The method of embodiment 11, wherein the solution comprising the first polyelectrolyte comprises 0.1 weight equivalents of poly-arginine in relation to the nanoparticle. Embodiment 14. The method of any one of embodiments 2-13, wherein the second polyelectrolyte is poly-glutamate. Embodiment 15. The method of embodiment 14, wherein the solution comprising the second polyelectrolyte comprises less than 0.6 weight equivalents of poly-glutamate in relation to the nanoparticle. Embodiment 16. The method of embodiment 15, wherein the solution comprising the second polyelectrolyte comprises between 0.6 and 0.4 weight equivalents of poly-glutamate in relation to the nanoparticle. Embodiment 17. The method of embodiment 16, wherein the solution comprising the second polyelectrolyte comprises 0.5 weight equivalents of poly-glutamate in relation to the nanoparticle. Embodiment 18. The method of any one of embodiments 1-17, wherein the nanoparticle comprises a liposome. Embodiment 19. The method of any one of embodiments 1-18, wherein the composition further comprises at least one additional pharmaceutical agent. Embodiment 20. The method of embodiment 19, wherein the at least one additional pharmaceutical agent comprises a protein, or fragment thereof, a small molecule, or a nucleotide. Embodiment 21. The method of embodiment 19 or 20, wherein the at least one additional pharmaceutical agent comprises a protein. Embodiment 22. The method of any one of embodiments 19-21, wherein the at least one additional pharmaceutical agent comprises a cytokine. Embodiment 23. The method of any one of embodiments 19-22, wherein the at least one additional pharmaceutical agent comprises IL-12. Embodiment 24. The method of any one of embodiments 1-23, wherein the method does not comprise purification by ultrafiltration. Embodiment 25. The method of any one of embodiments 1-24, wherein said microfluidic mixing comprises the use of at least one microfluidic chip. Embodiment 26. The method of embodiment 25, wherein the at least one microfluidic chip comprises a herring bone-shaped channel. Embodiment 27. The method of any one of embodiments 2-26, wherein said microfluidic mixing comprises the use of at least two microfluidic chips comprising a herring bone-shaped channel. Embodiment 28. The method of embodiment 27, wherein the at least two microfluidic chips are connected in series. Embodiment 29. The method of any one of embodiments 1-28, wherein each microfluidic mixing step independently comprises a flow rate of at least 1 mL/min. Embodiment 30. The method of any one of embodiments 1-29, wherein each microfluidic mixing step independently comprises a flow rate of at least 3 mL/min. Embodiment 31. The method of any one of embodiments 1-30, wherein the method does not comprise sonication or agitation. Embodiment 32. The method of any one of embodiments 1-31, wherein the layered nanoparticle has a polydispersity index of less than 0.25. Embodiment 33. The method of any one of embodiments 1-32, wherein the layered nanoparticle is smaller than 200 nm in size. Embodiment 34. The method of any one of embodiments 1-33, wherein the layered nanoparticle is between 110-130 nm in size. Embodiment 35. The method of any one of embodiments 1-34, wherein the solution comprising the nanoparticle has a concentration of at least 0.5 mg/mL. Embodiment 36. The method of any one of embodiments 1-35, wherein the solution comprising the nanoparticle has a concentration of about 5 mg/mL. Embodiment 37. A kit comprising: the solution comprising the nanoparticle any one of embodiments 1-3, the solution comprising the first polyelectrolyte of any one of embodiments 1-3, and the solution comprising the second polyelectrolyte of any one of embodiments 2-3; the microfluidic chips of embodiment 25 or 27; and instructions for its use. References: [1] H. Park, A. Otte, K. Park, Journal of Controlled Release 2022, 342, 53. [2] A. Yusuf, A. R. Z. Almotairy, H. Henidi, O. Y. Alshehri, M. S. Aldughaim, Polymers (Basel) 2023, 15, 1596. [3] M. J. Mitchell, M. M. Billingsley, R. M. Haley, M. E. Wechsler, N. A. Peppas, R. Langer, Nat Rev Drug Discov 2021, 20, 101. [4] P. T. Hammond, Advanced Materials 2004, 16, 1271. [5] D. Alkekhia, P. T. Hammond, A. Shukla, Annu Rev Biomed Eng 2020, 22, 1. [6] J. J. Richardson, J. Cui, M. Björnmalm, J. A. Braunger, H. 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Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. [00181] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. [00182] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art. [00183] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

CLAIMS What is claimed: 1. A method of making a composition comprising a layered nanoparticle, comprising: a first step of microfluidic mixing of a solution comprising a nanoparticle with a solution comprising a first polyelectrolyte to form a solution comprising a first- layered nanoparticle.
2. The method of claim 1, further comprising: a second step of microfluidic mixing of the solution comprising the first- layered nanoparticle with a solution comprising a second polyelectrolyte to form a solution comprising a second-layered nanoparticle.
3. The method of claim 1 or 2, wherein the first step results in charge conversion from the nanoparticle to the first-layered nanoparticle.
4. The method of claim 2 or 3, wherein the second step results in charge conversion from the nanoparticle to the second-layered nanoparticle.
5. The method of any one of claims 1-4, wherein the first and second polyelectrolytes independently comprise one or more of hyaluronic acid, poly-glutamate, polyaspartic acid, poly-amino acid composite, block co-polymers of poly amino acids and poly- ethylene glycol, poly-arginine, poly-lysine, poly-acrylic acid, dextran sulfate, heparin sulfate, heparin folate, nucleic acids, or a fluorescently labeled derivative thereof.
6. The method of any one of claims 1-5, further comprising determining an amount of polyelectrolyte needed to achieve a plateau onset point (POP).
7. The method of claim 6, wherein the solution comprising the first polyelectrolyte comprises at least 80% of the amount of polyelectrolyte needed to achieve the POP.
8. The method of claim 6, wherein the solution comprising the first polyelectrolyte comprises at least 90% of the amount of polyelectrolyte needed to achieve the POP.
9. The method of claim 6, wherein the solution comprising the first polyelectrolyte comprises the amount of polyelectrolyte needed to achieve the POP.
10. The method of any one of claims 6-9, wherein the solution comprising the second polyelectrolyte comprises at least 80% of the amount of polyelectrolyte needed to achieve the POP.
11. The method of any one of claims 6-10, wherein the solution comprising the second polyelectrolyte comprises a least 90% of the amount of polyelectrolyte needed to achieve the POP.
12. The method of any one of claims 6-11, wherein the solution comprising the second polyelectrolyte comprises the amount of polyelectrolyte needed to achieve the POP.
13. The method of any one of claims 1-12, wherein the solution comprising the first- layered nanoparticle is substantially free of unbound first polyelectrolyte after mixing.
14. The method of any one of claims 2-13, wherein the solution comprising the second- layered nanoparticle is substantially free of unbound second polyelectrolyte after mixing.
15. The method of any one of claims 1-14, wherein the solution comprising the first- layered nanoparticle does not comprise an excess of the first polyelectrolyte in relation to the nanoparticle.
16. The method of any one of claims 2-15, wherein the solution comprising the second- layered nanoparticle does not comprise an excess of the second polyelectrolyte in relation to the first-layered nanoparticle.
17. The method of any one of claims 1-16, wherein the nanoparticle comprises a surface charge that is opposite the charge of the first polyelectrolyte.
18. The method of any one of claims 2-17, wherein the first polyelectrolyte and second polyelectrolyte comprise opposite charges.
19. The method of any one of claims 1-18, wherein the first polyelectrolyte is poly- arginine.
20. The method of claim 19, wherein the solution comprising the first polyelectrolyte comprises between about 0.05 to 0.3 weight equivalents of poly-arginine in relation to the nanoparticle.
21. The method of claim 19 or 20, wherein the solution comprising the first polyelectrolyte comprises between about 0.1 and about 0.3 weight equivalents of poly-arginine in relation to the nanoparticle.
22. The method of claim 19, wherein the solution comprising the first polyelectrolyte comprises about 0.1 weight equivalents of poly-arginine in relation to the nanoparticle.
23. The method of any one of claims 2-22, wherein the second polyelectrolyte is selected from the group consisting of poly-glutamate, hyaluronic acid, polyaspartic acid, and polyacrylic acid.
24. The method of any one of claims 2-23, wherein the second polyelectrolyte is poly- glutamate. 25. The method of claim 24, wherein the solution comprising the second polyelectrolyte comprises between about 0.
25 to about 0.8 weight equivalents of poly-glutamate in relation to the nanoparticle.
26. The method of claim 25, wherein the solution comprising the second polyelectrolyte comprises between about 0.4 and about 0.6 weight equivalents of poly-glutamate in relation to the nanoparticle.
27. The method of claim 25 or 26, wherein the solution comprising the second polyelectrolyte comprises about 0.5 weight equivalents of poly-glutamate in relation to the nanoparticle.
28. The method of any one of claims 2-23, wherein the second polyelectrolyte is hyaluronic acid.
29. The method of claim 28, wherein the solution comprising the second polyelectrolyte comprises about 0.3 to about 0.5 weight equivalents of hyaluronic acid in relation to the nanoparticle.
30. The method of claim 28 or 29, wherein the solution comprising the second polyelectrolyte comprises about 0.4 weight equivalents of hyaluronic acid in relation to the nanoparticle.
31. The method of any one of claims 2-23, wherein the second polyelectrolyte is polyaspartic acid.
32. The method of claim 31, wherein the solution comprising the second polyelectrolyte comprises about 0.2 to about 0.4 weight equivalents of polyaspartic acid in relation to the nanoparticle.
33. The method of claim 31 or 32, wherein the solution comprising the second polyelectrolyte comprises about 0.3 weight equivalents of polyaspartic acid in relation to the nanoparticle.
34. The method of any one of 2-23, wherein the second polyelectrolyte is polyacrylic acid.
35. The method of claim 34, wherein the solution comprising the second polyelectrolyte comprises about 0.1 to about 0.3 weight equivalents of polyacrylic acid in relation to the nanoparticle.
36. The method of claim 34 or 35, wherein the solution comprising the second polyelectrolyte comprises about 0.15 to about 0.2 weight equivalents of polyacrylic acid in relation to the nanoparticle.
37. The method of any one of claims 1-36, wherein the nanoparticle is selected from the group consisting of lipid nanoparticles, liposomes, solid nanoparticles, metallic nanoparticles, polymeric nanoparticles, nanotubes, quantum dots, micelles, and lipid nano discs.
38. The method of claim 37, wherein the nanoparticle is a liposome.
39. The method of claim 37, wherein the nanoparticle is a latex bead.
40. The method of any one of claims 1-35, wherein the nanoparticle comprises an negative charge.
41. The method of claim 39, wherein the latex bead is carboxy-modified.
42. The method of any one of claims 1-41, wherein the composition further comprises at least one additional pharmaceutical agent.
43. The method of claim42, wherein the at least one additional pharmaceutical agent comprises a protein, or fragment thereof, a small molecule, or a nucleotide.
44. The method of claim 42 or 43, wherein the at least one additional pharmaceutical agent is a protein.
45. The method of any one of claims 42-44, wherein the at least one additional pharmaceutical agent comprises a cytokine.
46. The method of any one of claims 42-45, wherein the at least one additional pharmaceutical agent comprises IL-12.
47. The method of any one of claims 1-46, wherein the method does not comprise purification by ultrafiltration.
48. The method of any one of claims 1-47, wherein said microfluidic mixing comprises the use of at least one microfluidic mixing chip.
49. The method of claim 48, wherein the at least one microfluidic mixing chip comprises a bifurcating channel.
50. The method of any one of claims 2-49, wherein said microfluidic mixing comprises the use of at least two microfluidic mixing chips comprising a bifurcating channel.
51. The method of claim 50, wherein the at least two microfluidic mixing chips are connected in series.
52. The method of any one of claims 1-51, wherein each microfluidic mixing step independently comprises a flow rate of at least 1 mL/min.
53. The method of any one of claims 1-52, wherein each microfluidic mixing step independently comprises a flow rate of at least 3 mL/min.
54. The method of any one of claims 1-53, wherein the method does not comprise sonication or agitation.
55. The method of any one of claims 1-54, wherein the layered nanoparticle has a polydispersity index of about 0.05 to about 0.15.
56. The method of any one of claims 1-55, wherein the layered nanoparticle has a polydispersity index of less than 0.25.
57. The method of any one of claims 1-56, wherein the layered nanoparticle is smaller than 400 nm in size.
58. The method of any one of claims 1-56, wherein the layered nanoparticle is between 100-140 nm in size.
59. The method of any one of claims 1-58, wherein the solution comprising the nanoparticle has a concentration of at least 0.5 mg/mL.
60. The method of any one of claims 1-59, wherein the solution comprising the nanoparticle has a concentration of about 5 mg/mL.
61. The method of any one of claims 1-60, wherein the solution comprising the nanoparticle has a concentration of about 1 mg/mL.
62. A kit comprising: the solution comprising the nanoparticle any one of claims 1-3 or 36-41; the solution comprising the first polyelectrolyte of any one of claims 1-22; the solution comprising the second polyelectrolyte of any one of claims 2-36; the microfluidic mixing chips of any one of claims 48-51; and instructions for its use.
63. A kit comprising: 1) a solution comprising a nanoparticle; 2) a solution comprising a first polyelectrolyte; 3) a solution comprising a second polyelectrolyte; 4) one or more microfluidic mixing chips; and instructions for its use.
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