Controlled assembly of lipid-based nanoparticles from surfactant micelles

Abstract

The disclosure provides particles, including anisotropic particles, and compositions, methods and kits thereof that are useful, e.g., for delivering an agent or in treating or preventing diseases, such as proliferative diseases. The disclosure also provides methods of preparing plurality of such particles and liposomes that provide control over size and morphology.

Description

CONTROLLED ASSEMBLY OF LIPID-BASED NANOPARTICLES FROM SURFACTANT MICELLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S.S.N. 63 / 653,644, filed May 30, 2024, titled “Controlled Assembly of Lipid- Based Nanoparticles from Surfactant Micelles,” which is incorporated herein by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under AI161297, CA235375, CA274651, and AI048240 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Particles such as liposomes, nanoparticles, and lipid nanoparticles have been used to deliver agents to cells due to their ability to encapsulate drugs and accumulate in target tissues. However, most methods of synthetizing particles fail to controllably generate particles at controllable sizes and low poly dispersity, especially via scalable approaches. In addition, while considerable effort has been placed on developing spherical lipid-based nanocarriers, few techniques reliably afford high aspect ratio lipid nanocarriers. To date, altering lipid-based particle shape has required additional agents, such as polymers, proteins, lipid-polymer conjugates, or detergents, and has only been accomplished through non- scalable methodologies and / or processes that do not result in particles stable under physiological conditions.SUMMARY OF THE DISCLOSURE

[0004] In one aspect, provided herein is a particle, wherein the particle comprises:(a) a polyvalent phospholipid;(b) a helper phospholipid; and(c) optionally, a sterol; wherein the particle has anisotropic dimensions.

[0005] In another aspect, provided herein is a method of preparing a plurality of particles having anisotropic dimensions provided herein, the method comprising:(a) preparing a mixture of a polyvalent phospholipid, a helper phospholipid, and a detergent;(b) allowing the mixture to equilibrate afford a plurality of micelles;(c) diluting the micelles with a buffer to a target detergent concentration, thereby forming a plurality of particles having anisotropic dimensions; and(d) separating the plurality of particles.

[0006] In another aspect, provided herein is a method of preparing a plurality of liposomes, the method comprising:(a) preparing a mixture of a monoanionic phospholipid, a helper phospholipid, and a detergent;(b) allowing the mixture to equilibrate afford a plurality of micelles;(c) diluting the micelles with a buffer to a target detergent concentration to promote self-assembly of a plurality of bicelles having anisotropic dimensions, wherein the bicelles undergo bicelle fusion to afford a plurality of liposomes; and(d) separating the plurality of liposomes.

[0007] In another aspect, provided herein is a liposome prepared according to a method provided herein.

[0008] In another aspect, provided herein is a pharmaceutical composition comprising a plurality of particles provided herein, and a pharmaceutically acceptable excipient.

[0009] In another aspect, provided herein is a pharmaceutical composition comprising a plurality of liposomes provided herein, and a pharmaceutically acceptable excipient.

[0010] In another aspect, provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a particle provided herein or a pharmaceutical composition provided herein.

[0011] In another aspect, provided herein is a method of delivering an agent to a target cell in a subject, cell, or biological sample, comprising contacting the target cell with a particle provided herein.

[0012] In another aspect, provided herein is a kit comprising: a particle provided herein, or a pharmaceutical composition provided herein; and instructions for using the particle or the pharmaceutical composition.

[0013] 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 specificembodiments, 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which constitute a part of this specification, illustrate several embodiments of the present disclosure and together with the description, provide nonlimiting examples of the disclosure. The figures are exemplary and do not limit the scope of the present disclosure.

[0015] FIGs. 1 A-1D show the effect of detergent concentration during nanoparticle formation on equilibrium size of nanoparticles. FIG. 1 A is a schematic showing liposome assembly from mixed micelle dilution. FIG. IB shows the size of samples diluted with PBS to various concentrations of MEGA- 10 starting from an initial mixture of 10 mg / mL of 6:3: 1 molar mixture of DSPC:cholesterol:POPG in 10% MEGA-10. FIGs. 1C show size kinetics of mixed micelles diluted to 0.1%, 0.02% and 0.01% MEGA-10. FIG. ID shows PDI kinetics of mixed micelles diluted to 0.1%, 0.02% and 0.01% MEGA-10.

[0016] FIGs. 2A-2F show that varying detergent concentration and temperature controls equilibrium size of liposomes. FIG. 2A shows the DLS intensity -weighted size (Z-avg) and PDI after overnight incubation of a 10 mg / mL of 6:3: 1 molar mixture of DSPC:cholesterol:POPG in 10% MEGA-10 diluted to various final detergent concentrations. FIG. 2B shows the DLS intensity-weighted size (Z-avg) and PDI after overnight incubation of a 20 mg / mL of 6:3: 1 molar mixture of DSPC: cholesterol :POPG in 10% octylglucoside diluted to various final detergent concentrations. FIG. 2C shows the effect of dilution samples equilibrated at 0.1% or 0.04% MEGA-10 overnight to 0.01% MEGA-10 on particle size (Z-avg) and PDI; samples were allowed to equilibrate at 0.01% overnight. FIG. 2D shows the size and PDI determined by DLS of nanoparticles resulting from four independent samples of 10 mg / mL of 6:3: 1 molar mixture of DSPC:cholesterol:POPG in 10% MEGA-10 diluted to either 0.1% or 0.01% MEGA-10 with PBS and equilibrated. FIG. 2E shows the particle size and PDI of samples diluted to various MEGA- 10 concentrations and incubated at room temperature (RT), 37 °C, or 4 °C overnight. FIG. 2F shows the count rate as a function of temperature of liposomes composed of 6:3 : 1 DSPC:cholesterol:POPG molar ratio as analyzed via DLS for structural changes; a phase transition of DSPC was observed at 55 °C.

[0017] FIGs. 3 A-3B show that intermediates formed during liposome self-assembly comprise multiple species. FIG. 3A shows the DLS intensity-weighted size (Z-avg) and PDI ofsamples taken during self-assembly of liposomes at 0.1% MEGA-10 before (pre) and after (post) dilution to 0.01% MEGA-10. FIG. 3B shows the volume-weighted distribution of samples taken during self-assembly of liposomes at 0.1% MEGA-10 before (pre) and after (post) dilution to 0.01% MEGA-10.

[0018] FIGs. 4A-4D show that solution ionic strength and lipid composition regulate selfassembly from lipid / detergent micelles into liposomes. FIG. 4A shows the particle size (Z- avg) and PDI after lipid / detergent micelle dilution (10 mg / mL of 6:3: 1 molar mixture of DSPC:cholesterol:POPG in 10% MEGA-10) with 10 mM HEPES. FIG. 4B shows the Z-avg and PDI of neutral lipid / detergent micelles (10 mg / mL of 7:3 molar mixture of DSPGcholesterol in 10% MEGA-10) diluted with either PBS or 10 mM HEPES. FIG. 4C shows the Z-avg and PDI of 9: 1 DSPGPOPG mixture diluted to various final MEGA-10 concentrations and incubated overnight at room temperature. FIG. 4D shows the Z-avg and PDI of anionic lipid / detergent micelles charged with DSPG either containing or lacking cholesterol (10 mg / mL of either 6:3: 1 or 9: 1 molar mixture of DSPC:cholesterol:DPSG or DSPGDSPG in 10% MEGA-10) which were diluted with either PBS or 10 mM HEPES.

[0019] FIGs. 5A-5F show that FRET analysis of diluted mixed micelles reveals intermediates with high rates of lipid exchange. FIG. 5 A is a schematic for dilution of a mixture of donor or acceptor FRET micelles. FIG. 5B depicts a schematic for separately diluting donor-only or acceptor-only micelles and then mixing the diluted samples (sepFRET). FIG. 5C shows normalized FRET efficiency immediately after (0 hours) mixing donor and acceptor FRET micelles and sepFRET micelles mixed after incubating for either 0 hours, 1 hour, or 24 hours. FIG. 5D shows the time course of normalized FRET efficiency since mixing donor and acceptor micelles at 0.1% MEGA-10 for FRET micelles of sepFRET micelles (0 hours, 1 hour, and 24 hour). FIG. 5E shows the normalized FRET efficiency two days after mixing donor and acceptor (48) of FRET micelles and sepFRET (0 hours, 1 hour, and 24 hours). FIG. 5F shows the time course of normalized FRET efficiency since mixing donor and acceptor micelles at 0.0125% MEGA-10 for FRET micelles of sepFRET micelles (0 hours, 1 hour, and 24 hour).

[0020] FIG. 6 shows that FRET analysis of lipid exchange reveals that assembled liposomes in region iv (as described in Example 1) are stable whereas particles in region iii (as described in Example 1) have high rates of lipid exchange even after no change in particle size. Data are shown as normalized FRET efficiency from samples containing 1 mol% of dye diluted with PBS to various concentrations of MEGA- 10 and mixed after set incubation periods.

[0021] FIGs. 7A-7D are Cryo-TEM micrographs of particles incubated in region iii (as described in Example 1) that show formation of large disc assemblies and coexistence of micelles with fully assembled bilayer vesicles. The Cryo-TEM micrographs were obtained for 10 mg / mL 6:3: 1 DSPC:Chol:POPG sample in 10% MEGA-10 rapidly diluted to 0.1% MEGA- 10 with 10 mM HEPES 150 mM NaCl and frozen for 5 minutes (FIG. 7A), 2 hours (FIG. 7B), 5 hours (FIG. 7C), and 24 hours (FIG. 7D) after dilution. FIG. 7E is a schematic representation of primary events for samples allow to incubate at each region (as described in Example 1) from FIGs. 1A-1D. FIG. 7F is a schematic representation of a proposed model for the driving force for bicelle fusion to free detergents and phase separate into high curvature assemblies.

[0022] FIGs. 8A-8E show that the detergent to lipid ratio affects liposome assembly only in the mixed micelle coexistence region with bilayers. FIG. 8A shows the Z-avg size and PDI measured via DLS of samples of 10% MEGA- 10 loaded with either 50 mg / mL, 25 mg / mL, 10 mg / mL, or 1 mg / mL of the 6:3: 1 DSPC:cholesterol:POPG molar ratio composition to generate 2: 1, 4: 1, 10: l, or l00: l mass ratio mixtures then diluted with PBS to various concentrations of MEGA- 10 then incubated overnight at room temperature. FIG. 8B shows the Z-avg size and PDI as a function of varied lipid to detergent mass ratios at specified MEGA- 10 concentrations. FIG. 8C shows the Z-avg size and PDI obtained from diluting samples equilibrated at 0.1% MEGA-10 overnight then diluted to further to 0.0125%. FIG. 8D shows the Z-avg size and PDI obtained from diluting samples equilibrated at 0.2% MEGA-10 overnight then diluted to further to 0.01% MEGA-10. FIG. 8E is a schematic showing the proposed effect of varying the lipid to detergent mass ratio when samples are diluted to the mixed micelle region.

[0023] FIGs. 9A-9B are chromatograms showing that TFF of assembled lipid vesicles enables efficient removal of MEGA- 10. FIG. 9A is an ELSD chromatogram of MEGA- 10 at increasing concentrations. FIG. 9B is an ELSD chromatogram of final purified particles incubated at either 0.1%, 0.02%, or 0.004% MEGA-10.

[0024] FIGs. 10A-10I show that purified lipid nanoparticles maintain size and monodispersity. FIG. 10A shows DLS intensity and number distribution for liposomes generated by diluting a lipid mix (of 10 mg / mL of 6:3 : 1 molar mixture of DSPC:cholesterol:POPG in 10% MEGA-10) to 0.1%, 0.02% and 0.004% of detergent. FIG. 10B shows the size and PDI of samples from FIG. 10 A. FIG. 10C shows the zeta potential of samples from FIG. 10A; the variation in measured zeta potentials can be explained by the approximation of the Henry’s function as a constant in the instrument given that for a givenzeta potential, increased particle size increases electrophoretic mobility.24FIGs. 10D-10F are representative cryoTEM images of samples from samples generated at 0.004% (FIG. 10D), 0.02% (FIG. 10E), and 0.1% (FIG. 10F). Internal shading on large liposomes is due to particle protrusion from ice. FIG. 10G is a histogram of particles measured on cryoTEM micrographs from samples in FIG. 10A. FIG. 10H is a representative cryoTEM micrograph from liposomes generated via thin film hydration followed by extrusion on a 50 nm poresized membrane. FIG. 101 is a normalized histogram of samples from FIG. 10A and extruded liposome sample from FIG. 10H; the parentheses indicate PDI based on the measured particle sizes.

[0025] FIGs. 11 A-l ID show the characteristics of liposomes generated from lipid film hydration followed by extrusion with a 50 nm pore sized membrane. FIG. 11 A shows the DLS intensity-based and number-based size distribution. FIG. 1 IB shows the Z-avg, number-average, and PDI. FIG. 11C is a CryoTEM micrograph showing large liposomes after extrusion. FIG. 1 ID shows CryoTEM micrograph analysis of particle size from extruded sample.

[0026] FIGs. 12A-12B show controlled assembly of liposomes reveals a linear sizedependence effect on NP uptake in macrophages in vitro. FIG. 12A shows the intensity weighted size (Z-avg) and PDI of liposomes generated for this experiment. FIG. 12B shows the % NP uptake by RAW264.7 macrophage cells as a function of NP diameter after 4 or 24 hours of incubation with NPs.

[0027] FIGs. 13A-13G show the effect of solution ionic strength on self-assembly of lipid / detergent micelles. FIG. 13 A shows the effect of ionic strength on particle size upon lipid / detergent micelle dilution (10 mg / mL of 6:3: 1 molar mixture of DSPC:cholesterol:POPG in 10% MEGA-10). All solutions contained 10 mM HEPES for buffer control. FIG. 13B shows intensity over elution time for samples containing pure MEGA-10, PBS buffer, or purified lisCND and purified liposomes diluted with 200 mM NaCl. FIG. 13C shows the Z-avg, number average, and PDI of purified samples from lipid / detergent micelles diluted to 0.1% MEGA-10 overnight followed by dilution to 0.02% prior to TFF. FIGs. 13D-13G are CryoTEM micrographs of purified samples from dilution of lipid / detergent micelles using 200 mM NaCl (FIG. 13D), 0 mM NaCl (FIG. 13E), 0 mM NaCl but then added to IX PBS (FIG. 13F). FIG 13G are cryoTEM micrograph of purified 0 mM NaCl particles but then added to IX PBS in presence of bovine serum albumin then purified.

[0028] FIGs. 14A-14D show that the ionic strength of the dilution buffer regulated lipid nanoparticle structure. FIG. 14A shows the effect of ionic strength on PDI upon lipid / detergent micelle dilution (10 mg / mL of 6:3: 1 molar mixture of DSPC:cholesterol:POPG in 10% MEGA-10). All solutions contained 10 mM HEPES for buffer control. FIG. 14B shows Zeta potential measurements of lisCNDs. FIG. 14C shows the Z-avg and PDI of LNDs and lisCNDs after dilution to 0.02% MEGA-10 in lOmM HEPES and after TFF purification. FIG. 14D shows the DLS size and PDI of lisCNDs after purification and after 6 months of storage at 4 °C in deionized water.

[0029] FIGs. 15A-15F show that highly anionic lipid compositions yield minute liposomes. FIG. 15A shows the size and PDI of PBS-diluted lipid / detergent micelles containing 10 mg / mL of lipids in 10% MEGA-10 composed of either 5:3:2, 4:3:3, or 3:3:4 molar ratios of DSPC:cholesterol:POPG. FIG. 15B shows the size and PDI of lipid / detergent micelles with 3:3:4 molar ratios of DSPC:cholesterol:POPG diluted to 0.05% MEGA-10 then purified via TFF. FIG. 15C shows the zeta potential of purified 3:3:4 molar ratio nanoparticles. FIG. 15D is a chromatogram (RP-HPLC coupled with ELSD) of purified liposomes with 3:3:4 molar ratio showing undetectable levels of residual MEGA-10. FIG. 15E is a histogram of measured sizes from cryoTEM micrographs. FIG. 15F is a representative cryoTEM micrograph of TFF-purified sample from (FIG. 15B).

[0030] FIGs. 16A-16G show that the charge density of anionic lipid DOPE-glutaryl enables synthesis of CNDs stable at physiological ionic strength buffers. FIG. 16A depicts the chemical structures of POPG and DOPE-glutaryl. FIG. 16B shows the DLS size (Z-avg) of particles from varying compositions of DPSC, cholesterol and DOPE-glutaryl diluted to specific MEGA- 10 concentrations (all samples contained mol% cholesterol and the mol% indicated of DOPE-glutaryl with the remainder begin DSPC). FIG. 16C shows the DLS Z- avg, PDI and number average (#-avg) of samples with increasing DOPE-glutaryl mol% incubated at 0.1% MEGA-10. FIG. 16D shows the DLS Z-avg, #-avg, and PDI of purified CNDs composed with 10 mol% DOPE-glutaryl compared to extrusion-based liposomes with 10 mol% POPG (both samples contained 30% cholesterol and 60% DSPC). FIG. 16E shows the zeta potential of CNDs and liposomes from FIG. 16D. FIG. 16F shows negative stain TEM (NS-TEM) of CNDs from FIG. 16D. FIG. 16G depicts the proposed structure of CNDs composed of DOPE-glutaryl, DSPC, and cholesterol.

[0031] FIGs. 17A-17C show characterization data for DOPE-glutaryl NPs. FIG. 17A shows the PDI of particles obtained from varying compositions of DPSC, cholesterol and DOPE- glutaryl diluted to specific MEGA- 10 concentrations (all samples contained mol% cholesteroland the mol% indicated of DOPE-glutaryl with the remainder begin DSPC). FIG. 17B is a ELSD RP-HPLC chromatogram of purified CND sample. FIG. 17C shows the DLS count rate for liposomes and CNDs measured either without or with polarized light filters.

[0032] FIGs. 18A-18F show that CNDs produced better tumor accumulation than liposomes in a subcutaneous mouse model. FIG. 18A depicts an in vivo study timeline in which mice were dosed with 1 nmol of cyanine-5 labeled NPs (1 mol%). FIG. 18B shows tumor radiant efficiency measured in vivo via IVIS as a function of time. FIG. 18C shows the area under the curve (AUC) for liposomes and CNDs. FIG. 18D shows the serum fluorescence of CND- and liposome-dosed animals at 4 and 16 hours after dosing. FIG. 18E shows the recovered radiant efficiency from tumor, liver, and spleen tissues 24 hours after dosing mice with either CNDs or liposomes. FIG. 18F shows the total NP radiant efficiency of ex vivo tumors measured on IVIS.

[0033] FIGs. 19A-19D show that deposition of poly electrolyte layers composed of PLR and PLE onto CNDs enabled improved association of CNDs with ovarian cancer cells in vitro. FIG. 19A is a schematic of LbL technique used to generate liposome-LbL and CND-LbL. FIG. 19B shows the size and PDI of CND and liposomes before and after LbL modification. FIG. 19C shows the association of NP formulations with HM-1 cells relative to liposomes. FIG. 19D is confocal images of HM-1 cells after 4 hours of incubation with NPs.

[0034] FIGs. 20A-20C show the characteristics of LbL-CNDs and LbL-lisCNDs. FIG. 20A shows the zeta potential of liposomes and CNDs upon layering with PLR and PLE. FIG. 20B shows the size and zeta potential of lisCNDs upon layering with PLR and PLE. FIG. 20C shows the in vitro HM-1 association of NPs relative to that of liposomes 24 hours after dosing cells.

[0035] FIGs. 21 A-21F show that LbL-CNDs NPs efficiently targeted metastatic ovarian cancer in vivo. FIG. 21 A depicts an in vivo timeline for treatment of fluorescently-labeled NPs administered intraperitoneally in an ovarian cancer model. FIG. 2 IB shows the total radiant efficiency of NP fluorescence from peritoneum. FIG. 21C shows the AUC of peritoneal fluorescence readings from FIG. 2 IB. FIG. 2 ID shows the ex vivo weight normalized NP fluorescence in liver, spleen, UGT, and omentum. FIG. 2 IE shows the Spearman’s correlation coefficient between weight-normalized NP fluorescence and weight- normalized BLI readings. Error bars (s.e.m.) derived from parameter estimates for each group. FIG. 2 IF shows the slope of linear fit between weight-normalized NP fluorescence and weight-normalized BLI readings. Error bars (s.e.m.) represent variation between each animal in respective treatment groups. Error bars represent SEM, n = 4. Statisticalcomparisons in FIGs. 2 IB and 2 ID were performed using two-way analysis of variance (ANOVA). One-way ANOVA was used in FIG. 21C and FIG. 2 IF with Tukey’s multiplecomparisons test. Spearman’s correlation significance for FIG. 2 IE was performed based on a t-test analysis with the null hypothesis of no (r=0) correlation. Asterisks denote p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.DEFINITIONS

[0036] 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 (e.g., lipid nanoparticles), macroscopic particles, pellets, crystals, aggregates, composites, pulverized, milled or otherwise disrupted matrices, and cross-linked protein or polysaccharide particles. A particle may be composed of a single substance or multiple substances. In certain embodiments, the particle is a viral particle. In other embodiments, the particle is a liposome. In certain embodiments, the particle is a micelle. In certain embodiments, the particle is not a viral particle. 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.

[0037] The term “anisotropic” refers to particles that exhibit different sizes when measured in different directions.

[0038] 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 (pm) (e.g., between about 10 nm and about 300 nm, between about 10 nm and about 100 nm, between about 10 nm and about 80 nm, between about 10 nm and about 50 nm, between about 10 nm and about 30 nm, or between about 20 nm and about 200 nm), inclusive.

[0039] The term “microparticle” refers to a particle having an average (e.g., mean) dimension (e.g., diameter) of between about 0.5 micrometer (pm) and about 1 millimeter (mm) (e.g., between about 0.5 pm and about 100 pm, between about 0.5 pm and about 30 pm, between about 0.5 pm and about 10 pm, or between about 0.5 pm and about 3 pm), inclusive.

[0040] The term “polymer” refers to a compound comprising two or more covalently connected repeating units. In certain embodiments, a polymer is naturally occurring. In certain embodiments, a polymer is synthetic (z.e., not naturally occurring).

[0041] 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 embodiment, a 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 charge. The net charge of a given polyelectrolyte may depend on the surrounding chemical conditions, e.g., on the pH. Exemplary polyelectrolytes for use in polymeric coatings in the composition disclosed herein are but not limited to: poly(L-arginine) (PLR), poly-L-lysine (PLL), polyethylenimine (PEI), poly(P- amino esters), poly-L-glutamic acid (PLE), polyarginine, polyglutamic acid, polylysine, heparin folate, heparin sulfate, fucoidan, sulfated-P-cyclodextrin, hyaluronic acid (HA), polyglutamic acid-block-polyethylene glycol, poly-L-aspartic acid (PLD), polyaspartic acid, polystyrene sulfonate (SPS), polyacrylic acid (PAA), linear poly(ethylene imine) (LPEI), poly(diallyldimethyl ammonium chloride) (PDAC), polyallylamine hydrochloride (PAH), poly(L-lactide-co-L-lysine), polyserine ester, poly(4-hydroxy-L-proline ester), poly[a-(4- aminobutyl)-L-glycolic acid], sodium polystyrene sulfonate, dextran sulfate (DXS), alginate, and chondroitin sulfate.

[0042] The term “cation” or “cationic” used herein refer to a species which has a net positive charge. The term “anion” or “anionic” used herein refer to a species which has a net negative charge.

[0043] The term “sterol” refers to a subgroup of steroids also known as steroid alcohols, z.e., a steroid containing at least one hydroxyl group. Sterols are usually divided into two classes: (1) plant sterols also known as “phytosterols,” and (2) animal sterols also known as “zoosterols.” The term “sterol” includes, but is not limited to, cholesterol, sitosterol, campesterol, stigmasterol, brassicasterol (including dihydrobrassicasterol), desmosterol, chalinosterol, poriferasterol, clionasterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, clerosterol, nervisterol, lathosterol, stellasterol, spinasterol, chondrillasterol, peposterol, avenasterol, isoavenasterol, fecosterol, pollinastasterol, and all natural or synthesized forms and derivatives thereof, including isomers.

[0044] As used herein, the term “salt” refers to any and all salts, and encompasses pharmaceutically acceptable salts. Salts include ionic compounds that result from the neutralization reaction of an acid and a base. A salt is composed of one or more cations (positively charged ions) and one or more anions (negative ions) so that the salt is electrically neutral (without a net charge). Salts of the present disclosure include those derived from inorganic and organic acids and bases. Examples of acid addition salts are salts of an aminogroup 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 known in the art such as ion exchange. Other salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, 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, -toluenesulfonate, undecanoate, valerate, hippurate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N (Ci 4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further salts include ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0045] The term “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, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts 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 known 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, di gluconate, dodecyl sulfate, 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+(CI-4 alkyl)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, lower alkyl sulfonate, and aryl sulfonate.

[0046] An “amino acid” refers to natural and unnatural D / L alpha-amino acids, as well as natural and unnatural beta- and gamma- amino acids. A “peptide” refers to two amino acids joined by a peptide bond. A “polypeptide” refers to three or more amino acids joined by peptide bonds. An “amino acid side chain” refers to the group(s) pended to the alpha carbon (if an alpha amino acid), alpha and beta carbon (if a beta amino acid), or the alpha, beta, and gamma carbon (if a gamma amino acid).

[0047] A “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, 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 (z.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 famesyl group, an isofamesyl 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.

[0048] The terms “composition” and “formulation” are used interchangeably.

[0049] A “subject” to which administration is contemplated refers to a human (z.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. Incertain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal may be a male or female at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal. The term “patient” refers to a human subject in need of treatment of a disease.

[0050] The term “biological sample” refers to any sample including tissue samples (such as tissue sections and needle biopsies of a tissue); cell samples (e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); samples of whole organisms (such as samples of yeasts or bacteria); or cell fractions, fragments or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample.

[0051] The term “target tissue” refers to any biological tissue of a subject (including a group of cells, a body part, or an organ) or a part thereof, including blood and / or lymph vessels, which is the object to which an agent, particle, and / or composition of the present disclosure is delivered. A target tissue may be an abnormal or unhealthy tissue, which may need to be treated. A target tissue may also be a normal or healthy tissue that is under a higher than normal risk of becoming abnormal or unhealthy, which may need to be prevented. In certain embodiments, the target tissue is the liver. In certain embodiments, the target tissue is the lung. A “non-target tissue” is any biological tissue of a subject (including a group of cells, a body part, or an organ) or a part thereof, including blood and / or lymph vessels, which is not a target tissue.

[0052] A “target cell” refers to a cell in a subject (in vivo) or ex vivo to which an agent, particle, and / or composition of the present disclosure is delivered. A target cell may be an abnormal or unhealthy, which may need to be treated. A target cell may also be a normal or healthy but is under a higher-than-normal risk of becoming abnormal or unhealthy, which may need to be prevented. In certain embodiments, the target cell is a cancer cell. In certain embodiments, the target cell is an ovarian cancer cell (e.g., HM-1 cell). In certain embodiments, the target cell is a colon cancer cell. In certain embodiments, the target cell isa brain cancer cell. In certain embodiments, the target cell is a skin cancer cell. In certain embodiments, the target cell is a head and neck cancer cell. In certain embodiments, the target cell is a lung cancer cell.

[0053] The terms “administer,” “administering,” or “administration” refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a particle described herein, or a composition thereof, in or on a subject.

[0054] The term “intravenous injection” is used herein to refer to a mode of administration wherein the composition is administered through an injection into the vein or veins.

[0055] The term “intraperitoneal injection” is used herein to refer to a mode of administration wherein the composition is administered through an injection into the peritoneum.

[0056] The terms “condition,” “disease,” and “disorder” are used interchangeably.

[0057] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0058] The term “prevent,” “preventing,” or “prevention” refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.

[0059] An “effective amount” of a particle or agent described herein refers to an amount sufficient to elicit the desired biological response. An effective amount of a particle or agent described herein may vary depending on such factors as the desired biological endpoint, severity of side effects, disease, or disorder, the identity, pharmacokinetics, and pharmacodynamics of the particular particle or agent, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age and health or general condition of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a particle or agent described herein in a single dose. In certain embodiments, an effectiveamount is the combined amounts of a particle or agent described herein in multiple doses. In certain embodiments, the desired dosage is delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage is delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

[0060] In certain embodiments, an effective amount of a particle or agent for administration one or more times a day to a 70 kg adult human comprises about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a particle or agent per unit dosage form.

[0061] In certain embodiments, the particle or agent of the disclosure is administered at dosage levels sufficient to deliver from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 40 mg / kg, preferably from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, and more preferably from about 1 mg / kg to about 25 mg / kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

[0062] It will be appreciated that dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0063] A “therapeutically effective amount” of a particle or agent described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a particle or agent means an amount of particle or agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient for delivering an agent to a subject or a cell. In certain embodiments, a therapeutically effective amount is an amount sufficient for deliveringan agent to a subject or a cell. In certain embodiments, a therapeutically effective amount is an amount sufficient for treating a disease, disorder, or condition. In certain embodiments, a therapeutically effective amount is an amount sufficient for delivering an agent to a subject or a cell and treating a disease, disorder, or condition.

[0064] A “prophylactically effective amount” of a particle or agent described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a particle or agent means an amount of a particle or agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. In certain embodiments, a prophylactically effective amount is an amount sufficient for delivering an agent to a subject or a cell. In certain embodiments, a prophylactically effective amount is an amount sufficient for preventing a disease, disorder, or condition. In certain embodiments, a prophylactically effective amount is an amount sufficient for delivering an agent to a subject or a cell and preventing a disease, disorder, or condition.

[0065] A “proliferative disease” refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology, Cambridge University Press: Cambridge, UK, 1990). A proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.

[0066] The term “angiogenesis” refers to the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels in a developing embryo form through vasculogenesis, after which angiogenesis is responsible for most blood vessel growth during normal or abnormal development. Angiogenesis is a vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. However, angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors inthe treatment of cancer. Angiogenesis may be chemically stimulated by angiogenic proteins, such as growth factors (e.g., VEGF). “Pathological angiogenesis” refers to abnormal (e.g., excessive or insufficient) angiogenesis that amounts to and / or is associated with a disease.

[0067] The terms “neoplasm” and “tumor” are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A neoplasm or tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A “benign neoplasm” is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin. In addition, a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor’s neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.” An exemplary pre-malignant neoplasm is a teratoma. In contrast, a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites. The term “metastasis,” “metastatic,” or “metastasize” refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. For example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.

[0068] The term “cancer” refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See e.g., Stedman ’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast,mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi’s sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma); Ewing’s sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g. , head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B- cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom’s macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a. k.a. Wilms’ tumor, renal cellcarcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget’s disease of the penis and scrotum); pineal oma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’s disease of the vulva).

[0069] 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.

[0070] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0071] Lipid-based nanoparticles are promising vehicles for the delivery of therapeutic agents. Since their first Food and Drug Administration (FDA) approval in 1995, the development of these nanocarriers has greatly expanded due to their ability to efficiently package therapeutics and improve accumulation at target tissues.1,2Nanoparticles (NP) can serve as carriers for insoluble drugs or as therapeutic delivery vehicles for substances which have inherent poor pharmacokinetic properties. Nanomedicine is a promising approach to treat cancer,1as NPs tend to passively accumulate in tumors via mechanisms such as the enhanced permeability and retention (EPR) effect and may be functionalized to target tumors. Accordingly, NPs can improve the effectiveness of a drug while also potentially mitigating side effects by accumulating tumor tissue and sparing healthy tissue.

[0072] The size, shape, rigidity, and surface chemistry of NPs are all parameters that contribute to regulating pharmacokinetics, biodistribution, tumor penetration, and tumor cell uptake.2 4While there are many strategies to generate nanocarriers, most methods involve kinetically trapped species which often yields minimal control on their size, morphology, and polydispersity.3Moreover, the requirement of rapid mixing in many of these systems presents a major challenge to scale-up manufacturing.3

[0073] One of the main limitations of NPs is their poor tumor penetration as particles tend to accumulate on the borders preventing further NP entrance into tumors.4,5In order to overcome this issue, recent studies have indicated that sub 100 nm particles with high aspect ratios morphologies may improve tumor penetration.4,5However, the most widely studied cancer nanomedicines have consisted of spherical nanoparticles.4In the case of lipid-based nanocarriers, this is in part due to the difficulties in controlling the self-assembly of lipids into non-spherical shapes. Some approaches to alter lipid-based nanoparticles from spherical to discoidal have included the addition of lipid-polymer conjugates, proteins, synthetic amphiphilic polymers, and mixtures of surfactant tail lengths.6 10Indeed, it was demonstrated that lipid nanodics (LNDs) may have greater tumor penetration and accumulation in vivo compared to state-of-the-art spherical liposomes.7Unfortunately, the use of polymer conjugates such as polyethylene glycol (PEG) or other additives to induce disc formation can limit the usefulness of the therapeutics due to altered membrane properties, low biodegradability, clearance receptors, or immunogenicity.10,11

[0074] The inventors have surprisingly discovered that anisotropic discoidal particles (“nanodiscs”) can be obtained using a detergent-assisted assembly method that does not require the use of PEG or other additives that can impart deleterious properties to theparticles. In particular, the inventors obtained isolable charge-stabilized nanodiscs (CNDs) that were stable in solution under physiological conditions from the use of polyvalent phospholipids in conjunction with helper phospholipids (and optionally a sterol). The prepared CNDs exhibited improved tumor accumulation relative to comparable liposomes for improved cancer treatments.

[0075] The inventors also surprisingly discovered that nanodisc intermediates formed from monoanionic phospholipids in conjunction with a helper phospholipid (and optionally a sterol) could be used to afford large liposomes with improved control over particle size and low dispersity, which were not previously obtainable.Particles, Pharmaceutical Compositions, Kits, and Administration

[0076] In one aspect, provided herein is a particle, wherein the particle comprises:(a) a polyvalent phospholipid;(b) a helper phospholipid; and(c) optionally, a sterol; wherein the particle has anisotropic dimensions.

[0077] In another aspect, provided herein is a liposome prepared according to a method provided herein.

[0078] In another aspect, provided herein is a pharmaceutical composition comprising a plurality of particles provided herein, and a pharmaceutically acceptable excipient.

[0079] In another aspect, provided herein is a pharmaceutical composition comprising a plurality of liposomes provided herein, and a pharmaceutically acceptable excipient.

[0080] In another aspect, provided herein is a kit comprising: a particle provided herein, or a pharmaceutical composition provided herein; and instructions for using the particle or the pharmaceutical composition.

[0081] In some embodiments, the helper phospholipid is a neutral phospholipid or monovalent phospholipid. In some embodiments, the helper phospholipid is a monovalent phospholipid. In some embodiments, the helper phospholipid is a monoanionic phospholipid. In some embodiments, the helper phospholipid is a neutral phospholipid.

[0082] In some embodiments, the particle is not spherical. In some embodiments, wherein the particle is disc-shaped. In some embodiments, the particle has a largest cross-sectional dimension that is about 1.2- to about 10-fold larger than its smallest cross-sectional dimension. In some embodiments, the particle has a largest cross-sectional dimension that is about 1.2- to about 3-fold larger than its smallest cross-sectional dimension. In someembodiments, the particle has a largest cross-sectional dimension that is about 1.5- to about 2-fold larger than its smallest cross-sectional dimension. In some embodiments, the particle has a largest cross-sectional dimension that is about 3- to about 6-fold larger than its smallest cross-sectional dimension. In some embodiments, the particle has a largest cross-sectional dimension that is about 6- to about 10-fold larger than its smallest cross-sectional dimension. In some embodiments, the particle has a largest cross-sectional dimension of about 20 nm to about 50 nm. In some embodiments, the particle has a smallest cross-sectional dimension of about 3 nm to about 15 nm. In some embodiments, the particle has a smallest cross-sectional dimension of about 5 nm to about 10 nm.

[0083] In some embodiments, the particle comprises a sterol. In some embodiments, the sterol is campesterol, desmosterol, stigmasterol, lanosterol, sitosterol, or cholesterol. In some embodiments, the sterol is cholesterol.

[0084] In some embodiments, the particle comprises about 1 mol% to about 50 mol% of the polyvalent phospholipid. In some embodiments, the particle comprises about 5 mol% to about 50 mol% of the polyvalent phospholipid. In some embodiments, the particle comprises about 2 mol% to about 15 mol% of the polyvalent phospholipid. In some embodiments, the particle comprises at least about 5 mol% of the polyvalent phospholipid. In some embodiments, the particle comprises at least about 7.5 mol% of the polyvalent phospholipid. In some embodiments, the particle comprises about 10 mol% of the polyvalent phospholipid.

[0085] In some embodiments, the particle comprises about 20 mol% to about 95 mol% of the helper phospholipid In some embodiments, the particle comprises about 60 mol% of the helper phospholipid.

[0086] In some embodiments, the particle comprises about 0 mol% to about 50 mol% of the sterol. In some embodiments, the particle comprises about 30 mol% of the sterol.

[0087] In some embodiments, the particle comprises a molar ratio of about 6:3: 1, about 5:3:2, about 4:3:3, or about 3:3:4 of the helper phospholipid:the sterokthe polyvalent phospholipid. In some embodiments, the particle comprises a molar ratio of about 6:3: 1 of the helper phospholipid:the sterol :the polyvalent phospholipid. In some embodiments, the particle comprises a molar ratio of about 5:3:2 of the helper phospholipid:the sterokthe polyvalent phospholipid. In some embodiments, the particle comprises a molar ratio of about 4:3:3 of the helper phospholipid:the sterol :the polyvalent phospholipid. In some embodiments, the particle comprises a molar ratio of about 3:3:4 of the helper phospholipid:the sterokthe polyvalent phospholipid.

[0088] In some embodiments, the polyvalent phospholipid is divalent. In some embodiments, the polyvalent phospholipid is trivalent. In some embodiments, the polyvalent phospholipid has a valency greater than or equal to 10. In some embodiments, the polyvalent phospholipid has a valency greater than or equal to 50. In some embodiments, the polyvalent phospholipid has a valency greater than or equal to 100. In some embodiments, the polyvalent phospholipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl). In some embodiments, the polyvalent phospholipid is a polyanionic phospholipid.

[0089] In some embodiments, the helper phospholipid is l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-Dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), soy phosphatidylcholine, and egg phosphatidylcholine. In some embodiments, the helper phospholipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0090] In some embodiments, the particle further comprises a polymer coating. In some embodiments, the polymer coating is non-covalently adsorbed onto the surface of the particle. In some embodiments, the polymer coating comprises a polyelectrolyte. In some embodiments, the polymer coating comprises a mixture of polyelectrolytes. In some embodiments, the polymer coating comprises poly-L-lysine (PLL), polyethylenimine (PEI), poly(diallyldimethylammonium chloride) (PDAC), poly(P-amino esters) (PBAE), poly(allylamine hydrochloride) (PAH), poly-L-aspartic acid (PLD), poly-hyaluronic acid (HA), poly-acrylic acid (PAA), poly(sodium 4-styrenesulfonate), dextran sulfate, heparin sulfate, a folate-conjugated polymer, poly-L-arginine (PLR), poly-L-glutamate (PLE), PEOPLE, or PEG-PLD. In some embodiments, the polymer coating comprises poly-L-arginine and / or poly-L-glutamate.

[0091] In some embodiments, the particle is a lipid-based particle. In some embodiments, the particle further comprises a membrane scaffolding protein.

[0092] In some embodiments, the particle does not comprise one or more of a synthetic amphiphilic polymer, a protein, or a neutral lipid-polymer conjugate. In some embodiments, the particle does not comprise one or more of a synthetic amphiphilic polymer, a protein, a neutral lipid-polymer conjugate, or a detergent. In some embodiments, the particle does not comprise any non-lipid components.

[0093] In some embodiments, the particle is stable in solution under physiological conditions. In some embodiments, the particle is stable in physiological fluid. In some embodiments, theparticle is stable in solution under physiological pH. In some embodiments, the particle is stable in solution under physiological ionic strength. In some embodiments, the particle is stable in solution for at least one week. In some embodiments, the particle is stable in solution for at least two weeks. In some embodiments, the particle is stable in solution for at least one month. In some embodiments, wherein the particle is stable in solution for up to 6 months. In some embodiments, the particle is stable at up to 4 °C. In some embodiments, the particle is stable at up to 10 °C. In some embodiments, the particle is stable at up to 15 °C. In some embodiments, the particle is stable at up to 22 °C.

[0094] In some embodiments, the liposome is spherical. In some embodiments, the liposome has a diameter of about 50 nm to about 1 pm. In some embodiments, the liposome has a diameter of about 50 nm to about 750 nm. In some embodiments, the liposome has a diameter of about 50 nm to about 500 nm. In some embodiments, the liposome has a diameter of about 50 nm to about 250 nm. In some embodiments, the liposome has a diameter of about 100 nm to about 1 pm. In some embodiments, the liposome has a diameter of about 250 nm to about 1 pm. In some embodiments, the liposome has a diameter of about 500 nm to about 1 pm. In some embodiments, the liposome has a diameter of about 750 nm to about 1 pm.

[0095] In some embodiments, the liposome has a poly dispersity of less than about 0.5. In some embodiments, the liposome has a polydispersity of less than about 0.4. In some embodiments, the liposome has a poly dispersity of less than about 0.3. In some embodiments, the liposome has a poly dispersity of less than about 0.25. In some embodiments, the liposome has a poly dispersity of less than about 0.2. In some embodiments, the liposome has a poly dispersity of less than about 0.15. In some embodiments, the liposome has a poly dispersity of less than about 0.1.

[0096] The present disclosure provides pharmaceutical compositions comprising a particle disclosed herein and a pharmaceutically acceptable excipient. The present disclosure also provides pharmaceutical compositions comprising a liposome disclosed herein and a pharmaceutically acceptable excipient.

[0097] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutical agent. In some embodiments, the pharmaceutical composition the additional pharmaceutical agent is encapsulated by the particle. In some embodiments, the additional pharmaceutical agent is attached to the surface of the particle. In some embodiments, the additional pharmaceutical agent is a cancer therapeutic. In some embodiments, the cancer therapeutic is a platinum-based therapeutic, a taxol-basedtherapeutic, a microtuble inhibitor, or doxorubicin. In some embodiments, the additional pharmaceutical agent is a therapeutic protein.

[0098] In certain embodiments, the particle described herein is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is an amount effective for treating a proliferative disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a proliferative disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for reducing the risk of developing a disease (e.g., proliferative disease) in a subject in need thereof.

[0099] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmaceutics. In general, such preparatory methods include bringing the particle described herein into association with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping, and / or packaging the product into a desired single- or multi-dose unit.

[0100] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0101] Relative amounts of the particle, the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0102] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents or fillers, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

[0103] Exemplary diluents or fillers include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, starches (such as dry starch, cornstarch), sugars (such as powdered sugar), calcium trisulfate, carboxymethylcellulose calcium, dextrate, dextrin, dextrose, fructose, lactitol, lactose, magnesium carbonate, magnesium, maltitol, maltodextrin, maltose, sucrose, glucose, mannitol, silicic acid, xylitol, and mixtures thereof.

[0104] Exemplary granulating and / or dispersing agents include potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0105] Exemplary surface active agents and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij® 30)), poly(vinyl-pyrrolidone), di ethylene glycolmonolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or mixtures thereof.

[0106] Exemplary disintegrating agents or disintegrants include agar, algin, alginic acid, sodium alginate, silicates, sodium carbonate, calcium carbonate, carboxymethylcellulose, cellulose, clay, colloidal silicon dioxide, croscarmellose sodium, crospovidone, rubber, magnesium silicate, methylcellulose, potassium krillin, hydroxypropylcellulose (e.g., low substituted Hydroxypropylcellulose), crosslinked polyvinylpyrrolidone, hydroxypropylcellulose, and starch (e.g., sodium glycolate starch, potato or tapioca starch).

[0107] Exemplary binding agents include starch (e.g., glycolate starch, cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0108] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0109] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0110] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof.

[0111] Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0112] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0113] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0114] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, betacarotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0115] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, NeoIone®, Kathon®, and Euxyl®.

[0116] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen- free water, isotonic saline, Ringer’s solution, ethyl alcohol, and mixtures thereof.

[0117] Exemplary lubricating agents include agar, ethyl oleate, ethyl laurate, glycerin, blyceryl palmitostearate, magnesium oxide, magnesium stearate, mannitol, poloxamer, glycol, sodium stearyl, sorbitol, zinc stearate, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0118] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0119] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0120] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed includingsynthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0121] In some embodiments, injectable preparations of the compositions disclosed herein are in the form of a ready-to-use (“RTU”) preparation that can be directly administered to a subject. In some embodiments, the RTU preparation is a suspension. In some embodiments, the RTU preparation is a solution. In some embodiments, the RTU preparation is an emulsion. In some embodiments, injectable preparations of the compositions disclosed herein are in the form of a solid that is reconstituted prior to administration. In some embodiments, the solid is a lyophilized solid. In some embodiments, injectable preparations of the compositions disclosed herein are in the form of a liquid or suspension that is diluted prior to administration.

[0122] In some embodiments, the pharmaceutical compositions disclosed herein comprise a bulking agent. Bulking agents can be used, e.g., to improve the appearance of a solid composition, to provide visible “bulk” to demonstrate product quality or to facilitate preparation, e.g., of a solid composition prepared for reconstitution prior to administration. Bulking agents can be used for low dose (high potency) drugs that do not have the necessary bulk to support their own structure or provide a visible composition in a unit dosage form. Bulking agents are used in lyophilized formulations. Bulking agents provide a desirable structure for a lyophilized cake comprising pores that provide the means for vapor to escape from the product during lyophilization cycles, and facilitate dissolution on reconstitution. In some embodiments, the bulking agent is mannitol, lactose, sucrose, dextran, trehalose, povidone, dextran, glycine, isoleucine, methionine, or a cyclodextrin (e.g., (2- hydroxypropyl)-P-cyclodextrin).

[0123] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0124] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.

[0125] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

[0126] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

[0127] Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmaceutics. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0128] The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such assucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.

[0129] Dosage forms for topical and / or transdermal administration of a particle described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and / or any needed preservatives and / or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and / or by dispersing the active ingredient in a polymer matrix and / or gel.

[0130] Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin.Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and / or via a needle which pierces the stratum comeum and produces a jet which reaches the dermis are suitable. Ballistic powder / particle delivery devices which use compressed gas to accelerate the particle in powder form through the outer layers of the skin to the dermis are suitable.

[0131] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid preparations such as liniments, lotions, oil-in-water and / or water-in-oil emulsions such as creams, ointments, and / or pastes, and / or solutions and / or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w / w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additional ingredients described herein.

[0132] A pharmaceutical composition described herein can be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and / or using a self-propelling solvent / powder dispensing container such as a device comprising the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0133] Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and / or solid anionic surfactant and / or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

[0134] Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations can be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and / or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and / or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

[0135] Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

[0136] Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w / w) to as much as 100% (w / w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition described herein can be prepared, packaged, and / or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w / w) active ingredient, the balance comprising an orally dissolvable and / or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

[0137] A pharmaceutical composition described herein can be prepared, packaged, and / or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w / w) solution and / or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and / or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and / or in a liposomal preparation. Ear drops and / or eye drops are also contemplated as being within the scope of this disclosure.

[0138] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilledveterinary pharmacologist can design and / or perform such modification with ordinary experimentation.

[0139] Particles provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

[0140] The particles and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and / or lymph supply, and / or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the particle or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.

[0141] The exact amount of a particle or agent required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular particle or agent, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of a particle or agentdescribed herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 pg and 1 pg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a particle or agent described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a particle or agent described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a particle or agent described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a particle or agent described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a particle or agent described herein.

[0142] Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, achild or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0143] A particle or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and / or prophylactically active agents). The particles or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and / or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, and / or in reducing the risk to develop a disease in a subject in need thereof), improve bioavailability, improve safety, reduce drug resistance, reduce and / or modify metabolism, inhibit excretion, and / or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and / or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a particle described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the particle and the additional pharmaceutical agent, but not both. In some embodiments, the additional pharmaceutical agent achieves a desired effect for the same disorder. In some embodiments, the additional pharmaceutical agent achieves different effects.

[0144] The particle or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g, combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g, compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and / or preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder). Each additional pharmaceutical agent may be administered at a dose and / or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each otherand / or with the particle or composition described herein in a single dose or composition or administered separately in different doses or compositions. The particular combination to employ in a regimen will take into account compatibility of the particle or agent described herein with the additional pharmaceutical agent(s) and / or the desired therapeutic and / or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

[0145] The additional pharmaceutical agents include, but are not limited to, antiproliferative agents, anti-cancer agents, anti-angiogenesis agents, steroidal or non-steroidal anti-inflammatory agents, immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, anesthetics, anti-coagulants, inhibitors of an enzyme, steroidal agents, steroidal or antihistamine, antigens, vaccines, antibodies, decongestant, sedatives, opioids, analgesics, anti-pyretics, hormones, and prostaglandins. In certain embodiments, the additional pharmaceutical agent is an anti-proliferative agent. In certain embodiments, the additional pharmaceutical agent is an anti-cancer agent. In certain embodiments, the additional pharmaceutical agent is an anti-viral agent. In certain embodiments, the additional pharmaceutical agent is an binder or inhibitor of a protein kinase. In certain embodiments, the additional pharmaceutical agent is selected from the group consisting of epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g., tyrosine protein kinase inhibitors), modulators of protein stability (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acids, and other agents that promote differentiation. In certain embodiments, the particles described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy. Additional pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved by the US Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins,lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins and cells.

[0146] Anti-cancer agents encompass biotherapeutic anti-cancer agents as well as chemotherapeutic agents. Exemplary biotherapeutic anti-cancer agents include, but are not limited to, immune therapies such as interferons, cytokines (e.g., tumor necrosis factor, interferon a, interferon y), vaccines, hematopoietic growth factors, monoclonal serotherapy, immunostimulants and / or immunodulatory agents (e.g., IL-1, 2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) and antibodies (e.g., HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR (tositumomab), anti-PDl (pembrolizumab, nivolumab, cemiplimab), anti-PDLl, anti-CTLA4 (ipilimumab, tremelimumab), anti-TIM-3, anti -LAG-3, anti- NKG2A, anti-CD73, anti-A2aR, anti-B7-H3, and anti-B7-H4).

[0147] Exemplary chemotherapeutic agents include, but are not limited to, anti-estrogens (e.g, tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g, goscrclin and leuprolide), anti-androgens (e.g., flutamide and bicalutamide), photodynamic therapies (e.g., vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2- DMHA)), nitrogen mustards (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g., carmustine (BCNU) and lomustine (CCNU)), alkyl sulphonates (e.g., busulfan and treosulfan), triazenes (e.g., dacarbazine, temozolomide), platinum containing compounds (e.g., cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), taxoids (e.g., paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel -EC- 1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g, ’2’ -paclitaxel methyl 2-glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g., etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors (e.g., methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonuclotide reductase inhibitors (e.g., hydroxyurea and deferoxamine), uracil analogs (e.g., 5 -fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil,capecitabine), cytosine analogs (e.g., cytarabine (ara C), cytosine arabinoside, and fludarabine), purine analogs (e.g., mercaptopurine and Thioguanine), Vitamin D3 analogs (e.g., EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., l-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g., staurosporine), actinomycin (e.g., actinomycin D, dactinomycin), bleomycin (e.g., bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g., daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g., verapamil), Ca2+ATPase inhibitors (e.g., thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AGO 13736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK / ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and / or XL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin,, aminopterin, and hexamethyl melamine.

[0148] Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or particle described herein and a container (e.g., a vial, ampule, bottle, syringe, and / or dispenser package, or other suitablecontainer). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or particle described herein. In some embodiments, the pharmaceutical composition or particle described herein provided in the first container and the second container are combined to form one unit dosage form.

[0149] Thus, in one aspect, provided are kits including a first container comprising a particle or pharmaceutical composition described herein. In certain embodiments, the kits are useful for treating a disease (e.g., proliferative disease) in a subject in need thereof. In certain embodiments, the kits are useful for preventing a disease (e.g., proliferative disease) in a subject in need thereof. In certain embodiments, the kits are useful for reducing the risk of developing a disease (e.g., proliferative disease) in a subject in need thereof.

[0150] In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for treating a disease (e.g., proliferative disease) in a subject in need thereof. In certain embodiments, the kits and instructions provide for preventing a disease (e.g., proliferative disease) in a subject in need thereof. In certain embodiments, the kits and instructions provide for reducing the risk of developing a disease (e.g., proliferative disease) in a subject in need thereof. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.Methods of Treatment and Related Uses

[0151] In another aspect, provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a particle provided herein, or a pharmaceutical composition provided herein.

[0152] In another aspect, provided herein is a method of delivering an agent to a target cell in a subject, cell, or biological sample, comprising contacting the target cell with a particle provided herein.

[0153] In some embodiments, the disease is a disease, disorder, or condition provided herein. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is cancer. In some embodiments, the disease is ovarian cancer. In some embodiments, the cancer is metastatic.

[0154] In some embodiments, the particle is administered via injection. In some embodiments, the particle is administered via intravenous injection or intraperitoneal injection. In some embodiments, the particle is administered via intravenous injection. In some embodiments, the particle is administered via intraperitoneal injection.

[0155] In certain embodiments, the subject is a human. In certain embodiments, the subject is a human of at least 18 years of age. In certain embodiments, the subject is a human under 18 years of age. In certain embodiments, the subject is a mammal. In another embodiment, the subject is a research animal. In some embodiments, the subject is a rodent.

[0156] In some embodiments, the subject is a mouse.

[0157] In some embodiments, the agent is released from the particle. In some embodiments, the cell is in vivo. In some embodiments, wherein the cell is ex vivo or in vitro. In some embodiments, wherein the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the cell is a tumor cell.Additional Methods and Uses

[0158] In another aspect, provided herein is a method of preparing a plurality of particles having anisotropic dimensions provided herein, the method comprising:(a) preparing a mixture of a polyvalent phospholipid, a helper phospholipid, and a detergent;(b) allowing the mixture to equilibrate afford a plurality of micelles;(c) diluting the micelles with a buffer to a target detergent concentration, thereby forming a plurality of particles having anisotropic dimensions; and(d) separating the plurality of particles.

[0159] In another aspect, provided herein is a method of preparing a plurality of liposomes, the method comprising:(a) preparing a mixture of a monoanionic phospholipid, a helper phospholipid, and a detergent;(b) allowing the mixture to equilibrate afford a plurality of micelles;(c) diluting the micelles with a buffer to a target detergent concentration to promote self-assembly of a plurality of bicelles having anisotropic dimensions, wherein the bicelles undergo bicelle fusion to afford a plurality of liposomes; and(d) separating the plurality of liposomes.

[0160] In some embodiments, the polyvalent phospholipid is a polyvalent phospholipid provided herein. In some embodiments, the polyvalent phospholipid is 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine-N-(glutaryl).

[0161] In some embodiments, the monoanionic phospholipid is a monoanionic phospholipid provided herein. In some embodiments, the monoanionic phospholipid is l-palmitoyl-2- oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol).

[0162] In some embodiments, the helper phospholipid is a helper phospholipid provided herein. In some embodiments, the helper phospholipid is l,2-distearoyl-sn-glycero-3- phosphocholine.

[0163] In some embodiments, the separating of step (d) comprises ultrafiltration, density gradient separation, size exclusion chromatography, use of detergent depletion agents, adsorption chromatography, or ion exchange chromatography. In some embodiments, the separating of step (d) comprises ultrafiltration. In some embodiments, the separating of step (d) comprises tangential flow filtration.

[0164] In some embodiments, the mixture of step (a) further comprises a sterol. In some embodiments, the sterol is a sterol provided herein. In some embodiments, the sterol is cholesterol.

[0165] In some embodiments, the buffer is PBS, HEPES saline, or HEPES. In some embodiments, the buffer is PBS. In some embodiments, the buffer is HEPES saline or HEPES. In some embodiments, the buffer is HEPES saline. In some embodiments, the buffer is HEPES.

[0166] In some embodiments, the detergent is ionic. In some embodiments, the detergent is non-ionic. In some embodiments, the detergent is CHAPs, Tween 20, Tween 80, triton X- 100, SDS, DDM / CHS, CHAPSO, one or more bile salts, MEGA- 10, or octylglucoside. In some embodiments, the detergent is MEGA- 10 or octylglucoside. In some embodiments, the detergent is MEGA- 10. In some embodiments, the detergent is octylglucoside.

[0167] In some embodiments, the mixture of step (a) comprises about 10- to 200-fold more detergent than the pure detergent critical micelle concentration. In some embodiments, the mixture of step (a) comprises about 50- to 200-fold more detergent than the pure detergent critical micelle concentration. In some embodiments, the mixture of step (a) comprises about 100- to 200-fold more detergent than the pure detergent critical micelle concentration. In some embodiments, the mixture of step (a) comprises about 150- to 200-fold more detergent than the pure detergent critical micelle concentration. In some embodiments, the mixture of step (a) comprises about 100- to 200-fold more detergent than the pure detergent criticalmicelle concentration. In some embodiments, the mixture of step (a) comprises about 120- to 200-fold more detergent than the pure detergent critical micelle concentration. In some embodiments, the mixture of step (a) comprises about 150- to 200-fold more detergent than the pure detergent critical micelle concentration. In some embodiments, the mixture of step (a) comprises about 180- to 200-fold more detergent than the pure detergent critical micelle concentration. In some embodiments, the mixture of step (a) comprises about 100- to 180- fold more detergent than the pure detergent critical micelle concentration. In some embodiments, the mixture of step (a) comprises about 100- to 150-fold more detergent than the pure detergent critical micelle concentration. In some embodiments, the mixture of step (a) comprises about 100- to 120-fold more detergent than the pure detergent critical micelle concentration.

[0168] In some embodiments, the mixture of step (a) comprises about 1% to about 20% (w / v) detergent in water. In some embodiments, the mixture of step (a) comprises about 5% to about 15% (w / v) detergent in water. In some embodiments, the mixture of step (a) comprises about 8% to about 12% (w / v) detergent in water. In some embodiments, the mixture of step (a) comprises about 10% (w / v) detergent in water.

[0169] In some embodiments, the dilution is performed to below the critical micelle concentration. In some embodiments, the dilution is performed to reach the critical micelle concentration. In some embodiments, the dilution is performed to above the critical micelle concentration.

[0170] In some embodiments, the target detergent concentration of step (c) is about 0.05% to about 0.3% of the buffer. In some embodiments, the target detergent concentration of step (c) is about 0.05% to about 0.1% of the buffer. In some embodiments, the target detergent concentration of step (c) is about 0.5% to about 0.15% of the buffer. In some embodiments, the target detergent concentration of step (c) is about 0.1% to about 0.2% of the buffer. In some embodiments, the target detergent concentration of step (c) is about 0.15% to about 0.25% of the buffer. In some embodiments, the target detergent concentration of step (c) is about 0.2% to about 0.3% of the buffer.

[0171] In some embodiments, the method further comprises depositing a polymer coating onto the particle using layer-by-layer assembly. In some embodiments, the layer-by-layer assembly comprises the steps of:(e) mixing the plurality of particles or liposomes in a buffer with a polymer to deposit the polymer onto the particles or liposomes, forming a polymer coating; and(f) optionally removing excess polymer.

[0172] In some embodiments, the polymer coating is a polymer coating provided herein. In some embodiments, the polymer coating comprises a polyelectrolyte. In some embodiments, the polymer coating comprises poly-L-arginine or poly-L-glutamate. In some embodiments, the polymer coating comprises poly-L-arginine. In some embodiments, the polymer coating comprises poly-L-glutamate.

[0173] In some embodiments, the method further comprises a step of loading an agent into the particle. In some embodiments, the agent is added during step (a). In some embodiments, the agent is added during step (b). In some embodiments, the agent is added during step (c). In some embodiments, the agent is added during step (d). In some embodiments, the agent is added prior to step (c). In some embodiments, the agent is added after step (c). In some embodiments, the agent is added after step (d). In some embodiments, the agent is an agent provided herein. In some embodiments, the agent is an additional pharmaceutical agent. In some embodiments, the agent is an additional pharmaceutical agent provided herein.EXAMPLES

[0174] 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 particles, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting in their scope.Example 1: Precise and scalable self-assembly of liposomes via surfactant-stabilized intermediates

[0175] As disclosed herein, a surfactant-assisted liposome assembly method was used to carefully control liposome size from 50 nm to 1 pm with monodispersed populations. The inventors discovered that the detergent removal method for liposome synthesis could be used to control liposome size. Nanoparticle self-assembly was performed by diluting a lipid / detergent mixture (mixed micelles) to below the critical micelle concentration (CMC) of the detergent. In addition to precisely controlling liposome size in from ~1 pm down to 50 nm, a model for the growth of liposomes was provided based on the observed phase separation into lipid rich and detergent rich phases in solution. It was also shown that tangential flow filtration (TFF), a scalable size-based separations technique, purified out detergent from the liposomes. The removal of more than 99.9% of detergent was achieved via TFF without disturbing assembled liposome monodispersity. Finally, it was determinedthat these varying size nanoparticles are differentially recognized by macrophages, demonstrating the utility of controlled liposome size.Concentration of detergent enables precise control of liposome size with minimal polydispersity

[0176] Studies on liposome formation from nonionic detergent micelles suggested that the rate of detergent removal may dictate final liposome size4,10The inventors explored the effect of rapid dilution of a lipid mixture solubilized in a nonionic detergent to various final total detergent concentrations (FIG. 1A). N-decanoyl-N-methylglucamine (MEGA-10) was chosen due to its high critical micelle concentration (CMC), which facilitated detergent removal. Samples were diluted with a phosphate-buffered saline (PBS), a physiological buffer. The initial lipid composition was a 6:3 : 1 molar ratio of l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol, and l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac- glycerol) (POPG), as these lipids were previously used in therapeutic liposomes.1 1 13After dilution, sample size and polydispersity index (PDI) was measured via DLS before and after overnight incubation to ensure final self-assembly.

[0177] The final concentration of MEGA- 10 during incubation determined the final particle size with diameters ranging from 50-500 nm and minimal polydispersity (FIG. IB). Four regions were determined: a micelle-predominant region at high detergent concentrations (region i), followed by a mixed micelle region with a low size (<100 nm) and high PDI (>0.1) (region ii). After the detergent CMC (~0.1-0.2%), a region with large size (>200 nm) and low PDI (<0.1) was seen (region iii) which followed by a low size (<200 nm) and low PDI (<0.1) region (region iv). Interestingly, when diluted closer to the transition region between (ii) and (iii), lipid vesicles could self-assembled into >1 pm liposomes (FIG. 2A). While previous studies have found similar behavior of detergent / liposome mixtures,14these reports have not demonstrated a large liposome with low polydispersity such as seen in region (iii). Based on these results but without wishing to be bound by any particular theory, the inventors posited that it was possible that, close to a detergent CMC, mixtures of mixed micelles and bilayers enabled a controlled self-assembly of large liposomes.

[0178] To validate that this observed size control was not restricted to MEGA-10, the same protocol was repeated using a different nonionic surfactant n-octyl-P-d-glucoside (octylglucoside, CMC~0.6-0.7%). Like MEGA-10 mixed micelle dilution, four distinct regions (FIG. 2B) and control of liposome size based on MEGA- 10 concentration during incubation were observed. Importantly, after particle self-assembly at regions (iii) and (iv), further dilution did not cause a change in size, indicating that residual detergent was notstabilizing these constructs (FIG. 2C). Further, it was determined that the final particle size after incubation was reproducible (within 10-20%) across independent batches of lipids in MEGA- 10 (FIG. 2D). However, reducing the temperature reduced nanoparticle size (FIG. 2E). Assessment of phase transition temperatures for the 6:3 : 1 DSPC: cholesterol :POPG liposome via DLS15 did not indicate any phase transition between 4 °C and 37 °C (FIG. 2F). Thus, the importance of temperature may be related to membrane rigidity, or an energetic barrier required for increasing liposome size,

[0179] The kinetics of lipid vesicle assembly were characterized during the incubations at different detergent concentrations (FIG. 1C). It was determined that the kinetics of selfassembly could be described by the Hill Equation where the fits of EC50 for both 0.01% and 0.02% MEGA-10 (region iv) occurred within 1-2 hours, whereas 0.1% MEGA-10 (region iii) occurred at -5.5 hours, indicating a more delayed assembly for larger species. Furthermore, while 0.01% had a Hillslope of <1, 0.02% and 0.1% neared values of 2, suggesting a cooperative behavior during assembly of larger liposomes. While the PDI at 0.02% decreased near its ECso, for 0.1% it was at its maximum, reaching low values only at the end of self-assembly. Interestingly, when attempting to further dilute the sample during the incubation at region iii to 0.01% midway through the assembly process, it was determined that monodisperse particles were generated if particles were less than -150 nm (FIGs. 3A- 3B). Dilution of the sample at >200 nm led to a subsequent decrease in particle size and high PDI. Without wishing to be bound by any particular theory, the inventors posit that this result indicated that either smaller mixed micelles coalesced into vesicles or that larger vesicles broke into smaller species.Electrostatic interactions and lipid membrane rigidity control growth of liposomes

[0180] To explore whether the buffer ionic strength or lipid composition affected the observed control over the size of liposomes, the rapid dilution to varying concentrations of detergent was repeated but using HEPES buffer (10 mM) without added salts instead of PBS. As shown in FIG. 4A, there was no appreciable change in the size of mixed micelles regardless of final detergent concentration, indicating that electrostatic repulsion may be one of the factors that limited the growth of liposomes. To confirm the effect of electrostatic repulsion, it was assessed whether the nanoparticle charge was required for controlled liposome assembly by diluting mixed micelles without POPG (neutral liposomes). While it was possible to generate varying-sized liposomes with minimal poly dispersity in region iv, liposomes at 0.1% and 0.05% MEGA-10 (region iii) had high poly dispersity, implicating charge in controlled formation of large monodisperse liposomes (FIG. 4B) in region iii.

[0181] The effect of removing cholesterol (chol) or replacing POPG with 1,2-distearoyl-sn- glycero-3-phospho-(l'-rac-glycerol) (DSPG), a saturated anionic lipid, was explored. Removal of cholesterol from DSPC / POPG / chol mixed micelles altered the observed trends, with nanoparticle size increasing from 20 nm to 60 nm when diluted to between 0.2% to <0.01% (FIG. 4C). Thus, instead of large liposomes, small monodisperse liposomes were generated at 0.1 and 0.05% MEGA-10 concentrations (region iii) and region iv consisted of samples with high poly dispersity. Dilution of DSPG / DSPC / chol mixed micelles behaved like POPG / DSPC / chol mixed micelles (FIG. 4D). However, liposomes at intermediate MEGA- 10 concentrations (region iii) were smaller than liposomes with POPG composition when diluted with PBS.

[0182] On the other hand, removal of cholesterol from the saturated lipidic composition (DSPC / DSPG only) led to a highly polydisperse and large particle sample at intermediate and low MEGA- 10 concentrations when diluted with PBS. However, no significant effect of cholesterol was seen when diluted with HEPES-only buffer as the sample did not show substantial size changes regardless of final detergent concentration. While unsaturated lipid compositions or saturated compositions with cholesterol were observed to form liposomes, the inventors posit that the high membrane rigidity of saturated lipids without cholesterol may prevent liposomes from proper self-assembly. Moreover, unsaturated liposomes appeared to form only with the use of some amount of detergent. Without wishing to be bound by any particular theory, the inventors posit that this result was potentially due to the positive curvature imposed by detergents.Self-assembly of large liposomes occurs through phase separation into lipid rich and detergent rich phases

[0183] To better understand the mechanism of liposome self-assembly, fluorescence resonance energy transfer (FRET) was used to evaluate lipid exchange amongst mixed micelles or assembled particles. Fluorescently tagged lipids were included in the formulation (6:3: 1 molar ratio of DSPC:cholesterol:POPG) with MEGA-10. Micelles were generated with either donor-only or acceptor-only fluorophores which were mixed to generate FRET micelles (FIG. 5A). Moreover, it was also assessed whether mixing separately diluted donor-only or acceptor-only micelles during the self-assembly period (~24 hours based on FIG. 1C) could also result in FRET (sepFRET micelles FIG. 5 A).

[0184] FRET micelles diluted to or below 0.1% MEGA-10 had increased FRET efficiency as the total surface area of the sample decreased due to detergent partitioning to the aqueous phase (FIGs. 5A, 6). Further, sepFRET micelles behaved very similar to FRET micelleswhen donors and acceptors were mixed right after dilution (sepFRET-0 hour), indicating that the initial intermediates underwent rapid lipid mixing during self-assembly (FIG. 6). However, after one hour or more of independent self-assembly of donor and acceptor samples, sepFRET micelles showed low FRET efficiency at <0.1% MEGA-10 (FIGs. 5C- 5D). Thus, liposomes did not undergo significant lipid exchange events after final assembly in region iv. It was also determined that there tended to be a decrease in FRET efficiency after donor and acceptor mixing which could be attributed to increased inter-lipid distance from vesicle growth (FIG. 5D). However, sepFRET micelles converged to similar levels of FRET efficiency to that of FRET micelles at 0.1% MEGA-10 (FIGs. 5E-5F). This observation further suggested the coexistence of mixed micelles and bilayers at 0.1% MEGA- 10, which could facilitate lipid exchange even when the particles have reached their final size.

[0185] Cryo-TEM was used to follow the assembly of liposomes at 0.1% MEGA-10 (region iii) when diluted with HEPES and 150 mM NaCl. As show in FIGs. 7A-7D, the sample started as small micelles and bicelles (~15 nm) that coalesced to larger (>100 nm) discs and liposomes within 1-2 hours. At 5 hours, smaller discs were no longer seen, and the sample was composed of primarily large (>300 nm) discs and liposomes. Finally, after 24 hours, only large liposomal and disc species could be seen as well as many micellar aggregates. The presence of these micellar aggregates in cryoTEM micrographs confirmed mixed micelle coexistence with bilayers. Interestingly, some of these liposomes contained an additional bilayer which did not fully cover the liposome surface, resembling a lamellar phase which suggests a path for multilam ellar liposomes to be formed in region iii.

[0186] Based on these results and previously described models of liposome self-assembly,18it was possible to determine the primary events observed in each of the identified regions from FIG. 1 (FIG. 7E). When mixed micelles in region i were diluted, detergent partitioning into aqueous phase caused mixed micelles to coalesce into bicelles (surfactant-stabilized nanodiscs), leading to the formation of region ii. As further detergent partitions into the aqueous phase, bicelle fusion was driven as less detergent was available to stabilize the outer edges of discoidal assemblies. At a critical bicelle size, bilayer bending enables vesicle assembly, leading to the formation of liposomes in regions iii and iv. When mixed micelles were diluted to region iii, phase separation began, generating a detergent-rich mixed micelle phase and lipid-rich bilayer phase. This phase separation was likely favored by the preference of detergents for high curvature assemblies which could be achieved by forming small spherical micelles. However, formation of detergent-rich micelles appeared to occurthrough fusion of bicelles into larger species to reduce the amount of detergent required to stabilize the disc rims (FIG. 7F). However, the larger the bilayer discs, the lower the amounts of detergent released upon further fusion, enabling the system to reach a terminal size.

[0187] In addition to bicelle fusion, if bilayers formed vesicles during the self-assembly period, detergents released into the internal aqueous volume cannot freely diffuse to the external volume leading to imbalances between the inner and outer leaflet due to continued detergent depletion in the outer leaflet.19,20These imbalances should promote fusion or vesicle rupture and become more pronounced as the size of the liposome grows.19Without wishing to be bound by any particular theory, the inventors posit that this leaflet imbalance- driven fusion was the primary cause for liposome fusion when diluted to region iv due to rapid detergent depletion from bilayers (i.e., lack of detergent-stabilized bicelles) and lack of mixed micelle coexistence.21Under this scenario, the final liposome size was dictated by the number of fusion events required to normalize the leaflets imbalances such that higher dilutions allow for more detergent partitioning prior to vesicle closure and subsequently, smaller liposome size.

[0188] As bicelles require a certain number of detergent molecules to stabilize their outer edges, the inventors posited that it should be possible to favor the formation of fully closed vesicles over discs by increasing the total lipid concentration. Thus, to further validate the model where detergent-stabilized discs promoted large vesicle growth in region iii, the lipid to detergent ratio was altered prior to dilution. While previous reports on liposome assembly from dilution of mixed micelles indicated that the ratio between lipid and detergent controlled liposome size,22,23it was unexpectedly determined that there was almost no difference in liposome size in region (iv) (FIGs. 8A-8B). This result was consistent with this region being governed by the imbalances in leaflets, which would not be influenced by lipid concentration. However, an effect of lipid concentration in region iii was observed. While 100: 1, 10: 1, and 4: 1 generated large liposomes (>100 nm) at 0.1 % MEGA-10, only 10: 1 showed low poly dispersity (PDI<0.1). Moreover, a 2: 1 ratio generated minute (~50 nm) species at 0.1% MEGA- 10 suggesting that high lipid concentrations indeed could limit the formation of detergent-stabilized discs, preventing bilayer fusion.

[0189] The inventors posited that after incubation at 0.1% MEGA-10, dilution to region iv would generate monodisperse liposomes at high detergent to lipid ratios whereas a mixture of large and small liposomes would be formed at low detergent to lipid ratios. Without wishing to be bound by any particular theory, this could occur since as low detergent to lipid ratios, some number of detergent-containing vesicles would be formed and not participate in thebilayer fusion events. Thus, samples were diluted 100: 1 and 4: 1 then incubated at 0.1% MEGA-10 to 0.025% and allowed them to equilibrate overnight. While 100: 1 decreased its poly dispersity and increased its size, 4: 1 increased its poly dispersity and decreased its size (FIG. 8C). It was also determined that dilution of 2: 1 samples incubated at 0.1% MEGA-10 to region iv promoted the assembly of similar sized liposomes as samples diluted directly to region iv from 10% MEGA- 10, indicating that these detergent-containing vesicles may be intermediates for the assembly of liposomes in region iv. This was also identified by diluting 10: 1, 4: 1 and 2: 1 after incubation at 0.2% MEGA-10 to region iv and observing that they also generated similarly sized liposomes (FIG. 8D). Accordingly, the observation for liposome assembly in region iii was depicted in FIG. 8E.Purification of assembled nanoparticles via tangential flow filtration (TFF) removes detergent without affecting liposome structure

[0190] Having established this method for self-assembly of liposomes, the approach was evaluated for production of varied sized liposomes. For biomedical applications, the presence of detergent could interfere with the desired nanoparticle effect. Thus, to determine detergent could fully be removed from the sample, TFF was used to concentrate and purify samples previously diluted to 0.1%. 0.02% and 0.004% MEGA-10 (FIGs. 10A-10B). Analysis of particles via reverse-phase high-pressure liquid-chromatography (RP-HPLC) coupled with evaporative light scattering detector (ELSD) showed no detectable levels of MEGA-10 (<1% mass composition), indicating removal of >99.9% of detergent from the sample (FIGs. 9A-9B). There also was no appreciable difference in final particle size of the small-scale experiments (-0.05 mg) to the larger experiments (5 mg) and particles maintained their size and monodispersity with yield of 70-80% after purification. Zeta potential measurements indicated negative surface charge on these particles due to the presence of POPG (FIG. 10C). Analysis via cryoTEM revealed that the nanoparticles were primarily unilamellar liposomes from detergent-assembled vesicles (FIGs. 10D-10G). Liposomes generated via the conventional thin-film hydration followed by extrusion through a 50 nm pore-sized membrane (FIGs. 11 A-l ID) were more polydisperse than detergent assembled liposomes. After extrusion both large and many smaller nanoparticles (FIGs. 1 OH- 101 and FIGs. 11C-1 ID) were present, demonstrating the unique monodispersity of the detergent- assembled liposomes.Purified liposomes maintain biophysical interactions with macrophages

[0191] Different sized nanoparticles have different biophysical interaction with cells. While conflicting results have been presented,25most studies have indicated that macrophagespreferentially uptake larger liposomes in vitro.26 29However, prior work has failed to present clear relationship between liposome size and uptake, potentially due to the use of thin film hydration followed by extrusion where it is difficult to control liposome size. Given that the method presented here enabled synthesis of large and monodisperse liposomes, the effect of liposome size on macrophage uptake was evaluated.

[0192] To investigate this trend, a library of varied-sized liposomes was generated (FIG. 12A). These liposomes were then incubated with RAW 264.7 macrophages and quantified total lipid uptake. Macrophages were found to have increased uptake of large liposomes (FIG. 12A). However, the assembly method facilitated identification of a linear relationship between liposome size and liposome uptake at either 4 or 24 hours (FIG. 12B) of incubation.

[0193] Lipid nanoparticles such as liposomes are useful as delivery vehicles for both the current and next generation of therapeutics. The inventors unexpectedly discovered that the size of charged liposomes could be precisely controlled. Additionally, models for their controlled assembly which has been lacking in the literature were identified.20

[0194] Although previous work using glycocholate and egg phosphatidylcholine similarly found that dilution of micellar mixtures could control liposome size, these studies only achieved size ranges of only -100-50 nm22,23Furthermore, while the results contradicted the concept that the rate of detergent removal either via controlled dilution or controlled dialysis governed liposome size,4,10,30’31the rate of detergent removal likely altered the residence time of the lipidic mixture at each concentration of detergent, leading to the control in liposome size observed previously.

[0195] Taken together, the findings presented here demonstrate a new method of detergent- aided assembly of monodisperse liposomes via precise and scalable techniques.Example 2: Charge-stabilized nanodiscs as a new class of lipid nanoparticles

[0196] A bottom-up approach was employed to generate a new class of lipid nanodiscs starting from detergent micelles. While dilution of lipid / detergent mixture (mixed micelles) has been known to form liposomes,12the inventors surprisingly discovered that the lipid head groups comprising more than one anionic group could stabilize lipid nanodics without nonlipid components. Without wishing to be bound by any particular theory, the inventors posit that the stabilization in part depends on the buffer ionic strength and the lipid headgroup charge valency. The ability to generate CNDs was observed with lipids comprising more than one anionic group, whereas monovalent lipids generated limit-size nanoliposomal assemblies rather than nanodiscs. Using tangential flow filtration (TFF), a scalable size-based separations technique, excess detergent was removed to yield discoidal lipid nanoparticles, which were termed charge-stabilized nanodiscs (CNDs). This approach of detergent dilution followed by TFF was used to produce nanoparticulate vaccine adjuvants at clinical scales. The resulting CNDs were shown to have better tumor accumulation properties than liposomes in subcutaneous tumors. To exploit the increased NP surface area, CNDs were modified by the layer-by-layer (LbL) technique to further improve their tumor targeting properties in a metastatic model of ovarian cancer.Electrostatic interactions control formation of low-ionic strength charge-stabilized nanodiscs (lisCNDs)

[0197] Unilamellar liposomes can be assembled from mixed micelle detergents by diluting the sample to below the detergent critical micelle concentration (CMC).12The assembly of liposomes from mixed micelles was characterized in Example 1. The effect of buffer ionic strength was assessed on rapid dilution of a lipid mixture solubilized in 10% (w / v) N- decanoyl-N-methylglucamine (MEGA- 10) micelles to various final MEGA- 10 concentrations. After dilution, samples were allowed to equilibrate overnight prior to size measurements via dynamic light scattering. MEGA- 10 was chosen due to its high critical micelle concentration (CMC) which facilitates detergent removal. The initial lipid composition was a negatively charged 6:3: 1 molar ratio of l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol, and l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac- glycerol) (POPG) as these lipids were previously used in therapeutic liposomes.14 16

[0198] It was determined that from 0-25 mM of NaCl, there was no appreciable change in the size of lipidic nanoparticles regardless of final detergent concentration (FIG. 13 A). It was also observed that from 50-500 mM of NaCl, increasing the ionic strength increased the resulting nanoparticle size, but at >200 mM of NaCl samples had high poly dispersity (PDI>0.2, FIG. 14A). Based on these observations, the resulting nanoparticle characteristics were further assessed following dilution with either a high ionic strength (200 mM NaCl) versus low ionic strength (0 mM NaCl) buffer at 0.1% MEGA-10. Thus, 5 mg of lipids were diluted in 10% MEGA-10 with both solutions to 0.1% MEGA-10 then used tangential flow filtration (TFF) to readily concentrate and purify out MEGA- 10.13After purification, high pressure liquid chromatography (HPLC) coupled with evaporative light scattering detector was used to quantify residual MEGA- 10. There was no detectable MEGA- 10 in the sample (<1% by mass composition), indicating >99.9% removal of MEAG-10 (FIG. 13B). After TFF processing, 200 mM NaCl diluted mixed micelles maintained their large size and low PDI (<0.1) whereas, 0 mM NaCl afforded low size and moderately high PDI (~0.2) (FIG.13C). Interestingly, addition of PBS to the purified sample from the 0 mM NaCl sample increased its size and reduced its PDI, indicating an effect of ionic strength on this sample.

[0199] The morphology of the purified samples was then determined via cryogenic transmission electron micrography (cryoTEM). While it was clear that micrographs of the samples from 200 mM NaCl were liposomes (FIG. 13D), samples from low ionic strength buffer surprisingly resembled disc-like structures similar to bicelles or LNDs (FIG. 13E). After PBS treatment to these negatively charged discoidal nanoparticles, they assembled into small liposomes (FIG. 13F, 14B). Without wishing to be bound by any particular theory, the inventors posited that this was possible because these were electrostatically stabilized nanoparticles, termed low-ionic strength charge-stabilized nanodiscs (lisCNDs). It was determined that use of neutral lipid compositions yielded liposomes at low ionic strength. From the assembly process of liposomes from mixed micelles, bicelles are known to be intermediates.17Without wishing to be bound by any particular theory, the inventors posited bicelles may have enabled the formation of lisCNDs which, due to their large Debye length in low ionic strength buffer may have prevented liposomes assembly upon detergent depletion.

[0200] To validate that lisCNDs were discs and not appeared as discs on cryoTEM from artifacts of sample preparation, lisCNDs were mixed with a model protein - bovine serum albumin (BSA) - and evaluated if the disc to liposome transition could encapsulate it. If lisCNDs were liposomes, their internal aqueous compartment would already have been separated from the external buffer and no BSA would be internalized. It was determined that BSA appeared internalized when mixed with lisCNDs prior to liposome transition as evidenced by the internal high-density spots in cryo-TEM, confirming disc-like morphology (FIG. 13G).18Further, the lack of size change after mixed micelle dilution with low ionic strength buffer was similarly observed when using a lipid composition known to generated PEG-stabilized LNDs (FIG. 14B).7lisCNDs were also found to be stable when stored at 4 °C as 6 month-old samples did not show signs of aggregation (FIG. 14D).High density of charged lipids yields minute liposomes, but not disc-shaped nanoparticles

[0201] Having successfully generated lipid-based nanodiscs devoid of non-lipid components, approaches to synthesizing CNDs that would be stable in physiological buffers for biomedical applications were explored. The first approach towards this goal was to increase the molar fraction of negatively charged phospholipids in the formulation, such that increased NP charge could act similarly to low ionic strength buffer. MEGA- 10 micelles were generated with molar lipid compositions of 5:3:2, 4:3:3, and 3 :3:4 of DSPC:cholesterol:POPG and diluted them using a physiological buffer, phosphate bufferedsaline (PBS) (FIG. 15A). The only formulation with sub 100 nm particles was 3:3:4, but dilution to low MEGA- 10 concentration yielded high PDI (>0.2). Based on these results, the morphology of 3:3:4 molar ratios when diluted to 0.05% MEGA-10 was assessed. This approach formed small (<60 nm) particles with low poly dispersity (PDI<0.05). After TFF processing of the sample, it maintained its small size and low PDI (FIG. 15B) and was found to have a negative surface charge (FIG. 15C). These findings were validated via RP-HPLC- ELSD and there was no detectable level of MEGA- 10 in the purified sample (FIG. 15D). However, the sample was found to be composed of minute liposomes when assessed via cryoTEM, indicating that surface charge alone was not sufficient to induce disc morphology (FIGs. 15E-15F).Lipid charge valency enables synthesis of CNDs stable in physiological buffer

[0202] Surface charge density did not appear sufficient to induce disc formation. Without wishing to be bound by any particular theory, the inventors posited that the surface charge did not act to prevent bicelle-bicelle fusion, but that instead, the charged lipid headgroups prompted lipid-lipid repulsion on bilayers. This repulsion could act similarly to how PEGylated lipids sterically repel each other leading to formation of LNDs when a sufficient amount of PEGylated lipids is incorporated.19Without wishing to be bound by any particular theory, the inventors posited that this charge-induced repulsion would only be significant at low ionic strengths, as charge screening at high ionic strengths would limit charge-charge interactions. Moreover, the inventors posited that the large hydration shell of the anionic lipids in low ionic strength likely resulted in a cone-shaped lipid which could stabilize the edge of lisCNDs.

[0203] To overcome this charge-shielding effect of increasing ionic strength, the inventors posited that lipids which contain two charges in close-proximity could increase the lipid headgroup charge density such that stable CNDs could be formed in a physiological buffer (PBS). A commercially available lipid - l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (glutaryl) (sodium salt) (DOPE-glutaryl) was evaluated (FIG. 16 A). The anionic POPG lipid was replaced with DOPE-glutaryl and tested at increasing molar ratios prior to mixed micelle dilution with PBS.

[0204] While low amounts of DOPE-glutaryl (<2.5 mol%) led to large particles (>100 nm) when diluted to or below 0.1% MEGA-10, incorporating more than 5 mol% DOPE-glutaryl, small (Z-avg<50 nm) particles could be generated (FIG. 16B). The effect of DOPE-glutaryl was clearly observed at 0.1% MEGA-10 in which mixed micelles may coexist with the particles to facilitate assembly (FIG. 16C).13Indeed, dilution of >5 mol% DOPE-glutarylcompositions to below 0.1% MEGA caused particle size and poly dispersity to grow (FIG. 17A). Given that these were promising results towards the target nanodisc-shaped NPs, a larger batch size of 10 mol% DOPE-glutaryl NPs diluted to 0.1% MEGA-10 was prepared. After overnight incubation, particles were diluted to 0.02% MEGA-10 and purified via TFF. RP-HPLC analysis of the sample via ELSD demonstrated that TFF purification led to undetectable levels of MEGA-10 with >99.9% removal of detergent and <1% by mass composition (FIG. 17B).

[0205] In addition to their smaller size compared to liposomes, the purified particles had moderately larger PDI ~0.2 as appropriate for an anisotropic material such as nanodiscs (FIG. 16D). The particle anisotropy was confirmed by evaluating the contribution of polarized (vertical) versus horizontal (depolarized) scattered light which showed these particles had relatively high depolarized scattering compared to liposomes (FIG. 17B).20Further, compared to 10% POPG liposomes, the purified particles exhibited marginally lower zeta potential (FIG. 16E). Importantly, when characterized via negative stain TEM, it was evident that these particles were nanodisc shaped based on the presence of the characteristic nanodisc “edge-on” and “face-on” particles on micrographs (FIG. 16F).21Given that these particles were stable in physiological buffer (PBS), they were termed charge-stabilized nanodiscs (CNDs). The proposed structure is shown in FIG. 16G.CNDs have higher tumor accumulation in vivo compared to liposomes

[0206] To determine if CNDs maintained the desired tumor accumulating properties seen previously with PEG-stabilized LNDs7, intravenous injection of CNDs and extruded liposomes were compared in mice bearing subcutaneous tumors (FIG. 18 A). While liposomes were larger than CNDs, 50-60 nm liposomes are one of the most common NPs for drug delivery and smaller liposomes could not be generated without significantly modifying the composition of the liposomes. As shown in FIGs. 18B-18C, there was a significant increase in the tumor accumulation of NPs even though serum NP fluorescence was similar to that of liposomes (FIG. 18D). Moreover, while there was no significant difference in NP accumulation in major clearance organs (liver and spleen), CNDs were preferentially accumulated in tumors (FIG. 18E). Indeed, when tumors were extracted ex vivo, CNDs resulted in a more than five-fold increase in tumor accumulation relative to liposomes (FIG. 14F). These results validate that the nanodisc shaped lipidic CNDs promoted better tumor accumulation in vivo compared to state-of-the-art 50-60 nm liposomes.Deposition of thin polyelectrolyte films via layer-by-layer (LbL) technique enables increased tumor cell association of CND-LbL compared to liposome-LbL in vitro

[0207] Previous work has shown that the deposition of thin polymer films onto nanoparticles via the layer-by-layer (LbL) technique can enable controlled drug release and tumor targeting.22The choice of polymer chemistry regulates the overall LbL-NP properties, enabling a wide range of particle characteristics.23Association of LbL-NPs with cells is a surface-driven phenomena. The inventors posited that the increased surface area of CNDs could generate improved LbL-NP formulations relative to spherical liposomes. This was assessed by depositing a bilayer of poly-L-arginine (PLR) and poly-L-glutamate (PLE) onto NPs which has been previously shown to target ovarian cancer cells and controls subcellular trafficking such that NPs accumulate on the cell surface.23 25Thus, CNDs were layered with PLR and PLE and the resulting nanoparticles were compared to layered liposomes (FIG.19A).

[0208] Addition of the polyelectrolyte layers led to characteristic changes in zeta potential (FIG. 20A) with slight increases in the final particle size while maintaining low PDI (<0.2) in both CNDs and liposomes (FIG. 19B). Interestingly, while lisCND could also be modified via the LbL technique (FIG. 20B), only CND-LbL presented improved association with ovarian cancer cells in vitro relative to liposomes-LbL FIGs. 19C and 20C). Without wishing to be bound by any particular theory, the inventors posited that this could be due to the instability of lisCND which were found to have lower uptake than unlayered liposomes whereas CND had equal or higher uptake by ovarian cancer cells. It was determined that the properties of cell-membrane accumulation from PLR / PLE LbL were maintained in CND-LbL when cells were imaged on a confocal microscope (FIG. 19D).Deposition of thin polyelectrolyte films via layer-by-layer (LbL) technique enables increased tumor cell association of CND-LbL compared to liposome-LbL in vitro

[0209] Based on the improved in vitro ovarian cancer binding of LbL-CNDs compared to LbL-liposomes, the in vivo tumor-targeting properties of LbL-CNDs were examined in a model of metastatic ovarian cancer. Luciferase-expressing HM-1 cells were injected intraperitoneally (i.p.) into mice and allowed to establish for 14 days, followed by i.p. administration of fluorescent liposomes or CNDs, with or without the LbL coating (FIG. 21A).

[0210] Mice given LbL-CNDs had significantly higher (~2-fold) in vivo peritoneal fluorescence 4 hours after dosing compared to other groups indicating a better retention of NPs in the i.p. space (FIGs. 21B-C). While accumulation in the major clearance tissues(spleen and liver) was low, ex vivo measurement of total NP fluorescence in the main sites of metastasis development (i.e., omentum and UGT35) revealed significantly improved accumulation of LbL-CNDs in tumor tissue with a 10-fold or more increase relative to bare liposomes (FIG. 2 ID). Further, as all peritoneal organs present some level of tumor burden which could be measured via bioluminescence intensity (BLI) readings, the correlation between the tumor burden of an organ to its NP fluorescence reading was analyzed. While bare liposome fluorescence did not correlate with tissue tumor burden, LbL-liposomes showed a significant correlation, consistent with the tumor-targeting properties of LbL-coated NPs (FIG. 2 IE). Moreover, both CND and LbL-CND accumulation were correlated with tumor burden, albeit CND-LbL treatment yielded a more confident fit. Indeed, LbL modification of CNDs increased the amount of NP accumulation per BLI reading ~4-fold (FIG. 2 IF). These results indicate that combining the nanodisc morphology with an ovarian cancer-targeting LbL coating substantially enhances the delivery of lipid nanoparticles to metastatic ovarian cancer.

[0211] Lipid nanoparticles are important delivery vehicles for current and new generation therapeutics. Here it was shown that through the rational disassembly of mixed micelles, nanodisc intermediates can be stabilized to generate a new class of lipid nanoparticles - charge-stabilized nanodiscs (CNDs). It was also determined that assembly from detergent micelles enabled synthesis of minute liposomes. These minute liposomes with minimal poly dispersity are close to the limit size of nanoliposomal systems, making them advantageous for biomedical applications.26Achieving this small of a liposome size via conventional methods has been challenging.27Even with optimized conditions, limit-size nanoliposomes have previously been reported only via high poly dispersity (PDI>0.1).26’27

[0212] Previous studies have shown that PEG, proteins, synthetic polymers, or mixtures of short and long-tailed surfactants (such as detergents and lipids) can generate disc-like structures.6 9However, these added components can alter the particle characteristics, and in the case of bicelles, they are limited by their sensitivity to dilution which did not occur with CNDs.8

[0213] Taken together, the method provided herein facilitated generation of a new and controlled lipid-based assembly. The novel CNDs demonstrated improved tumor accumulating properties compared to standard liposomes and could be surface modified via the LbL technique to further promote tumor targeting.Example 3: Experimental Protocols

[0214] Materials: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), Cholesterol, 1- palmitoyl-2-oleoyl-sn-glycero-3-phospho-(r-rac-glycerol) (sodium salt) (POPG), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (sodium salt) (DOPE-glutaryl), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (DOPE-DBCO), 1,2- distearoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DSPG), and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-(Cyanine 5) (DSPE-cy5) 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 were purchased from Alamanda Polymers. N-decanoyl-N- methylglucamine (MEGA- 10) and n-octyl-P-d-glucoside (octylglucoside) were purchased from Sigma Aldrich.

[0215] Generation deter gent / lipid mixture: Lipid stock solutions were made in chloroform and them measured into glass vials and left drying on a desiccator overnight. For solubilization in MEGA- 10 micelles, a 10% MEGA- 10 solution was made in deionized water. The 10% MEGA- 10 solution was then added to the dried lipids and left in a water bath sonicator at 50 to 60 °C until all lipids were solubilized. The lipid / detergent mixture was allowed to equilibrate at room temperature prior to dilution. The same process was performed with octylglucoside instead of MEGA- 10.

[0216] Synthesis of nanoparticles via dilution: The lipid / detergent micelles were diluted by rapidly adding buffer to the micelles to reach the target detergent concentration.

[0217] Purification via TFF: Generally, 5 mg of the lipid nanoparticle mixtures were diluted to the target MEGA- 10 concentration and left to self-assemble overnight. Then samples were diluted to a minimum of 0.02% MEGA-10 to ensure minimal effect of the detergent on the NPs. Samples were then placed on a KrosFlo KR2i TFF system (Repligen) system using either a 50 kDa mPES membrane with a surface area of 75 cm2 (D02-E050-10- N) for particles <100 nm or a 100 kDa mPES membrane with a surface area of 115 cm2 for particles >100 nm (D02-E100-05-N). Samples underwent 10 diafiltration volumes against the buffer used for their assembly. Particle yield was either accessed based on recovered nanoparticle fluorescence or total lipid content measured via the Steward assay.28

[0218] Analysis of lipid transfer via FRET: 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. Lipid / detergent micelles of desired composition were generated with 1 mol% of either DOPE-TMR (donor-micelles) or DOPE-630 / 650 (acceptor-micelles). For FRET-micelles, donor micelles and acceptor micelles were mixed 1 : 1. FRET- micelles, donor-micelles, and acceptor-micelles were diluted to target MEGA- 10 concentration. After dilution of donor and acceptor micelles, the diluted samples of equal final MEGA- 10 concentration were mixed at 0 hours, 1 hour and 24 hours after dilution.FRET efficiency was then measured at 0 hours, 1 hour, 24 and 48 hours after mixing of donor and acceptor micelles. FRET efficiency was calculated based on the equation for the corrected FRET efficiency (FRETN) described previously.29

[0219] Layer-by-Layer (LbL) deposition: For synthesis of lisCND-LbL, particles were mixed with excess polymers in 10 mM HEPES while for CND-LbL, particles were first buffer exchanged into 50 mM HEPES and 40 mM NaCl then mixed with excess PLR in deionized water followed by PLE also in deionized water. After PLR deposition, samples were purified into deionized water on a 30 kDa mPES membrane with surface area of 20 cm2(C02-E030-05-N) pre-equilibrated with excess PLR. After purification of PLR excess PLE and then purified into deionized water on a 50 kDa mPES membrane with a surface area of 75 cm2(D02-E050-10-N). Liposome-LbL controls were generated using the same conditions. Fluorescently-labeled lisCNDs contained 0.2 mol% of DOPE-630 / 650 while CNDs contained 1 mol% of DSPE-cy5.

[0220] 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 21 OOF microscope (200 kV). For cryo-TEM, particles were buffer exchanged into deionized water via either dialysis or TFF. The microscopes were with a magnification range of 10, 000-60, 000X. Cryo-TEM micrographs were analyzed on ImageJ to measure particles diameter. Particles with 1 mol% DSPE-cy5 were characterized on a Wyatt Dyna Pro Plate Reader.

[0221] RAW 264.7 Cells

[0222] Cell Culture: RAW 264.7 macrophages 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.

[0223] In vitro cellular association: The day before dosing, RAW 264.7 cells were plated on a tissue-culture 96-well plate at a density of 25k cells per well. The next day, wells were dosed with NPs to 0.01 mg / mL and left for the target incubation time (4 hours or 24 hours). For analysis of association, the supernatant was removed from the well and diluted 5X withDMSO. Cells were then washed three times with PBS then dissolved with DMSO.Fluorescence of NPs associated with cells as well as supernatant NP fluorescence were quantified to determine total uptake.

[0224] OV2944-HM-1 Cells

[0225] Cell Culture: OV2944-HM-1 cells were acquired through Riken BRC and were cultured in a-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.

[0226] 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 hours or 24 hours). 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. 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 lOk / 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 Imaged. Slides were imaged on a Olympus FV1200 Laser Scanning Confocal Microscope.

[0227] Mice: C57B1 / 6 and B6C3F1 mice were purchased from Jackson Laboratories.Female mice were used between 8-12 weeks of age unless otherwise noted. 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.

[0228] Subcutaneous tumor model: C57B1 / 6 mice were implanted with subcutaneous MC38 tumors by injecting 106 cells into the right flank. One week after tumor inoculation, mice were injected intravenously via the tail vein with NPs containing 1 mol% DSPE-cyanine5 (1 nmol dye injected per mouse). In vivo tumor radiant efficiency was measured on an In Vivo Imaging System (IVIS, Perkin Elmer) and serum was collected via cheek bleeds. After thefinal timepoint (24 hours), mice were euthanized and the major NP clearance organs - liver and spleen - as well as tumor were removed and had their radiant efficiency measured ex vivo on an IVIS. Data were analyzed using Living Image software. Background fluorescence measurements were made for each organ based on signal from mice treated with PBS. Recovered fluorescence efficiency was calculated as described previously.23

[0229] Intraperitoneal ovarian cancer model: B6C3F1 mice were inoculated with firefly luciferase-expressing OV2944-HM1 (HM-1) cells through intraperitoneal (i.p.) injection of 106 cells in PBS. Two weeks after tumor inoculation, mice were injected with 0.75 nmol of 1 mol% DSPE-cy5 NPs. Analysis was done similar to subcutaneous tumor model with the exception that peritoneal radiant efficiency was measured instead of tumor fluorescence.Main tumor tissue was considered the omental adipose tissue which is the primary metastatic niche of ovarian cancer.30For correlation analysis, the weight-normalized bioluminescence flux (p / s / g) and radiant efficiency ([p / s] / [pW / cm2] / g) for each organ (excluding main tumor) were analyzed on Graphpad Prism 9 for their correlation via the Pearson’s coefficient.

[0230] 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 for time-based analysis or Sidak posthoc for other ANOVA analysis.References:1. Mundekkad, D. & Cho, W. C. Nanoparticles in Clinical Translation for Cancer Therapy. IntJMol Sci 23, 1685 (2022).2. Albanese, A., Tang, P. S. & Chan, W. C. W. The Effect of Nanoparticle Size, Shape, and Surface Chemistry on Biological Systems. Annu Rev Biomed Eng 14, 1-16 (2012).3. Zhu, X., Vo, C., Taylor, M. & Smith, B. R. Non-spherical micro- and nanoparticles in nanomedicine. Mater Horiz 6, 1094-1121 (2019).4. Ding, J. el al. Engineered nanomedicines with enhanced tumor penetration. Nano Today 29, 100800 (2019).5. Sun, Q., Ojha, T., Kiessling, F., Lammers, T. & Shi, Y. Enhancing Tumor Penetration of Nanomedicines. Biomacromolecules 18, 1449-1459 (2017).6. Smith, A. A. A. et al. Lipid Nanodiscs via Ordered Copolymers. Chem 6, 2782- 2795 (2020).7. Dane, E. L. etal. STING agonist delivery by tumour-penetrating PEG-lipid nanodiscs primes robust anticancer immunity. Nat Mater 21, 710-720 (2022).8. Lu, Z. et al. Bicelles at Low Concentrations. MolPharm 9, 752-761 (2012).9. Patel, H. etal. Characterization of apolipoprotein A-I peptide phospholipid interaction and its effect on HDL nanodisc assembly. Int J Nanomedicine Volume 14, 3069-3086 (2019).10. Bariwal, J., Ma, H., Altenberg, G. A. & Liang, H. Nanodiscs: a versatile nanocarrier platform for cancer diagnosis and treatment. Chem Soc Rev 51, 1702-1728 (2022).11. Kozma, G. T., Shimizu, T., Ishida, T. & Szebeni, J. Anti-PEG antibodies: Properties, formation, testing and role in adverse immune reactions to PEGylated nanobiopharmaceuticals. Adv Drug Deliv Rev 154-155, 163-175 (2020).12. Jiskoot, W ., Teerlink, T., Beuvery, E. C. & Crommelin, D. J. A. Preparation of liposomes via detergent removal from mixed micelles by dilution. Pharm Weekbl Sci 8, 259- 265 (1986).13. Pires, I. S. et al. Controlled lipid self-assembly for scalable manufacturing of nextgeneration immune stimulating complexes. Chemical Engineering Journal 464, 142664 (2023).14. Barberio, A. E. et al. Layer-by-layer interleukin- 12 nanoparticles drive a safe and effective response in ovarian tumors. Bioeng Transl Med (2022) doi: 10.1002 / btm2.10453.15. Barberio, A. E. et al. Cancer Cell Coating Nanoparticles for Optimal Tumor-Specific Cytokine Delivery. ACS Nano 14, 11238-11253 (2020).16. Correa, S. et al. Highly Scalable, Closed-Loop Synthesis of Drug-Loaded, Layer-by- Layer Nanoparticles. Adv Funct Mater 26, 991-1003 (2016).17. Ollivon, M., Lesieur, S., Grabielle-Madelmont, C. & Paternostre, M. Vesicle reconstitution from lipid-detergent mixed micelles. Biochimica et Biophysica Acta (BBA) - Biomembranes 1508, 34-50 (2000).18. Sej wal, K. et al. Proteoliposomes - a system to study membrane proteins under buffer gradients by cryo-EM. Nanotechnol Rev 6, 57-74 (2017).19. Viitala, L. et al. Shape and Phase Transitions in a PEGylated Phospholipid System. Langmuir 35, 3999-4010 (2019).20. Lehner, D., Lindner, H. & Glatter, O. Determination of the Translational and Rotational Diffusion Coefficients of Rodlike Particles Using Depolarized Dynamic Light Scattering. Langmuir 16, 1689-1695 (2000).21. Asadi, J. et al. Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals. Micron 99, 40-48 (2017).22. Alkekhia, D., Hammond, P. T. & Shukla, A. Layer-by-Layer Biomaterials for Drug Delivery. Annu Rev Biomed Eng 22, 1-24 (2020).23. Correa, S. etal. Tuning Nanoparticle Interactions with Ovarian Cancer through Layer- by-Layer Modification of Surface Chemistry. ACS Nano 14, 2224-2237 (2020).24. Barberio, A. E. et al. Layer-by-layer interleukin- 12 nanoparticles drive a safe and effective response in ovarian tumors. Bioeng Transl Med (2022) doi: 10.1002 / btm2.10453.25. Barberio, A. E. et al. Cancer Cell Coating Nanoparticles for Optimal Tumor-Specific Cytokine Delivery. ACS Nano 14, 11238-11253 (2020).26. Zhigaltsev, I. V., Tam, Y. K., Leung, A. K. K. & Cullis, P. R. Production of limit size nanoliposomal systems with potential utility as ultra-small drug delivery agents. J Liposome Res 1-7 (2015) doi: 10.3109 / 08982104.2015.1025411.27. Zhigaltsev, I. V. et al. Bottom-Up Design and Synthesis of Limit Size Lipid Nanoparticle Systems with Aqueous and Triglyceride Cores Using Millisecond Microfluidic Mixing. Langmuir 28, 3633-3640 (2012).28. Stewart, J. C. M. Colorimetric determination of phospholipids with ammonium ferrothiocyanate. AnalBiochem 104, 10-14 (1980).29. Edidin, M. Fluorescence Resonance Energy Transfer: Techniques for Measuring Molecular Conformation and Molecular Proximity. Curr Protoc Immunol 57, (2003).30. Krishnan, V. et al. Omental macrophages secrete chemokine ligands that promote ovarian cancer colonization of the omentum via CCR1. Commun Biol 3, 524 (2020).EQUIVALENTS AND SCOPE

[0231] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. 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.

[0232] 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 verbaherein. 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.

[0233] 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.

[0234] 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

CLAIMSWhat is claimed is:

1. A particle, wherein the particle comprises:(a) a polyvalent phospholipid;(b) a helper phospholipid; and(c) optionally, a sterol; wherein the particle has anisotropic dimensions.

2. The particle of claim 1, wherein the particle is not spherical.

3. The particle of claim 1 or 2, wherein the particle is disc-shaped.

4. The particle of any one of claims 1-3, wherein the particle has a largest cross- sectional dimension that is about 1.2- to about 10-fold larger than its smallest cross-sectional dimension.

5. The particle of any one of claims 1-4, wherein the particle has a largest cross- sectional dimension that is about 1.2- to about 3-fold larger than its smallest cross-sectional dimension.

6. The particle of any one of claims 1-5, wherein the particle has a largest cross- sectional dimension that is about 1.5- to about 2-fold larger than its smallest cross-sectional dimension.

7. The particle of any one of claims 1-4, wherein the particle has a largest cross- sectional dimension that is about 3- to about 6-fold larger than its smallest cross-sectional dimension.

8. The particle of any one of claims 1-4, wherein the particle has a largest cross- sectional dimension that is about 6- to about 10-fold larger than its smallest cross-sectional dimension.

9. The particle of any one of claims 1-6, wherein the particle has a largest cross- sectional dimension of about 20 nm to about 50 nm.

10. The particle of any one of claims 1-9, wherein the particle has a smallest cross- sectional dimension of about 3 nm to about 15 nm.

11. The particle of any one of claims 1-10, wherein the particle has a smallest cross- sectional dimension of about 5 nm to about 10 nm.

12. The particle of any one of claims 1-11, wherein the particle comprises a sterol.

13. The particle of any one of claims 1-12, wherein the sterol is campesterol, desmosterol, stigmasterol, lanosterol, sitosterol, or cholesterol.

14. The particle of any one of claims 1-13, wherein the sterol is cholesterol.

15. The particle of any one of claims 1-14, wherein the particle comprises about 1 mol% to about 50 mol% of the polyvalent phospholipid.

16. The particle of any one of claims 1-15, wherein the particle comprises about 5 mol% to about 50 mol% of the polyvalent phospholipid.

17. The particle of any one of claims 1-15, wherein the particle comprises about 2 mol% to about 15 mol% of the polyvalent phospholipid.

18. The particle of any one of claims 1-17, wherein the particle comprises at least about 5 mol% of the polyvalent phospholipid.

19. The particle of any one of claims 1-18, wherein the particle comprises at least about 7.5 mol% of the polyvalent phospholipid.

20. The particle of any one of claims 1-19, wherein the particle comprises about 10 mol% of the polyvalent phospholipid.

21. The particle of any one of claims 1-20, wherein the particle comprises about 20 mol% to about 95 mol% of the helper phospholipid22. The particle of any one of claims 1-21, wherein the particle comprises about 60 mol% of the helper phospholipid23. The particle of any one of claims 1-22, wherein the particle comprises about 0 mol% to about 50 mol% of the sterol.

24. The particle of any one of claims 1-23, wherein the particle comprises about 30 mol% of the sterol.

25. The particle of any one of claims 1-24, wherein the particle comprises a molar ratio of about 6:3: 1, about 5:3:2, about 4:3:3, or about 3:3:4 of the helper phospholipid:the sterokthe polyvalent phospholipid.

26. The particle of any one of claims 1-25, wherein the polyvalent phospholipid is divalent.

27. The particle of any one of claims 1-25, wherein the polyvalent phospholipid is trivalent.

28. The particle of any one of claims 1-25, wherein the polyvalent phospholipid has a valency greater than or equal to 10.

29. The particle of any one of claims 1-25 or 28, wherein the polyvalent phospholipid has a valency greater than or equal to 50.

30. The particle of any one of claims 1-25, 28, or 29, wherein the polyvalent phospholipid has a valency greater than or equal to 100.

31. The particle of claim any one of claims 1-26, wherein the polyvalent phospholipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl).

32. The particle of claim any one of claims 1-31, wherein the polyvalent phospholipid is a polyanionic phospholipid.

33. The particle of any one of claims 1-32, wherein the helper phospholipid is 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-Dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), soy phosphatidylcholine, and egg phosphatidylcholine.

34. The particle of any one of claims 1-33, wherein the helper phospholipid is 1,2- di stearoyl -sn-gly cero-3 -phosphocholine (D SPC) .

35. The particle of any one of claims 1-34, further comprising a polymer coating.

36. The particle of claim 35, wherein the polymer coating is non-covalently adsorbed onto the surface of the particle.

37. The particle of claim 35 or 36, wherein the polymer coating comprises a polyelectrolyte.

38. The particle of any one of claims 35-37, wherein the polymer coating comprises a mixture of polyelectrolytes.

39. The particle of any one of claims 35-38, wherein the polymer coating comprises poly- L-lysine (PLL), polyethylenimine (PEI), poly(diallyldimethylammonium chloride) (PDAC), poly(P-amino esters) (PBAE), poly(allylamine hydrochloride) (PAH), poly-L-aspartic acid (PLD), poly-hyaluronic acid (HA), poly-acrylic acid (PAA), poly(sodium 4- styrenesulfonate), dextran sulfate, heparin sulfate, a folate-conjugated polymer, poly-L- arginine (PLR), poly-L-glutamate (PLE), PEG-PLE, or PEG-PLD.

40. The particle of any one of claims 35-39, wherein the polymer coating comprises poly- L-arginine and / or poly-L-glutamate.

41. The particle of any one of claims 1-40, wherein the particle is a lipid-based particle.

42. The particle of any one of claims 1-41, wherein the particle further comprises a membrane scaffolding protein.

43. The particle of any one of claims 1-41, wherein the particle does not comprise one or more of a synthetic amphiphilic polymer, a protein, or a neutral lipid-polymer conjugate.

44. The particle of any one of claims 1-41, wherein the particle does not comprise one or more of a synthetic amphiphilic polymer, a protein, a neutral lipid-polymer conjugate, or a detergent.

45. The particle of any one of claims 1-41, wherein the particle does not comprise any non-lipid components.

46. The particle of any one of claims 1-45, wherein the particle is stable in solution under physiological conditions.

47. The particle of claim 46, wherein the particle is stable in physiological fluid.

48. The particle of claim 46 or 47, wherein the particle is stable in solution under physiological pH.

49. The particle of any one of claims 46-48, wherein the particle is stable in solution under physiological ionic strength.

50. The particle of any one of claims 1-49, wherein the particle is stable in solution for at least one week.

51. The particle of any one of claims 1-50, wherein the particle is stable in solution for at least one month.

52. The particle of any one of claims 1-51, wherein the particle is stable in solution for up to 6 months.

53. The particle of any one of claims 1-52, wherein the particle is stable at up to 4 °C.

54. The particle of any one of claims 1-53, wherein the particle is stable at up to 22 °C.

55. A method of preparing a plurality of particles having anisotropic dimensions according to any one of claims 1-54, the method comprising:(a) preparing a mixture of a polyvalent phospholipid, a helper phospholipid, and a detergent;(b) allowing the mixture to equilibrate afford a plurality of micelles;(c) diluting the micelles with a buffer to a target detergent concentration, thereby forming a plurality of particles having anisotropic dimensions; and(d) separating the plurality of particles.

56. The method of claim 55, wherein the polyvalent phospholipid is 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine-N-(glutaryl).

57. A method of preparing a plurality of liposomes, the method comprising:(a) preparing a mixture of a monoanionic phospholipid, a helper phospholipid, and a detergent;(b) allowing the mixture to equilibrate afford a plurality of micelles;(c) diluting the micelles with a buffer to a target detergent concentration to promote self-assembly of a plurality of bicelles having anisotropic dimensions, wherein the bicelles undergo bicelle fusion to afford a plurality of liposomes; and(d) separating the plurality of liposomes.

58. The method of claim 57, wherein the monoanionic phospholipid is l-palmitoyl-2- oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol).

59. The method of any one of claims 55-58, wherein the helper phospholipid is 1,2- distearoyl-sn-glycero-3-phosphocholine.

60. The method of any one of claims 55-59, wherein the separating of step (d) comprises ultrafiltration, density gradient separation, size exclusion chromatography, use of detergent depletion agents, adsorption chromatography, or ion exchange chromatography.

61. The method of claim 60, wherein the separating of step (d) comprises ultrafiltration.

62. The method of claim 61, wherein the separating of step (d) comprises tangential flow filtration.

63. The method of any one of claims 55-62, wherein the mixture of step (a) further comprises a sterol.

64. The method of any one of claims 55-63, wherein the buffer is PBS, HEPES saline, or HEPES.

65. The method of any one of claims 55-64, wherein the detergent is ionic.

66. The method of any one of claims 55-64, wherein the detergent is non-ionic.

67. The method of any one of claims 55-64, wherein the detergent is CHAPs, Tween 20,Tween 80, triton X-100, SDS, DDM / CHS, CHAPSO, one or more bile salts, MEGA- 10, or octylglucoside.

68. The method of any one of claims 55-64, wherein the detergent is MEGA-10 or octylglucoside.

69. The method of any one of claims 55-68, wherein the mixture of step (a) comprises about 10- to 200-fold more detergent than the pure detergent critical micelle concentration.

70. The method of any one of claims 55-69, wherein the mixture of step (a) comprises about 1% to about 20% (w / v) detergent in water.

71. The method of any one of claims 55-70, wherein the mixture of step (a) comprises about 10% (w / v) detergent in water.

72. The method of any one of claims 55-71, wherein the dilution is performed to below the critical micelle concentration.

73. The method of any one of claims 55-71, wherein the dilution is performed to reach the critical micelle concentration.

74. The method of any one of claims 55-71, wherein the dilution is performed to above the critical micelle concentration.

75. The method of any one of claims 55-74, wherein the target detergent concentration of step (c) is about 0.05% to about 0.3% of the buffer.

76. The method of any one of claims 55-75, wherein the target detergent concentration of step (c) is about 0.05% to about 0.1% of the buffer.

77. The method of any one of claims 55-75, wherein the target detergent concentration of step (c) is about 0.1% to about 0.2% of the buffer.

78. The method of any one of claims 55-77, further comprising depositing a polymer coating onto the particle using layer-by-layer assembly.

79. The method of claim 78, wherein the layer-by-layer assembly comprises the steps of:(e) mixing the plurality of particles or liposomes in a buffer with a polymer to deposit the polymer onto the particles or liposomes, forming a polymer coating; and(f) optionally removing excess polymer.

80. The method of claim 78 or 79, wherein the polymer coating comprises a polyelectrolyte.

81. The method of any one of claims 78-80, wherein the polymer coating comprises poly- L-arginine or poly-L-glutamate.

82. The method of any one of claims 55-81, further comprising a step of loading an agent into the particle.

83. The method of claim 82, wherein the agent is added during step (a).

84. The method of claim 82, wherein the agent is added during step (b).

85. The method of claim 82, wherein the agent is added prior to step (c).

86. The method of claim 82, wherein the agent is added after step (c).

87. The method of claim 82, wherein the agent is added after step (d).

88. The method of any one of claims 82-87, wherein the agent is an additional pharmaceutical agent.

89. A liposome prepared according to the method of any one of claims 57-88.

90. The liposome of claim 89, wherein the liposome is spherical.

91. The liposome of claim 89 or 90, wherein the liposome has a diameter of about 50 nm to about 1 pm.

92. The liposome of any one of claims 89-91, wherein the liposome has a poly dispersity of less than about 0.2.

93. The liposome of any one of claims 89-92, wherein the liposome has a poly dispersity of less than about 0.1.

94. A pharmaceutical composition comprising a plurality of particles of any one of claims 1-54, and a pharmaceutically acceptable excipient.

95. A pharmaceutical composition comprising a plurality of liposomes of any one of claims 89-93, and a pharmaceutically acceptable excipient.

96. The pharmaceutical composition of claim 94 or 95, further comprising an additional pharmaceutical agent.

97. The pharmaceutical composition of claim 96, wherein the additional pharmaceutical agent is encapsulated by the particle.

98. The pharmaceutical composition of claim 96, wherein the additional pharmaceutical agent is attached to the surface of the particle.

99. The pharmaceutical composition of any one of claims 96-98, wherein the additional pharmaceutical agent is a cancer therapeutic.

100. The pharmaceutical composition of claim 99, wherein the cancer therapeutic is a platinum-based therapeutic, a taxol-based therapeutic, a microtuble inhibitor, or doxorubicin.

101. The pharmaceutical composition of any one of claims 96-99, wherein the additional pharmaceutical agent is a therapeutic protein.

102. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a particle of any one of claims 1-54 or a pharmaceutical composition of any one of claims 94-101.

103. The method of claim 102, wherein the disease is a proliferative disease.

104. The method of claim 102 or 103, wherein the disease is cancer.

105. The method of any one of claims 102-104, wherein the disease is ovarian cancer.

106. The method of claim 104 or 105, wherein the cancer is metastatic.

107. The method of any one of claims 102-106, wherein the particle is administered via intravenous injection or intraperitoneal injection.

108. A method of delivering an agent to a target cell in a subject, cell, or biological sample, comprising contacting the target cell with a particle of any one of claims 1-54.

109. The method of claim 108, wherein the agent is released from the particle.

110. The method of claim 108 or 109, wherein the cell is ex vivo.

111. The method of claim 108 or 109, wherein the cell is in vivo.

112. The method of any one of claims 108-111, wherein the cell is a tumor cell.

113. A kit comprising: a particle of any one of claims 1-54, or a pharmaceutical composition of any one of claims 94-101; and instructions for using the particle or the pharmaceutical composition.

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