Multivesicular lipid nanoparticle tolerizing vaccine for in vivo systemic immune tolerance induction

Abstract

The multivesicular lipid nanoparticle composition comprises a plurality of multivesicular lipids. Each multivesicular lipid nanoparticle comprises a carrier phospholipid micelle comprising a carrier phospholipid layer and having an average diameter of less than 1 micron; and at least one sub-chamber phospholipid reverse micelle comprising a sub-chamber phospholipid layer and having an average diameter of less than 100 nm. The carrier micelle nanoparticle encapsulates at least one sub-chamber reverse micelle nanoparticle. Characteristically, the phospholipid tail group of the carrier micelle is covalently bonded to the phospholipid tail group of at least one sub-chamber reverse micelle nanoparticle.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 299,669, filed January 14, 2022, the disclosure of which is incorporated herein by reference in its entirety. [Technical field]

[0002] The present invention relates generally to the controlled encapsulation and delivery of small molecule drugs, nucleotides, and biomolecules, which can be characterized as biologics, to immune cells to induce immune tolerance states in vivo and in vitro. In particular, the present invention relates to improvements in gene delivery, uptake, and stimuli-responsive expression for cellular reprogramming of immune cells to treat and prevent autoimmune diseases, allergies, and food hypersensitivity. [Background technology]

[0003] Vertebrates have evolved complex immune systems to defend themselves against pathogens, parasites, and harmful environmental substances. Normally, these immune systems can distinguish between these threats and the body's own cells and tissues (and other harmless substances). Cells and tissues that the immune system determines to be "self" are said to be "tolerant". This self-tolerance is initiated during the organism's early development (central tolerance) and is maintained throughout life by a wide variety of cellular and molecular mechanisms, including the action of regulatory T cells (Tregs) (peripheral tolerance). When these mechanisms fail and components of cells and tissues are no longer recognized as "self", they become autoantigens, leading to autoimmunity and autoimmune diseases. Similarly, peripheral tolerance mechanisms can train the immune system to recognize common environmental substances (such as house dust and pollen) as harmless, which are tolerated by the immune system. When this process goes awry, allergies and food sensitivities result. Tolerance to common allergens can sometimes be restored by crude methods such as "allergy shots," in which the patient's immune system is repeatedly stimulated with small amounts of the allergen. Recently, this method has been formalized and has been used successfully, for example, in efforts to minimize peanut allergy by repeatedly feeding children with minute amounts of peanut. However, this approach is not feasible for many allergens, is not targeted in any way, and cannot induce a tolerogenic effect strong enough to treat or prevent autoimmune diseases. To achieve this, a truly tolerogenic vaccine is needed. Whereas standard vaccines activate cells of the immune system and provide examples of pathogens to attack, tolerizing vaccines put specific cells of the immune system into a tolerizing state and provide examples of self-antigens to be tolerated. The driving force behind this innovation is the development of tolerogenic vaccine technology that can be broadly applied to induce systemic, antigen-specific immune tolerance to any self-antigen or allergen.

[0004] Liposomes and lipid nanoparticle systems are soft material-based nanoparticles that have been used as carriers within the extracellular environment, either as naturally occurring extracellular vesicles (EVs) or as vesicles synthesized from phospholipid formulations. Naturally occurring exosomes are known to encapsulate and deliver microRNAs and proteins to signal relative health and oxidative stress to other cells. Recent innovations have utilized liposome-based carriers to deliver cytotoxic drugs to diseased tissues and cells. Interest in the delivery of nucleotides and other gene editing tools such as CRISPR / Cas9 has boosted the inherent potential of liposomal systems as biomimetic carriers that are naturally endocytosed by healthy and diseased cells and tissues. Critical improvements in the encapsulation and stabilization of biomolecules packaged in vesicles for eventual delivery have been the driving force behind this innovation. Current lipid nanoparticle vaccines are hampered by their instability at room temperature, necessitating a complex and expensive logistics chain that includes storage at sub-zero temperatures. Once thawed, lipid nanoparticle vaccines must be stored refrigerated and used within a few days. A key feature of this innovation is its extreme stability, with lipid nanoparticles stable for months at high ambient temperatures rather than just a few days in the refrigerator.

[0005] Prior art on this subject is the use of cellular and molecular methods to induce immune tolerance in vivo. Essentially, all of these methods are similar in that they seek to deliver protein / peptide autoantigens (or nucleic acids encoding the autoantigens) to immune cells in a tolerant context. The aim of these techniques is to induce antigen presentation of the autoantigen by tolerant immune cells such as tolerant dendritic cells (tolDCs). The tolDCs then mediate peripheral immune tolerance to the autoantigen by various mechanisms, including the induction of regulatory T cells (Tregs). Cell-free methods that have been attempted so far include the delivery of nucleic acids encoding the autoantigens in vivo, either directly as plasmid DNA vaccines or indirectly via viral vectors or nanoparticles. Alternatively, the protein structure of the autoantigen itself can be delivered directly in vivo, either as the whole autoantigen protein or as small fragments of it (peptide vaccines). These cell-free methods have the advantage of being relatively easy to manufacture and administer, but have drawbacks as they do not necessarily deliver the payload in the correct way to the correct immune cell type to elicit a therapeutic tolerance response. Furthermore, current cell-free methods can generally only deliver a single class of payload (e.g., autoantigen protein or mRNA encoding an autoantigen protein), a significant limitation that prevents the simultaneous engineering of immune cells with multiple different classes of payloads to optimize tolerogenic immune responses (e.g., co-delivering autoantigen-encoding mRNA and cytokine proteins within the same nanoparticle). Cell-based methods have attempted to engineer and expand Tregs or tolDCs ex vivo and then reinfuse them into patients. These methods have the advantage of ensuring the production of the correct tolerogenic cell type, but each treatment is patient-specific, expensive, and difficult to manufacture. Currently, no compositions of matter or methods exist for the delivery of multiple payloads designed to modify T cells or DCs into therapeutically relevant Tregs or tolDCs in vivo.

[0006] Structurally, lipid nanoparticle carriers are formed by layers of phospholipids and other lipids. The phospholipid head groups are oriented to be exposed to the aqueous environment, making them ideal for functionalizing small molecule components to improve cellular uptake. The phospholipids and other lipids are weakly bound in the bilayer by van der Waals interactions. A major drawback is that lipid nanoparticles dissipate before fully circulating due to the weak lipid binding. Naturally occurring microvesicles (MVs), known as multivesicle liposomes (MVLs), are large (>1 μm) unilamellar liposomes that are known to encapsulate multiple smaller vesicles (200 nm) and form non-concentric chambers. These chambers contain biomolecules such as nucleotides, small molecules, metabolites, and proteins. Synthetically formulated MVLs have been demonstrated to encapsulate and deliver anticancer drugs and other small molecule chemotherapeutic agents. 1, 2 In the prior art, the release of biologically active therapeutic agents was controlled by the difference in salt concentration between the smaller vesicle compartments and the larger encapsulated vesicles. Other soft material-based nanoparticles are polymer, peptide, and protein-based materials. Overview

[0007] In at least one embodiment, the multivesicular lipid nanoparticle composition comprises a plurality of multivesicular lipid nanoparticles. Each multivesicular lipid nanoparticle comprises a carrier lipid nanoparticle comprising a carrier micelle. The carrier micelle is comprised of a carrier lipid selected from the group consisting of phospholipids, phosphorus-free lipids, and combinations thereof, and the carrier lipid nanoparticle has an average diameter of less than 1 micron. The multivesicular lipid nanoparticle composition also comprises at least one antechamber lipid nanoparticle comprising an antechamber reverse micelle. The antechamber reverse micelle is comprised of antechamber lipid selected from the group consisting of phospholipids, phosphorus-free lipids, and combinations thereof. Characteristically, the at least one antechamber lipid nanoparticle has an average diameter of less than 100 nm, with the carrier lipid nanoparticle encapsulating the at least one antechamber lipid nanoparticle.

[0008] In another embodiment, a multivesicular lipid nanoparticle composition is provided that includes a plurality of multivesicular lipid nanoparticles. Each multivesicular lipid nanoparticle includes a carrier micelle lipid nanoparticle that includes a carrier phospholipid layer that is composed of carrier phospholipids and has an average diameter of less than 1 micron; and at least one sub-chamber reverse micelle lipid nanoparticle that includes a sub-chamber phospholipid layer that is composed of sub-chamber phospholipids and has an average diameter of about 100 nm or less. The carrier lipid nanoparticle micelle encapsulates at least one sub-chamber reverse micelle nanoparticle. Critical to this innovation is that a portion of the carrier micelle phospholipid tail is physically cross-linked with a portion of the sub-chamber reverse micelle phospholipid tail, which greatly improves the stability and release kinetics of the present invention compared to similar technologies that lack these stabilizing cross-links. This multivesicular lipid nanoparticle composition is referred to herein as SNIPR (Sub-Nanoparticle Intracellular Payload Release).

[0009] In another aspect, a method for forming the multivesicular lipid nanoparticle composition is provided. The method includes forming a plurality of lipid nanoparticles, the plurality of carrier micelle lipid nanoparticles having an average nanoparticle diameter of less than about 1 micron, and the plurality of sub-chamber reverse micelle lipid nanoparticles having an average diameter of less than 100 nanometers. The plurality of lipid nanoparticles is formed from a lipid nanoparticle-forming composition that includes one or more payloads encapsulated in either the carrier micelles or the sub-chamber reverse micelles, depending on the physicochemical properties of the payload. The plurality of lipid nanoparticles are then subjected to a stabilization cross-linking process that covalently bonds the tail group of a particular phospholipid in the carrier micelle lipid nanoparticles to the tail group of a particular phospholipid in the sub-chamber reverse micelle lipid nanoparticles.

[0010] In another aspect, a method is provided for forming a multivesicular lipid composition comprising a plurality of multivesicular lipids, each multivesicular lipid nanoparticle comprising a carrier phospholipid micelle comprising a carrier phospholipid layer comprised of a carrier phospholipid and having an average diameter of less than 1 micron, and at least one antechamber phospholipid reverse micelle comprising an antechamber phospholipid layer comprised of antechamber phospholipid and having an average diameter of less than 100 nm, the carrier phospholipid micelle encapsulating at least one antechamber phospholipid reverse micelle. The method includes the steps of: a) combining an organic solvent in which the carrier phospholipid, the annular phospholipid, and any hydrophobic therapeutic / diagnostic payload are dissolved with an aqueous solvent in which the hydrophilic therapeutic / diagnostic payload is dissolved; and b) forming a plurality of sub-chamber phospholipid reverse micelles, the plurality of sub-chamber phospholipid reverse micelles having an average nanoparticle diameter of less than 100 nm, the plurality of sub-chamber phospholipid reverse micelles being formed from a first MVL-forming composition comprising a hydrophilic therapeutic / diagnostic payload; and c) forming a plurality of carrier phospholipid micelles having an average nanoparticle diameter of less than 1 micron, said plurality of carrier phospholipid micelles being formed from a second MVL-forming composition comprising a plurality of antechamber phospholipid reverse micelles such that the antechamber phospholipid reverse micelles are encapsulated by the carrier phospholipid micelles (it should be understood that steps b and c can occur simultaneously, particularly during the mixing process); and d) covalently bonding phospholipid tail groups of a plurality of carrier phospholipid micelles and a plurality of antechamber phospholipid reverse micelles by a chemical reaction catalyzed by application of actinic radiation.

[0011] In another embodiment, a conjugated tail group covalent bond attaches the antechamber phospholipid reverse micelles to the inner surface of the carrier lipid phospholipid micelle.

[0012] In another embodiment, a multivesicular lipid nanoparticle composition is provided that delivers a payload designed to induce antigen-specific immune tolerance. Each tolerogenic multivesicular lipid nanoparticle comprises a carrier micelle lipid nanoparticle that includes a carrier phospholipid layer composed of a carrier phospholipid and optionally other lipids that do not contain phosphorus. The carrier phospholipid layer has an average diameter of less than 1 micron; and at least one sub-chamber reverse micelle lipid nanoparticle that includes a sub-chamber phospholipid bilayer having an average diameter equal to or less than about 100 nm, comprising a sub-chamber phospholipid. The carrier lipid nanoparticle micelle encapsulates at least one sub-chamber reverse micelle nanoparticle. In one variation, the sub-chamber reverse micelle nanoparticle encapsulates one or more hydrophilic payloads (e.g., nucleic acids, proteins, or peptides) designed to induce presentation of autoantigen epitopes by antigen-presenting cells that take up the lipid nanoparticle composition. In another variation, the sub-chamber reverse micelle nanoparticles encapsulate one or more hydrophilic payloads (e.g., nucleic acids, proteins, or peptides) designed to induce presentation of autoantigen epitopes by antigen-presenting cells that take up the lipid nanoparticle composition, and the lipid nanoparticle composition also encapsulates one or more payloads (e.g., nucleic acids, proteins, peptides, or small molecule drugs) that induce a tolerogenic phenotype in antigen-presenting cells that take up the lipid nanoparticle composition. In this variation, a hydrophilic payload that induces a tolerogenic phenotype is encapsulated in the sub-chamber reverse micelle, and a hydrophobic payload that induces a tolerogenic phenotype is encapsulated in a carrier micelle lipid nanoparticle having at least one sub-chamber reverse micelle. SNIPR nanoparticles containing a payload designed to induce antigen-specific tolerance in this manner are referred to herein as Xavine. TM They are called nanoparticles. [Brief description of the drawings]

[0013] For a further understanding of the nature, objects, and advantages of the present invention, reference should be made to the following detailed description taken in conjunction with the following drawings, in which like reference numerals represent like elements, and in which: [Figure 1]Schematic of multivesicular SNIPR lipid nanoparticles. [Diagram 2] Schematic diagram of the principal device components (phospholipids and other components) of a SNIPR lipid nanoparticle in one embodiment. [Diagram 3] 3A, 3B, 3C, 3D, 3E, 3F: Schematic of principle device components and examples of payloads. Multiple different payloads can be loaded into the hydrophilic core of the lipid nanoparticle sub-chamber and / or the hydrophobic core of the main lipid nanoparticle. These payloads include, but are not limited to, one or more small molecule drugs in the sub-chamber (A), one or more polynucleotides in the sub-chamber (B), an antibody in the sub-chamber, and one or more small molecule drugs in the main lipid nanoparticle (C), one or more small molecule drugs in the main lipid nanoparticle and one or more small molecule drugs in the sub-chamber (D), different small molecule drugs in each sub-chamber (E), one or more polynucleotides and one or more small molecule drugs in each sub-chamber (F). [Figure 4] Schematic diagram of the principle device components and example payloads for the XavineTM application. [Diagram 5] Schematic fabrication process of SNIPR and XavineTM lipid nanoparticles. [Figure 6] Schematic diagram of phospholipid head-tail interactions. [Figure 7] Modeling of phospholipid bilayers with and without PEG. Minimum free energy5 as a function of curvature for lipid / PEG-lipid bilayers containing 9.15% (solid line), 9.5% (dashed line), and 9.7% (dotted line) PEG-lipid. [Figure 8A] [Figure 8B] Figures 8A and 8B: Analysis of the physical properties of empty and DNA-loaded SNIPR nanoparticles. (A) Average empty SNIPR nanoparticle diameter, polydispersity, and nanoparticle concentration. (B) Average pDNA-loaded SNIPR nanoparticle diameter, polydispersity, and nanoparticle concentration. [Figure 9A] [Figure 9B]Figures 9A and 9B: Visualization and quantification of pDNA loaded onto SNIPR invention after fractionation by size exclusion chromatography. (A) SYBR Safe agarose gel visualization of DNA encapsulated within SNIPR nanoparticles in fractions eluted from a size exclusion column. (B) Quantification of pDNA encapsulated within SNIPR by quantitative real-time PCR in the same fractions eluted from a size exclusion chromatography (SEC) column. [Figure 10A-1] [Figure 10A-2] [Figure 10A-3] [Figure 10B-1.10B-2] [Figure 10B-3] [Figure 10C] [Figure 10D] [Figure 10E-1] [Figure 10E-2] [Figure 10F] Figures 10A-1, 10A-2, 10A-3, 10B-1, 10B-2, 10B-3, 10C, 10D, 10E-1, 10E-2, and 10F: Determination of cellular uptake mechanisms of SNIPR lipid nanoparticles. (A) Quantification of uptake of fluorescently labeled SNIPR nanoparticles by human THP-1 cells at two time points, 30 min and 2 h, across a range of SNIPR concentrations. (B) Quantification of uptake of fluorescently labeled SNIPR nanoparticles by human HEK-293 cell line 24 h after treatment with various SNIPR concentrations. (C) Quantification of uptake of fluorescently labeled SNIPR nanoparticles by various primary immune cells (T cells, natural killer cells, monocytes, dendritic cells, B cells) across different SNIPR concentrations. (D) Overview of uptake mechanisms commonly used by lipid nanoparticles for cellular uptake. (E) Transfection of T cell lines with pDNA-loaded SNIPR nanoparticles is inhibited by the caveolin-mediated uptake inhibitor Methyl-β-cyclodextrin (MbC).(F) Transfection of liver cell lines with pDNA-loaded SNIPR nanoparticles is inhibited by the caveolin-mediated uptake inhibitor MbC. [Figure 11A] [Fig. 11B.11C] [Figure 11D] [Fig. 11E.11F] Figures 11A, 11B, 11C, 11D, 11E, and 11F: Quantification of transfection efficiency with plasmid-loaded SNIPR lipid nanoparticles in multiple cell types. (A) Fluorescence imaging of EGFP expression in human HEK-293 cells treated with SNIPR[DNA] nanoparticles loaded with a plasmid encoding EGFP at various concentrations. (B) Flow cytometric quantification of transfection efficiency (percentage of EGFP positive cells) in HEK-293 cell line cells treated with SNIPR[pDNA-EGFP]. (C) Flow cytometric quantification of EGFP expression intensity (mean fluorescence intensity, MFI of EGFP positive cells) in HEK-293 cell line cells treated with SNIPR[pDNA-EGFP]. (D) Flow cytometric quantification of uptake of fluorescently labeled SNIPR nanoparticles by primary mouse bone marrow-derived macrophages (BMM) cells over a range of SNIPR concentrations after 24 hours. (E) Fluorescence imaging of DiI-labeled SNIPR[pDNA-EGFP] nanoparticle uptake in BMM cells 24 hours after treatment. (F) Fluorescence imaging of EGFP expression in BMM cells 6 days after treatment with DiI-labeled SNIPR[pDNA-EGFP] nanoparticles. [Figure 12-1] [Figure 12-2] [Fig. 12-3.12-4.12-5.12-6] Figures 12-1, 12-2, 12-3, 12-4, 12-5, and 12-6: Comparison of cell viability and transfection efficiency of cells treated with SNIPR or commercial DNA transfection reagents. [Fig. 13-1. 13-2. 13-3. 13-4] [Figure 13-5.13-6] Figures 13-1, 13-2, 13-3, 13-4, 13-5, and 13-6: Evaluation of transfection efficiency with SNIPR[pDNA-EGFP] nanoparticles after storage at different ambient temperatures (-20°C, 4°C, 25°C, or 37°C) for different periods of time (0, 2, 4, 8, 16, or 32 weeks). [Figure 14]Quantification of immunogenicity of SNIPR nanoparticles in vitro. [Figure 15] Maximum tolerated dose test of SNIPR nanoparticles in mice. [Figure 16A] [Figure 16B] 16A and 16B: In vivo immunogenicity and efficacy evaluation of SNIPR-based Xavine™ nanoparticles in mice. (A) Quantification of serum TNFα levels in treated mice 6 hours after the first dose. (B) Quantification of splenic antigen-specific Foxp3+ regulatory T cells in treated mice. [Figure 17A-1, 17A-2, 17A-3] [Fig. 17B-1, 17B-2, 17B-3] Figures 17A-1, 17A-2, 17A-3, 17B-1, 17B-2, and 17B-3: Quantification of the tolerogenic phenotype of human dendritic cells induced by SNIPR-based XavineTM nanoparticles in vitro. [Figure 18-1] [Figure 18-2.18-3] Figures 18-1, 18-2, and 18-3: Biodistribution of SNIPR-based XavineTM nanoparticles in mice. [Figure 19A.19B] Figures 19A and 19B: Quantification of MOG autoantigen expression in the spleens of mice administered SNIPR-based XavineTM nanoparticles. [Figure 20] Quantification of IL-10 secretion by splenocytes isolated from mice treated with SNIPR-based XavineTM nanoparticles. [Figure 21A.21B] Figures 21A and 21B: Evaluation of the effect of SNIPR-based XavineTM nanoparticle treatment on regulatory T cell populations in mice. (A) Quantification of CD4+Foxp3+ Treg populations in splenocytes of mice treated with two different SNIPR-based XavineTM candidates. (B) Quantification of MOG antigen-specific Treg populations in mice treated with two different SNIPR-based XavineTM candidates. [Figure 22]1 shows the effect of MOG peptide restimulation on IFNg release by splenocytes isolated from mice treated with two SNIPR-based Xavine™ candidates. [Figure 23A] [Figure 23B] [Figure 23C] [Figure 23D-1] [Figure 23D-2] Figures 23A, 23B, 23C, 23D-1, and 23D-2: Evaluation of mRNA delivery by SNIPR-023 variant nanoparticles. (A) Schematic of the experimental design. (B) Flow cytometric quantification of DiI-labeled SNIPR-023[mDNA-EGFP] nanoparticle uptake in HEK-293 cells 24 hours after treatment. (C) Flow cytometric quantification of transfection efficiency (percentage of EGFP+ cells) in HEK-293 cells 24 and 72 hours after DiI-labeled SNIPR-023[mDNA-EGFP] nanoparticle treatment. (D) Fluorescence imaging of DiI-labeled SNIPR-023[mDNA-EGFP] nanoparticle uptake and resulting EGFP expression in HEK-293 cells 24 hours after treatment. [Figure 24A] [Figure 24B-1] [Figure 24B-2] [Figure 24C] Figures 24A, 24B-1, 24B-2, and 24C: Various SNIPR lipid nanoparticles co-encapsulating plasmid DNA encoding mCherry and RNA encoding EGFP are efficiently taken up into HEK293 cells and mediate dual transfection. Detailed Description of the Invention

[0014] Reference will now be made in detail to the presently preferred compositions, embodiments and methods of the present invention, which constitute the best mode of practicing the invention currently known to the inventors. The figures are not necessarily to scale. However, it should be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various alternative forms. Thus, the specific details disclosed herein are not to be construed as limiting, but merely as a representative basis for any aspect of the invention and / or for teaching those skilled in the art how to employ the invention in various ways.

[0015] Except in the examples or where otherwise expressly indicated, all numerical amounts herein expressing amounts of materials or conditions of reaction and / or use are understood to be modified by the word "about" in describing the broadest scope of the invention. Practice within the numerical limits described is generally preferred. Also, in the compounds herein, unless expressly indicated to the contrary, C-H bonds are not limited to alkyl, lower alkyl, C 1-6 Alkyl, C 6-10 Aryl, C 6-10 Heteroaryl, -NO2, -NH2, -N(R'R")2, -N(R'R"R"')3 + L-, Cl, F, Br, -CF3, -CCl3, -CN, -SO3H, -PO3H2, -COOH, -CO2R', -COR', -CHO, -OH, -OR', -OM + , -SO3 - M + , -PO3 - M + , -COO - M + , -CF2H, -CF2R', -CFH3, and -CFR'R", where R', R" and R"' are C 1-10 Alkyl group or C 6-18aryl groups; single letters (e.g., "n" or "o") represent 1, 2, 3, 4, 5, and percent, "parts of," and ratio values ​​represent weights; the term "polymer" includes "oligomer," "copolymer," "terpolymer," and the like, and molecular weights listed for polymers refer to weight average molecular weights unless otherwise specified; in the context of this invention, when a group or group of materials is described as suitable or preferred for a given purpose, it is meant that mixtures of any two or more members of that group or group are similarly suitable or preferred; description of components in chemical terms refers to the components at the time they are added in any combination specified herein, and does not necessarily exclude chemical interactions between the components of the mixture once mixed; the initial definition of an acronym or other abbreviation applies to all subsequent uses of the same abbreviation in this specification, mutatis mutandis, to normal grammatical variations of the abbreviation originally defined.

[0016] It is also to be understood that the invention is not limited to the specific embodiments and methods described below, as specific components and / or conditions may, of course, vary. Further, the terminology used herein is used only for the purpose of describing particular embodiments of the invention, and is not intended to be limiting in any way.

[0017] It should also be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" constitute plural references unless the context clearly dictates otherwise. For example, reference to a singular element is intended to consist of multiple elements.

[0018] The term "comprising" is synonymous with "including," "having," "containing," or "featuring." These terms are inclusive and open-ended and do not exclude additional, non-repetitive elements or method steps.

[0019] The term "consisting of" excludes any element, step, or ingredient not specified in the claim. When this term appears in a clause in the body of the claim rather than immediately following a preamble, it limits only the elements listed in that clause and does not exclude other elements from the claim as a whole.

[0020] The phrase "consisting essentially of" limits the scope of a claim to those specified materials or steps that, in addition to those specified materials or steps, do not materially affect the basic and novel characteristics of the claimed subject matter.

[0021] The term "composed of" means "including," "comprising," or "consisting of." Typically, this phrase is used to indicate that an object is formed from a material.

[0022] With respect to the terms "comprising," "consisting," and "consisting essentially of," when one of these three terms is used in this specification, the presently disclosed and claimed subject matter may include use of either of the other two terms.

[0023] It should also be understood that ranges of integers explicitly include all intervening integers. For example, the integer range 1 to 10 explicitly includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range 1 to 100 includes 1, 2, 3, 4..... 97, 98, 99, 100.

[0024] Throughout this application, when publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0025] By "reverse micelle" is meant a micelle in which the roles of the nonpolar and polar phases are reversed and the orientation of the surfactant molecules is reversed so that their head groups point toward the enclosed volume containing the polar phase.

[0026] By "tolerogenic payload" is meant a payload (proteins, peptides, cytokines, polynucleotides, small molecules, and combinations thereof) that induces a tolerogenic phenotype upon uptake into one or more cell types of the immune system.

[0027] For non-spherical bodies, "mean diameter" means twice the average shortest distance from the center of mass of the object to the surface. For spheres, diameter has its usual meaning. "Mean diameter" is sometimes simply called "diameter".

[0028] Abbreviation:

[0029] "CL" means cardiolipin.

[0030] "DLS" means dynamic light scattering.

[0031] "EV" means extracellular vesicles.

[0032] "MVL" means multivesicular lipid nanoparticles.

[0033] "PC" means phosphatidylcholine.

[0034] "PC" means phosphatidylcholine.

[0035] "PE" means phosphatidylethanolamine.

[0036] "PEG" means polyethylene glycol.

[0037] "PS" means phosphatidylserine.

[0038] "PA" means phosphatidic acid.

[0039] "PI" means phosphatidylinositol.

[0040] "PG" means phosphatidylglycerol.

[0041] "siRNA" means silencing RNA.

[0042] "SNIPR" stands for Sub-Nanoparticle Intracellular Payload Release.

[0043] Generally, a multivesicular lipid nanoparticle composition is provided that includes a plurality of multivesicular lipid nanoparticles. Each multivesicular lipid nanoparticle includes a carrier lipid nanoparticle. The carrier lipid nanoparticle includes a carrier micelle (i.e., a carrier lipid nanoparticle micelle). The carrier micelle is comprised of a carrier lipid selected from the group consisting of phospholipids, phosphorus-free lipids, and combinations thereof, and the carrier lipid nanoparticle has an average diameter of less than 1 micron. The multivesicular lipid nanoparticle composition also includes at least one antechamber lipid nanoparticle (e.g., a micelle or reverse micelle) that includes antechamber reverse micelles. The antechamber reverse micelles are comprised of antechamber lipids selected from the group consisting of phospholipids, phosphorus-free lipids, and combinations thereof. Characteristically, at least one antechamber lipid nanoparticle has an average diameter of less than 100 nm, with the carrier lipid nanoparticle encapsulating the at least one antechamber lipid nanoparticle.

[0044] Referring to FIG. 1, a schematic diagram of a multivesicular lipid nanoparticle of the present invention is shown. The multivesicular lipid nanoparticles designed herein are referred to as "SNIPR". The multivesicular lipid nanoparticle 10 comprises a carrier micelle nanoparticle 12 and a sub-chamber reverse micelle nanoparticle 14. The carrier micelle nanoparticle 12 is a sac-like structure encapsulating at least one sub-chamber reverse micelle nanoparticle 14. In one embodiment, the carrier micelle nanoparticle 12 encapsulates a plurality of sub-chamber reverse micelle nanoparticles 14 (e.g., 1-100 or 2-10). In a variant, the carrier micelle 12 is comprised of a carrier lipid selected from the group consisting of phospholipids, phosphorus-free lipids, and combinations thereof. In one embodiment, the carrier micelle nanoparticle 12 comprises a carrier phospholipid monolayer 16 comprising a carrier phospholipid. Typically, the carrier micelle nanoparticle 12 has a diameter (e.g., the average diameter of a plurality of particles or an individual particle diameter) of less than 1 micron. In some embodiments, the carrier micelle nanoparticles 12 have a diameter (e.g., average diameter of the plurality of particles or individual particle diameter) of less than 1 μm, 800 nm, 600 nm, 500 nm, 400 nm, or 300 nm, in order of increasing preference. In further embodiments, the carrier micelle nanoparticles 12 have a diameter (e.g., average diameter of the plurality of particles or individual particle diameter) of greater than 500 nm, 400 nm, 300 nm, 100 nm, 150 nm, or 200 nm, in order of decreasing preference. In a variation, the antechamber reverse micelle 14 is comprised of antechamber lipids selected from the group consisting of phospholipids, phosphorus-free lipids, and combinations thereof. In one embodiment, the antechamber reverse micelle nanoparticles 14 include antechamber phospholipid monolayer 18 that includes antechamber phospholipids. Typically, the antechamber reverse micelle nanoparticles 14 have a diameter (e.g., average diameter of the plurality of particles or individual nanoparticle diameter) of about 100 nm or less. In one embodiment, the sub-chamber reverse micelle nanoparticles 14 have a diameter (eg, the average diameter of a plurality of nanoparticles or the diameter of an individual nanoparticle) of about 20 nm to about 100 nm.In one embodiment, the antechamber reverse micelle nanoparticles have a diameter (e.g., average diameter of the plurality of nanoparticles or diameter of an individual nanoparticle) of less than 150 nm, 125 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, or 50 nm, in order of increasing preference. In a further embodiment, the antechamber reverse micelle nanoparticles 14 have a diameter (e.g., average diameter of the plurality of nanoparticles or diameter of an individual nanoparticle) of greater than 10 nm, 15 nm, 20 nm, 30 nm, 40 nm, or 50 nm, in order of increasing preference. The multivesicular lipid nanoparticles 10 include an exterior hydrophilic surface 20 and an interior hydrophobic surface 22. In some embodiments, polyethylene glycol groups (PEG) are attached to the exterior surface 20 of the multivesicular micelles 10. In some embodiments, PEG groups are attached to the interior surface of the antechamber reverse micelles 14. In some embodiments, cholesterol is incorporated into the interior hydrophobic surface 22 and the antechamber reverse micelle nanoparticles 14. Characteristically, an irreversible bond 24 (e.g., a covalent bond via acrylate polymerization) attaches the antechamber reverse micelle nanoparticle 14 to the inner surface 22 of the carrier micelle nanoparticle 12. In some embodiments, a reversible bond 24 (e.g., a disulfide bond, a pH-sensitive bond, an electrostatic interaction, etc.) attaches the antechamber reverse micelle nanoparticle 14 to the inner surface 22 of the carrier micelle nanoparticle 12. The carrier micelle nanoparticle 12 can also encapsulate a payload 28. Similarly, the antechamber reverse micelle nanoparticle 14, which is also saccular, can independently encapsulate a payload 30, which may be the same as or different from the payload 28. It should be appreciated that the irreversible or reversible attachment of the antechamber reverse micelles to the inner surface of the carrier micelle provides a significant improvement in the stability of the SNIPR multivesicular lipid nanoparticles compared to other lipid nanoparticle formulations and is a key feature of the present innovation.

[0045] As described above, the therapeutic / diagnostic payload is encapsulated by at least one of the antechamber reverse micelles and / or carrier micelles. In a variant, the therapeutic / diagnostic payload is selected from the group consisting of small molecule drugs, polynucleotides, proteins, peptides, ribonucleoproteins, and combinations thereof. Examples of therapeutic / diagnostic payloads include fluorescent proteins, peptides, cytokines, antibodies, and combinations thereof. Examples of therapeutic immune tolerance-inducing payloads include immune tolerance-inducing proteins, peptides, cytokines, polynucleotides, small molecules, and combinations thereof. Examples of therapeutic payloads include autoantigen proteins, autoantigen peptides, polynucleotides encoding autoantigen proteins, polynucleotides encoding autoantigen peptides, and combinations thereof. Further examples of therapeutic payloads include one or more fusion proteins comprising one or more autoantigen epitopes, polynucleotides encoding fusion proteins comprising one or more autoantigen epitopes, and combinations thereof. Further examples of therapeutic payloads include immune tolerance-inducing proteins, peptides, cytokines, polynucleotides, small molecules, autoantigenic proteins, autoantigenic peptides, fusion proteins comprising one or more autoantigenic epitopes, polynucleotides encoding autoantigenic proteins, polynucleotides encoding autoantigenic peptides, polynucleotides encoding fusion proteins comprising one or more autoantigenic epitopes, and combinations thereof.

[0046] FIG. 2 shows the chemical structures of the components that make up one variation of the SNIPR multivesicular lipid nanoparticle.

[0047] FIG. 3 illustrates a method and apparatus for delivering biomolecules of interest (e.g., polynucleotides, small molecule drugs, or other biologics) to healthy and / or diseased cells and / or tissues using the multivesicular lipid nanoparticles of FIG. 1. Advantageously, the multivesicular lipid nanoparticles provide a highly ordered, densely packed structure. In one embodiment, the multivesicular lipid nanoparticles comprise a novel combination of four components of phospholipids, ionizable lipids, cholesterol, and optionally lipophilic dyes (FIG. 2). Advantageously, the SNIPR multivesicular lipid nanoparticles can be used to deliver the following therapeutic / diagnostic assets (i.e., payloads): (i) small molecule drugs, (ii) polynucleotides such as plasmid DNA (pDNA), silencing RNA (siRNA), messenger RNA (mRNA), (iii) and macromolecular proteins such as fluorescent proteins, antigenic proteins, and allergens, cytokines, antibodies; and combinations thereof. The abbreviation SNIPR[payload] refers to a multivesicular lipid nanoparticle containing a payload, which may be any compound, drug, polynucleotide, or other biological agent that can be delivered by the multivesicular lipid nanoparticle.

[0048] FIG. 4 illustrates a method and apparatus for using the multivesicular lipid nanoparticles of FIG. 1 to deliver a biomolecule of interest (e.g., a polynucleotide, small molecule drug, or other biologic) to immune cells and / or tissues for the purpose of inducing immune tolerance to one or more antigens in a subject identified as in need of such induction. Multivesicular lipid nanoparticles designed to encapsulate and so deliver a therapeutic antigen-specific tolerance-inducing payload to immune cells and / or tissues are referred to herein as "Xaviers' nanoparticles." TM ", "Xavine TM "Multivesicular lipid nanoparticles" or "Xavine TM MVL. Advantageously, Xavine TM The size of MVL (e.g., 10-200 nm) is appropriate for uptake by dendritic cells via caveolin-mediated endocytosis. TMMVL fuses with endosomes and delivers the payload to the cytoplasm of dendritic cells (DCs), where it can exert its therapeutic effect. In contrast, liposomes and lipid nanoparticle structures of other sizes are taken up by different cellular mechanisms (such as clathrin-mediated endocytosis or micropinocytosis), resulting in trafficking to and destruction by lysosomes in the cell, without the desired therapeutic effect. Advantageously, Xavine TM MVL can be used to deliver the following therapeutic assets (i.e., payloads): (i) small molecule drugs, (ii) polynucleotides such as plasmid DNA (pDNA), silencing RNA (siRNA), and messenger RNA (mRNA), (iii) macromolecular proteins such as antigenic proteins, allergens, cytokines, and antibodies, and combinations thereof. This is advantageous because combinations of different payloads are required to elicit the desired therapeutic effect to treat or prevent autoimmune diseases, allergies, and food hypersensitivity. This is the most important aspect of the present invention, because Xavine TM MVL can deliver one or more tolerizing signals to immune cells and / or tissues simultaneously with one or more payloads that induce antigen-presenting cells to present one or more self-antigen epitopes. This is in contrast to current technology, where antigen-presenting cells rely only on defaulting to a tolerized state when they are forced to present self-antigen epitopes in the absence of inflammatory signals. For example, administering a tolerizing vaccine when a patient has an undetected viral or bacterial infection could later trigger self-antigen presentation in an inflammatory environment, exacerbating autoimmunity instead of improving it. Another example where current passive tolerizing vaccines may be administered in a strongly pro-inflammatory environment is when they are administered to patients with ongoing autoimmune disease, which is the very patient population that is being treated. Table 1 shows Xavine, a vaccine that aims to actively induce a tolerizing state in antigen-presenting cells. TMThe payload of MVL is shown in Table 2. Xavine is a marker for the induction of epitope presentation of autoantigens known to be pathogenic in autoimmune diseases. TM A list of MVL payloads.

[0049] Table 1. Xavine TM Tolerance factor payloads available for multivesicular lipid nanoparticles. [Table 1]

[0050] Table 2. Xavine TM Autoantigen expression and presentation payloads available for multivesicular lipid nanoparticles. [Table 2] JPEG2025502274000004.jpg216167JPEG2025502274000005.jpg205167JPEG2025502274000006.jpg162167

[0051] As described above, the carrier micelles and the antechamber reverse micelles can be independently comprised of antechamber lipids selected from the group consisting of phospholipids, phosphorus-free lipids, and combinations thereof. In a variation, the carrier micelles and / or at least one antechamber lipid nanoparticle comprises a lipid described by the formula: JPEG2025502274000007.jpg12167 here HD is head group; LK is Linking Group; TL, tail base; n is an integer representing the number of tail groups. In one embodiment, n is 1, 2, 3, or 4. It should be understood that when there is more than one tail group, the tail groups can be the same or different. Typically, the tail group comprises a branched or unbranched C6-25 aliphatic chain. In some embodiments, the tail group comprises one or more carbon-carbon double bonds that can be cis or trans. In some embodiments, the lipid comprises multiple aliphatic chains that are symmetrical to each other. In some embodiments, one or more carbon atoms of the aliphatic chain can be substituted with O, N, S, or carbonyl. In some embodiments, the aliphatic chain can comprise one or more ester groups. In one embodiment, when there is more than one tail group, the tail groups can be the same or different.

[0052] In one embodiment, the head group HD comprises a moiety selected from the group consisting of: JPEG2025502274000008.jpg197166JPEG2025502274000009.jpg230167JPEG2025502274000010.jpg184167JPEG2025502274000011.jpg32167Here, the wavy lines represent the attachment points to the lipid structure moieties.

[0053] Examples of lipids include: JPEG2025502274000012.jpg184167JPEG2025502274000013.jpg176167JPEG2025502274000014.jpg205167JPEG2025502274000015.jpg75167 and combinations of these.

[0054] As mentioned above, the size of these lipid nanoparticles is controlled by the length of the tail group of the phospholipids used to construct the lipid monolayer. The following formula shows the general structure of a phospholipid: JPEG2025502274000016.jpg48167

[0055] where O=C-R2, O=C-R3 are tail groups; and X is a head group substituent (e.g., PC, PE, PS, PA, PI, PG, or CL). As described above, the tail group can include a branched or unbranched C6-25 aliphatic chain. Thus, R2 and R3 are independently C6-25 aliphatic chains. In some embodiments, the tail group includes one or more carbon-carbon double bonds that can be cis or trans. In some embodiments, the lipid includes multiple aliphatic chains that are symmetrical to each other. In some embodiments, one or more carbon atoms of the aliphatic chain can be substituted with O, N, S, or carbonyl. In some embodiments, the aliphatic chain can include one or more ester groups. In some embodiments, the lipid nanoparticle phospholipid monolayer is a combination of phospholipids having 14 to 17 carbons. At least a portion of the tail groups of the lipid nanoparticle phospholipid can include double bonds, particularly cis double bonds. Furthermore, at least a portion of the tail groups for the lipid nanoparticle phospholipids can include photoactive groups that can be crosslinked when the phospholipid is included in a phospholipid monolayer. Examples of such photoactive groups include moieties with ethylenic unsaturation, such as acrylate groups. Furthermore, at least a portion of the head groups of the lipid nanoparticle phospholipids can include ionizable structures that have a positive charge in a low pH environment (such as an acidic buffer or in an intracellular endosome) when the phospholipid is included in a phospholipid bilayer, but a neutral charge in a neutral pH environment (such as a neutral buffer or an extracellular environment in the body). Furthermore, at least a portion of the head groups of the lipid nanoparticle phospholipids can include structures that have a positive charge regardless of the pH of the environment when the phospholipid is included in a phospholipid bilayer.

[0056] Chemical modification of the phospholipid tail groups can promote and / or stabilize the interlayer association between the compartments of MVLs. This key structural mechanism stabilizes the payload-encapsulated SNIPR multivesicular lipid nanoparticles and extends their time to release. The key novelty of our innovation lies in the configuration of the primary device, in which the outer surface of the subcompartmental reverse micelle nanoparticles is thus covalently linked to the inner surface of the carrier micelle. In particular in the primary device, the bilayer composition is stabilized by a network of UV-conjugated tail group covalent bonds. As a result, the lipid bilayer surface of the lipid nanoparticles is composed of bilayer rafts that shift based on relieving the particle's structural distortion. While in nature, cells undergo this process to increase the stability of the structure through the action of transmembrane protein complexes, our system achieves a similar effect based on a series of covalent bonds between the phospholipid tail groups.

[0057] The lipid nanoparticles include phospholipids or residues thereof. The lipids used to form the lipid nanoparticle bilayer are shown in Table 3. In this context, the term "residue" refers to the reaction products (e.g., crosslinks) of these phospholipids when such groups are included in the phospholipid bilayer.

[0058] Table 3. Phospholipids used in multivesicular lipid nanoparticles. [Table 3] JPEG2025502274000018.jpg192163

[0059] The primary device of lipid nanoparticles consists of four unique phospholipids, an ionizable lipid, cholesterol, and an optional lipophilic dye, organized in a molar ratio that promotes a monodisperse distribution of lipid nanoparticles (100-300 nm in diameter). The principle device consisted of: (1) 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, (2) 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (chloride salt), (3) 1-palmitoyl-2-[16-(acryloyloxy)palmitoyl]-sn-glycero-3-phosphorylcholine, (4) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), (5) 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxane, (6) cholest-5-en-3β-ol, and (7) (2Z)-2-[(E)-3-(3,3-dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole perchlorate.In variants, the residues are present in an amount between 9 mole percent and 95 mole percent of the total amount of phospholipid; 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))](chloride salt) or a residue thereof is present in an amount between about 0 mole percent and about 1 mole percent of the total amount of phospholipid; 1-palmitoyl-2-[16-(acryloyloxy)palmitoyl]-sn-glycero-3-phosphorylcholine or a residue thereof is present in an amount between about 1 mole percent and about 10 mole percent of the total amount of phospholipid; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt) or a residue thereof is present in an amount between about 0 mole percent and about 1.5 mole percent of the total amount of phospholipid; the xolane or residue thereof is present in an amount of about 4 mole percent to about 50 mole percent of the total amount of phospholipid; the cholest-5-en-3β-ol or residue thereof is present in an amount of about 0 mole percent to about 38.5 mole percent of the total amount of phospholipid; the (2Z)-2-[(E)-3-(3,3-dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole perchlorate or residue thereof is present in an amount of about 0 mole percent to about 0.1 mole percent of the total amount of phospholipid; and the (2E)-3-octadecyl-2-[(E)-3-(3-octadecyl-1,3-benzoxazol-3-ium-2-yl)prop-2-enylidene]-1,3-benzoxazole perchlorate or residue thereof is present in an amount of about 0 mole percent to about 0.1 mole percent of the total amount of phospholipid.

[0060] The organization and stability of lipid nanoparticle devices is facilitated by crosslinking of phospholipid tail groups (e.g., 16:0 acrylate PC phospholipid tail groups) via reactive terminal olefins on at least a subset of the phospholipid tail groups upon formation of lipid nanoparticles in aqueous solution. Crosslinking of the tail groups is driven by a photoinitiator (e.g., 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone [HEM]), which upon exposure to UV light forms a radical intermediate at the carbon atoms in the reactive terminal olefin, specifically the carbon atom furthest from the carbonyl group (e.g., 16:0 acrylate PC, carbon-16 of the reactive terminal olefin). The radical intermediate reacts in a chain fashion with other adjacent tail groups that contain terminal olefins until there are no more reactive terminal olefins. A critical aspect of this organization is the ratio of phospholipids with and without reactive terminal olefins. Too much crosslinking results in lipid nanoparticles that are unable to release their payload, whereas too little results in unstable lipid nanoparticles that release their payload prematurely. Additionally, the organization and stability of lipid nanoparticles encapsulating payloads is facilitated by enhancing the charge characteristics of the 16:0 lysyl-PG phospholipid head groups and the ionizable lipids (e.g., D-Lin-KC2-DMA). During the formation of lipid nanoparticles, the positively charged 16:0 lysyl-PG head groups and the D-Lin-KC2-DMA ionizable head groups are attracted to the negatively charged payload (e.g., the phosphate backbone of nucleic acids). Upon association of the lipid nanoparticle monolayer, the payload molecules are captured or encapsulated in the newly formed lipid nanoparticles. As a result, lipid nanoparticles facilitate high encapsulation efficiency (>60%) of payload molecules. The central importance of this innovation is the ratio of permanently positively charged head groups (16:0 lysyl-PG phospholipids) to transiently positively charged head groups (D-Lin-KC2-DMA lipids). Without sufficient positive charges, lipid nanoparticles cannot initiate and maintain association with negatively charged payloads such as nucleic acids during assembly, nor can they fuse efficiently with endosomes for proper payload release.However, lipid nanoparticles with a high positive charge are toxic and can associate with negatively charged cell membranes, a mechanism that facilitates uptake by disrupting them before payload release. The presence of ionizable head groups (e.g., on D-Lin-KC2-DMA lipids) allows for the induction of a temporary strong positive charge during preparation (facilitating payload encapsulation) and within endosomes (facilitating escape from endosomes and delivery of payload to the cytoplasm), while allowing the lipid nanoparticles to assume a neutral charge in other environments, such as inside the body (thereby minimizing toxicity and destructive uptake by cells). The presence of a constitutively positively charged head group (on the 16:0 lysyl-PG phospholipid) maintains the association between the lipid bilayer and the negatively charged payload regardless of the local environment, and also increases the likelihood that the lipid bilayer will adopt a hexagonal formation in endosomes, facilitating endosomal fusion and release of the payload into the cytoplasm. The presence of cholesterol in the lipid nanoparticle lipid bilayer also facilitates endosomal fusion and payload transport to the cytoplasm.

[0061] The structural organization of phospholipids in lipid nanoparticles is relevant to certain embodiments of the invention. In particular, the length of the tail group drives the curvature, and therefore the diameter, of the liposomal nanoparticle. Phospholipids containing tail groups with shorter carbon lengths tend to form shorter angles with other similar phospholipids (Figure 6). As the non-polar tail groups become shorter, the curvature of the bilayer is driven by the opposing forces of the head groups. 3 Another important aspect of the structural organization is the permeability of lipid nanoparticles, which is driven by the cis or trans conformation of unsaturated phospholipids. From first principles, as Gillespe et al. showed, these parameters control the release rate of the payload from lipid nanoparticles. Another important aspect of the structural organization is the covalent attachment of water-soluble poly(ethylene glycol) (PEG) to the surface of lipid nanoparticles. As shown in Figure 7, the curvature of the bilayer functionalized with PEG is energetically more stable compared to the bilayer not functionalized with PEG. 5 .

[0062] In another embodiment, a method of forming a multivesicular lipid nanoparticle composition as described above is provided. The method includes forming a plurality of multivesicular lipid nanoparticles, the plurality of multivesicular lipid nanoparticles having an average nanoparticle diameter of less than about 1 micron. The plurality of multivesicular lipid nanoparticles is formed from a combination of two MVL-forming compositions comprising one or more payloads. The first MVL-forming composition comprises the hydrophobic components of the MVL composition (e.g., phospholipids and any hydrophobic payload(s) are combined in a non-aqueous solvent such as ethanol, methanol, DMSO, or DMF). More specifically, the aqueous fraction of the MVL-forming composition comprises one or more additional hydrophilic therapeutic / diagnostic payloads, and / or the organic fraction of the anterior chamber lipid-forming composition comprises one or more hydrophobic therapeutic / diagnostic payload(s). The second MVL-forming composition comprises the hydrophilic components of the MVL composition (typically one or more hydrophilic payloads) in an aqueous solution of appropriate pH and molarity to induce a positive charge on the ionizable lipid head groups and promote the spontaneous formation of multivesicular lipid nanoparticles when mixed with the first MVL composition. In an alternative embodiment, the first multivesicular lipid nanoparticle-forming composition is selected from the group consisting of 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (chloride salt), 1-palmitoyl-2-[16-(acryloyloxy)palmitoyl]-sn-glycero-3-phosphorylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[ Methoxy(polyethylene glycol)-2000] (ammonium salt), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, cholest-5-en-3β-ol, and (2Z)-2-[(E)-3-(3,3-dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole perchlorate, which are typically mixed together in a solvent. One or more hydrophobic payloads are optionally combined with the first multivesicular lipid nanoparticle-forming composition.In this variation, the second multivesicular lipid nanoparticle-forming composition comprises a 25 mM acetic acid solution (prepared to pH 4 with acetic acid and sodium acetate) and, optionally, one or more hydrophilic payload(s). The first and second multivesicular lipid nanoparticle-forming compositions are then mixed in a 1:3 ratio via a mixing robot such as the NanoAssemblr device (Precision Nanosystems, Inc.). The multivesicular lipid nanoparticle raw material is then crosslinked with actinic radiation (e.g., UV light), washed, concentrated, and filter-sterilized prior to use.

[0063] In a variant, Xavine TM Nanoparticles are produced using an improved version of the above method. In this variation, the payload is one or more hydrophilic biomolecules that induce the presentation of one or more antigens on the cell surface of treated antigen-presenting cells. In a further variation, the first payload is one or more hydrophilic biomolecules that induce the presentation of one or more antigens on the cell surface of treated antigen-presenting cells, and the second payload is one or more hydrophilic biomolecules that induce a tolerogenic phenotype in antigen-presenting cells (e.g., pDNA encoding IL-10, TGFb1, or IL-27), and / or one or more hydrophobic biomolecules that induce a tolerogenic phenotype in antigen-presenting cells (e.g., rapamycin).

[0064] The following examples illustrate various embodiments of the invention, and those of ordinary skill in the art will recognize many variations that are within the spirit and scope of the claims.

[0065] method

[0066] solution:

[0067] The following solutions can be made up in advance and stored as needed:

[0068] 1. 1x Phosphate Buffer (1x PBS)

[0069] 2. 125 mM acetate buffer, pH 4.0 (5x)

[0070] a. Dissolve 465 mg of sodium acetate in 180 mL of water.

[0071] b. To the sodium acetate solution, add 1109 μL of glacial acetic acid (17.4 M).

[0072] c. Adjust the pH to 4.0 with glacial acetic acid or 10N sodium hydroxide.

[0073] d. Dilute the solution to 200 μL.

[0074] 3. 25 mM acetate buffer, pH

[0075] a. Dilute 10 mL of 5x acetate buffer to 50 mL with water.

[0076] b. Lipid stock (prepared in ethanol, stored at -20°C)

[0077] 4. 100 mg / mL 16:1-(Δ9-cis)-PC lipid

[0078] 5. 5 mg / mL 16:0 lysyl-PG lipid

[0079] 6. 10 mg / mL 16:0-16:0 acrylate PC lipid

[0080] 7. 10 mg / mL 16:0 PEG2000 PE lipid

[0081] 8. 100 mg / mL D-Lin-KC2-DMA ionizable lipid

[0082] 9. 10 mg / mL cholesterol

[0083] 10. 1 mg / mL DiI - 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate ('DiI'; DiI C18 (3))

[0084] The following solutions should be made up immediately before use:

[0085] 11. 1% (v / v) Tetramethylethylenediamine (TEMED)

[0086] a. Add 3 μL of TEMED to 297 μL of 25 mM sodium acetate buffer.

[0087] b. Vortex thoroughly and store at room temperature away from light.

[0088] 12. 0.2% (w / v) 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (HEM)

[0089] a. Weigh out 10 mg of HEM and dissolve it in 500 μL of ethanol.

[0090] b. Dilute a 10 μL aliquot to 100 μL with ethanol.

[0091] c. Vortex thoroughly and store at room temperature away from light.

[0092] Preparation of starting materials:

[0093] The lipid formulation is determined according to the following table: [Table 4]

[0094] 13. To a microcentrifuge tube, add the following volumes from the lipid stock: (Only 375 μL is used in the final product, but an excess is needed to operate the Precision Nanosystems NanoAssembler):

[0095] a. 323 μL of ethanol

[0096] b. 12.12 μL of 16:1-(Δ9-cis)-PC lipid

[0097] b. 30.7 μL of 16:0 PG lipid

[0098] c. 13.34 μL of 16:0-16:0 acrylic acid PC lipid

[0099] d. 68.5 μL of 16:0 PEG2000 PE lipid

[0100] e. 53.5 μL of D-Lin-KC2-DMA ionizable lipid

[0101] f. 234.0 μL of Cholesterol

[0102] g. 15.5 μL of DiI

[0103] 14. Vortex the lipid mixture thoroughly and store at room temperature, away from light, for 3 hours.

[0104] 15. Under sterile conditions, prepare 1500 μL of plasmid "payload" solution. (Note that only 1125 μL will be used for the final product, but an excess amount is required for operation of the Precision Nanosystems NanoAssembler).

[0105] a. The final concentration of the plasmid is 203 μg / ml, resulting in an N / P ratio of 6:1, and the final concentration of the acetate buffer is 25 mM, "1x".

[0106] b. Determine the concentration of the plasmid stock solution.

[0107] c. To a microcentrifuge tube, add 304.5±5% μg of plasmid, 300 μL of "5x" 125 mM acetate buffer, and enough water to bring the final volume to 1500 μL.

[0108] 16. Insert the cartridge into a microfluidic mixing device (such as a Precision NanoSystems Nano Assembler).

[0109] 17. Load a 3mL BD syringe with "1x" 25mM acetate buffer and insert it into the left "aqueous" channel of the cartridge.

[0110] 18. Load a 1mL BD syringe with ethanol and insert it into the right "organic" channel of the cartridge.

[0111] 19. Rinse and prepare the cartridge using the following settings: [Table 5]

[0112] 20. Take up the plasmid solution into a 3mL BD syringe and insert into the left "aqueous" channel of the cartridge.

[0113] 21. Take the lipid solution into a 1 mL BD syringe and insert it into the right "organic" channel of the cartridge.

[0114] 22. Prepare a 1500 μL sample using the following settings: [Table 6]

[0115] 23. Collect samples into microcentrifuge tubes and gently vortex for 15 seconds.

[0116] 24. Add 26 μL of 1% TEMED solution.

[0117] 25. Add 10.12 μL of 0.2% HEM solution.

[0118] 26. Gently vortex the solution for 15 seconds.

[0119] 27. Place the sample under the UV light for 90 seconds.

[0120] 28. Gently vortex the solution for 15 seconds.

[0121] 29. Add 10 mL of 1x PBS to a 50 kDa Amicon-Ultra 15 centrifugal filter.

[0122] 30. Add the SNIPR solution to the centrifugal filter.

[0123] 31. Centrifuge the samples at 1000rcf for 30 minutes.

[0124] 32. Dilute the residue to 2 mL with 1x PBS and load onto an IZON qEV2 35 nm SEC column.

[0125] 33. Elute the sample with 1xPBS, collect and discard the first 14 mL fraction.

[0126] 34. Collect the next 15 mL fraction and sterilize by filtering through a 45 μm PES syringe filter.

[0127] 35. Add the SNIPR solution to a 50 kDa Amicon-Ultra 15 centrifugal filter.

[0128] 36. Centrifuge the sample at 1000 rcf for 30 minutes and collect the residue.

[0129] result

[0130] Figures 8A and 8B are plots of the hydrodynamic diameter of SNIPR [EMPTY] and SNIPR [pDNA] nanoparticles. The SNIPR manufacturing protocol produces SNIPR nanoparticles with an average hydrodynamic diameter of approximately 190 nm and low polydispersity. A batch of empty SNIPR (SNIPR [EMPTY]) nanoparticles and a batch of SNIPR nanoparticles containing plasmid DNA (SNIPR [pDNA]) were produced using the standard SNIPR manufacturing protocol. The hydrodynamic size and polydispersity of the produced SNIPR nanoparticles were quantified by variable resistive pulse detection (TRPS) using a qNano Gold nanoparticle analyzer (Izon Science, New Zealand). Figure 8A shows the analysis of SNIPR [EMPTY] nanoparticles produced without encapsulating a payload. The figure reveals a strong signal indicating an average diameter of 192 nm. The concentration of the produced SNIPR [EMPTY] nanoparticles was 1.79x1011 LNP / ml. Figure 8B shows the analysis of SNIPR [pDNA] nanoparticles, demonstrating that although the final concentration was somewhat lower (1.02x1011 LNP / ml), their size (mean diameter 193 nm) and polydispersity were virtually identical to the SNIPR [EMPTY] nanoparticles mentioned above.

[0131] Figures 9A and 9B provide visualization and quantification of pDNA-encapsulated SNIPR[pDNA] nanoparticles. Figure 9A shows the standard SNIPR manufacturing protocol that was used to produce a batch of SNIPR[pDNA] nanoparticles containing plasmid DNA encoding EGFP (pEGFP). After SNIPR[pDNA] stabilization by UV crosslinking, an aliquot of the product was taken before passing through a SEC column ("pre" or "prefilter SNIPR"). The remaining product was passed through a SEC column, and aliquots were taken from each elution fraction (1-13). The aliquots were visualized by gel electrophoresis (1% agarose gel containing SYBR Safe at the manufacturer's recommended concentration). Plasmid DNA encapsulated within SNIPR was predominantly encapsulated in wells that encapsulated elution fractions 1 and 2, and in small amounts in wells that encapsulated elution fraction 3 (arrows indicate SNIPR[pDNA]). Almost no unencapsulated pDNA was found in any of the elution fractions, with the majority in elution fraction 1 (arrows labeled "free pDNA" and "free pDNA (supercoiled)"), indicating very high encapsulation efficiency of pDNA within SNIPR lipid nanoparticles. Lane key: Lane 1 = 1 kb size ladder, Lane 2 = unspiked, Lanes 3-15 = elution fractions 1-13, respectively, Lane 16 = prefiltered SNIPR, Lane 17 = input DNA (pEGFP from an aliquot of the aqueous input fraction used to produce this SNIPR batch), Lanes 18-20 = unspiked. In Figure 9B, the total pDNA encapsulated within SNIPR[pDNA] was calculated by QPCR. After stabilizing SNIPR[pDNA] by UV crosslinking, an aliquot of the product was taken ("Pre") before passing it through the SEC column. The remaining product was passed through the SEC column and aliquots were taken from each elution fraction (1-13). This aliquot was used as a template in a QPCR assay designed to amplify a sequence within the EGFP reporter gene encoded by SNIPR-encapsulated pEGFP. Serial dilutions of the input pEGFP plasmid (without SNIPR encapsulation) were also used as template in the same QPCR assay to establish a standard curve of pEGFP concentrations.All QPCR reactions were performed on the same plate, under the same reaction conditions, and with the same primers and QPCR reaction mix (SYBR Green qPCR, MedChemExpress, Cat no. HY-K0523) to ensure consistency. The amount of plasmid DNA in each elution fraction was calculated from the standard curve using Prism statistical analysis software (GraphPad Software, San Diego, CA). Consistent with the results shown in (A), the majority of the detected plasmids were contained in elution fractions 1 and 2, with a small amount in elution fraction 3. Almost no pDNA was detected in the other elution fractions. Key: C1-C13 = elution fractions 1-13, respectively, Pre = prefilter SNIPR.

[0132] Figures 10A-1, 10A-2, 10A-3, 10B-1, 10B-2, 10B-3, 10C, 10D, 10E-1, 10E-2, 10F: Caveolin-mediated uptake is optimal for SNIPR transfection. In Figure 10A, THP-1 cells were treated with increasing concentrations of DiIC18(3)-labeled SNIPR nanoparticles (red) for 30 min and 2 h. Cells were washed extensively and co-stained with nuclear dye (2.5 uM Hoechst-3342, blue). THP-1 cells were imaged and uptake efficiency was analyzed by flow cytometry. In Figure 10B, HEK-293 cells were treated with fluorescently labeled DNA-loaded SNIPR-012 or fluorescently labeled empty SNIPR-012. Cells were harvested 24 hours after treatment and uptake was assessed by the percentage of fluorescent dye (DiI) positive cells quantified by flow cytometry. In Figure 10C, total primary human peripheral blood mononuclear cells (hPBMCs) were treated with DiIC18(3)-labeled SNIPR, labeled with antibodies to detect NK cells, T cells, monocytes, dendritic cells, and B cells, and uptake was analyzed by flow cytometry. Figure 10D shows that there are three major endocytic pathways for uptake of substances smaller than 0.8 μM: 1) receptor-independent pathway / pinocytosis, 2) clathrin-mediated endocytosis, and 3) caveolin-mediated endocytosis. Uptake of SNIPR nanoparticles via caveolin reduces the chance of lysosomal degradation and increases the probability of endosomal fusion and transfection success. It should also be understood that these particles undergo uptake of LDLR via APOE. For Figure 10E, Jurkat cells were treated with pathway-specific endocytosis inhibitors, namely methyl-β-cyclodextrin (MbC, 15 μM), chlorpromazine (CP, 30 μM), or LY-294002 (LY, 20 μM) for 1 h. Pretreated Jurkat cells were incubated with DiIC18(3)-labeled SNIPR prepared in RPMI medium alone or RPMI medium supplemented with 10% FBS for 2 h. To inhibit ATP-dependent endocytosis, one group was incubated at 4o C. Cells were fixed and uptake was analyzed by flow cytometry (n=3).In Figure 10F, HepG2 cells were pretreated with caveolin inhibitor (15 μM methyl-β-cyclodextrin, MbC) or vesicles for 1 h and then incubated with vesicle control, SNIPR[pEGFP] (SNIPR) or GenJet transfection agent (GenJet) containing 0.5 μg pEGFP for 48 h. SNIPR[pEGFP] was prepared in FBS-free or 10% FBS-supplemented medium and incubated at 37o C for 1 h. Cells were stained with nuclear dye (2.5 μM Hoechst-3342, blue) and imaged for EGFP (green) expression analysis.

[0133] Figures 11A, 11B, 11C, 11D, 11E, and 11F show that SNIPR nanoparticles successfully transfect multiple cell types by delivering plasmid DNA. SNIPR-012 lipid nanoparticles encapsulating EGFP-encoding plasmid DNA (SNIPR[DNA]) are efficiently taken up by HEK293 cells and mediate transfection. HEK-293 were treated with SNIPR[DNA] containing EGFP-encoding plasmid DNA, or with a negative control. For 11A, EGFP expression was imaged by fluorescence microscopy at 72 and 48 h (data not shown). Additionally, cells were harvested at 48 and 72 h to assess transfection efficiency by quantifying the percentage of cells expressing EGFP (Figure 11B) and mean fluorescence intensity (MFI, Figure 11C) by flow cytometry. Figure 11D shows that SNIPR lipid nanoparticles encapsulating plasmid DNA (SNIPR[DNA]) encoding EGFP and a fluorescent dye (DiI) are efficiently taken up by and mediate transfection in mouse bone marrow-derived macrophages (BMMs). BMMs were treated in vitro for 24 h with fluorescently labeled SNIPR-012 containing pEGFP (blue triangles, pCMV-EGFP), or with an equal amount of empty SNIPR nanoparticles (red squares, MT). Cells were harvested and analyzed for the intracellular presence of the fluorescent dye DiI18 (3) tagged to the LNPs. As for Figure 11E, images represent BMMs containing DiI-labeled SNIPR, which were quantified in the graph. Figure 11F provides fluorescent microscopy images revealing that after 6 days of treatment, the difficult-to-transduce BMMs are expressing the EGFP payload.

[0134] Figure 12-1, Figure 12-2, Figure 12-3, Figure 12-4, Figure 12-5, and Figure 12-6 show that SNIPR lipid nanoparticles encapsulating plasmid DNA mediate highly efficient transfection of cells without the loss of viability observed with commercial cationic transfection agents. HEK-293 cells were treated with SNIPR-012D, Lipofectamine 3000 (Invitrogen), GenJet (SignaGen) or Viafect (Promega). Six different pEGFP concentrations were selected per group according to the manufacturer's protocol to cover the range of EGFP expression. Cells were harvested 24 h after treatment and subjected to viability analysis by MTT reduction assay. Transfection efficiency was analyzed as the percentage of EGFP-positive cells at the peak of expression (24 h for cationic transfection agents and 72 h for SNIPR-012D). Simple regression and Pearson's correlation were used to compare groups. Data obtained from three independent experiments.

[0135] Figure 13-1, Figure 13-2, Figure 13-3, Figure 13-4, Figure 13-5, and Figure 13-6 show that SNIPR lipid nanoparticles encapsulating plasmid DNA are extremely stable and retain the ability to transfect cells even after storage at ambient temperatures of 4°C, 25°C, and 37°C for at least 32 weeks. Four batches of SNIPR-12 encapsulating pEGFP plasmid were manufactured and dispensed into 200 μl aliquots. One aliquot from each batch was stored in sterile PBS at -20°C, +4°C, +25°C, and +37°C for 0 weeks (i.e., immediate use), 2 weeks, 4 weeks, 8 weeks, 16 weeks, and 32 weeks. No stabilizing excipients were added to the sterile PBS. For each storage time point, HEK293 cells were treated separately with one of the stored aliquots or with an equal volume of sterile PBS. Cells were imaged by fluorescence microscopy 63 hours after treatment to confirm EGFP (green) expression. The percentage of treated cells that were EGFP positive was quantified by flow cytometry. Bar graphs show the mean percentage of EGFP positive HEK cells per treatment group (n = 4 replicates per time point and storage temperature). Error bars show SEM. Filled circles show the mean percentage of EGFP positive HEK cells per replicate at each time point and storage temperature. The color of the filled circle indicates the storage temperature. Treatment with SNIPR[pDNA] LNPs stored under any of the experimental conditions did not result in a statistically significant change in the percentage of EGFP positive cells. There was a notable trend towards reduced transfection in cells treated with SNIPR[pDNA] stored at -20°C for more than 4 weeks.

[0136] Figure 14 shows that empty SNIPR nanoparticles do not induce TNFa release by macrophages even after 48 hours of exposure. As expected, the highly immunogenic positive control substances lipopolysaccharide (LPS) and CpG oligodeoxynucleotide (ODN B) strongly induced TNFa release by mouse bone marrow-derived macrophages. In contrast, empty SNIPR LNPs, equivalent to the dose required to deliver 500 nanograms or 1000 nanograms of DNA (MT500 and MT1000, respectively), did not induce TNFa release from cells exposed for 4 hours, 24 hours, or 48 hours. As expected, the negative vehicle control (PBS) also did not cause TNFa release. TNFa release was quantified by ELISA. Bar graphs show the average TNFa release per treatment group (n = 3 replicates per treatment group). Error bars show SEM. Filled circles show the average TNFa release for individual replicates of each treatment group. The color of the filled circle indicates the treatment group.

[0137] Figure 15 shows that mice receiving four doses of empty SNIPR nanoparticles over a two-week period did not experience weight loss. Groups of mice (n=8 per group) were treated with MS-Xavine delivering low, medium, and high doses of DNA (4 μg / dose, 12 μg / dose, and 36 μg / dose, respectively). TM LNP or empty Xavine in the same amount as used in the 36 μg / dose group TM Mice were treated four times with LNP or vehicle control (sterile saline for injection). Body weight of mice was monitored throughout the experiment. Mice in the high-dose group showed a strong and statistically significant weight loss compared to the vehicle-treated control group (*** p < 0.001 at the final time point). This weight loss was below the humane endpoint of 20% weight loss mandated by our IACUC protocol. Mice in the mid-dose group showed a moderate weight loss that disappeared by the end of the experiment. In contrast, MS-Xavine TM Mice receiving low dose and empty LNPs did not exhibit weight loss compared to the vehicle-only control group at any time during the experiment.

[0138] Figures 16A and 16B show that MS-Xavine was administered in sufficient amounts to induce an increase in antigen-specific regulatory T cells in vivo. TM The nanoparticles administered to mice induced low IFNa release 6 hours after administration. Mice were treated with MS-Xavine nanoparticles delivering low, medium, and high doses of DNA. TM LNP (4 μg / dose, 12 μg / dose, 36 μg / dose, respectively) or blank Xavine in the same amount as used in the 36 μg / dose group TM Mice were treated four times with LNP, or vehicle control (sterile saline for injection). For Figure 16A, plasma samples were taken from each mouse 6 hours after the first dose and IFNa levels were quantified by ELISA. TM Treated mice showed a strong and statistically significant increase in IFNa secretion compared to saline and empty LNP controls (IFNa release was due to the Xavine but not the SNIPR LNP delivery system). TM (Shows that low doses of MS-Xavine are associated with a DNA payload.) TM Mice treated with MS-Xavine also showed increased IFNa secretion compared to controls, but this was not statistically significant. TM Mice from the treatment groups were euthanized and their spleens were harvested. Spleen cells were analyzed by flow cytometry. Spleen cells from mice in the low and mid dose groups showed a robust and statistically significant increase in MS-specific (MOG tetramer positive) CD4+ T cells that were FOXP3+ regulatory T cells (Tregs) compared to spleen cells from mice treated with vehicle control.

[0139] Figures 17A-1, 17A-2, 17A-3, 17B-1, 17B-2, and 17B-3 show the prototype MS-Xavine TM We show that nanoparticles induce tolerogenic antigen-presenting cells (APCs). Primary human CD14-derived dendritic cells (DCs) were cultured with MS-Xavine. TMCells were treated with a single dose for 72 hours. Cells were stained with the amine-reactive dye EF506 and fluorescently labeled antibodies against human HLA-DR, CD83, CD45, ILT3, ILT4, and MERTK, and marker expression was analyzed by flow cytometry.

[0140] Figure 18-1, Figure 18-2, and Figure 18-3 show that SNIPR nanoparticles labeled with near-infrared dyes were primarily biodistributed in the liver and spleen of mice within 24 hours. Plasmid DNA-loaded SNIPR LNPs were labeled with near-infrared (nIR) dyes (DiR) and administered via tail vein injection to groups of naive mice (n=4 per group). Each mouse received a single dose of nIR-labeled SNIPR transfected with 12μg of plasmid DNA encoding the autoantigen MOG. Twenty-four hours after administration, the mice were euthanized, and their organs were removed and fixed in paraformaldehyde. After fixation, the nIR signal in each organ was quantified using an iBright FL1000 imager system (ThermoFisher Scientific). The administered nIR-labeled SNIPR primarily accumulated in the two major lymphoid organs, the liver and spleen.

[0141] Figures 19A and 19B show that MS-Xavine nanoparticles delivering plasmid DNA encoding the multiple sclerosis autoantigen MOG induce dose-dependent expression of MOG protein in the spleens of treated mice. A.) Groups of naïve mice (n=5) were treated four times with three doses of MOG-encoding plasmid DNA (low=4 μg / dose, medium=12 μg / dose, high=36 μg / dose) or empty SNIPR LNPs (equivalent to the 36 μg / dose group). Protein was isolated in triplicate from membrane fractions of spleen cells harvested from each mouse and quantitative Western blotted. B.) MOG-6xHis protein expression was normalized to expression of a housekeeping control (actin). Normalized MOG-6xHis expression increased in an MS-Xavine dose-dependent manner and was statistically significantly higher in the medium and high dose groups compared to the empty SNIPR control (** p <0.01).

[0142] Figure 20 shows that IL-10 secretion was strongly upregulated in spleen cells from mice treated with MS-Xavine nanoparticles delivering plasmid DNA encoding the multiple sclerosis autoantigen MOG and IL-10. A.) Naive mice groups (n=5) were treated four times with MS-Xavine containing DNA encoding MOG alone (MS-Xavine Ag) or DNA encoding MOG and IL-10 (MS-Xavine Ag+TolX). A scrambled insert plasmid was used to control for the total amount of DNA administered (12 μg / dose / mouse). Spleen cells isolated from mice with or without MOG restimulation were cultured and IL-10 secretion was quantified by ELISA. Mice treated with MS-Xavine Ag+Tol mutant secreted statistically significantly more IL-10 than negative control mice treated with sterile PBS for injection, regardless of MOG restimulation (*** p < 0.001 with MOG restimulation, ** p < 0.01 without MOG restimulation), indicating that MS-Xavine nanoparticles successfully delivered IL-10 plasmid to splenocytes in treated animals, resulting in proper expression, translation, and secretion of IL-10.

[0143] FIG. 21 shows that the addition of tolerizing factors (IL-10 plasmid) to MS-Xavine increases the total number of antigen-specific Tregs, but not their percentage within the antigen-specific CD4+ T cell population. A.) Spleen cells isolated from the same mice as above (FIG. 20) for analysis by flow cytometry. Bar graphs show the mean percentage of CD4+ T cells that were positive for the Treg-specific marker FOXP3 in each treatment group (n=5 or 6 mice per treatment group). Error bars show SEM. Filled circles show the mean percentage of Treg-specific marker FOXP3-positive CD4+ T cells in individual animals. Color of filled circle indicates treatment group (white=saline vehicle control; purple=MS-Xavine (Ag-only variant); green=MS-Xavine (Ag+TolX variant)). Both variants of MS-Xavine induced a statistically significant increase in splenic CD4+FOXP3+ Tregs (*p < 0.05). B.) Mice treated with the Ag+TolX variant showed a statistically significant increase in MOG-specific CD4+FOXP3+ Tregs compared to both vehicle controls and the Ag-only variant. Interestingly, this was within a general increase in MOG-specific CD4+ T cells; the proportion of MOG-specific CD4+FOXP3+ Tregs was approximately 20% in mice treated with either MS-Xavine. Bar graphs show the mean percentage of T cells in each treatment group that were positive for the markers indicated in the y-axis legend of each graph (n = 5 or 6 animals per treatment group). Error bars show SEM. Filled circles show the mean percentage of T cells in individual animals that were positive for the markers indicated in the y-axis legend of each chart.

[0144] Figure 22 shows that restimulation with MOG does not induce IFNg secretion by MOG-reactive splenocytes isolated from mice treated with either of the MS-Xavine variants, and addition of IL-10 tolerizing factor completely inhibits IFNg secretion. Splenocytes isolated from the same mice as above (Figure 20) were cultured and restimulated with MOG peptide or vehicle control. (A) As expected, cells isolated from the spleens of MOG-induced experimental autoimmune encephalitis (EAE) positive control mice robustly increased IFNg expression upon restimulation with MOG (*** p < 0.0001). Spleen cells from mice administered the Ag-only MS-Xavine variants showed no change in IFNg secretion upon MOG restimulation, suggesting that the increase in MOG-specific CD4+ cells observed in these mice is not inflammatory Tresp cells. Interestingly, spleen cells from mice treated with Ag+Tol MS-Xavine did not secrete IFNg, suggesting that they were exposed to an immunosuppressive microenvironment.

[0145] Figures 23A, 23B, 23C, 23D-1, and 23D-2 show that SNIPR-023 lipid nanoparticles encapsulating messenger RNA encoding EGFP and labeled with a fluorescent dye (DiI) efficiently take up into HEK293 cells and mediate transfection. In Figure 23A, HEK-293 were treated with DiI-labeled SNIPR-023 containing mRNA encoding EGFP (SNIPR-023[mRNA]) or empty SNIPR LNPs (SNIPR-23[Empty]). For Figure 23B, cells 24 hours after treatment were assessed for LNP uptake via flow cytometry by quantifying the percentage of cells positive for fluorescently labeled DiI. For Figure 23C, HEK-293 cells were also assessed for transfection efficiency by quantifying the percentage of cells expressing EGFP by flow cytometry. Cells were harvested 72 hours after treatment and EGFP expression was assessed by flow cytometry. Figures 23D-1 and 23D-2 are representative fluorescence micrographs showing both SNIPR[mRNA] and SNIPR[Empty] uptake (red spots), and EGFP expression only in cells treated with SNIPR[mRNA].

[0146] Figures 24A, 24B-1, 24B-2, and 24C show that various SNIPR lipid nanoparticles co-encapsulating plasmid DNA encoding mCherry and RNA encoding EGFP are efficiently taken up by HEK293 cells and mediate dual transfection. In Figure 24A, HEK-293 cells were treated with six different SNIPR[pDNA+mRNA] formulations (SNIPR AF) containing pDNA and mRNA encoding mCherry and EGFP at a 5:1 pDNA:mRNA ratio (SNIPRA-C) or a 3:1 pDNA:mRNA ratio (SNIPR DF). SNIPR AF formulations also differed by the ratio of the two ionizable lipids included in the formulation, with the following pairs of formulations containing the same ratio of ionizable lipids: SNIPR A and D, SNIPR B and E, and SNIPR C and F. For Figure 24B-1, HEK-293 cells were assessed for transfection efficiency by quantifying EGFP mRNA expression (24 hours after treatment) via quantitative PCR. For Figure 24B-2, HEK-293 cells were assessed for transfection efficiency by quantifying mCherry pDNA expression (72 hours after treatment) via quantitative PCR. The transfection efficiency of pDNA and mRNA was influenced by both the ratio of pDNA:mRNA and the ratio of ionizable lipids contained in the various formulations. Figure 24C is a representative fluorescence micrograph showing the dose response of simultaneous dual transfection of mCherry pDNA and EGFP mRNA in cells treated with SNIPR D and SNIPR F formulations.

[0147] Although exemplary embodiments have been described above, it is not intended that these embodiments describe all possible forms of the invention. Rather, the words used herein are words of description rather than limitation, and it will be understood that various changes may be made without departing from the spirit and scope of the invention. Moreover, features of various embodiments may be combined to form further embodiments of the invention.

[0148] References

[0149] Sankaram, Mantripagada B., and Sinil Kim. Preparation of Multivesicular Eiposomes for Controlled Release of Active Agents. Mantripragada B. Sankaram, assignee. Patent CA2199004 C. 13 Sept. 1994.

[0150] Kim, Sinil, and Stephen B. Howell. Multivesicular liposomes with controlled release of active agents encapsulated in the presence of a hydrochloride. Sinil Kim, assignee. Patent US6071534 A. 15 Feb. 1988.

[0151] Rovira-Bru, M., Thompson, D.H., Szleifer, I. “Size and Structure of Spontaneously Forming Eiposomes in Lipid / PEG-Eipid Mixtures.” Biophysical Journal. 2002. 83(5); 2419-2439.

[0152] Gillespie DT. Exact stochastic simulation of coupled chemical reactions. J Phys Chem.1977;81(25):2340-236E doi:10.1021 / jl00540a008.

[0153] Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. The Lipid Bilayer.Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK26871 / .

Claims

1. 1. A multivesicular lipid nanoparticle composition comprising a plurality of multivesicular lipid nanoparticles, each multivesicular lipid nanoparticle comprises a carrier lipid nanoparticle comprising a carrier phospholipid and a carrier phospholipid micelle having an average diameter of less than 1 micron; at least one antechamber lipid nanoparticle comprising an antechamber phospholipid reverse micelle having an average diameter of less than 100 nm; A multivesicular lipid nanoparticle composition, wherein said carrier lipid nanoparticle encapsulates at least one annular lipid nanoparticle.

2. The multivesicular lipid nanoparticle composition of claim 1, wherein the diameter of the subcompartment phospholipid reverse micelles is 20 to 100 nm.

3. 3. The multivesicular lipid nanoparticle composition of claim 2, wherein said carrier phospholipid micelles have a diameter of 100-300 nm.

4. 2. The multivesicular lipid nanoparticle composition of claim 1, wherein said carrier phospholipid micelle and said antechamber phospholipid reverse micelle each independently comprise a covalent bond of a conjugated tail group.

5. 2. The multivesicular lipid nanoparticle composition of claim 1, wherein the antechamber phospholipid reverse micelles comprise 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine or a residue thereof; 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (chloride salt) or a residue thereof; 1-palmitoyl-2-[16-(acryloyloxy)palmitoyl]-sn-glycero-3-phosphorylcholine or a residue thereof; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) or a residue thereof; 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or a residue thereof; and cholest-5-en-3β-ol or a residue thereof.

6. 2. The multivesicular lipid nanoparticle composition of claim 1, wherein the carrier phospholipid micelle comprises 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine or a residue thereof; 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (chloride salt) or a residue thereof; 1-palmitoyl-2-[16-(acryloyloxy)palmitoyl]-sn-glycero-3-phosphorylcholine or a residue thereof; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) or a residue thereof; 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or a residue thereof; and cholest-5-en-3β-ol or a residue thereof.

7. 2. The multivesicular lipid nanoparticle composition of claim 1, further comprising a therapeutic / diagnostic payload encapsulated by at least one of said annular phospholipid reverse micelles and / or said carrier phospholipid micelles.

8. 8. The multivesicular lipid nanoparticle composition of claim 7, wherein the therapeutic / diagnostic payload is selected from the group consisting of small molecule drugs, polynucleotides, proteins, peptides, ribonucleoproteins, and combinations thereof.

9. 8. The multivesicular lipid nanoparticle composition of claim 7, wherein the therapeutic / diagnostic payload is selected from the group consisting of a fluorescent protein, a peptide, a cytokine, an antibody, and combinations thereof.

10. The multivesicular lipid nanoparticle composition of claim 7, wherein the therapeutic / diagnostic payload comprises a therapeutic tolerogenic payload selected from the group consisting of tolerogenic proteins, peptides, cytokines, polynucleotides, small molecules, and combinations thereof.

11. The multivesicular lipid nanoparticle composition of claim 7, wherein the therapeutic / diagnostic payload is a therapeutic payload selected from the group consisting of an autoantigenic protein, an autoantigenic peptide, a polynucleotide encoding an autoantigenic protein, a polynucleotide encoding an autoantigenic peptide, and combinations thereof.

12. The multivesicular lipid nanoparticle composition of claim 7, wherein the therapeutic / diagnostic payload is a therapeutic payload selected from the group consisting of one or more fusion proteins comprising one or more autoantigen epitopes, a polynucleotide encoding a fusion protein comprising one or more autoantigen epitopes, and combinations thereof.

13. The multivesicular lipid nanoparticle composition of claim 7, wherein the therapeutic / diagnostic payload is a therapeutic payload selected from the group consisting of an immune alleviation-inducing protein, a peptide, a cytokine, a polynucleotide, a small molecule, an autoantigenic protein, an autoantigenic peptide, one or more fusion proteins comprising one or more autoantigenic epitopes, a polynucleotide encoding an autoantigenic protein, a polynucleotide encoding an autoantigenic peptide, a polynucleotide encoding a fusion protein comprising one or more autoantigenic epitopes, and combinations thereof.

14. The multivesicular lipid nanoparticle composition of claim 7, wherein the therapeutic / diagnostic payload comprises a therapeutic immune tolerance-inducing payload selected from the group consisting of interleukin 10 (IL-10), interleukin 27 (IL-27), transforming growth factor beta 1 (TGFb1), hepatocyte growth factor (VGF), vasoactive intestinal peptide (VIP), vitamin D3, corticosteroids (including cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, hydrocortisone), rapamycin, cyclosporine, tacrolimus, aspirin, and combinations thereof.

15. The multivesicular lipid nanoparticle composition of claim 7, wherein the therapeutic / diagnostic payload comprises an immune tolerance-inducing payload selected from the group consisting of myelin basic protein (MBP); myelin oligodendrocyte glycoprotein (MOG); myelin proteolipid protein (PLP); myelin associated glycoprotein (MAG); myelin associated oligodendrocyte basic protein (MOBP); 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase); S100 calcium binding protein B (S100β); transaldopa; tyrosine kinase (TALDO1); nicotinic acetylcholine receptor (nAChR); muscle-specific tyrosine kinase (MuSK); lipoprotein-associated protein 4 (LRP4); insulin; islet antigen 2 (IA-2); glutamic acid decarboxylase 65 kilodalton isoform (GAD65); glutamic acid decarboxylase 67 kilodalton isoform (GAD67); solute carrier family 30 zinc transporter 8 (ZnT8); islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP); chromogranin A; aquaposide phospho-4 (AQP-4); myelin oligodendrocyte glycoprotein (MOG); N-methyl-D-aspartate receptor (NMDAR); alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR); gamma aminobutyric acid isotype A receptor (GABAAR); gamma aminobutyric acid isotype B receptor (GABABR); glycine receptor (GlyR); dipeptidyl peptidase-like protein-6 (DPPX); glutamate receptor, ionotropic, kainate 1 (GRIK1, also known as GluR5); Voltage-gated potassium channel complex (VGKC complex); paraneoplastic antigen Ma1 (PNMA1); paraneoplastic antigen Ma2 (PNMA2); zic family member 4 (Zic4); glutamic acid decarboxylase 65 kilodalton isoform (GAD65); glutamic acid decarboxylase 67 kilodalton isoform (GAD67); collapsin response mediator protein-5 (CRMP5); amphiphysin; modified citrullinated vimentin (MCV); type II collagen (COL2A1); 65-kDa heat shock protein (HSP65);Cartilage glycoprotein-39 (HC-gp39); aggrecan G1 (ACAN); immunoglobulin heavy constant gamma 1 (IGHG1); acetyl-CoA acetyltransferase 2 (ACAT2); fructose bisphosphate aldolase A (ALDOA); carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1); eukaryotic translation elongation factor 1 gamma (EEF1G); inosine monophosphate dehydrogenase 1 (IMPDH1); keratin 19 (KRT19); lactate dehydrogenase-B (LDHB); macrophage migration inhibitory factor (MIF); transketolase (TKT); slow muscle alpha (α)-tropomyosin (TPM3); vimentin (VIM); calcium / calmodulin-dependent protein kinase II gamma (CAMK2G); calcium-regulated thermostable protein 1 (CARHSP1); eukaryotic translation translational elongation factor 1 delta (EEF1D); interferon-inducible protein 35 (IFI35); interleukin enhancer-binding factor 2 (ILF2); protein kinase C AMP-dependent type I regulatory subunit alpha (PRKAR1A); proteasome activator subunit 3 (PSME3); ribosomal protein lateral stalk subunit P1 (RPLP1); RUN and FYVE domain-containing 1 (RUFY1); small RNA-binding exonuclease protective factor La (SSB); signal transducer and activator of transcription 1 (STAT1); tropomyosin 1 (TPM1); citrullinated antigen; carbamylated antigen; thyroglobulin (Tg); thyroid peroxidase (TPO); thyrotropin receptor (TSHR) A-subunit; tissue transglutaminase (TG2); Thyrotropin receptor (TSHR); gamma-enolase (ENO2); o-enolase (ENO1); heat shock protein 90 (HSP90); osteopontin (SPP1); ubiquitin-conjugating enzyme E2 V1 (UBE2V1); eukaryotic translation initiation factor 2 (eIF2); ras-related protein rab-38 (RAB38); cardiac beta-myosin heavy chain (MYH7);vimentin (VIM); gastric H+ / K+ ATPase (ATP4A); trichohyalin (TCHH); tyrosinase-related protein-2 (TRP-2); platelet glycoprotein (GP) IIb / IIIa (ITGA2B); glycoprotein Ib complex component (GP1b-alpha, GP1b-beta), GP1X, GPV); cathelicidin antimicrobial peptide (CAMP); a disintegrin-like metalloprotease domain containing thrombospondin type 1 motif-like 5 (ADAMTSL5); phospholipase A2 group IVD (PLA2G4D); keratin 17 (KRT17); and combinations thereof.

16. Therapeutic / diagnostic payloads include interleukin 10 (IL-10), interleukin 27 (IL-27), transforming growth factor beta 1 (TGFb1), hepatocyte growth factor (VGF), vasoactive intestinal peptide (VIP), vitamin D3, corticosteroids (including cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, and hydrocortisone), rapamycin, cyclosporine, tacrolimus, aspirin, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOP), and vasoactive intestinal peptide (VIP). G), myelin proteolipid protein (PLP), myelin-associated glycoprotein (MAG), myelin-associated oligodendrocyte basic protein (MOBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), S100 calcium-binding protein B (S100β), transaldolase (TALDO1), nicotinic acetylcholine receptor (nAChR), muscle-specific tyrosine kinase (MuSK), lipoprotein-related protein 4 (LRP4), insulin, islet antigen 2 (IA-2), glutamate Decarboxylase 65 kilodalton isoform (GAD65), glutamic acid decarboxylase 67 kilodalton isoform (GAD67), solute carrier family 30 zinc transporter 8 (ZnT8), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), chromogranin A, aquaporin-4 (AQP-4), myelin oligodendrocyte glycoprotein (MOG), N-methyl-D-aspartate receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazole Propionate receptor (AMPAR), gamma aminobutyric acid isotype A receptor (GABAAR), gamma aminobutyric acid isotype B receptor (GABABR), glycine receptor (GlyR), dipeptidyl peptidase-like protein-6 (DPPX), glutamate receptor, ionotropic, kainate 1 (GRIK1, also known as GluR5), voltage-gated potassium channel complex (VGKC-complex), paraneoplastic antigen Ma1 (PNMA1), paraneoplastic antigen Ma2 (PNMA2), zic family member 4 (Zic4),Glutamic acid decarboxylase 65 kilodalton isoform (GAD65), glutamic acid decarboxylase 67 kilodalton isoform (GAD67), collapsin response mediator protein-5 (CRMP5), amphiphysin, modified citrullinated vimentin (MCV), type II collagen (COL2A1), 65-kDa heat shock protein (HSP65), cartilage glycoprotein-39 (HC-gp39), aggrecan G1 (ACAN), immunoglobulin heavy constant gamma 1 (IGHG1), acetyl-CoA acetyltransferase 2 (ACAT2), fructose bisphosphate aldolase A (ALDOA), carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), eukaryotic translation elongation factor 1 gamma (EEF1G), inosine monophosphate dehydrogenase 1 (IMPDH1), keratin 19 (KRT19), lactate dehydrogenase-B (LDHB), macrophage migration inhibitory factor (MIF), transketolase (TKT), slow muscle alpha (α)-tropomyosin (TPM3), vimentin (VIM), calcium / calmodulin-dependent protein kinase II gamma (CAMK2G), calcium-regulated thermostable protein 1 (CARHSP1), eukaryotic translation elongation factor 1 delta (EEF1D), interleukin-1 (IL-1), and IL-2. -feron-inducible protein 35 (IFI35), interleukin enhancer-binding factor 2 (ILF2), protein kinase C AMP-dependent type I regulatory subunit alpha (PRKAR1A), proteasome activator subunit 3 (PSME3), ribosomal protein lateral stalk subunit P1 (RPLP1), RUN and FYVE domain-containing 1 (RUFY1), small RNA-binding exonuclease protective factor La (SSB), signal transducer and activator of transcription 1 (STAT1), and tropomyosin 1 (TPM1), citrullinated antigen, carbamylated antigen, thyroglobulin (Tg), thyroid peroxidase (TPO), thyrotropin receptor (TSHR) A subunit, tissue transglutaminase (TG2), thyrotropin receptor (TSHR), gamma-enolase (ENO2), o-enolase (ENO1), heat shock protein 90 (HSP90), osteopontin (SPP1),8. The multivesicular lipid nanoparticle composition of claim 7, wherein the therapeutic payload is selected from the group consisting of ubiquitin conjugating enzyme E2 V1 (UBE2V1), eukaryotic translation initiation factor 2 (eIF2), ras-related protein rab-38 (RAB38), cardiac beta myosin heavy chain (MYH7), vimentin (VIM), gastric H+ / K+ ATPase (ATP4A), trichohyalin (TCHH), tyrosinase-related protein-2 (TRP-2), platelet glycoprotein (GP) IIb / IIIa (ITGA2B), glycoprotein Ib complex component (GP1b-α, GP1b-β), GP1X, GPV), cathelicidin antimicrobial peptide (CAMP), diintegrin-like and metalloprotease domain containing thrombospondin type 1 motif-like 5 (ADAMTSL5), phospholipase A2 group IVD (PLA2G4D), keratin 17 (KRT17), and combinations thereof. ,

17. 2. The multivesicular lipid nanoparticle composition of claim 1, wherein the conjugated tail group covalent bond attaches the annular reverse micelle lipid nanoparticles to the inner surface of the carrier lipid nanoparticle.

18. 2. The multivesicular lipid nanoparticle composition of claim 1, wherein the carrier phospholipid micelles and the antechamber phospholipid reverse micelles are crosslinked to each other.

19. 1. A method of forming a multivesicular lipid composition comprising a plurality of multivesicular lipids, comprising: Each multivesicular lipid nanoparticle is a carrier phospholipid micelle comprising a carrier phospholipid layer having an average diameter of less than 1 micron; At least one sub-chamber phospholipid reverse micelle comprising a sub-chamber phospholipid layer having an average diameter of less than 100 nm, The carrier phospholipid micelle encapsulates at least one antechamber phospholipid reverse micelle, the method comprising: a) combining an organic solvent in which the carrier phospholipids and the ancillary phospholipids are dissolved with an aqueous solvent in which the hydrophilic therapeutic / diagnostic payload is dissolved; and b) forming a plurality of sub-chamber phospholipid reverse micelles, the plurality of sub-chamber phospholipid reverse micelles having an average nanoparticle diameter of less than 100 nm; the plurality of sub-chamber phospholipid reverse micelles are formed from a first MVL-forming composition that includes a hydrophilic therapeutic / diagnostic payload; and c) forming a plurality of carrier phospholipid micelles having an average nanoparticle diameter of less than 1 micron, the plurality of carrier phospholipid micelles being formed from a second MVL forming composition comprising a plurality of antechamber phospholipid reverse micelles such that the antechamber phospholipid reverse micelles are encapsulated by the carrier phospholipid micelles; d) covalently bonding the phospholipid tail groups of the plurality of carrier phospholipid micelles and the plurality of antechamber phospholipid reverse micelles by a chemical reaction catalyzed by application of actinic radiation.

20. 20. The method of claim 19, wherein the first MVL-forming composition comprises 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine or a residue thereof; 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (chloride salt) or a residue thereof, 1-palmitoyl-2-[16-(acryloyloxy)palmitoyl]-sn-glycero-3-phosphorylcholine or a residue thereof; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) or a residue thereof; 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or a residue thereof; and cholest-5-en-3β-ol or a residue thereof.

21. The method of claim 19, wherein the aqueous fraction of the first MVL-forming composition comprises one or more additional hydrophilic therapeutic / diagnostic payloads.

22. The method of claim 19, wherein the organic fraction of the first MVL-forming composition comprises one or more hydrophobic therapeutic / diagnostic payloads.

23. The method of claim 21, wherein the organic fraction of the first MVL-forming composition comprises one or more hydrophobic therapeutic / diagnostic payloads.

24. 20. The method of claim 19, wherein the hydrophilic therapeutic / diagnostic payload is selected from the group consisting of small molecule drugs, polynucleotides, proteins, peptides, ribonucleoproteins, and combinations thereof.

25. Hydrophilic therapeutic / diagnostic payloads include interleukin 10 (IL-10), interleukin 27 (IL-27), transforming growth factor beta 1 (TGFb1), hepatocyte growth factor (VGF), vasoactive intestinal peptide (VIP), vitamin D3, corticosteroids (cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, hydrocortisone, etc.), rapamycin, cyclosporine, tacrolimus, aspirin, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin proteolipid protein (PLP), myelin associated glycoprotein (MAG), myelin associated oligodendrocyte basic protein (MAG), myelin-associated glycoprotein (MAG), myelin-associated ... (MOBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), S100 calcium-binding protein B (S100β), transaldolase (TALDO1), nicotinic acetylcholine receptor (nAChR), muscle-specific tyrosine kinase (MuSK), lipoprotein-associated protein 4 (LRP4), insulin, islet antigen 2 (IA-2), glutamic acid decarboxylase 65 kilodalton isoform (GAD65), glutamic acid decarboxylase 67 kilodalton isoform (GAD67), solute carrier family 30 zinc transporter 8 (ZnT8); islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP),Chromogranin A, aquaporin-4 (AQP-4), myelin oligodendrocyte glycoprotein (MOG), N-methyl-D-aspartate receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), gamma aminobutyric acid isotype A receptor (GABAAR), gamma aminobutyric acid isotype B receptor (GABABR), glycine receptor (GlyR), dipeptidyl peptidase-like protein-6 (DPPX), glutamate receptor, ionotropic, kainate 1 (GRIK1, also known as GluR5), voltage-gated potassium channel complex (VGKC complex), paraneoplastic antigen Ma1 (PNMA1), paraneoplastic antigen Ma2 (PNMA2), zic family member 4 (Zic4), glutamic acid decarboxylase 65 kilodalton isoform (GAD65), glutamic acid decarboxylase 67 kilodalton isoform (GAD67), collapsin response mediator protein-5 (CRMP5), amphiphysin, modified citrullinated vimentin (MCV), collagen type II (COL2A1), 65-kDa heat shock protein (HSP65), cartilage glycoprotein-39 (HC-gp39), aggrecan G1 (ACAN), immunoglobulin heavy constant gamma 1 (IGHG1), acetyl-CoA acetyltransferase 2 (ACAT2), aldolase, fructose bisphosphate A (ALDOA), carcinoembryonic antigen cell adhesion molecule 1(CEACAM1), eukaryotic translation elongation factor 1 gamma (EEF1G), inosine monophosphate dehydrogenase 1 (IMPDH1), keratin 19 (KRT19), lactate dehydrogenase-B (LDHB), macrophage migration inhibitory factor (MIF), transketolase (TKT), slow muscle alpha (α)-tropomyosin (TPM3), vimentin (VIM), calcium / calmodulin-dependent protein kinase II gamma (CAMK2G), calcium-regulated thermostable protein 1 (CARHSP1), eukaryotic translation elongation factor 1 delta (EEF1D), interleukin-1 (IL-1), and IL-2. -feron-inducible protein 35 (IFI35), interleukin enhancer-binding factor 2 (ILF2), protein kinase C AMP-dependent type I regulatory subunit alpha (PRKAR1A), proteasome activator subunit 3 (PSME3), ribosomal protein lateral stalk subunit P1 (RPLP1), RUN and FYVE domain-containing 1 (RUFY1), small RNA-binding exonuclease protective factor La (SSB), signal transducer and activator of transcription 1 (STAT1), and tropomyosin 1 (TPM1), citrullinated antigen, carbamylated antigen, thyroglobulin (Tg), thyroid peroxidase (TPO), thyrotropin receptor (TSHR) A subunit, tissue transglutaminase (TG2), thyrotropin receptor (TSHR), gamma-enolase (ENO2), o-enolase (ENO1), heat shock protein 90 (HSP90), osteopontin (SPP1), ubiquitin-conjugating enzyme E2 V1 (UBE2V1), eukaryotic translation initiation factor 2 (eIF2), ras-related protein rab-38 (RAB38), cardiac beta-myosin heavy chain (MYH7), vimentin (VIM), gastric H+ / K+ ATPase (ATP4A), trichohyalin (TCHH), tyrosinase-related protein-2 (TRP-2), platelet glycoprotein (GP) IIb / IIIa (ITGA2B), a component of the glycoprotein Ib complex20. The method of claim 19, wherein the peptide is selected from the group consisting of: (GP1b-α, GP1b-β), GP1X, GPV), cathelicidin antimicrobial peptide (CAMP), diintegrin-like and metalloprotease domain containing thrombospondin type 1 motif-like 5 (ADAMTSL5), phospholipase A2 group IVD (PLA2G4D), keratin 17 (KRT17), and combinations thereof.

26. 1. A multivesicular lipid nanoparticle composition comprising a plurality of multivesicular lipid nanoparticles, each multivesicular lipid nanoparticle comprising: a carrier lipid nanoparticle comprising a carrier micelle comprising a carrier lipid selected from the group consisting of a phospholipid, a phosphorus-free lipid, and combinations thereof, the carrier lipid nanoparticle having an average diameter of less than 1 micron; and At least one antechamber lipid micelle nanoparticle comprising an antechamber reverse micelle comprising an antechamber lipid selected from the group consisting of phospholipids, phosphorus-free lipids, and combinations thereof, wherein at least one antechamber lipid micelle nanoparticle has an average diameter of less than 100 nm, and said carrier lipid nanoparticle encapsulates the at least one antechamber lipid nanoparticle.

27. 27. The multivesicular lipid nanoparticle composition of claim 26, wherein the carrier micelles and / or the antechamber reverse micelles independently comprise a lipid described by the formula: where HD is a head group; LK is the linking group; TL is the tail group; n is an integer representing the number of tail groups.

28. 28. The multivesicular lipid nanoparticle composition of claim 27, wherein when multiple tail groups are present, the tail groups can be the same or different.

29. The tail group may be branched or unbranched C 6-25 28. The multivesicular lipid nanoparticle composition of claim 27, comprising an aliphatic chain.

30. 30. The multivesicular lipid nanoparticle composition of claim 29, wherein the tail group comprises one or more carbon-carbon double bonds, which may be cis or trans.

31. The branched or unbranched C 6-25 30. The multivesicular lipid nanoparticle composition of claim 29, wherein one or more carbon atoms in the aliphatic chain may be substituted with O, N, S, or carbonyl.

32. The branched or unbranched C 6-25 30. The multivesicular lipid nanoparticle composition of claim 29, wherein the aliphatic chain may include one or more ester groups.

33. 28. The multivesicular lipid nanoparticle composition of claim 27, wherein the head group comprises a moiety selected from the group consisting of:

33. The multivesicular lipid nanoparticle composition of claim 26, wherein the carrier micelles and / or the antechamber reverse micelles each independently comprise a lipid having a formula described by:

34. The multivesicular lipid nanoparticle composition of claim 26, wherein the diameter of the sub-chamber reverse micelles is 20 to 100 nm.

35. 35. The multivesicular lipid nanoparticle composition of claim 34, wherein the carrier micelles have a diameter of 100-300 nm.

36. 27. The multivesicular lipid nanoparticle composition of claim 26, wherein said carrier micelle and said antechamber reverse micelle each independently comprise a covalent bond of a conjugated tail group.

37. 27. The multivesicular lipid nanoparticle composition of claim 26, wherein the antechamber reverse micelles comprise 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine or a residue thereof; 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (chloride salt) or a residue thereof; 1-palmitoyl-2-[16-(acryloyloxy)palmitoyl]-sn-glycero-3-phosphorylcholine or a residue thereof; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) or a residue thereof; 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or a residue thereof; or cholest-5-en-3β-ol or a residue thereof.

38. 27. The multivesicular lipid nanoparticle composition of claim 26, wherein the carrier micelles comprise 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine or a residue thereof; 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (chloride salt) or a residue thereof; 1-palmitoyl-2-[16-(acryloyloxy)palmitoyl]-sn-glycero-3-phosphorylcholine or a residue thereof; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) or a residue thereof; 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or a residue thereof; or cholest-5-en-3β-ol or a residue thereof.

39. 27. The multivesicular lipid nanoparticle composition of claim 26, further comprising a therapeutic / diagnostic payload encapsulated by at least one annular phospholipid reverse micelle and / or carrier micelle.

40. 40. The multivesicular lipid nanoparticle composition of claim 39, wherein the therapeutic / diagnostic payload is selected from the group consisting of a small molecule drug, a polynucleotide, a protein, a peptide, a ribonucleoprotein, and combinations thereof.

41. 40. The multivesicular lipid nanoparticle composition of claim 39, wherein the therapeutic / diagnostic payload is selected from the group consisting of a fluorescent protein, a peptide, a cytokine, an antibody, and combinations thereof.

42. The multivesicular lipid nanoparticle composition of claim 39, wherein the therapeutic / diagnostic payload comprises a therapeutic tolerogenic payload selected from the group consisting of a tolerogenic protein, a peptide, a cytokine, a polynucleotide, a small molecule, and combinations thereof.

43. The multivesicular lipid nanoparticle composition of claim 39, wherein the therapeutic / diagnostic payload is a therapeutic payload selected from the group consisting of an autoantigenic protein, an autoantigenic peptide, a polynucleotide encoding an autoantigenic protein, a polynucleotide encoding an autoantigenic peptide, and combinations thereof.

44. The multivesicular lipid nanoparticle composition of claim 39, wherein the therapeutic / diagnostic payload is a therapeutic payload selected from the group consisting of one or more fusion proteins comprising one or more autoantigen epitopes, a polynucleotide encoding a fusion protein comprising one or more autoantigen epitopes, and combinations thereof.

45. The multivesicular lipid nanoparticle composition of claim 39, wherein the therapeutic / diagnostic payload is a therapeutic payload selected from the group consisting of an immune tolerance-inducing protein, a peptide, a cytokine, a polynucleotide, a small molecule, an autoantigenic protein, an autoantigenic peptide, a fusion protein comprising one or more autoantigenic epitopes, a polynucleotide encoding an autoantigenic protein, a polynucleotide encoding an autoantigenic peptide, a polynucleotide encoding a fusion protein comprising one or more autoantigenic epitopes, and combinations thereof.

46. The multivesicular lipid nanoparticle composition of claim 39, wherein the therapeutic / diagnostic payload comprises a therapeutic immune tolerance-inducing payload selected from the group consisting of interleukin 10 (IL-10), interleukin 27 (IL-27), transforming growth factor beta 1 (TGFb1), hepatocyte growth factor (VGF), vasoactive intestinal peptide (VIP), vitamin D3, corticosteroids (including cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, hydrocortisone), rapamycin, cyclosporine, tacrolimus, aspirin, and combinations thereof.

47. 40. The multivesicular lipid nanoparticle composition of claim 39, wherein the therapeutic / diagnostic payload comprises an immune tolerance-inducing payload selected from the group consisting of myelin basic protein (MBP); myelin oligodendrocyte glycoprotein (MOG); myelin proteolipid protein (PLP); myelin associated glycoprotein (MAG); myelin associated oligodendrocyte basic protein (MOBP); 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase); S100 calcium binding protein B (S100β); transaldopa. tyrosine kinase (TALDO1); nicotinic acetylcholine receptor (nAChR); muscle-specific tyrosine kinase (MuSK); lipoprotein-associated protein 4 (LRP4); insulin; islet antigen 2 (IA-2); glutamic acid decarboxylase 65 kilodalton isoform (GAD65); glutamic acid decarboxylase 67 kilodalton isoform (GAD67); solute carrier family 30 zinc transporter 8 (ZnT8); islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP); chromogranin A; aquaposide phospho-4 (AQP-4); myelin oligodendrocyte glycoprotein (MOG); N-methyl-D-aspartate receptor (NMDAR); alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR); gamma aminobutyric acid isotype A receptor (GABAAR); gamma aminobutyric acid isotype B receptor (GABABR); glycine receptor (GlyR); dipeptidyl peptidase-like protein-6 (DPPX); glutamate receptor, ionotropic, kainate 1 (GRIK1, also known as GluR5); Voltage-gated potassium channel complex (VGKC complex); paraneoplastic antigen Ma1 (PNMA1); paraneoplastic antigen Ma2 (PNMA2); zic family member 4 (Zic4); glutamic acid decarboxylase 65 kilodalton isoform (GAD65); glutamic acid decarboxylase 67 kilodalton isoform (GAD67); collapsin response mediator protein-5 (CRMP5); amphiphysin; modified citrullinated vimentin (MCV); type II collagen (COL2A1); 65-kDa heat shock protein (HSP65);Cartilage glycoprotein-39 (HC-gp39); aggrecan G1 (ACAN); immunoglobulin heavy constant gamma 1 (IGHG1); acetyl-CoA acetyltransferase 2 (ACAT2); fructose bisphosphate aldolase A (ALDOA); carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1); eukaryotic translation elongation factor 1 gamma (EEF1G); inosine monophosphate dehydrogenase 1 (IMPDH1); keratin 19 (KRT19); lactate dehydrogenase-B (LDHB); macrophage migration inhibitory factor (MIF); transketolase (TKT); slow muscle alpha (α)-tropomyosin (TPM3); vimentin (VIM); calcium / calmodulin-dependent protein kinase II gamma (CAMK2G); calcium-regulated thermostable protein 1 (CARHSP1); eukaryotic translation translational elongation factor 1 delta (EEF1D); interferon-inducible protein 35 (IFI35); interleukin enhancer-binding factor 2 (ILF2); protein kinase C AMP-dependent type I regulatory subunit alpha (PRKAR1A); proteasome activator subunit 3 (PSME3); ribosomal protein lateral stalk subunit P1 (RPLP1); RUN and FYVE domain-containing 1 (RUFY1); small RNA-binding exonuclease protective factor La (SSB); signal transducer and activator of transcription 1 (STAT1); tropomyosin 1 (TPM1); citrullinated antigen; carbamylated antigen; thyroglobulin (Tg); thyroid peroxidase (TPO); thyrotropin receptor (TSHR) A-subunit; tissue transglutaminase (TG2); Thyrotropin receptor (TSHR); gamma-enolase (ENO2); o-enolase (ENO1); heat shock protein 90 (HSP90); osteopontin (SPP1); ubiquitin-conjugating enzyme E2 V1 (UBE2V1); eukaryotic translation initiation factor 2 (eIF2); ras-related protein rab-38 (RAB38); cardiac beta-myosin heavy chain (MYH7);Vimentin (VIM); Gastric H+ / K+ ATPase (ATP4A); Trichohyalin (TCHH), Tyrosinase-related protein-2 (TRP-2), Platelet glycoprotein (GP) IIb / IIIa (ITGA2B), Glycoprotein Ib complex component (GP1b-α, GP1b-β), GP1X, GPV), Cathelicidin antimicrobial peptide (CAMP), Diintegrin-like and metalloprotease domain containing thrombospondin type 1 motif-like 5 (ADAMTSL5), Phospholipase A2 group IVD (PLA2G4D), Keratin 17 (KRT17); and combinations thereof.

48. The therapeutic / diagnostic payload is selected from the group consisting of interleukin 10 (IL-10), interleukin 27 (IL-27), transforming growth factor beta 1 (TGFb1), hepatocyte growth factor (VGF), vasoactive intestinal peptide (VIP), vitamin D3, corticosteroids (including cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, hydrocortisone), rapamycin, cyclosporine, tacrolimus, aspirin, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (M OG), myelin proteolipid protein (PLP), myelin-associated glycoprotein (MAG), myelin-associated oligodendrocyte basic protein (MOBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), S100 calcium-binding protein B (S100β), transaldolase (TALDO1), nicotinic acetylcholine receptor (nAChR), muscle-specific tyrosine kinase (MuSK), lipoprotein-related protein 4 (LRP4), insulin, islet antigen 2 (IA-2), glutamine Acid decarboxylase 65 kilodalton isoform (GAD65), glutamic acid decarboxylase 67 kilodalton isoform (GAD67), solute carrier family 30 zinc transporter 8 (ZnT8), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), chromogranin A, aquaporin-4 (AQP-4), myelin oligodendrocyte glycoprotein (MOG), N-methyl-D-aspartate receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazol-2-yltransferase (α-amino-3-hydroxy-5-methyl-4-isoxazol-2-yltransferase), and α-amino-3-hydroxy-5-methyl-4-isoxazol-2-yltransferase (α-amino-3-hydroxy-5-methyl-4-isoxazol-2-yltransferase). amphotericin A receptor (AMPAR), gamma aminobutyric acid isotype A receptor (GABAAR), gamma aminobutyric acid isotype B receptor (GABABR), glycine receptor (GlyR), dipeptidyl peptidase-like protein-6 (DPPX), glutamate receptor, ionotropic, kainate 1 (GRIK1, also known as GluR5), voltage-gated potassium channel complex (VGKC-complex), paraneoplastic antigen Ma1 (PNMA1), paraneoplastic antigen Ma2 (PNMA2), zic family member 4 (Zic4),Glutamic acid decarboxylase 65 kilodalton isoform (GAD65), glutamic acid decarboxylase 67 kilodalton isoform (GAD67), collapsin response mediator protein-5 (CRMP5), amphiphysin, modified citrullinated vimentin (MCV), type II collagen (COL2A1), 65-kDa heat shock protein (HSP65), cartilage glycoprotein-39 (HC-gp39), aggrecan G1 (ACAN), immunoglobulin heavy constant gamma 1 (IGHG1), acetyl-CoA acetyltransferase 2 (ACAT2), fructose bisphosphate aldolase A (ALDOA), carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), eukaryotic translation elongation factor 1 gamma (EEF1G), inosine monophosphate dehydrogenase 1 (IMPDH1), keratin 19 (KRT19), lactate dehydrogenase-B (LDHB), macrophage migration inhibitory factor (MIF), transketolase (TKT), slow muscle alpha (α)-tropomyosin (TPM3), vimentin (VIM), calcium / calmodulin-dependent protein kinase II gamma (CAMK2G), calcium-regulated thermostable protein 1 (CARHSP1), eukaryotic translation elongation factor 1 delta (EEF1D), interferon-inducible protein 35 (IFI35), interferon-induced ... Interleukin enhancer-binding factor 2 (ILF2), protein kinase C AMP-dependent type I regulatory subunit alpha (PRKAR1A), proteasome activator subunit 3 (PSME3), ribosomal protein lateral stalk subunit P1 (RPLP1), RUN and FYVE domain-containing 1 (RUFY1), small RNA-binding exonuclease protective factor La (SSB), signal transducer and activator of transcription 1 (STAT1), tropomyosin 1 (TPM1), citrullinated antigen, carbamylated antigen, thyroglobulin (Tg), thyroid peroxidase (TPO), thyrotropin receptor (TSHR) A subunit, tissue transglutaminase (TG2), thyrotropin receptor (TSHR), gamma-enolase (ENO2), o-enolase (ENO1), heat shock protein 90 (HSP90), osteopontin (SPP1), ubiquitin-conjugating enzyme E2 V1 (UBE2V1),40. The multivesicular lipid nanoparticle composition of claim 39, wherein the therapeutic payload is selected from the group consisting of eukaryotic translation initiation factor 2 (eIF2), ras-related protein rab-38 (RAB38), cardiac beta myosin heavy chain (MYH7), vimentin (VIM), gastric H+ / K+ ATPase (ATP4A), trichohyalin (TCHH), tyrosinase-related protein-2 (TRP-2), platelet glycoprotein (GP) IIb / IIIa (ITGA2B), glycoprotein Ib complex components (GP1b-α, GP1b-β), GP1X, GPV), cathelicidin antimicrobial peptide (CAMP), diintegrin-like and metalloprotease domain containing thrombospondin type 1 motif-like 5 (ADAMTSL5), phospholipase A2 group IVD (PLA2G4D), keratin 17 (KRT17), and combinations thereof.

49. 27. The multivesicular lipid nanoparticle composition of claim 26, wherein the conjugated tail group covalent bond attaches the annular reverse micelle lipid nanoparticle to the inner surface of the carrier lipid nanoparticle.

50. 27. The multivesicular lipid nanoparticle composition of claim 26, wherein the carrier micelles and the antechamber reverse micelles are crosslinked to each other.

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