WO2024039733A1

WO2024039733A1 - Methods of controlling lipid nanocrystal particle size and lipid nanocrystals produced by such methods

Info

Publication number: WO2024039733A1
Application number: PCT/US2023/030369
Authority: WO
Inventors: Hui Liu; Chris KARCH; Mariam MIKHAEL; Zohreh Sadat BADIEYAN
Original assignee: Matinas Biopharma Nanotechnologies, Inc.
Priority date: 2022-08-16
Filing date: 2023-08-16
Publication date: 2024-02-22

Abstract

This application relates generally to lipid nanocrystal particle size and aggregation, as well as related methods. Methods of controlling lipid nanociystal particle size are provided, as well as populations of lipid nanocrystals having a mean particle size of interest, such as a. mean particle size of less than about 200 nm.

Description

Attorney Docket No. MNY-01525 METHODS OF CONTROLLING LIPID NANOCRYSTAL PARTICLE SIZE AND LIPID NANOCRYSTALS PRODUCED BY SUCH METHODS Cross-Reference to Related Applications [0001] This application claims priority to U.S. Application No.63/371,530, filed August 16, 2022, and US Application No. 63/415,363, filed October 12, 2022, each of which are hereby incorporated by reference in their entirety for all purposes. Field [0002] This application relates generally to lipid nanocrystal particle size and aggregation, as well as related methods. Methods of controlling lipid nanocrystal particle size are provided, as well as populations of lipid nanocrystals having a mean particle size of interest, such as a mean particle size of less than about 200 nm. Background [0003] Lipid nanocrystal (LNC) delivery vehicles are a broad-based technology for the delivery of a wide range of bioactive therapeutic products. These delivery vehicles are stable phospholipid-cation precipitates composed of simple, naturally occurring materials, for example, phosphatidylserine and calcium. The entire lipid nanocrystal structure is a series of solid layers, which allows components within the interior of a lipid nanocrystal, such as a drug, to remain substantially intact, even if the outer layers of the lipid nanocrystal are exposed to harsh environmental conditions or enzymes. Thus, lipid nanocrystals are protected from, for instance, digestive enzymes in the stomach. [0004] Taking advantage of these unique properties, lipid nanocrystals have been used to mediate and enhance the bioavailability of a broad spectrum of beneficial, but difficult to formulate biopharmaceuticals, including compounds with poor water solubility, protein and peptide drugs, and large hydrophilic molecules. For example, lipid nanocrystal-mediated delivery of amphotericin B, large DNA constructs/plasmids, peptide formulations, and antibiotics has been achieved. [0005] The ability to produce lipid nanocrystals with a particle size of less than 200 nm is beneficial because the end product can easily pass through a 0.22 µm filter. Smaller sizes of lipid Attorney Docket No. MNY-01525 nanocrystals also enhance biodistribution of lipid nanocrystals following administration. However, due to their small size, lipid nanocrystals would inevitably undergo aggregation once released into aquatic environments, which could directly influence their bioavailability. Thus, there remains a need for producing lipid nanocrystals of smaller sizes, such as less than about 200 nm, with minimum aggregation potential. Summary [0006] Disclosed herein are methods of controlling particle size and aggregation of a population of lipid nanocrystals, as well as populations of lipid nanocrystals thus prepared. [0007] In one aspect, the present disclosure is directed to a method of preparing a population of lipid nanocrystals comprising a) preparing a solution of liposomes comprising a negatively charged phospholipid and a therapeutic agent in an aqueous medium; and b) adding a multivalent cation to the solution of liposomes, thereby forming the population of lipid nanocrystals. In some embodiments, the negatively charged phospholipid is in an amount ranging from about 50% to about 100% by weight of the liposomes. In some embodiments, the multivalent cation added to the solution of liposomes and the negatively charged phospholipid are in a molar ratio of from about 0.25:1 to about 4:1. In some embodiments, the solution of liposomes has a lipid concentration of from about 0.5 mg/mL to about 100 mg/mL. By adjusting the amount of negatively charged phospholipid, the molar ratio of multivalent cation to negatively charged phospholipid, and in some embodiments, the lipid concentration, it is possible to control the mean particle size of the lipid nanocrystals prepared by these methods. In some embodiments, the population of lipid nanocrystals thus prepared has a mean particle size no more than about 200 nm. [0008] In some aspects, the disclosure provides a method for preparing a population of lipid nanocrystals comprising: (i) providing a population of liposomes comprising (a) a lipid component comprising a negatively charged phospholipid, and (b) a therapeutic agent, wherein the negatively charged phospholipid is in an amount from about 50% to about 100% by weight of the lipid component; and (ii) contacting the population of liposomes with an amount of a multivalent cation, wherein the molar ratio of the multivalent cation and the negatively charged phospholipid is about 0.25:1 Attorney Docket No. MNY-01525 to about 4:1, thereby forming the population of lipid nanocrystals having a mean particle size of about 180-220 nm. In some embodiments, the mean particle size is about 200 nm. In some embodiments, the amount of the negatively charged phospholipid is about 100% by weight of the lipid component, and the molar ratio of the multivalent cation and the negatively charged phospholipid is about 0.25:1 to about 0.5:1. In some embodiments, the molar ratio of the multivalent cation and the negatively charged phospholipid is about 0.25:1. In some embodiments, the amount of the negatively charged phospholipid is about 75% by weight of the lipid component, and the molar ratio of the multivalent cation and the negatively charged phospholipid is about 1:1 to about 1.5:1. In some embodiments, the molar ratio of the multivalent cation and the negatively charged phospholipid is about 1.5:1. In some embodiments, the lipid component comprises about 25% of a neutrally charged lipid by weight of the lipid component. In some embodiments, the neutrally charged lipid comprises phosphatidylcholine. In some embodiments, the amount of the negatively charged phospholipid is about 50% by weight of the lipid component, and the molar ratio of the multivalent cation and the negatively charged phospholipid is about 1:1 to about 1.5:1. In some embodiments, the molar ratio of the multivalent cation and the negatively charged phospholipid is about 1.5:1. In some embodiments, the lipid component comprises about 50% of a neutrally charged lipid by weight of the lipid component. In some embodiments, the neutrally charged lipid comprises phosphatidylcholine. [0009] In another aspect, the present disclosure is directed to a population of lipid nanocrystals, wherein each lipid nanocrystal in the population comprises: a) a lipid component comprising a negatively charged phospholipid; b) a multivalent cation; and c) a therapeutic agent, and wherein the population of lipid nanocrystals has a mean particle size no more than about 200 nm. In some embodiments, the negatively charged phospholipid comprised in the lipid component of the lipid nanocrystal is in an amount ranging from about 50% to about 100% by weight of the lipid component. In some embodiments, the multivalent cation and the negatively charged phospholipid comprised in the lipid nanocrystal are in a molar ratio of from about 0.25:1 to about 4:1. [0010] In some aspects, the disclosure provides a population of lipid nanocrystals, wherein each lipid nanocrystal in the population comprises: a) a lipid component comprising a negatively charged phospholipid, wherein the negatively charged phospholipid is in an amount of about 100% by weight of the lipid component; b) a multivalent cation; and Attorney Docket No. MNY-01525 c) a therapeutic agent, wherein the multivalent cation and the negatively charged phospholipid are in a molar ratio of about 0.25:1 to about 0.5:1, and wherein the population of lipid nanocrystals has a mean particle size of about 180-220 nm. In some embodiments, the molar ratio is about 0.25:1. [0011] In some aspects, the disclosure provides a population of lipid nanocrystals, wherein each lipid nanocrystal in the population comprises: a) a lipid component comprising a negatively charged phospholipid, wherein the negatively charged phospholipid is in an amount of about 75% by weight of the lipid component; b) a multivalent cation; and c) a therapeutic agent, wherein the multivalent cation and the negatively charged phospholipid are in a molar ratio of about 1:1 to about 4:1, and wherein the population of lipid nanocrystals has a mean particle size of about 180-220 nm. In some embodiments, the molar ratio is about 1.5:1. In some embodiments, the lipid component comprises about 25% by weight of a neutrally charged lipid. In some embodiments, the neutrally charged lipid comprises phosphatidylcholine. [0012] In some aspects, the disclosure provides a population of lipid nanocrystals, wherein each lipid nanocrystal in the population comprises: a) a lipid component comprising a negatively charged phospholipid, wherein the negatively charged phospholipid is in an amount of about 50% by weight of the lipid component; b) a multivalent cation; and c) a therapeutic agent, wherein the multivalent cation and the negatively charged phospholipid are in a molar ratio of about 1:1 to about 4:1, and wherein the population of lipid nanocrystals has a mean particle size of about 180-220 nm. In some embodiments, the molar ratio is about 1.5:1. In some embodiments, the lipid component comprises about 50% by weight of a neutrally charged lipid. In some embodiments, the neutrally charged lipid comprises phosphatidylcholine. [0013] In some embodiments, the population of lipid nanocrystals of the disclosure does not aggregate over time. In some embodiments, the mean particle size of the population of lipid Attorney Docket No. MNY-01525 nanocrystals of the disclosure does not increase more than about 25% after storage at 4ºC for 21 days. In some embodiments, the population of lipid nanocrystals of the disclosure has a polydispersity index of about 1.0 or less, about 0.5 or less, or about 0.2 or less after storage at 4ºC for 7 days. [0014] In some embodiments, the negatively charged phospholipid comprised in the lipid nanocrystals of the disclosure comprises phosphatidylserine. In some embodiments, the lipid nanocrystals of the disclosure further comprise a neutrally charged lipid in an amount no more than about 50% by weight of the liposomes. In some embodiments, the neutrally charged lipid comprises phosphatidylcholine. [0015] In some embodiments, the multivalent cation is Ca⁺⁺, Zn⁺⁺, Ba⁺⁺, or Mg⁺⁺. In some embodiments, the multivalent cation is Ca⁺⁺. In some embodiments, the population of lipid nanocrystals of the disclosure has a concentration of the multivalent cation, such as Ca⁺⁺, of from about 1 mM to about 12 mM. [0016] In some embodiments, the population of lipid nanocrystals of the disclosure does not comprise a size regulating agent. [0017] In some embodiments, the population of lipid nanocrystals of the disclosure further comprises a cryoprotectant. In some embodiments, the cryoprotectant comprises sucrose at a concentration of from about 1% to about 10%. In such embodiments, disclosed herein is a method of preserving such a population of lipid nanocrystals comprising a cryoprotectant, the method comprising freezing the population of lipid nanocrystals. In some embodiments, the mean particle size of the population of lipid nanocrystals remains stable after one or more freeze-thaw cycles. [0018] In a further aspect, the present disclosure is directed to a method of preparing a population of lipid nanocrystals with a mean particle size of interest, the method comprising: a) preparing a solution of liposomes comprising a negatively charged phospholipid and a therapeutic agent in an aqueous medium; b) selecting an amount of a multivalent cation sufficient to form the population of lipid nanocrystals with the mean particle size of interest based on the percentage of the negatively charged phospholipid and the lipid concentration of the solution of liposomes; and c) adding the amount of multivalent cation to the solution of liposomes, thereby forming the population of lipid nanocrystals with the mean particle size of interest. In some embodiments, the mean particle size of interest is about 200 nm or less. In some embodiments, the negatively charged phospholipid comprised in the solution of liposomes is present in a percentage of about 50% to Attorney Docket No. MNY-01525 about 100% by weight of the liposomes. In some embodiments, the solution of liposomes has a lipid concentration of from about 0.5 mg/mL to about 100 mg/mL. In some embodiments, the multivalent cation added to the solution of liposomes and the negatively charged phospholipid are in a molar ratio of from about 0.25:1 to about 4:1. [0019] In some embodiments, the method of preparing a population of lipid nanocrystals disclosed herein further comprises spinning down the population of lipid nanocrystals and resuspending the population of lipid nanocrystals in a solution comprising the multivalent cation at a concentration that is lower than the concentration of the multivalent cation used to form the population of lipid nanocrystals. In some embodiments, the solution used to resuspend the population of lipid nanocrystals does not comprise the multivalent cation. [0020] In some embodiments, the method of preparing a population of lipid nanocrystals disclosed herein further comprises adding a cryoprotectant to the population of lipid nanocrystals and freezing the population of lipid nanocrystals. In some embodiments, the cryoprotectant comprises sucrose at a concentration of from about 1% to about 10%. In some embodiments, the mean particle size of the population of lipid nanocrystals remains stable after one or more freeze-thaw cycles. [0021] In some aspects, the disclosure provides a population of lipid nanocrystals produced by any of the methods disclosed herein. Brief Description of the Drawing [0022] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods and compositions disclosed herein. [0023] FIG.1A-1D depict the mean particle size and polydispersity index (PDI) of the lipid nanocrystals obtained at different time points from each of the formulations outlined in Table 1. FIG.1A: Median particle size of the lipid nanocrystals formed with liposomes containing 100% PS; FIG.1B: Median particle size of the lipid nanocrystals formed with liposomes containing 75% PS; FIG.1C: PDI of the lipid nanocrystals formed with liposomes containing 100% PS; FIG.1D: PDI of the lipid nanocrystals formed with liposomes containing 75% PS. [0024] FIG. 2A-2C depict the lipid composition in the supernatant at day 7 for each of the formulations outlined in Table 1. FIG. 2A: Lipid composition in the supernatant of the Attorney Docket No. MNY-01525 formulations using liposomes containing 100% PS at day 7; FIG. 2B: Lipid composition in the supernatant of the formulations using liposomes containing 75% PS and 25% PC at day 7; FIG. 2C: Percent of each lipid (i.e., PS and PC) in the supernatant of the formulations using liposomes containing 75% PS and 25% PC at day 7. [0025] FIG.3A-3C depict the effects of phosphatidylserine (PS) concentration in liposomes to the amount of calcium needed for forming lipid nanocrystal s. FIG.3A: Median particle size; FIG.3B: polydispersity index (PDI); FIG.3C: Light microscopy image. [0026] FIG. 4A-4F depict the amount of free lipids present in the supernatant after lipid nanocrystal formation as determined by HPLC. FIG.4A-4C: Concentration of free lipids (FIG. 4A: Total lipids; FIG.4B: PS; FIG.4C: PC); FIG.4D-4F: Percentage of PS vs. PC (FIG.4D: 100% PS; FIG.4E: 75% PS; FIG.4F: 50% PS). [0027] FIG. 5A-5B depict the effects of calcium concentration on particle size and aggregation. FIG.5A: Effect of adding extra calcium to lipid nanocrystal formulation; FIG.5B: Data of the chart shown in FIG.5A. [0028] FIG. 6A-6B depict the effects of the starting lipid concentration on particle size and aggregation. FIG.6A: Starting lipid concentration effect (blue line: 100% PS; orange line: 75% PS); FIG.6B: Microscopic images of lipid nanocrystals prepared with liposomes containing 75% PS in varying starting concentrations. [0029] FIG.7A-7C depict the results of factorial experiments on lipid nanocrystal particle size control. FIG.7A: Actual-by-predicted plot on particle size; FIG.7B: Effect summary; FIG.7C: Prediction profiler. [0030] FIG. 8A-8G depict plots on factorial particle size. FIG. 8A: 3-dimensional (3D) scatterplot of the factorial particle size; FIG. 8B: Bubble plot of calcium eq. by %PS sized by particle size; FIG.8C: Bubble plot of lipid concentration (mg/mL) by calcium eq. sized by particle size; FIG. 8D: Bubble plot of lipid concentration (mg/mL) by %PS sized by particle size; FIG. 8E: Contour plot for particle size (nm) of %PS by calcium eq.; FIG.8F: Contour plot for particle size (nm) of lipid concentration (mg/mL) by %PS; FIG.8G: Contour plot for particle size (nm) of lipid concentration (mg/mL) by calcium eq. [0031] FIG. 9A-9B depict prediction based on calcium eq. and %PS as the only significant variables. FIG.9A: Actual-by-predicted plot on particle size; FIG.9B: Effect summary and lack of fit calculation. Attorney Docket No. MNY-01525 [0032] FIG.10A-10D depict the effect of freeze-thaw cycles on particle size and homogeneity of lipid nanocrystals with or without cryoprotectant. FIG.10A: lipid nanocrystals made of 100% PS without cryoprotectant; FIG. 10B: lipid nanocrystals made of 75% PS and 25% PC without cryoprotectant; FIG.10C: lipid nanocrystals made of 100% PS with 5% sucrose; FIG.10D: lipid nanocrystals made of 75% PS and 25% PC with 5% sucrose. [0033] FIG.11A-11D depict the effect of aqueous calcium concentration on lipid nanocrystal stability at 4ºC. FIG.11A: particle size of lipid nanocrystal made of 100% PS in different CaCl2 concentrations; FIG. 11B: HPLC results showing free lipids in the supernatant of the lipid nanocrystals made of 100% PS; FIG.11C: particle size of lipid nanocrystal made of 75% PS and 25% PC in different CaCl2 concentrations; FIG. 11D: HPLC results showing free lipids in the supernatant of the lipid nanocrystals made of 75% PS and 25% PC. Detailed Description [0034] Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings and discussed in the detailed description that follows. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the disclosure, and should not be interpreted as limiting the scope of the disclosure. Definitions [0035] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term. [0036] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. Attorney Docket No. MNY-01525 [0037] The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amount and the like, “about” is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value as such variations are appropriate to perform the disclosed methods and/or to make and use the disclosed compositions. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range. [0038] The term “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. [0039] The terms “at least,” “no more than” or “more than” prior to a number or series of numbers (e.g., “at least two”) is understood to include the number adjacent to the term “at least,” “no more than” or “more than,” and all subsequent numbers or integers that could logically be included, as clear from context. When the term “at least,” “no more than” or “more than” is present before a series of numbers or a range, it is understood that “at least,” “no more than” or “more than” can modify each of the numbers in the series or range. [0040] As used herein, the term “in some embodiments,” “in other embodiments,” or the like, refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise. [0041] “Mean particle size,” as used herein, generally refers to the statistical mean particle size (diameter) of the particles in a population of particles. The diameter of an essentially spherical particle may be referred to as the physical or hydrodynamic diameter. The diameter of a non- spherical particle may refer preferentially to the hydrodynamic diameter. As used herein, the Attorney Docket No. MNY-01525 diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as dynamic light scattering. Dynamic light scattering measures the size of particles suspended in liquid by measuring temporal fluctuations in the intensity of scattered light that reflects the diffusion of the particles. In some embodiments, a sample is contacted with a laser and the scattered light is collected by a detector. The fluctuations are characterized by computing the intensity correlation function which provides the diffusion coefficient of the particles. The diffusion coefficient (D) is related to the radius of the particles by means of the Stokes-Einstein Equation: D= kB7^^ʌ^r, wherein kB is the Boltzmann-Konstant, T is temperature, ^ is viscosity. Instrumentation suitable for dynamic light scattering analysis are known to those of skill in the art. [0042] The term “multivalent cation” refers to a divalent cation or higher valency cation, or any compound that has at least two positive charges, including mineral cations such as calcium, barium, zinc, iron and magnesium and other elements capable of forming ions or other structures having multiple positive charges capable of chelating and bridging negatively charged lipids. A “divalent metal cation,” as used herein, refers to a metal having two positive charges. [0043] The term “neutrally charged lipid” refers to a lipid that has a net charge of about zero at physiological pH. The neutrally charged lipid may comprise a single type of neutrally charged lipid, or a mixture of two or more different, neutrally charged, lipids. The neutrally charged lipid can be natural, such as soy-based, or synthetic. Examples of neutrally charged lipids include, but are not limited to, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and dioleoylphosphatidylethanolamine (DOPE). [0044] “Polydispersity index” or “PDI,” as used herein, refers to a measure of the heterogeneity of a sample based on size. Polydispersity can occur due to size distribution in a sample or agglomeration or aggregation of the sample during isolation or analysis. PDI is defined as Mw/Mn where Mw is the weight average molar mass and

is the number average molar mass. PDI can be obtained from instruments that use dynamic light scattering (DLS) or determined from electron micrographs. [0045] The term “size regulating agent,” as used herein, refers to an agent that reduces the particle size of a lipid nanocrystal, such as a lipid-anchored polynucleotide, a lipid-anchored sugar, a lipid-anchored polypeptide, or a bile salt (such as oxycholate or deoxycholate). Attorney Docket No. MNY-01525 [0046] The term “therapeutic agent” refers to an agent that can be administered to prevent or treat a disease or disorder. Therapeutic agents include, but are not limited to, a nucleic acid, a nucleic acid analog, a small molecule, a peptidomimetic, a protein, peptide, carbohydrate or sugar, lipid, or surfactant, or a combination thereof. [0047] The term “aqueous medium”, as used herein, refers to a solution wherein the solvent is water. [0048] The terms “Ca++”, “Ca²⁺”, and “aqueous calcium” as used herein are interchangeable. Lipid Nanocrystal Particle Size Control [0049] Disclosed herein are methods for regulating lipid nanocrystal particle size. Also disclosed are methods of reducing lipid nanocrystal mean particle size and thus, reducing aggregation potential to prevent lipid nanocrystal disintegration. [0050] At least three potential factors were identified by the inventors of this application as having the potential to impact lipid nanocrystal particle size and aggregation, namely the amount of negatively charged phospholipid (e.g., PS) in the lipid component, the amount of multivalent cation (e.g., calcium) used to precipitate the lipid nanocrystals, and the lipid concentration of the starting liposome composition. Accordingly, provided herein are methods of preparing a population of lipid nanocrystals by manipulating these three factors to control particle size and aggregation. Lipid Nanocrystal Components [0051] As used herein, “lipid nanocrystals” refer to anhydrous, stable, multi-layered lipid crystals that spontaneously form upon the interaction of negatively charged lipids, such as phospholipids, and a multivalent cation, such as calcium (see, for example, U.S. Pat. Nos. 4,078,052; 5,643,574; 5,840,707; 5,994,318; 6,153,217; 6,592,894, as well as PCT Publ. Nos. WO 2004/091572; WO 2004/091578; WO 2005/110361, WO 2012/151517, and WO2014/022414, and U.S. Pat. Publ. 2010/0178325; each of which is incorporated fully herein by reference). These lipid nanocrystals are also referred to in the art as cochleates and these terms are used interchangeably in this application. A lipid nanocrystal or cochleate has a unique multilayered structure consisting of a large, continuous, solid, lipid bilayer sheet or strata rolled up in a spiral or as stacked sheets, with no internal aqueous space. This unique structure provides protection Attorney Docket No. MNY-01525 from degradation for associated, encapsulated or “encochleated” molecules. Divalent cation concentrations in vivo in serum and mucosal secretions are such that the lipid nanocrystal structure is maintained. Hence, the majority of lipid nanocrystal-associated molecules are present in the inner layers of a solid, stable, impermeable structure. Once within the interior of a cell, however, the low calcium concentration results in the opening of the lipid nanocrystal and release of the molecule that had been formulated into the lipid nanocrystals. Accordingly, lipid nanocrystal formulations remain intact in physiological fluids, including mucosal secretions, plasma and gastrointestinal fluid, thereby mediating the delivery of biologically active compounds by many routes of administration, including mucosal, intravenous and oral. [0052] Typical lipid nanocrystal structures of the present disclosure include a lipid strata comprising alternating divalent cations and lipid bilayers. Sequestered within the lipid strata of the lipid nanocrystal is a therapeutic agent. In some embodiments therefore, the lipid nanocrystal disclosed herein comprises a therapeutic agent encapsulated in the lipid nanocrystal. [0053] The lipid nanocrystal of the present disclosure can be, in some embodiments, a geode cochleate as described, for example, in U.S. Pat. Publ. 2013/0224284, the entire disclosure of which is incorporated herein by reference. Geode cochleates have a similar structure to lipid nanocrystals except that geode cochleates further comprise a lipid monolayer, where the lipid monolayer surrounds a hydrophobic domain, such as an oil, and a biological active, such as a therapeutic agent (e.g., a drug), dispersed within the hydrophobic domain. The lipid monolayer is sequestered within the lipid strata of the geode cochleate. [0054] Accordingly, the lipid nanocrystal of the disclosure comprises at least the following components: a) a lipid component; and b) a multivalent cation. In some embodiments, the lipid nanocrystal of the disclosure further comprises a therapeutic agent encapsulated in the lipid nanocrystal. [0055] In some embodiments, the lipid component comprises a head group and a tail group. In some embodiments, the lipid component is neutral (e.g., uncharged) or charged, (e.g., polar; positive or negative). In some embodiments, the lipid component is neutral (e.g., uncharged). In some embodiments, a lipid component is positively charged. In some embodiments, the lipid component is negatively charged. In some embodiments, the lipid component (e.g., lipid bilayers and monolayers, if present) of the lipid nanocrystal of the disclosure comprises a negatively charged lipid, such as a negatively charged phospholipid. As used herein, the term “negatively Attorney Docket No. MNY-01525 charged lipid” includes lipids having a head group bearing a formal negative charge in aqueous solution at an acidic, basic or physiological pH, and also includes lipids having a zwitterionic head group. “Negatively charged phospholipid,” as used herein, refers to a phospholipid that has a net negative charge at physiological pH. The negatively charged phospholipid may comprise a single type of negatively charged phospholipid, or a mixture of two or more different, negatively charged, phospholipids. In some embodiments, the lipid component of the lipid nanocrystal according to the disclosure comprises a single type of negatively charged phospholipid. In some embodiments, the lipid component of the lipid nanocrystal comprises a mixture of two or more different, negatively charged, phospholipids. [0056] The negatively charged phospholipid can be natural, such as soy-based, or synthetic. Examples of negatively charged phospholipids can include, but are not limited to, phosphatidylserine (PS), dioleoylphosphatidylserine (DOPS), phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylglycerol (PG). In some embodiments, the lipid component of the lipid nanocrystal according to the disclosure comprises a natural, such as soy- based, negatively charged phospholipid. In some embodiments, the lipid component of the lipid nanocrystal comprises a synthetic negatively charged phospholipid. In some embodiments, the lipid component of the lipid nanocrystal comprises soy-based PS (or “Soy PS”). In some embodiments, the lipid component of the lipid nanocrystal comprises synthetic PS. [0057] In some embodiments, the lipid component of the lipid nanocrystal according to the disclosure can also include non-negatively charged lipids (e.g., positive and/or neutral lipids). In some embodiments, a majority of the lipid component is negatively charged. In some embodiments, the lipid component comprises at about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% negatively charged phospholipid, such as PS (e.g., Soy PS). In some embodiments, the lipid component comprises at least about 50% negatively charged phospholipid, such as PS (e.g., Soy PS). In some embodiments, the lipid component comprises at least about 75% negatively charged phospholipid, such as PS (e.g., Soy PS). In some Attorney Docket No. MNY-01525 embodiments, the lipid component comprises at least about 85% negatively charged phospholipid, such as PS (e.g., Soy PS). In some embodiments, the lipid component comprises at least about 90%, 95% or even 99% negatively charged phospholipid, such as PS (e.g., Soy PS). In some embodiments, the lipid component comprises only negatively charged phospholipid (i.e., 100% negatively charged phospholipid), such as PS (e.g., Soy PS). In some embodiments, the negatively charged phospholipid, such as PS (e.g., Soy PS), is in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% by weight of the lipid component. In some embodiments, the negatively charged phospholipid, such as PS (e.g., Soy PS), is in an amount ranging from about 50% to about 100%, such as from about 55% to about 100%, from about 60% to about 100%, from about 65% to about 100%, from about 70% to about 100%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 50% to about 90%, from about 60% to about 80%, from about 70% to about 90%, or from about 75% to about 90% by weight of the lipid component. [0058] In some embodiments, non-negatively charged lipid can be natural, such as soy-based, or synthetic. Examples of such non-negatively charged phospholipids can include, but are not limited to, phosphatidylcholine (PC), phosphatidylethanolamine (PE), diphosphotidylglycerol (DPG), dioleoyl phosphatidic acid (DOPA), distearoyl phosphatidylserine (DSPS), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylgycerol (DPPG) and the like. In some embodiments, the lipid component of the lipid nanocrystal according to the disclosure comprises, a negatively charged phospholipid, such as PS (e.g., Soy PS), and a non-negatively charged lipid, such as PC. [0059] In some embodiments, the lipid chains of the phospholipids are from about 6 to about 26 carbon atoms, and the lipid chains can be saturated or unsaturated. Exemplary fatty acyl lipid chains include, but are not limited to, n-tetradecanoic, n-hexadecanoic acid, n-octadecanoic acid, n-eicosanoic acid, n-docosanoic acid, n-tetracosanoic acid, n-hexacosanoic acid, cis-9- hexadecenoic acid, cis-9-octadecenoic acid, cis,cis-9,12-octadecedienoic acid, all-cis-9,12,15- Attorney Docket No. MNY-01525 octadecetrienoic acid, all-cis-5,8,11,14-eicosatetraenoic acid, all-cis-4,7,10,13,16,19- docosahexaenoic acid, 2,4,6,8-tetramethyl decanoic acid, and lactobacillic acid, and the like. [0060] In some embodiments, the lipid component of the lipid nanocrystals disclosed herein comprises pharmaceutical grade lipids, such as phospholipids, such as soy phospholipids, such as soy phosphatidylserine. As used herein, “pharmaceutical grade” refers to components, such as phospholipids, that are under strict quality and purity testing (greater than 99% pure), and do not contain binders, fillers, dyes, excipients, or unknown substances. [0061] In other embodiments, the lipid component comprises nutraceutical grade lipids, such as phospholipids, such as soy phospholipids, such as soy phosphatidylserine. As used herein, “nutraceutical grade” refers to a classification introduced in 1990 by the Food and Nutrition Board of the United States Institute of Medicine to describe functional food products that offer medical and/or health benefits. [0062] Pharmaceutical grade and nutraceutical grade lipids comprising about 40% to about 74% phosphatidylserine, such as about 40% to about 74% soy phosphatidylserine, are commercially available from e.g., American Lecithin Company or Lipoid LLC, e.g. LIPOID® PS 70, LIPOID® PS 50 or ALCOLEC®PS 50 P. [0063] In some embodiments, a multivalent compound is used to precipitate the lipid nanocrystals disclosed herein from the liposome starting materials. In some embodiments, a compound may be used as a source of cations, e.g., monovalent cations, divalent cations, trivalent cations. In some embodiments, a compound is a source of monovalent cations. In some embodiments, a compound is a source of divalent cations. In some embodiments, a compound is a source of trivalent cations. In some embodiments, a compound is a multivalent compound. In some embodiments, the multivalent compound is a source of multivalent cations (e.g., divalent cations). Exemplary multivalent cations include, but are not limited to, Ca⁺⁺, Zn⁺⁺, Ba⁺⁺, and Mg⁺⁺. Exemplary sources of these cations include, but are not limited to, the chloride, acetate, carbonate, citrate, gluconate, oxide, sulfate, nitrate, hydroxide, and lactate salts of calcium, zinc, barium, and magnesium. In some embodiments, CaCl₂ is a source of divalent cations. Depending on the multivalent compound used for the precipitation, the multivalent cation comprised in the lipid nanocrystals disclosed herein may vary. In some embodiments, the multivalent cation comprised in the lipid nanocrystals disclosed here is Ca⁺⁺. In some embodiments, the multivalent cation is Attorney Docket No. MNY-01525 Zn⁺⁺. In some embodiments, the multivalent cation is Ba⁺⁺. In some embodiments, the multivalent cation is Mg⁺⁺. Controlling Lipid Nanocrystal Size and Aggregation [0064] In some embodiments, the method comprises preparing a solution of liposomes having a lipid concentration of from about 0.5 mg/mL to about 100 mg/mL and comprising a negatively charged phospholipid in an amount ranging from about 50% to about 100% by weight of the liposomes and adding a multivalent cation to the solution of liposomes to precipitate the lipid nanocrystals, wherein the multivalent cation added to the solution of liposomes and the negatively charged phospholipid are in a molar ratio of from about 0.25:1 to about 4:1. In some embodiments, the solution of liposomes further comprises a therapeutic agent in an aqueous medium. [0065] In some embodiments, the amount of the negatively charged phospholipid in the solution of liposomes can be any amount between from about 50% to about 100%, such as from about 55% to about 100%, from about 60% to about 100%, from about 65% to about 100%, from about 70% to about 100%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 50% to about 90%, from about 60% to about 80%, from about 70% to about 90%, or from about 75% to about 90%, by weight of the liposomes. In some embodiments, the amount of the negatively charged phospholipid in the solution of liposomes is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% by weight of the liposomes. Examples of negatively charged phospholipids can include, but are not limited to, phosphatidylserine (PS), dioleoylphosphatidylserine (DOPS), phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylglycerol (PG). Negatively charged phospholipids useful for the present disclosure can be natural, such as soy-based, or synthetic. In some embodiments, the negatively charged phospholipid comprised in the solution of liposomes comprises PS, such as Soy PS. In some embodiments, the negatively charged phospholipid comprised in the solution of liposomes comprises synthetic PS. [0066] In some embodiments, the solution of liposomes comprises both a negatively charged phospholipid and a neutrally charged lipid. In some embodiments, the neutrally charged lipid comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, Attorney Docket No. MNY-01525 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or up to 50% by weight of the liposomes. In some embodiments, the liposomes comprise less than about 100% negatively charged phospholipid and further comprises a neutrally charged lipid in an amount no more than about 50%, such as about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5%, by weight of the liposomes. Examples of such neutrally charged lipids can include, but are not limited to, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (DPG), dioleoyl phosphatidic acid (DOPA), distearoyl phosphatidylserine (DSPS), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylgycerol (DPPG) and the like. Neutrally charged lipids useful for the present disclosure can be natural, such as soy-based, or synthetic. In some embodiments, the neutrally charged lipid comprised in the solution of liposomes comprises PC, such as soy PC. In some embodiments, the neutrally charged lipid comprised in the solution of liposomes comprises synthetic PC. [0067] The amount of multivalent cation, such as Ca⁺⁺, added to the solution of liposomes should be in an amount sufficient to precipitate the lipid nanocrystals and generally is expressed in term of the molar ratio between the multivalent cation added and the negatively charged phospholipid comprised in the solution of liposomes. In some embodiments, the molar ratio of the multivalent cation, such as Ca⁺⁺, added to the solution of liposomes and the negatively charged phospholipid is from about 0.25:1 to about 4:1, such as from about 0.5:1 to about 4%, from about 1:1 to about 4:1, from about 1.5:1 to about 4:1, from about 2:1 to about 4:1, from about 2.5:1 to about 4:1, from about 3:1 to about 4:1, from about 0.5:1 to about 2:1, or from about 1:1 to about 2.5:1. In some embodiments, the molar ratio of the multivalent cation, such as Ca⁺⁺, added to the solution of liposomes and the negatively charged phospholipid is about 0.25:1, about 0.5:1, about 0.75:1, about 0.8:1, about 1:1, about 1.25:1, about 1.5:1, about 1.75:1, about 2:1, about 2.25:1, about 2.5:1, about 2.75:1, about 3:1, about 3.25:1, about 3.5:1, about 3.75:1, or about 4:1. [0068] The amount of multivalent cation, such as Ca⁺⁺, added to the solution of liposomes can also be expressed in term of its final concentration in the population of lipid nanocrystals. In some embodiments, the population of lipid nanocrystals has a concentration of the multivalent cation, such as Ca⁺⁺, of from about 1 mM to about 12 mM, such as from about 1.5 mM to about 10 mM, from about 2 mM to about 10 mM, from about 2.5 mM to about 8 mM, from about 3 mM to about 6 mM, or from about 3.5 mM to about 6 mM. In some embodiments, the population of lipid Attorney Docket No. MNY-01525 nanocrystals has a concentration of the multivalent cation, such as Ca⁺⁺, of about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, about 10 mM, about 10.5 mM, about 11 mM, about 11.5 mM, or about 12 mM. [0069] In some embodiments, the population of lipid nanocrystals has a concentration of Ca⁺⁺ of from about 1 mM to about 12 mM, such as from about 1.5 mM to about 10 mM, from about 2 mM to about 10 mM, from about 2.5 mM to about 8 mM, from about 3 mM to about 6 mM, or from about 3.5 mM to about 6 mM. In some embodiments, the population of lipid nanocrystals has a concentration of Ca⁺⁺ of about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, about 10 mM, about 10.5 mM, about 11 mM, about 11.5 mM, or about 12 mM. [0070] In some embodiments, the solution of liposomes used for preparing lipid nanocrystals of the disclosure has a lipid concentration of from about 0.5 mg/mL to about 100 mg/mL, such as from about 0.75 mg/mL to about 95 mg/mL, from about 1 mg/mL to about 90 mg/mL, from about 1.5 mg/mL to about 85 mg/mL, from about 2 mg/mL to about 80 mg/mL, from about 3 mg/mL to about 60 mg/mL, from about 4 mg/mL to about 50 mg/mL, from about 1 mg/mL to about 30 mg/mL, from about 1 mg/mL to about 20 mg/mL, or from about 0.5 mg/mL to about 10 mg/mL. In some embodiments, the solution of liposomes has a lipid concentration of about 0.5 mg/mL, about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, about 90 mg/mL, about 95 mg/mL, or about 100 mg/mL. [0071] In some embodiments, the solution of liposomes used for preparing lipid nanocrystals of the disclosure has a phosphatidylcholine (PC) concentration of from about 0.5 mg/mL to about 100 mg/mL, such as from about 0.75 mg/mL to about 95 mg/mL, from about 1 mg/mL to about 90 mg/mL, from about 1.5 mg/mL to about 85 mg/mL, from about 2 mg/mL to about 80 mg/mL, from about 3 mg/mL to about 60 mg/mL, from about 4 mg/mL to about 50 mg/mL, from about 1 Attorney Docket No. MNY-01525 mg/mL to about 30 mg/mL, from about 1 mg/mL to about 20 mg/mL, or from about 0.5 mg/mL to about 10 mg/mL. In some embodiments, the solution of liposomes has a PC concentration of about 0.5 mg/mL, about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, about 90 mg/mL, about 95 mg/mL, or about 100 mg/mL. [0072] In some embodiments, the solution of liposomes used for preparing lipid nanocrystals of the disclosure has a phosphatidylserine (PS) concentration of from about 0.5 mg/mL to about 100 mg/mL, such as from about 0.75 mg/mL to about 95 mg/mL, from about 1 mg/mL to about 90 mg/mL, from about 1.5 mg/mL to about 85 mg/mL, from about 2 mg/mL to about 80 mg/mL, from about 3 mg/mL to about 60 mg/mL, from about 4 mg/mL to about 50 mg/mL, from about 1 mg/mL to about 30 mg/mL, from about 1 mg/mL to about 20 mg/mL, or from about 0.5 mg/mL to about 10 mg/mL. In some embodiments, the solution of liposomes has a PS concentration of about 0.5 mg/mL, about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, about 90 mg/mL, about 95 mg/mL, or about 100 mg/mL. [0073] In some embodiments, particle size is predicted based on the following variables: molar ratio of multivalent cation (calcium) and negatively charged lipid (“calcium eq.”), percentage of negatively charged lipid, and starting lipid concentration (mg/mL). In some embodiments, the following formula predicts particle size (Formula I): 227.45 + 97.510714286 ή

+ 96.560714286 ή

Attorney Docket No. MNY-01525

In some embodiments, particle size is predicted based on calcium eq. and percentage of negatively charged lipid. In some embodiments, starting lipid concentration does not predict particle size. In some embodiments, the following formula predicts particle size (Formula II): _{205.13214286 + 79.901785714 ή ^} ^{(^^^^^^^ ^^.െ1.25)}

5

74.242857143 0.7 ^

Analysis of Lipid Nanocrystal Populations [0075] In some embodiments, the population of lipid nanocrystals prepared by the disclosed methods has a mean particle size no more than about 300 nm, such as no more than about 250 nm, no more than about 200 nm, about 190 nm, about 180 nm, about 170 nm, about 160 nm, about 150 nm, about 140 nm, about 130 nm, about 120 nm, about 110 nm, about 100 nm, or less than about 100 nm. The mean particle size of the lipid nanocrystals can be determined using any methods known in the art, such as dynamic light scattering. In some embodiments, the population of lipid nanocrystals prepared by the disclosed methods, despite of the small particle size, does not aggregate over time. [0076] In some embodiments, the size of a lipid nanocrystal or population of lipid nanocrystals is determined by dynamic light scattering. In some embodiments, the size of a lipid nanocrystal or population of lipid nanocrystals is determined by electron microscopy. In some embodiments, the size of a lipid nanocrystal or population of lipid nanocrystals is determined by atomic force microscopy. In some embodiments, the size of a lipid nanocrystal or population of lipid nanocrystals is determined by tunable resistive pulse sensing. In some embodiments, the size of a lipid nanocrystal or population of lipid nanocrystals is determined by disc centrifugation. Attorney Docket No. MNY-01525 [0077] In some embodiments, aggregation of a population of lipid nanocrystals is determined by observation. In some embodiments, aggregation of a population of lipid nanocrystals is determined by particle size assessed over time. [0078] In some embodiments, the disclosure provides a method of preparing a population of lipid nanocrystals with a mean particle size of interest, the method comprising: a) preparing a solution of liposomes comprising a negatively charged phospholipid, as described herein elsewhere, and a therapeutic agent in an aqueous medium, wherein the negatively charged phospholipid is present in a percentage of about 50% to about 100% by weight of the liposomes and wherein the solution of liposomes has a lipid concentration of from about 0.5 mg/mL to about 100 mg/mL; b) selecting an amount of a multivalent cation sufficient to form the population of lipid nanocrystals with the mean particle size of interest based on the percentage of the negatively charged phospholipid and the lipid concentration of the solution of liposomes; and c) adding the amount of multivalent cation to the solution of liposomes, thereby forming the population of lipid nanocrystals with a mean particle size of interest. In some embodiments, the mean particle size of interest is about 200 nm or less. In some embodiments, the multivalent cation added to the solution of liposomes and the negatively charged phospholipid are in a molar ratio of from about 0.25:1 to about 4:1. [0079] The particle size of the population of lipid nanocrystals prepared by the disclosed methods is generally stable over time. In some embodiments therefore, the mean particle size of the population of lipid nanocrystals prepared by the disclosed methods does not increase more than about 25% after storage at 4ºC for 21 days. In some embodiments, the mean particle size of the population of lipid nanocrystals prepared by the disclosed methods does not increase more than about 20% after storage at 4ºC for 21 days. In some embodiments, the mean particle size of the population of lipid nanocrystals prepared by the disclosed methods does not increase more than about 15% after storage at 4ºC for 21 days. In some embodiments, the mean particle size of the population of lipid nanocrystals prepared by the disclosed methods does not increase more than about 10% after storage at 4ºC for 21 days. [0080] In some embodiments, the population of lipid nanocrystals prepared by the disclosed methods has a polydispersity index (PDI) of about 1.0 or less, such as about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, or about 0.1. In some embodiments, the population of lipid nanocrystals prepared by the disclosed methods are stored at about 1ºC, 2ºC, Attorney Docket No. MNY-01525 3ºC, 4ºC, 5ºC, 6ºC, 7ºC, or at about 8ºC. In some embodiments, the population of lipid nanocrystals are stored for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the population of lipid nanocrystals are stored for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some embodiments, the population of lipid nanocrystals are stored for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or more. In some embodiments, the population of lipid nanocrystals prepared by the disclosed methods has a polydispersity index of about 0.5 or less after storage at 4ºC for 7 days. In some embodiments, the population of lipid nanocrystals has a polydispersity index of about 0.2 or less after storage at 4ºC for 7 days. In some embodiments, the population of lipid nanocrystals prepared by the disclosed methods has a polydispersity index of about 0.5 or less after storage at 4ºC for 21 days. In some embodiments, the population of lipid nanocrystals has a polydispersity index of about 0.2 or less after storage at 4ºC for 21 days. PDI can be obtained using known techniques, including, for example, from instruments that use dynamic light scattering (DLS) or determined from electron micrographs. [0081] Because of the ability to regulate mean particle size and the stability in particle size achieved by the methods disclosed herein, the populations of lipid nanocrystals of the disclosure can be made without the need for a size regulating agent to inhibit aggregation or to produce populations of lipid nanocrystals with low mean particle sizes, such as particle sizes of no more than 250 nm or no more than 200 nm, which may be desired for pharmaceutical use. In some embodiments therefore, the population of lipid nanocrystals of the disclosure does not comprise a size regulating agent. [0082] As used herein, a “size regulating agent” refers to agent that reduces the particle size of a lipid nanocrystal, such as a lipid-anchored polynucleotide, a lipid-anchored sugar, a lipid- anchored polypeptide, or a bile salt (such as oxycholate or deoxycholate). Examples of size regulating agents are disclosed in, for instance, PCT Publ. No. WO 2016/141203, which is incorporated fully herein by reference. [0083] As is known in the art, a series of extreme, rapid temperature changes, such as multiple freeze-thaw cycles, may have a negative effect on particle size and homogeneity of lipid nanocrystals. In some embodiments, a population of lipid nanocrystals of the disclosure is stored in the presence of one or more cryoprotectants. A “cryoprotectant,” as used herein, refers to a substance that is used to protect lipid nanocrystals from freezing damage (e.g., due to ice Attorney Docket No. MNY-01525 formation) or damages caused by a series of extreme, rapid temperature changes. Exemplary cryoprotectants are low molecular weight molecules including, but not limited to, glycerol, propylene glycol, ethylene glycol, dimethyl sulfoxide (DMSO), 2-Methyl-2,4-pentanediol (MPD), sucrose, and trehalose. In embodiments, the population of lipid nanocrystals of the disclosure can be stored in the presence of about 1%-10% sucrose. In embodiments, the population of lipid nanocrystals can be stored in the presence of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or about 10% sucrose. In some embodiments, the population of lipid nanocrystals are stored in the presence of about 1%, about 2.5%, about 5%, about 7.5% or about 10% sucrose. [0084] Since the amount of multivalent cation (e.g., calcium) used to precipitate the lipid nanocrystals was identified as being one of the potential factors to impact lipid nanocrystal particle size and aggregation, it is generally believed that the aqueous calcium concentration in the population of lipid nanocrystals has to be maintained at a certain level to stabilize lipid nanocrystals. However, it was unexpectedly found that, once the lipid nanocrystals form, aqueous calcium is not necessary to stabilize lipid nanocrystals. Thus, in some embodiments, the population of lipid nanocrystals of the disclosure is maintained at an aqueous calcium concentration of from about 0 to about 5 mM, such as about 0 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM. [0085] In some embodiments, a population of lipid nanocrystals comprises an aqueous medium. Exemplary aqueous mediums include, but are not limited to, acid solutions, basic solutions, and salt solutions. In some embodiments, a salt solution is a buffering solutions. In some embodiments, an aqueous medium is an acid solution. In some embodiments, an aqueous medium is a basic solution. In some embodiments, an aqueous medium is a salt solution. In some embodiments, an aqueous medium is a buffering solution. In some embodiments, buffering solutions comprise buffer components. Exemplary buffer components include, but are not limited to, phosphate, bicarbonate, tris, acetate, HEPES, TES, MES, and the like. Any of the aforementioned buffer components may be used in free base and/or conjugate salt form. In some embodiments, a buffering solution contains non-buffering components. Exemplary non-buffering components include, but are not limited to, detergents (e.g., TRITON™ X-100), chelators (e.g., EDTA, EGTA), stabilizers (e.g., hydroxypropyl cellulose, hydroxypropylmethyl cellulose), chaotrophs (e.g., urea, ethanol, sodium dodecyl sulfate), cations, anions, salts (e.g., sodium Attorney Docket No. MNY-01525 chloride), sugars (e.g., sucrose), cyclodextrins (e.g., Į-F\FORGH[WULQ^^ ȕ-cyclodextrin, and Ȗ- cyclodextrin), glycols (e.g., polyethylene glycol), amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), surfactants (e.g., sodium dodecyl sulfate) and osmolytes (e.g., betaine, taurine).Accordingly, in one aspect, provided herein is a method of preparing a population of lipid nanocrystals, the method comprising; a) preparing a solution of liposomes comprising a negatively charged phospholipid and a therapeutic agent in an aqueous medium; and b) adding a multivalent cation to the solution of liposomes, thereby forming the population of lipid nanocrystals, wherein the negatively charged phospholipid is in an amount ranging from about 50% to about 100% by weight of the liposomes, and wherein the multivalent cation added to the solution of liposomes and the negatively charged phospholipid are in a molar ratio of from about 0.25:1 to about 4:1. In some embodiments, the solution of liposomes used for preparing the population of lipid nanocrystals has a lipid concentration of from about 0.5 mg/mL to about 100 mg/mL. In some embodiments, the population of lipid nanocrystals prepared by the method of the disclosure has a mean particle size of no more than about 200 nm. Population of Lipid Nanocrystals [0086] In another aspect, provided herein is a population of lipid nanocrystals, wherein each lipid nanocrystal in the population comprises: a) a lipid component comprising a negatively charged phospholipid; b) a multivalent cation; and c) a therapeutic agent, wherein the negatively charged phospholipid is in an amount ranging from about 50% to about 100% by weight of the lipid component, wherein the multivalent cation and the negatively charged phospholipid are in a molar ratio of from about 0.25:1 to about 4:1, and wherein the population of lipid nanocrystals has a mean particle size no more than about 200 nm. [0087] In some embodiments, the population of lipid nanocrystals according to the disclosure does not aggregate over time. In some embodiments, the mean particle size of the population of lipid nanocrystals according to the disclosure does not increase more than about 25%, such as about 20%, about 15%, or about 10%, after storage at 4ºC for 21 days. In some embodiments, the population of lipid nanocrystals according to the disclosure has a polydispersity index of about 1.0 or less after storage at 4ºC for 7 days. In some embodiments, the population of lipid nanocrystals Attorney Docket No. MNY-01525 according to the disclosure has a polydispersity index of about 0.5 or less after storage at 4ºC for 7 days. In some embodiments, the population of lipid nanocrystals according to the disclosure has a polydispersity index of about 0.2 or less after storage at 4ºC for 7 days. [0088] In some embodiments, the negatively charged phospholipid comprised in the solution of liposomes used for preparing the lipid nanocrystals of the disclosure, or the negatively charged phospholipid comprised in the lipid component of the lipid nanocrystals of the disclosure, comprises phosphatidylserine (PS), such as natural PS (e.g., Soy PS) or synthetic PS. In some embodiments, the solution of liposomes used for preparing the lipid nanocrystals, or the lipid component of the lipid nanocrystals of the disclosure, further comprises a neutrally charged lipid in an amount no more than about 50% by weight of the liposomes or the lipid component. In some embodiments, the neutrally charged lipid comprises phosphatidylcholine (PC), such as natural PC (e.g., soy PC) or synthetic PC. [0089] In some embodiments, the multivalent cation used for preparing the lipid nanocrystals of the disclosure, or the multivalent cation comprised in the lipid nanocrystals of the disclosure, is Ca⁺⁺, Zn⁺⁺, Ba⁺⁺, or Mg⁺⁺. In some embodiments, the multivalent cation is Ca⁺⁺. In some embodiments, the multivalent cation is Zn⁺⁺. In some embodiments, the multivalent cation is Ba⁺⁺. In some embodiments, the multivalent cation is Mg⁺⁺. In some embodiments, the population of lipid nanocrystals according to the disclosure has a concentration of the multivalent cation, such as Ca⁺⁺, Zn⁺⁺, Ba⁺⁺, or Mg⁺⁺, of from about 1 mM to about 12 mM. [0090] In some embodiments, a population of lipid nanocrystals of the disclosure comprises an excipient that contributes to the stability of the lipid nanocrystal, encapsulation efficiency, and/or achieving target particle sizes (e.g., a size regulating agent), such as one or more of serum albumin (e.g., human serum albumin or bovine serum albumin), casein, vitamin E, cholesterol, or any combination thereof. In some embodiments, a population of lipid nanocrystals does not comprise a size regulating agent. [0091] In some embodiments, the liposomes for preparing the lipid nanocrystals of the disclosure can be prepared using any method known in the art. In some embodiments, the liposomes are prepared by stirring the lipids comprising the phospholipids, such as phosphatidylserine, e.g., soy phosphatidylserine, and a chelating agent, e.g., ethylenediaminetetraacetic acid (EDTA), in purified water, thereby forming the liposomes. In some embodiments, the formed liposomes are stirred for about 1, 2, 3 or 4 or more hours (e.g., at Attorney Docket No. MNY-01525 room temperature). In some embodiments, the formed liposomes are stirred for about 4 hours (e.g., at room temperature). [0092] In some embodiments, the liposomes are homogenized or filtered after stirring at room temperature, as disclosed in, for example, U.S. 2021/0038722, which is herein incorporated by reference in its entirety. In some embodiments, the liposomes may be homogenized by passing the liposomes through a homogenizer, such as a PandaPlus 2000 homogenizer (GEA Inc.). In some embodiments, the liposomes are filtered through a 5 µm pre-rinsed filter, such as a syringe 5 µm filter, e.g., obtained from Fisher Scientific, code # SLSVO25LS to remove any insoluble material and produce a more uniform population of liposomes. In some embodiments, the liposomes prepared from pharmaceutical grade soy phosphatidylserine are filtered twice and then mixed with water, e.g., purified water to form a liposomal suspension. In some embodiments, the liposomes prepared from pharmaceutical grade soy phosphatidylserine are homogenized twice and then mixed with water, e.g., purified water to form a liposomal suspension. [0093] In some embodiments, the liposomal suspension is in a buffered environment having a pH of about 6.5-8.0. In some embodiments, the liposomal suspension is in a buffered environment having a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or about 8.0. In some embodiments, the liposomal suspension is in a buffered environment having a pH of about 7.0. In some embodiments, the suspension of liposomes is buffered with phosphate. [0094] In some embodiments, the liposomes, prepared as described herein, are stored in water, such as sterile or purified water in a liposomal suspension. In some embodiments, the liposomal suspension is combined with a therapeutic agent (e.g., drug). [0095] In some embodiments, the therapeutic agent is generally solubilized by combining it with one or more solubilizing agents, such as benzyl alcohol, polyethylene glycol (e.g., PEG 400 or PEG 300), N-methyl pyrrolidone, modified cyclodextrin or a combination thereof to achieve a concentration of the therapeutic agent in the solubilizing agent ranging from about 1 mg/mL to about 200 mg/mL. [0096] In some embodiments, an excipient can be used in the method disclosed herein for preparing the lipid nanocrystal. In such embodiments, the excipient is added to the prepared liposomes either before or after the solubilized therapeutic agent is added to the liposomes. In some embodiments, the excipient is added to the liposomes before the solubilized therapeutic agent Attorney Docket No. MNY-01525 is added to the liposomes. In some embodiments, the excipient, is added to the liposomes after the solubilized therapeutic agent is added to the liposomes. Therapeutic Agents [0097] In some embodiments, the disclosure provides a method of producing a population of lipid nanocrystals comprising one or more therapeutic agents (e.g., a drug). In some embodiments, the disclosure provides a population of lipid nanocrystals comprising one or more therapeutic agents produced by a method described herein. In some embodiments, the drug is a small molecule or a biologic (e.g., a nucleic acid or an amino acid). In some embodiments, the nucleic acid is DNA and/or RNA. In some embodiments, the amino acid is a protein or peptide. In some embodiments, the one or more therapeutic agents comprise any combination of small molecule, DNA, RNA, protein, and/or peptide. In some embodiments, the therapeutic agent is an antibiotic. In some embodiments, the therapeutic agent is an antifungal. Nucleic Acids [0098] In some embodiments, the therapeutic agent is an RNA molecule. Exemplary RNA molecules include, but are not limited to, an oligonucleotide (e.g., RNAi oligonucleotide), small hairpin RNA (shRNA) molecule, a small interfering RNA (siRNA), a microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNAs (rRNA), an aptamer, small nuclear RNA (snRNA), piwi-interacting RNA (piRNA), non-coding RNA (ncRNA), long non- coding RNA, (lncRNA), and fragments of any of the foregoing. In some embodiments, the RNA molecule is single-stranded, double-stranded, or partially single- or double-stranded. [0099] In some embodiments, the therapeutic agent is a DNA molecule. Exemplary DNA molecules include, but are not limited to, an oligonucleotide (e.g., an antisense oligonucleotide), an aptamer, a plasmid, and fragments of any of the foregoing. [0100] In some embodiments, the nucleic acid molecule comprises both DNA and RNA. [0101] In some embodiments, the nucleic acid is a virus. In some embodiments, the virus is an adeno-associated virus (AAV), a retrovirus, a lentivirus, a herpes simplex virus, or other useful virus. In some embodiments, the virus is engineered or naturally occurring. Attorney Docket No. MNY-01525 Polypeptide [0102] In some embodiments, the one or more therapeutic agents comprises a protein or peptide. Exemplary therapeutic agents include, but are not limited to, an antibody, an antibody fragment, a hormone, a ligand, or an immunoglobulin. In some embodiments, the protein or peptide is naturally occurring or is synthetic. In some embodiments, the protein comprises an engineered variant of a protein (e.g., recombinant protein), or fragment thereof. In some embodiments, the protein is subjected to other modifications, e.g., post-translational modifications, including but not limited to: glycosylation, acylation, prenylation, lipoylation, alkylation, amidation, acetylation, methylation, formylation, butyrylation, carboxylation, phosphorylation, malonylation, hydroxylation, iodination, propionylation, S-nitrosylation, S-glutationylation, succinylation, sulfation, glycation, carbamylation, carbonylation, biotinylation, carbamylation, oxidation, pegylation, sumoylation, ubiquitination, ubiquitylation, racemization, etc. One or more modifications may be made to the protein or peptide. Small Molecule [0103] In some embodiments, the one or more therapeutic agents comprises a small molecule (e.g., a molecule having a molecular weight of less than 900 Daltons). In some embodiments, the small molecule increases or decreases the expression level and/or activity level of a polypeptide. In some embodiments, the small molecule inhibits the normal cellular function of a polypeptide. In some embodiments, the small molecule prevents protein-protein interactions. In some embodiments, the small molecule is an allosteric therapeutic agent. Antibiotics [0104] In some embodiments, the one or more therapeutics agents comprise an antibiotic. Antibiotics include drugs that are effective against bacteria, e.g., by killing or preventing growth. Exemplary antibiotics include, but are not limited to, pencillins (e.g., amoxicillin), fluoroquinolones (e.g., ciprofloxacin), cephalosporins (e.g., cefuroxime), macrolides (e.g., erythromycin), beta-lactams (e.g., amoxicillin, carbapenems), tetracyclines (e.g., doxycycline), trimethoprim-sulfamethoxazole, lincosamides (e.g., lincomycin), and urinary anti-infectives (e.g., fosfomycin), and the like. Attorney Docket No. MNY-01525 Antifungals [0105] In some embodiments, the one or more therapeutics agents comprise an antifungal. Antifungals include drugs that are effective against fungi, e.g., by killing or preventing growth. Exemplary antifungals include, but are not limited to, amphotericin B, azole derivates (e.g., fluconazole), echinocandins (e.g., anidulafungin), and flucytosine. Combinations [0106] It will be appreciated that one or more therapeutic agents (e.g., peptides, RNA molecules, DNA molecules, antibiotics, antifungals) are listed as examples and that a population of lipid nanocrystals may comprise a combination of therapeutic agent types. For example, in some embodiments, a protein or peptide co-administered with a small molecule (e.g., a molecule having a molecular weight of less than 900 Daltons), an RNA molecule, a DNA molecule, or a complexed molecule (e.g., protein-nucleic acid molecule). In some embodiments, an RNA molecule is co- administered with a small molecule, a DNA molecule, or a complexed molecule (e.g., protein- nucleic acid molecule). In some embodiments, a small molecule is co-administered with a DNA molecule or a complexed molecule (e.g., protein-nucleic acid molecule). In some embodiments, an antibiotic is co-administered with a DNA molecule of a complexed molecule (e.g., protein- nucleic acid molecule). In some embodiments, an antifungal is co-administered with a DNA molecule of a complexed molecule (e.g., protein-nucleic acid molecule). In some embodiments, an antibiotic is co-administered with a RNA molecule of a complexed molecule (e.g., protein- nucleic acid molecule). In some embodiments, an antifungal is co-administered with a RNA molecule of a complexed molecule (e.g., protein-nucleic acid molecule).These combinations are non-limiting examples of different combinations of agents. Pharmaceutical compositions [0107] The lipid nanocrystals according to the disclosure can be used as a delivery agent for delivering a therapeutic agent (e.g., drug). Accordingly, in some embodiments, the disclosure is also directed to a pharmaceutical composition comprising the lipid nanocrystals as described herein. In some embodiments, the one or more therapeutic agents is delivered to the subject enterally. For example, in some embodiments, the one or more therapeutic agents is administered to the subject orally, nasally, rectally, sublingually, sub-labially, buccally, topically, or through an Attorney Docket No. MNY-01525 enema. In some embodiments, the pharmaceutical composition comprising the lipid nanocrystals of the disclosure is formulated for oral administration. Exemplary preparation forms for the present pharmaceutical compositions include, but not limited to, for example, tablets, capsules, soft capsules, granules, powders, suspensions, emulsions, microemulsions, nanoemulsions, unit dosage forms, solutions, and syrups. In some embodiments, the pharmaceutical composition comprising the lipid nanocrystals of the disclosure is formulated for parenteral administration, such as orally, topically, transdermally, transmucosally, subcutaneous, intramuscular, intravenous, or intrathecal administration. [0108] After lipid nanocrystal formation, which occurs upon addition of a multivalent cation as described herein, a pH of the formed lipid nanocrystals may be adjusted to pH about 7.0, e.g. with a 5N HCL solution. [0109] In some embodiments, one or more preservatives, such as sorbate, sodium methylparaben and/or propylparaben are added to the lipid nanocrystals formed in the method disclosed herein. In some embodiments, the pH of the lipid nanocrystals is adjusted to pH about 7.0., e.g., with a 5N HCL solution after the addition of the preservatives. [0110] In some embodiments, one or more cryoprotectants, such as sucrose, are added to the lipid nanocrystals formed in the method disclosed herein to preserve or stabilize the particle size of the lipid nanocrystals after one or more freeze-thaw cycles. In some embodiments, the one or more cryoprotectants added to the lipid nanocrystals of the disclosure comprise sucrose at a concentration of from about 1% to about 10%, such as about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the one or more cryoprotectants added to the lipid nanocrystals of the disclosure comprise about 5% sucrose. Accordingly, also disclosed is a method of preserving the population of lipid nanocrystals disclosed herein, said method comprising adding one or more cryoprotectants to the population of lipid nanocrystals and freezing the population of lipid nanocrystals. In such embodiments, the mean particle size of the population of lipid nanocrystals remains stable after one or more freeze- thaw cycles. In some embodiments, the mean particle size of the population of lipid nanocrystals according to the disclosure does not increase more than about 25%, such as about 20%, about 15%, or about 10%, after one or more freeze-thaw cycles. [0111] In some embodiments, the lipid nanocrystals formed in the method disclosed herein are stored as a whole suspension in a cation-containing buffer, or are concentrated by sedimentation, Attorney Docket No. MNY-01525 lyophilized or otherwise converted to a powder, and stored at room temperature. If desired, the lipid nanocrystals also can be reconstituted with liquid, such as purified or sterile water, prior to administration. [0112] In some embodiments, the lipid nanocrystals disclosed herein are stored in a solution comprising the multivalent cation, such as Ca⁺⁺, at a concentration that is lower than the concentration of the multivalent cation, such as Ca⁺⁺, used to form the population of lipid nanocrystals. In some embodiments, the solution used to store the population of lipid nanocrystals does not comprise the multivalent cation, such as Ca⁺⁺. In some embodiments, the lipid nanocrystals are made of 100% PS by adding sufficient amount of CaCl2 and the solution used to store the population of lipid nanocrystals may comprise 0 mM of CaCl₂. In some embodiments, the lipid nanocrystals are made of 75% PS and 25% PC by adding sufficient amount of CaCl₂ and the solution used to store the population of lipid nanocrystals may comprise from about 2 mM to about 5 mM of CaCl2. In some embodiments, the method disclosed herein for preparing the population of lipid nanocrystals further comprises spinning down the population of lipid nanocrystals and resuspending the population of lipid nanocrystals in the above-described solution to store the population of lipid nanocrystals. [0113] The pharmaceutical composition can include pharmaceutically acceptable carriers or excipients in addition to the excipients described herein for the preparation of lipid nanocrystals, such as a buffer (e.g., Tris, acetate, phosphate, TES, MES, HEPES) of various pH and ionic strength; an additive, such as gelatin to prevent absorption to surfaces; a protease inhibitor; a permeation enhancer; an anti-oxidant (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole); a stabilizer (e.g., hydroxypropyl cellulose, hydroxypropylmethyl cellulose); a viscosity increasing agent (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum); a sweetener (e.g. aspartame, citric acid); a preservative (e.g., sorbate, thimerosal, benzyl alcohol, parabens, such as sodium methylparaben and/or propylparaben); a flow-aid (e.g., colloidal silicon dioxide), a plasticizer (e.g., diethyl phthalate, triethyl citrate); an emulsifier (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate); a polymer coating (e.g., poloxamers or poloxamines, hypromellose acetate succinate); a coating and film forming agent (e.g., ethyl cellulose, acrylates, polymethacrylates, hypromellose acetate succinate); an adjuvant; a pharmaceutically acceptable carrier for liquid formulations, such as an aqueous (water, alcoholic/aqueous solution, emulsion or suspension, including saline and buffered media) or non- Attorney Docket No. MNY-01525 aqueous (e.g., propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate) solution, suspension, emulsion or oil; and a parenteral vehicle (for subcutaneous, intravenous, intraarterial, or intramuscular injection), including but not limited to, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer’s and fixed oils. [0114] The choice of carrier in the pharmaceutical composition may be determined in part by the particular method used to administer the composition. In some embodiments, the pharmaceutical composition contains preservatives. Exemplary preservatives include, but are not limited to, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. In some embodiments, the preservative or mixtures thereof are present in an amount of about 0.0001% to about 2% by weight of the total composition. [0115] In some embodiments, a composition disclosed herein further comprises buffering agents. Exemplary buffering agents include, for example, citric acid, Tris-HCl, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. In some embodiments, the buffering agent or mixtures thereof are present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins 21st ed. (May 1, 2005). Examples [0116] The examples provided below are simply for illustrative purposes. Those of skill in the art will be able to readily determine appropriate methods and equipment in order to produce lipid nanocrystals as described herein. Example 1. Determination of an Optimal Molar Ratio of Multivalent Cation to Negatively Charged Lipid for Lipid Nanocrystal Formation and Aggregation Inhibition [0117] The aim of this study was to test different molar ratios of calcium to negatively charged phospholipid (phosphatidylserine (PS) or PS and phosphatidylcholine (PC)) to determine if there is an optimal ratio that produces lipid nanocrystals while minimizing or eliminating aggregation. [0118] 8 mg/mL rhodamine-labeled liposomes at either 100% PS or 75% PS and 25% PC were used to prepare lipid nanocrystal formulations as outlined in Tables 1 and 2. The rhodamine was Attorney Docket No. MNY-01525 attached DOPE and 0.1% wt/wt of labeled DOPE was used to generate the labeled liposomes. Liposomes, 0.1M CaCl2 solution, and 1xTES buffer (2 mM TES, 2 mM Histidine, 100 mM sodium chloride, pH 7.4; TES: 10 mM Tris (pH 7.5); 10 mM EDTA (pH 8.0), 0.5% sodium dodecyl sulfate (SDS)) used for these experiments were all filtered through 0.22 µm filters before the experiments began. Table 1. Lipid nanocrystal formulations for determination of an optimal molar ratio of calcium and PS for lipid nanocrystal formation without causing aggregation; 100% PS.

Table 2: Lipid nanocrystal formulations for determination of an optimal molar ratio of calcium and PS for lipid nanocrystal formation without causing aggregation; 75% PS, 25% PC.

Attorney Docket No. MNY-01525

[0119] To form lipid nanocrystals, the calculated volumes of 1x TES buffer and 0.1 M CaCl₂ as outlined in Tables 1 and 2 were added to the top of the liposome-containing microcentrifuge tube slowly while the tube was slowly being vortexed for 10 seconds, followed by vigorously vortexed briefly to further combine everything. The lipid nanocrystal formulations were stored at 4ºC. Particle sizes and aggregation were monitored using dynamic light scattering (DLS) at day 0, day 1, day 7, and day 21 post-formulation. On day 7, a portion of each formulation was spun down at 16,000 rpm for 15 minutes and the supernatant was collected and used to determine free lipids that were present in the supernatant by high-performance liquid chromatography (HPLC). The column used was Water µ-Porasil™, 10µm, 3.9 x 300 mm or equivalent, the mobile phase used acetonitrile/methanol/phosphoric acid (80/20/1 v/v/v), the flow rate was 0.6 mL/min, the column temperature was 30⁰C, the detection wavelength was 203 nm and the run time was 25 minutes. [0120] The mean particle size and polydispersity index (PDI) of the lipid nanocrystals obtained at different time point from each formulation via DLS are summarized in FIG.1A-1D. As shown in FIG.1A and FIG.1B, the particle size at day 0 was generally small but increased when more calcium was added into the liposomes. The effect of calcium concentration in increasing particle size continued over time. [0121] Overall, liposomes containing 100% PS needed at least about 0.25 equal mole of calcium (i.e., molar ratio of calcium to PS = 0.25:1) to form lipid nanocrystals (FIG.1A), while liposomes containing 75% PS and 25% PC needed 1.5 equal mole of calcium (i.e., molar ratio of calcium to PS = 1.5:1) to form lipid nanocrystals (FIG. 1B). However, liposomes containing 100% PS formed lipid nanocrystals with particle sizes in the range of about 2-5 µm independent of the molar ratio of calcium to PS (FIG.1A), while liposomes containing 75% PS and 25% PC Attorney Docket No. MNY-01525 generally formed lipid nanocrystals with particle sizes less than about 200 nm (FIG.1B), as long as the molar ratio of calcium to PS was controlled. [0122] Moreover, it was observed that lipid nanocrystals formed with liposomes containing 100% PS aggregated very fast, and the aggregation increased when more calcium was added. Aggregation was determined by observation and monitoring particle size at different days. Conversely, lipid nanocrystals formed with liposomes containing 75% PS did not aggregate as much. Apparently, when more calcium is used for lipid nanocrystal formulation, it leads to bigger particle size and increased aggregation of the lipid nanocrystals. Nevertheless, this study shows that lipid nanocrystals having a mean particle size of about 200 nm or less could be achieved and maintained by carefully controlling the percentage of negatively charged lipid and the ratio of the multivalent cation to negatively charged lipid. [0123] FIG.2A-2C show the lipid composition in the supernatant at day 7. It appears that, at the same molar ratio of calcium to PS, more free lipid is present in the supernatant when liposomes containing 75% PS were used to form lipid nanocrystals (FIG.2B) as compared to formulations using liposomes containing 100% PS (FIG. 2A). Additionally, in the formulations using liposomes containing 75% PS, when more than 1.5 equal mole of calcium (e.g., molar ratio of calcium to PS = 1.75:1) was used, all PS was incorporated into the lipid nanocrystals, while 100% of PC was present as free lipid in the supernatant and, thus, not incorporated into the lipid nanocrystals (FIG.2C). Example 2. Varying the Amount of Negatively Charged Lipids and Multivalent Cation Impacts Lipid Nanocrystal Formation [0124] The aim of this study was to evaluate how varying the amount of phosphatidylserine (PS) and phosphatidylcholine (PC) in liposomes and varying the molar ratio of calcium to negatively charged phospholipids (PS and PC) impacts lipid nanocrystal formation. [0125] Soybean-derived PS (25 mg/mL; Soy PS) and soy PC (25 mg/mL) in chloroform were combined to produce 100 mg liposome at either 100% PS, 75% PS and 25% PC, or 50% PS and 50% PC. All contained 0.5% rhodamine. These mixtures were dried to a thin film using nitrogen gas. Liposomes were rehydrated with TES buffer (2 mM TES, 2 mM Histidine, 100 mM sodium chloride, pH 7.4; TES: 10 mM Tris (pH 7.5), 10 mM EDTA (pH 8.0), 0.5% sodium dodecyl sulfate (SDS)) to a final volume of 10 mL. Resulting liposomes were sequentially passed through the Attorney Docket No. MNY-01525 following filters: 0.8 µm, 0.45 µm, and 0.22 µm. Lipid composition was determined by high- performance liquid chromatography (HPLC) as described in Example 1. As shown in Table 3, the concentrations of PS in all three formulations were about 3 mg/mL (i.e., 2.53 mg/mL in 50% PS and 75% PS, and 3.3 mg/mL in 100% PS) resulting in final formulations with 2.5 mg/mL liposomes. Table 3. Lipid composition of various liposomes determined by HPLC.

[0126] Different formulations as summarized in Tables 4-6 were made by adding different ^{volumes of TES buffer and 0.1 M CaCl} ₂ ^{to achieve the desired molar ratio concentration. After} formulation, lipid nanocrystals were monitored for size and aggregation using dynamic light scattering (DLS) and light microscopy by diluting each sample in TES buffer (1/8^th dilution). This was repeated the following day, and one week thereafter. 50 µL of each formulation was also spun down at 16,400 rpm for 15 minutes in a bench top centrifuge, the supernatant was aspirated off and used to determine free lipids that were present in the supernatant by HPLC. Table 4. Lipid nanocrystal formulations prepared by varying the PS concentration in liposomes and different molar ratios of calcium to PS; 100% PS.

Table 5. Lipid nanocrystal formulations prepared by varying the PS concentration in liposomes and different molar ratios of calcium to PS; 75% PS, 25% PC. Attorney Docket No. MNY-01525

Table 6. Lipid nanocrystal formulations prepared by varying the PS concentration in liposomes and different molar ratios of calcium to PS; 50% PS, 50% PC.

[0127] The mean particle size and polydispersity index (PDI) of the lipid nanocrystals obtained from each formulation are shown in FIG.3A (median particle size) and FIG.3B (PDI) with their corresponding light microscopy image shown in FIG.3C. As shown in FIG. 3A-3B, when the liposome contained 100% of the negatively charged lipid (100% PS), the resulting lipid nanocrystals had larger particle size that increased with more calcium. In contrast, as shown in FIG. 3A-3B, the particle size of the lipid nanocrystals prepared from the liposomes containing lower percentages of negatively charged lipid (i.e., 75% PS and 50% PS) remained small and uniform and were generally not affected by increasing the molar ratio of calcium to negatively charged lipid (e.g., 0.5:1 to 4:1). The mean particle size and PDI obtained from the formulations prepared using liposomes containing 75% PS are provided in Table 7. Attorney Docket No. MNY-01525 Table 7. Mean particle size and PDI obtained from the formulations prepared using liposomes containing 75% PS.

[0128] The concentration of free lipids present in the supernatant of each formulation after lipid nanocrystal formation as determined by HPLC is shown in FIG.4A-4C with the calculated percentage of PS vs PC shown in FIG. 4D-4F. The lipid composition determined in the supernatant of the formulations prepared using liposomes containing 75% PS is provided in Table 8. These results show that PS is much preferred to be incorporated into lipid nanocrystals, while PC often left behind in aqueous liposome form. Table 8. Lipid composition determined in the supernatant of the formulations prepared using liposomes containing 75% PS.

[0129] Overall, the results show that the lower the percentage of negatively charged lipid in the liposomes, the higher the concentration of multivalent cation is needed to form lipid nanocrystals. For example, under the conditions of this experiment, when the liposomes contain 100% PS, a molar ratio of calcium to PS of less than about 0.5:1 is sufficient to form lipid nanocrystals. When the liposomes contain about 75% PS, a higher molar ratio of calcium to PS of about 1:1 was sufficient for lipid nanocrystals to form under the conditions of this experiment. Attorney Docket No. MNY-01525 When the liposomes contain about 50% PS, an increased molar ratio of calcium to PS of about 4:1 was sufficient for lipid nanocrystals to form under the conditions of this experiment. Example 3. Effects of Multivalent Cation Concentration on Particle Size and Aggregation [0130] Earlier studies showed that adding excipients, such as bile salt, bovine serum albumin (BSA), and serum, into lipid nanocrystal formulations could minimize or disrupt lipid nanocrystal aggregation. However, whether this effect is due to the presence of excipients or reduced multivalent cation concentration in the formulation due to dilution by the solution containing the excipients is unknown. It appears that aggregation is reversible based on multivalent cation concentration––from top down, disrupting aggregation by dilution, and from bottom up, inducing aggregation by adding multivalent cation. [0131] To study how the amount of multivalent cation impacts aggregation of the lipid nanocrystals, increasing amounts of calcium were added to a lipid nanocrystal sample prepared using liposomes containing 75% PS and 25% PC at 0.5 mg/mL starting PS concentration and the resulting mean particle size and PDI were measured. The results are shown in FIG.5A-5B. When the total calcium amount was about 7 mM or below, although the mean particle size increased proportionally with the total amount of calcium, the mean particle size nevertheless maintained below 200 nm while the PDI remained relatively low (between 0.1 to 0.2). However, when the total calcium amount increased to about 70 mM, the particle size increased to greater than 500 nm with a PDI of about 0.66. [0132] These results show that a low total calcium concentration (^ 7 mM) produces LNCs of a small mean particle size with minimal aggregation (as measured by PDI) whereas high total calcium concentrations (about 70 mM) increased both the mean particle size and aggregation. These results support that an excipient is not needed to prevent aggregation in LNC formulations if total calcium concentrations are kept low, approximately below about 7 mM. Example 4. Effects of the Starting Lipid Concentration on Particle Size and Aggregation [0133] The aim of this study was to evaluate how the starting lipid concentration impacts lipid nanocrystal particle size and aggregation. [0134] Liposomes of either 100% soybean-derived phosphatidylserine (Soy PS) or 75% Soy PS and soybean-derived phosphatidylcholine (Soy PC) were prepared with Avestin EmulsiFlex- Attorney Docket No. MNY-01525 C5 Homogenizer (0.5% rhodamine labelled). Liposomes prepared with either 100% Soy PS or 75% Soy PS and 25% soy PC both had a mean liposome size of about 60 nm and a total lipid concentration of about 8 mg/mL. Lipid nanocrystals were made using different starting lipid concentration (e.g., 8, 4, 2, 1, and 0.5 mg/mL) with a molar ratio of calcium and PS of 0.5:1 for 100% Soy PS and 1.5:1 for 75% Soy PS and 25% Soy PC. This molar ratio of calcium and PS was selected based on prior finding that virtually all liposomes convert to lipid nanocrystals at this molar ratio (see Example 1). For particle sizing, samples were diluted either with TES buffer or with the same diluent used in the lipid nanocrystal sample (i.e., TES and exact amount of calcium; for example, 300 µL TES and 15 µL of 0.1M CaCl2 in case of 8 mg/mL lipid sample). The results are shown in FIG.6A-6B and Table 9. Table 9. Particle size and PDI measured in lipid nanocrystal samples prepared with different starting lipid concentrations (data of the chart shown in FIG.6A).

[0135] Consistent with the prior findings, lipid nanocrystals prepared with liposomes containing 75% PS and 25% PC generally had smaller mean particle size and lower PDI as compared to lipid nanocrystals prepared with liposomes containing 100% PS. Furthermore, as the starting lipid concentration increased, so too did the particle size and this was more pronounced in the lipid nanocrystals prepared with liposomes containing 100% PS. Attorney Docket No. MNY-01525 Example 5. Lipid Nanocrystal Particle Size Control [0136] Based on the results of Examples 1-4, there are at least three factors that can impact lipid nanocrystal particle size and aggregation, namely the amount of PS in the liposomes (e.g., 100% PS, 75% PS, etc.), the molar ratio of calcium and PS (e.g., 0.5:1, 1:1, 2:1, etc.), and the starting lipid concentration (e.g., 1 mg/mL, 10 mg/mL, etc.). Experiments were designed to investigate how these three variables function to impact particle size and lipid nanocrystal yield. A complete factorial experimental design and the corresponding data on particle size and PDI are provided in Table 10, and the pattern key provided in Table 11. Table 10. Factorial experimental design and the corresponding data on particle size and PDI.

^a When % PS = 75, % PC = 25 Table 11. Factorial experimental design pattern key

Attorney Docket No. MNY-01525

[0137] A predicted particle size plot was made based on calcium eq., %PS, and lipid concentration, and the actual-by-predicted plot on particle size thus obtained is shown in FIG.7A. A prediction expression equation that can be used to calculate particle size based on calcium eq., % negatively charged lipid (e.g., PS), and starting lipid concentration (mg/mL) is as follows (Formula I): _{227.45 + 97.510714286 ή ^} ^{(^^^^^^^ ^^.െ1.25)} _{^ + 96.560714286 ή ൬} ^{(%^^ െ 0.875} ^

0.125

[0138] A summary of how all three variables affected particle size is provided in FIG. 7B. The p value demonstrates that both the amount of negatively charged lipid (PS) in the liposomes (“%PS”) and the molar ratio of multivalent cation (e.g., calcium) and negatively charged lipid (“Calcium eq.”) are significant variables, although starting lipid concentration also impacts particle size. The prediction profiler based on these data is shown in FIG.7C. Attorney Docket No. MNY-01525 [0139] FIG.8A depicts a 3-dimensional (3D) scatterplot of the factorial particle size based on the data provided in Table 10. The cut-off particle size in this 3D scatterplot is 450 nm and thus, the data obtained from Run #2 (100% PS, calcium eq.1, 10 mg/mL lipid concentration) and Run #12 (100% PS, calcium eq.2, 10 mg/mL lipid concentration) were excluded. FIG.8B-8D show the bubble plots of calcium eq. by %PS sized by particle size (FIG. 8B), lipid concentration (mg/mL) by calcium eq. sized by particle size (FIG. 8C), and lipid concentration (mg/mL) by %PS sized by particle size (FIG. 8D), respectively. The contour plots for particle size (nm) are shown in FIG.8E-8G. [0140] The p value calculated based on all three variables as shown in FIG.7B demonstrates that both the amount of negatively charged lipid (PS) in the liposomes (“%PS”) and the molar ratio of multivalent cation (calcium) and negatively charged lipid (“Calcium eq.”) are significant variables, although starting lipid concentration can also impact particle size. A predicted particle size plot was made based on calcium eq. and %PS as the only significant variables and the actual- by-predicted plot on particle size thus obtained is shown in FIG.9A with the effect summary and lack of fit calculation shown in FIG. 9B. A prediction expression equation that can be used to calculate particle size based on calcium eq. and % negatively charged lipid (PS) is as follows (Formula II): _{205.13214286 + 79.901785714 ή ^} ^{(^^^^^^^ ^^.െ1.25)} ^ + 74.242857143 0.75

[0141] The results demonstrate that both lipid composition in terms of the amount of negatively charged lipid (e.g., % PS and % PC) and the amount of multivalent cation (e.g., calcium) significantly impact lipid nanocrystal particle sizes and subsequent aggregation size and PDI. Although the starting lipid concentration was not a significant factor in primary particle size, it was still a variable that can used to regulate or control particle size of the lipid nanocrystal s, Attorney Docket No. MNY-01525 particularly when used in combination with the other two variables (amount of negatively charged lipid and amount of multivalent cation). A summary of this study is shown in Table 12. Table 12. Summary of lipid nanocrystal particle size control study.

Example 6. Effect of Multiple Freeze-Thaw Cycles with or without Cryoprotectant on Particle Size and Homogeneity of Lipid Nanocrystals [0142] To investigate what effect multiple freeze-thaw cycles would have on lipid nanocrystal particle size and homogeneity with or without cryoprotectant, two populations of lipid nanocrystals were prepared, one was made with 100% PS and the other one was made with 75% PS and 25% PC. As shown in FIG. 10A-10B, freeze-thaw cycles without cryoprotectant had a negative effect on particle size and homogeneity of lipid nanocrystals and this effect was more prominent for lipid nanocrystals made of 100% PS (FIG.10A) than those made of 75% PS and 25% PC (FIG.10B). However, the addition of 5% sucrose (a cryoprotectant) effectively stabilized lipid nanocrystals regardless of the composition, as shown in FIG.10C-10D. [0143] Overall, this experiment demonstrated that, in the absence of a cryoprotectant, such as sucrose, lipid nanocrystals rapidly aggregated with freeze-thaw cycles. However, in the presence of 5% sucrose, lipid nanocrystals were stabilized after one or more freeze-thaw cycles. Attorney Docket No. MNY-01525 Example 7. Effect of Aqueous Calcium Concentration on Lipid Nanocrystal Stability at 4ºC [0144] The aim of this study was to probe aqueous calcium boundary conditions to find out the low-end threshold to convert lipid nanocrystals to liposomes (i.e., lipid nanocrystal disintegration) and the high-end threshold to cause aggregation. [0145] Two groups of lipid nanocrystals were prepared, one was made with 100% PS and the other one was made with 75% PS and 25% PC. The molar ratio of PS to calcium was varied depending on the percentage of PS used in the formulation. For the group of lipid nanocrystals made with 100% PS, 5.8 mM of CaCl2 (equal to 0.5X mole of calcium relative to the moles of PS) was added to a liposome solution composed of 100% PS to form lipid nanocrystals. For the group of lipid nanocrystals made with 75% PS and 25% PC, 12 mM of CaCl₂ (equal to 1.5X mole of calcium relative to the moles of PS) was added to a liposome solution composed of 75% PS and 25% PC to form lipid nanocrystals. Each group was divided into 6 replicates, one with the calcium concentration unmodified and used as “as is” control and the other five were spun down followed by removing the supernatant and adding different concentrations of CaCl₂ (e.g., 0 mM, 0.5 mM, 1, mM, 2 mM, 10 mM, or 15 mM). Lipid nanocrystal stability was monitored immediately after the preparation (T0) and after 1 week, 4 weeks, and 6 weeks of storage at 4ºC. [0146] As shown in FIG. 11A, lipid nanocrystals made of 100% PS did not convert to liposomes even without aqueous CaCl2 (e.g., free and not coordinated by a liposome) in the resuspension media. In fact, the particle size was almost stable only when there was no aqueous CaCl₂ in the resuspension media. In all other cases where the aqueous CaCl₂concentration was more than 0.5 mM, aggregation occurred and the particle size and PDI increased over time. FIG. 11B depicts the HPLC results and shows that there were no free lipids in the supernatant of the lipid nanocrystals made of 100% PS. These results indicate that resuspending lipid nanocrystals made of 100% PS in 0 mM CaCl₂ leads to higher lipid nanocrystal stability. [0147] In the lipid nanocrystals made with 75% PS and 25% PC, the particle size was mostly stable over 6 weeks at all calcium levels, but 2-5 mM CaCl2 seems to be the optimum concentration for lipid nanocrystal stability (FIG.11C). Free PS in the supernatant was negligible, but about 4-5% of PC was released from lipid nanocrystals into the supernatant in the lower calcium concentrations after four to six weeks storage at 4ºC (FIG.11D). [0148] These results indicate that, once lipid nanocrystals form, aqueous CaCl₂ is not necessary to stabilize lipid nanocrystals. This is unexpected and surprising given that it was Attorney Docket No. MNY-01525 generally accepted by those having ordinary skill in the art that the aqueous CaCl₂ concentration had to be maintained at a certain level to stabilize lipid nanocrystals. [0149] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all constructs, methods, and/or component features, steps, elements, or other aspects thereof can be used in various combinations. [0150] 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 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 disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. In general, where embodiments or aspects of the disclosure, is/are referred to as comprising particular elements, features, etc., certain embodiments or aspects consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. [0151] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to Attorney Docket No. MNY-01525 the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.

Claims

Attorney Docket No. MNY-01525 We Claim: 1. A method of preparing a population of lipid nanocrystals, the method comprising: a) preparing a solution of liposomes comprising a negatively charged phospholipid and a therapeutic agent in an aqueous medium; and b) adding a multivalent cation to the solution of liposomes, thereby forming the population of lipid nanocrystals, wherein the negatively charged phospholipid is in an amount ranging from about 50% to about 100% by weight of the liposomes, and wherein the multivalent cation added to the solution of liposomes and the negatively charged phospholipid are in a molar ratio of from about 0.25:1 to about 4:1. 2. The method of claim 1, wherein the solution of liposomes has a lipid concentration of from about 0.5 mg/mL to about 100 mg/mL. 3. The method of claim 1 or 2, wherein the population of lipid nanocrystals has a mean particle size no more than about 200 nm. 4. The method of any one of claims 1-3, wherein the population of lipid nanocrystals does not aggregate over time. 5. The method of claim 4, wherein the mean particle size of the population of lipid nanocrystals does not increase more than about 25% after storage at 4ºC for 21 days. 6. The method of any one of claims 1-5, wherein the population of lipid nanocrystals has a polydispersity index of about 1.0 or less after storage at 4ºC for 7 days. 7. The method of claim 6, wherein the population of lipid nanocrystals has a polydispersity index of about 0.5 or less after storage at 4ºC for 7 days. Attorney Docket No. MNY-01525 8. The method of claim 7, wherein the population of lipid nanocrystals has a polydispersity index of about 0.2 or less after storage at 4ºC for 7 days. 9. The method of any one of claims 1-8, wherein the negatively charged phospholipid comprises phosphatidylserine. 10. The method of any one of claims 1-9, wherein the solution of liposomes further comprises a neutrally charged lipid in an amount no more than about 50% by weight of the liposomes. 11. The method of claim 10, wherein the neutrally charged lipid comprises phosphatidylcholine. 12. The method of any one of claims 1-11, wherein the multivalent cation is Ca⁺⁺, Zn⁺⁺, Ba⁺⁺, or Mg⁺⁺. 13. The method of claim 12, wherein the multivalent cation is Ca⁺⁺. 14. The method of any one of claims 1-13, wherein the population of lipid nanocrystals has a concentration of the multivalent cation of from about 1 mM to about 12 mM. 15. The method of any one of claims 1-14, wherein the population of lipid nanocrystals does not comprise a size regulating agent. 16. The method of any one of claims 1-15, further comprising spinning down the population of lipid nanocrystals and resuspending the population of lipid nanocrystals in a solution comprising the multivalent cation at a concentration that is lower than the concentration of the multivalent cation used to form the population of lipid nanocrystals. 17. The method of claim 16, wherein the solution used to resuspend the population of lipid nanocrystals does not comprise the multivalent cation. Attorney Docket No. MNY-01525 18. The method of any one of claims 1-17, further comprising adding a cryoprotectant to the population of lipid nanocrystals and freezing the population of lipid nanocrystals. 19. The method of claim 18, wherein the cryoprotectant comprises sucrose at a concentration of from about 1% to about 10%. 20. The method of claim 18 or 19, wherein particle size of the population of lipid nanocrystals remains stable after one or more freeze-thaw cycles. 21. A population of lipid nanocrystals, wherein each lipid nanocrystal in the population comprises: a) a lipid component comprising a negatively charged phospholipid; b) a multivalent cation; and c) a therapeutic agent, wherein the negatively charged phospholipid is in an amount ranging from about 50% to about 100% by weight of the lipid component, wherein the multivalent cation and the negatively charged phospholipid are in a molar ratio of from about 0.25:1 to about 4:1, and wherein the population of lipid nanocrystals has a mean particle size no more than about 200 nm. 22. A population of lipid nanocrystals, wherein each lipid nanocrystal in the population comprises: a) a lipid component comprising a negatively charged phospholipid; b) a multivalent cation; and c) a therapeutic agent, wherein the negatively charged phospholipid is in an amount of about 50% to about 100% by weight of the lipid component, wherein the multivalent cation and the negatively charged phospholipid are in a molar ratio of about 0.25:1 to about 4:1, and wherein the population of lipid nanocrystals has a mean particle size of about 180-220 nm. Attorney Docket No. MNY-01525 23. A population of lipid nanocrystals, wherein each lipid nanocrystal in the population comprises: a) a lipid component comprising a negatively charged phospholipid, wherein the negatively charged phospholipid is in an amount of about 100% by weight of the lipid component; b) a multivalent cation; and c) a therapeutic agent, wherein the multivalent cation and the negatively charged phospholipid are in a molar ratio of about 0.25:1 to about 0.5:1, and wherein the population of lipid nanocrystals has a mean particle size of about 180-220 nm. 24. The population of lipid nanocrystals of claim 23, wherein the molar ratio is about 0.25:1. 25. A population of lipid nanocrystals, wherein each lipid nanocrystal in the population comprises: a) a lipid component comprising a negatively charged phospholipid, wherein the negatively charged phospholipid is in an amount of about 75% by weight of the lipid component; b) a multivalent cation; and c) a therapeutic agent, wherein the multivalent cation and the negatively charged phospholipid are in a molar ratio of about 1:1 to about 4:1, and wherein the population of lipid nanocrystals has a mean particle size of about 180-220 nm. 26. The population of lipid nanocrystals of claim 25, wherein the molar ratio is about 1.5:1 27. The population of lipid nanocrystals of claim 25 or 26, wherein the lipid component comprises about 25% by weight of a neutrally charged lipid. 28. The population of lipid nanocrystals of claim 27, wherein the neutrally charged lipid comprises phosphatidylcholine. Attorney Docket No. MNY-01525 29. A population of lipid nanocrystals, wherein each lipid nanocrystal in the population comprises: a) a lipid component comprising a negatively charged phospholipid, wherein the negatively charged phospholipid is in an amount of about 50% by weight of the lipid component; b) a multivalent cation; and c) a therapeutic agent, wherein the multivalent cation and the negatively charged phospholipid are in a molar ratio of about 1:1 to about 4:1, and wherein the population of lipid nanocrystals has a mean particle size of about 180-220 nm. 30. The population of lipid nanocrystals of claim 29, wherein the molar ratio is about 1.5:1 31. The population of lipid nanocrystals of claim 29 or 30, wherein the lipid component comprises about 25% by weight of a neutrally charged lipid. 32. The population of lipid nanocrystals of claim 31, wherein the neutrally charged lipid comprises phosphatidylcholine. 33. The population of lipid nanocrystals of any one of claims 21-32, wherein the population of lipid nanocrystals does not aggregate over time. 34. The population of lipid nanocrystals of any one of claims 21-33, wherein the mean particle size of the population of lipid nanocrystals does not increase more than about 25% after storage at 4ºC for 21 days. 35. The population of lipid nanocrystals of any one of claims 21-34, wherein the population of lipid nanocrystals has a polydispersity index of about 1.0 or less after storage at 4ºC for 7 days. 36. The population of lipid nanocrystals of claim 35, wherein the population of lipid nanocrystals has a polydispersity index of about 0.5 or less after storage at 4ºC for 7 days. Attorney Docket No. MNY-01525 37. The population of lipid nanocrystals of claim 36, wherein the population of lipid nanocrystals has a polydispersity index of about 0.2 or less after storage at 4ºC for 7 days. 38. The population of lipid nanocrystals of any one of claims 21-37, wherein the negatively charged phospholipid comprises phosphatidylserine. 39. The population of lipid nanocrystals of any one of claims 21-38, wherein the solution of liposomes further comprises a neutrally charged lipid in an amount no more than about 50% by weight of the lipid component. 40. The population of lipid nanocrystals of claim 39, wherein the neutrally charged lipid comprises phosphatidylcholine. 41. The population of lipid nanocrystals of any one of claims 21-40, wherein the multivalent cation

42. The population of lipid nanocrystals of claim 41, wherein the multivalent cation is Ca⁺⁺. 43. The population of lipid nanocrystals of any one of claims 21-42, wherein the population of lipid nanocrystals has a concentration of the multivalent cation of from about 1 mM to about 12 mM. 44. The population of lipid nanocrystals of any one of claims 21-43, wherein the population of lipid nanocrystals does not comprise a size regulating agent. 45. The population of lipid nanocrystals of any one of claims 21-44, wherein the population of lipid nanocrystals further comprises a cryoprotectant. 46. The population of lipid nanocrystals of claim 45, wherein the cryoprotectant comprises sucrose at a concentration of from about 1% to about 10%. Attorney Docket No. MNY-01525 47. A method of preserving the population of lipid nanocrystals of claim 45 or 46, said method comprising freezing the population of lipid nanocrystals. 48. The method of claim 47, wherein the mean particle size of the population of lipid nanocrystals remains stable after one or more freeze-thaw cycles. 49. A composition comprising the population of lipid nanocrystals of any one of claims 21-46, wherein the composition comprises the multivalent cation at a concentration that is lower than the concentration of the multivalent cation comprised in the population of lipid nanocrystals. 50. The composition of claim 49, wherein the composition does not comprise the multivalent cation. 51. A method of preparing a population of lipid nanocrystals with a mean particle size of interest, the method comprising: a) preparing a solution of liposomes comprising a negatively charged phospholipid and a therapeutic agent in an aqueous medium, wherein the negatively charged phospholipid is present in a percentage of about 50% to about 100% by weight of the liposomes and wherein the solution of liposomes has a lipid concentration of from about 0.5 mg/mL to about 100 mg/mL; b) selecting an amount of a multivalent cation sufficient to form the population of lipid nanocrystals with the mean particle size of interest based on the percentage of the negatively charged phospholipid and the lipid concentration of the solution of liposomes; and c) adding the amount of multivalent cation to the solution of liposomes, thereby forming the population of lipid nanocrystals with a mean particle size of interest. 52. The method of claim 51, wherein the mean particle size of interest is about 200 nm or less. 53. The method of claim 51 or 52, wherein the multivalent cation added to the solution of liposomes and the negatively charged phospholipid are in a molar ratio of from about 0.25:1 to about 4:1. Attorney Docket No. MNY-01525 54. A method for preparing a population of lipid nanocrystals comprising: (i) providing a population of liposomes comprising (a) a lipid component comprising a negatively charged phospholipid, and (b) a therapeutic agent, wherein the negatively charged phospholipid is in an amount from about 50% to about 100% by weight of the lipid component; and (ii) contacting the population of liposomes with an amount of a multivalent cation, wherein the molar ratio of the multivalent cation and the negatively charged phospholipid is about 0.25:1 to about 4:1, thereby forming the population of lipid nanocrystals having a mean particle size of about 180-220 nm. 55. The method of claim 54, wherein the mean particle size is about 200 nm. 56. The method of claim 54 or 55, wherein the amount of the negatively charged phospholipid is about 100% by weight of the lipid component, and the molar ratio of the multivalent cation and the negatively charged phospholipid is about 0.25:1 to about 0.5:1. 57. The method of claim 56, wherein the molar ratio of the multivalent cation and the negatively charged phospholipid is about 0.25:1. 58. The method of claim 54 or 55, wherein the amount of the negatively charged phospholipid is about 75% by weight of the lipid component, and the molar ratio of the multivalent cation and the negatively charged phospholipid is about 1:1 to about 1.5:1. 59. The method of claim 58, wherein the molar ratio of the multivalent cation and the negatively charged phospholipid is about 1.5:1. 60. The method of claim 58 or 59, wherein the lipid component comprises about 25% of a neutrally charged lipid by weight of the lipid component. 61. The method of claim 60, wherein the neutrally charged lipid comprises phosphatidylcholine. Attorney Docket No. MNY-01525 62. The method of claim 54 or 55, wherein the amount of the negatively charged phospholipid is about 50% by weight of the lipid component, and the molar ratio of the multivalent cation and the negatively charged phospholipid is about 1:1 to about 1.5:1. 63. The method of claim 62, wherein the molar ratio of the multivalent cation and the negatively charged phospholipid is about 1.5:1. 64. The method of claim 62 or 63, wherein the lipid component comprises about 50% of a neutrally charged lipid by weight of the lipid component. 65. The method of claim 64, wherein the neutrally charged lipid comprises phosphatidylcholine. 66. the method of any one of claims 54-65, wherein the population of lipid nanocrystals does not aggregate over time. 67. The method of claim 66, wherein the mean particle size of the population of lipid nanocrystals does not increase more than about 25% after storage at 4ºC for 21 days. 68. The method of any one of claims 54-67, wherein the population of lipid nanocrystals has a polydispersity index of about 1.0 or less after storage at 4ºC for 7 days. 69. The method of claim 68, wherein the population of lipid nanocrystals has a polydispersity index of about 0.5 or less after storage at 4ºC for 7 days. 70. The method of claim 69, wherein the population of lipid nanocrystals has a polydispersity index of about 0.2 or less after storage at 4ºC for 7 days. 71. The method of any one of claims 54-70, wherein the negatively charged phospholipid comprises phosphatidylserine. Attorney Docket No. MNY-01525 72. The method of any one of claims 54-71, wherein the multivalent cation is Ca⁺⁺, Zn⁺⁺, Ba⁺⁺, or Mg⁺⁺. 73. The method of claim 72, wherein the multivalent cation is Ca⁺⁺. 74. The method of any one of claims 54-73, wherein the population of lipid nanocrystals has a concentration of the multivalent cation of from about 1 mM to about 12 mM. 75. The method of any one of claims 54-74, wherein the population of lipid nanocrystals does not comprise a size regulating agent. 76. The method of any one of claims 54-75, further comprising spinning down the population of lipid nanocrystals and resuspending the population of lipid nanocrystals in a solution comprising the multivalent cation at a concentration that is lower than the concentration of the multivalent cation used to form the population of lipid nanocrystals. 77. The method of claim 76, wherein the solution used to resuspend the population of lipid nanocrystals does not comprise the multivalent cation. 78. The method of any one of claims 54-77, further comprising adding a cryoprotectant to the population of lipid nanocrystals and freezing the population of lipid nanocrystals. 79. The method of claim 78, wherein the cryoprotectant comprises sucrose at a concentration of from about 1% to about 10%. 80. The method of claim 78 or 79, wherein particle size of the population of lipid nanocrystals remains stable after one or more freeze-thaw cycles. 81. A population of lipid nanocrystals produced by the method of any one of claims 54-80. Attorney Docket No. MNY-01525 82. A population of lipid nanocrystals produced by the method of any one of claims 1-20.