Multivalent nanoparticle compositions and uses thereof

Abstract

Described herein are multivalent nanoparticles. In some embodiments, the multivalent nanoparticles contain a programmable stochiometric amount of three or more polypeptides attached to a lipid shell of the multivalent nanoparticles. In some embodiments, one or more of the polypeptides is an antigenic polypeptide. Also described herein are formulations, such as vaccine formulations, that can contain a multivalent nanoparticle described herein. Also described herein are methods of using the multivalent nanoparticles described herein, such as in a vaccine or drug delivery.

Description

MULTIVALENT NANOPARTICLE COMPOSITIONS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 697,821, filed on September 23, 2024, entitled “Multivalent Nanoparticle Compositions and Uses Thereof,” the contents of which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. UG3DA048775 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] This application contains a sequence listing filed in electronic form as an xml file entitled “VTIP-PAT158-PCT01_ST26.xml”, created on September 23, 2025, and having a size of 18,800 bytes. The content of the sequence listing is incorporated herein in its entirety.TECHNICAL FIELD

[0004] The subject matter disclosed herein is generally directed to multivalent nanoparticle vaccines.BACKGROUND

[0005] Vaccination is the most effective way of protecting humans from various pathogens. Nanoparticle based vaccines (nanovaccines) have emerged as an area of research emphasis in vaccine development because of their ability to elicit strong immune responses. However, limitations of current nanovaccines such as, such as poor drug encapsulation, high burst release, phagocytic uptake, short half-life, aberrant immune reaction, uncontrolled tissue distribution, hypersensitivity, accelerated blood clearances, poor stability have hampered their clinical use. As such there still exists a need for improved nanoparticle vaccines capable of successfully presenting multiple antigens.

[0006] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0007] Described in certain example embodiments herein are nanoparticles including a polymer core; and a lipid shell, wherein the lipid shell comprises a first lipid operably coupled with a first orthogonal reaction handle; a second lipid coupled with a second orthogonal reaction handle; and a third lipid coupled with a third orthogonal reaction handle, wherein the first orthogonal reaction handle, the second orthogonal reaction handle, and the third orthogonal reaction handle are different from each other.

[0008] In certain example embodiments, the lipid shell comprises a programmable stochiometric ratio of a quantity of first lipid, the second lipid, and the third lipid.

[0009] In certain example embodiments, the polymer core comprises or consists of Poly(lactic-co-glycolic acid) (PLGA).

[0010] In certain example embodiments, the first lipid, the second lipid, and the third lipid each comprise or consist of the same lipid. In certain example embodiments, at least two of the first lipid, the second lipid, and the third lipid comprise or consist of a different lipid. In certain example embodiments, the first lipid, the second lipid, and the third lipid each comprises or consist of a different lipid. In certain example embodiments, the first lipid, the second lipid, the third lipid, or any combination thereof comprises or consists of a pegylated lipid. In certain example embodiments, the first lipid, the second lipid, the third lipid, or any combination thereof comprises or consists of l,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In certain example embodiments, the first lipid, the second lipid, the third lipid, or any combination thereof comprises or consists of pegylated DSPE.

[0011] In certain example embodiments, the first orthogonal reaction handle, the second orthogonal reaction handle, the third reaction handle is each individually selected from the group consisting of a mal eimide, a tetrazine, an azide, a terminal alkyne, a strained alkyne, trans-cyclooctene, a norbornene, cyclopropane, an aldehyde, a ketone, a vinyl sulfone, an aryl boronic acid, a boronate ester, a sulfonyl fluoride, an aryl fluorosulfate, triarylphosphine, a thiol, or any combination thereof.

[0012] In certain example embodiments, the lipid shell further comprises a first molecule of interest (MOI) comprising a fourth orthogonal reaction handle, a second MOI comprising afifth orthogonal reaction handle, and a third molecule comprising a sixth orthogonal reaction handle, and wherein the first MOI is covalently attached the first lipid via covalent binding between the first orthogonal reaction handle and the forth reaction handle, wherein the second MOI is covalently attached to the second lipid via covalent binding between the second orthogonal reaction handle and the fifth orthogonal reaction handle, and wherein the third MOI is covalently attached to the third lipid via covalent binding between the third orthogonal reaction handle and the sixth orthogonal reaction handle.

[0013] In certain example embodiments, the first MOI the second MOI, the third MOI, or any combination thereof is a polypeptide. In certain example embodiments, at least two of the first MOI, the second MOI, or the third MOI are different from each other. In certain example embodiments, the first MOI, the second MOI, and the third MOI are different from each other. In certain example embodiments, the first MOI, the second MOI, the third MOI, or any combination thereof is an antigen, optionally wherein the antigen is a polypeptide. In certain example embodiments, the first MOI, the second MOI, the third MOI, or any combination thereof is an immunostimulatory agent. In certain example embodiments, the first MOI, the second MOI, the third MOI, or any combination thereof is a therapeutic molecule, reporter molecule, or a barcode molecule.

[0014] In certain example embodiments, the nanoparticle includes a programmable stoichiometric ratio of the quantity of the first MOI, the second MOI, and third MOI. In certain example embodiments, the programmable stochiometric ratio of the quantity of the first MOI, the second MOI, and third molecule is effective to stimulate an immune response in a subject.

[0015] In certain example embodiments, the nanoparticle further includes cholesterol, DOTAP, or both.

[0016] Described in certain example embodiments herein are pharmaceutical formulation including a nanoparticle of the present description; and a pharmaceutically acceptable carrier.

[0017] Described in certain example embodiments herein are methods of administering a nanoparticle or a formulation thereof of the present description to a subject in need thereof. In some embodiments, the subject in need thereof has or is suspected of having a disease or condition. In some embodiments, the disease or condition is an infectious disease, a non- infectious disease, a mental health disease or condition, or a combination thereof.

[0018] Described in certain example embodiments herein are methods of stimulating an immune response in a subject in need thereof that includes administering a nanoparticle of thepresent description or a pharmaceutical formulation thereof to the subject in need thereof. In certain example embodiments, the immune response comprises B cell production. In certain example embodiments, the B cell is a memory B cell. In certain example embodiments, the immune response comprises T-cell production. In certain example embodiments, the T cell is a memory T cell. In certain example embodiments, the immune response includes an adaptive immune response. In certain example embodiments, the immune response includes an innate immune response. In certain example embodiments, the nanoparticle includes one or more antigenic molecules, optionally one or more antigenic polypeptides, and further comprising exposing the subject in need thereof to the one or more antigenic molecules one or more days after administering the nanoparticle. In certain example embodiments, the nanoparticle includes one or more therapeutic molecules. In certain example embodiments, the one or more therapeutic molecules comprise a psychoactive compound.

[0019] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0021] FIG. 1A-1E shows a schematic and representative transmission electron microscope (TEM) images of PLGA / lipid hNPs. FIG. 1A shows the schematic of PLGA / lipid hNPs and the molecular structure of functional lipids. The low (FIG. IB) and high (FIG. 1C) magnification TEM images of PLGA nanoparticles. The low (FIG. ID) and high (FIG. IE) magnification TEM images of PLGA / lipid hNPs. The nanoparticle size was measured by ImageJ 1.54g (n > 100).

[0022] FIG. 2A-2D shows strategies to tag model proteins with different fluorescent dyes and conversion of the amine group on surface exposed lysine residues. The strategies to tag the model proteins (FIG. 2A) OVA, (FIG. 2B) CRM, and (FIG. 2C) sKLH with different colors of fluorescent dyes of FITC, Rhodamine (Rd), and Cy5, and conversion of the amine group on the surface exposed lysine residues to different functional groups by Traut’s reagent, TCO-NHS, or DBCO-NHS, respectively, and the corresponding UV-vis absorption spectrum of each protein after the modifications are shown. FIG. 2D shows the estimated quantity of fluorescent dye labeled on each protein.

[0023] FIG. 3A-3E shows (FIG. 3A) strategies for conjugation of different proteins to the surface of hNPs and TEM images of (FIG. 3B) hNPs-OVA(FITC), (FIG. 3C) hNPs- OVA(FITC):CRM(Rd), (FIG. 3D) sKLH and (FIG. 3E) hNPs- O VA(FITC) : CRM(Rd) : sKLH(Cy 5).

[0024] FIG. 4A-4D shows the UV-vis absorption spectra of hNPs conjugated with different proteins that were labeled by different fluorescent dye (FIG. 4A) hNP-OVA(FITC), (FIG. 4B) hNP-OVA(FITC):CRM(Rd), and (FIG. 4C) hNP- OVA(FITC):CRM(Rd):sKLH(Cy5); and (FIG. 4D) the estimated quantity of protein loaded on the surface of hNPs at each step.

[0025] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION

[0026] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0028] All publications and patents cited in this specification are cited to disclose and describe the methods and / or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by references as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and / or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in thepublications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0029] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0030] Where a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’ . Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’ . In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0031] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0032] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.Definitions

[0033] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlett, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0034] Definitions of common terms and techniques in chemistry and organic chemistry can be found in Smith. Organic Synthesis, published by Academic Press. 2016; Tinoco et al. Physical Chemistry, 5thedition (2013) published by Pearson; Brown et al., Chemistry, The Central Science 14thed. (2017), published by Pearson, Clayden et al., Organic Chemistry, 2nded. 2012, published by Oxford University Press; Carey and Sunberg, Advanced Organic Chemistry, Part A: Structure and Mechanisms, 5thed. 2008, published by Springer; Carey and Sunberg, Advanced Organic Chemistry, Part B: Reactions and Synthesis, 5thed. 2010, published by Springer, and Vollhardt and Schore, Organic Chemistry, Structure and Function; 8thed. (2018) published by W.H. Freeman.

[0035] Definitions of common terms, analysis, and techniques in genetics can be found in e.g., Hartl and Clark. Principles of Population Genetics. 4thEd. 2006, published by Oxford University Press. Published by Booker. Genetics: Analysis and Principles, 7thEd. 2021, published by McGraw Hill; Isik et la., Genetic Data Analysis for Plant and Animal Breeding. First ed. 2017. published by Springer International Publishing AG; Green, E. L. Genetics and Probability in Animal Breeding Experiments. 2014, published by Palgrave; Bourdon, R. M. Understanding Animal Breeding. 2000 2ndEd. published by Prentice Hall; Pal and Chakravarty. Genetics and Breeding for Disease Resistance of Livestock. First Ed. 2019, published by Academic Press; Fasso, D. Classification of Genetic Variance in Animals. First Ed. 2015, published by Callisto Reference; Megahed, M. Handbook of Animal Breeding and Genetics, 2013, published by Omniscriptum Gmbh & Co. Kg., LAP Lambert Academic Publishing; Reece. Analysis of Genes and Genomes. 2004, published by John Wiley & Sons. Inc; Deonier et al., Computational Genome Analysis. 5thEd. 2005, published by Springer- Verlag, New York; Meneely, P. Genetic Analysis: Genes, Genomes, and Networks in Eukaryotes. 3rdEd. 2020, published by Oxford University Press.

[0036] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0037] As used herein, "about," "approximately," “substantially,” and the like, when used in connection with a measurable variable such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value including those within experimental error (which can be determined by e.g. given data set, art accepted standard, and / or with e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as variations of + / - 10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0038] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0039] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0040] As used herein, a “biological sample” refers to a sample obtained from, made by, secreted by, excreted by, or otherwise containing part of or from a biologic entity. A biologic sample can contain whole cells and / or live cells and / or cell debris, and / or cell products, and / or virus particles. The biological sample can contain (or be derived from) a “bodily fluid”. The biological sample can be obtained from an environment (e.g., water source, soil, air, and the like). Such samples are also referred to herein as environmental samples. As used herein “bodily fluid” refers to any non-solid excretion, secretion, or other fluid present in an organismand includes, without limitation unless otherwise specified or is apparent from the description herein, amniotic fluid, aqueous humor, vitreous humor, bile, blood or component thereof (e.g. plasma, serum, etc.), breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from an organism, for example by puncture, or other collecting or sampling procedures.

[0041] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0042] As used herein, “active agent” or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to. In other words, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed.

[0043] As used herein, “addiction” refers to a pathological (physical and / or mental) state, involving the progression of acute substance use to the development of substance-seeking behavior, the vulnerability to relapse, and the decreased, slowed ability to respond to naturally rewarding stimuli. The Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) has categorized three stages of addiction: preoccupation / anticipation, binge intoxication, and withdrawal / negative affect. These stages are characterized, respectively, everywhere by constant cravings and preoccupation with obtaining the substance; using more of the substance than necessary to experience the intoxicating effects; and experiencing tolerance, withdrawal symptoms, and decreased motivation for normal life activities. By the American Society of Addiction Medicine definition, substance addiction differs from substance dependence and substance tolerance. The term substance addiction is also used as a category which can include the same persons who can be given the diagnosis of substance dependence or substance abuse.

[0044] As used herein, “adjuvant” refers to an additional compound, composition, or ingredient that can facilitate stimulation an immune response in addition to the main antigen of a composition, formulation, or vaccine. Generally, an adjuvant can increase the immune response of an antigen as compared to the antigen alone. This can improve and / or facilitate any protective immunity developed in the recipient subject in response to the antigen. “Adjuvant” as used herein can refer to a component that potentiates the immune responses to an antigen and / or modulates it towards the desired immune response(s).

[0045] As used herein, “antibody” refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region and a light chain constant region. The VH and VL regions retain the binding specificity to the antigen and can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR). The CDRs are interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four framework regions, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Where fragments of antibodies are discussed, fragments, include but are not limited to fragments capable of epitope binding but are less than the full antibody. The term “antibody” as used herein include di and multi-valent antibodies. Methods of making antibodies, including poly and monoclonal antibodies to a known epitope are generally known in the art.

[0046] As used herein, “attached,” “attachment” and the like can refer to the formation of a covalent or non-covalent association (e.g. a bond) between two or more molecules or conjugation of two or more molecules. Where one type of attachment (e.g., covalent or non- covalent), the other is explicitly excluded as to that specified attachment. As used herein, “attached,” attachment” and the like can refer to direct association of two or more molecules together with no intermediate molecules between those that are attached together or to the indirect attachment of two or more molecules together that is mediated via one or more linkers. Where the association is non-covalent, this can encompass charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and / or combinations thereof. Where the association is covalent, this can encompass bonds where a pair of electrons is shared between one or more atoms in each molecule involved.

[0047] As used herein, “concentrated” refers to a molecule or population thereof, including but not limited to a polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than that of its naturally occurring counterpart.

[0048] As used herein, “control” refers to an alternative subject or sample used in an experiment for comparison purpose and included to minimize or distinguish the effect of variables other than an independent variable.

[0049] As used herein, “culturing” refers to maintaining cells under conditions in which they can proliferate and avoid senescence as a group of cells. “Culturing” can also include conditions in which the cells also or alternatively differentiate.

[0050] As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA can be in any form, including, but not limited to, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), or ribozymes.

[0051] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some, but not other features included in otherembodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0052] As used herein, the terms “Fc portion,” “Fc region,” and the like are used interchangeably herein and can refer to the fragment crystallizable region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. The IgG Fc region is composed of two identical protein fragments that are derived from the second and third constant domains of the IgG antibody's two heavy chains.

[0053] As used herein, “immunomodulator,” refers to an agent, such as a therapeutic agent, which is capable of modulating or regulating one or more immune function or response.

[0054] As used herein, “immune response” refers to the reaction of the molecules, components, pathways, organs, fluids and / or cells of the body to the presence of a substance that is foreign or recognized by the body as foreign to the body.

[0055] As used herein, “modulate or modulation of the immune response” refers to change in the immune response that results from the introduction of a composition, vaccine, or other compound or formulation described herein in a recipient subject as compared to a suitable control.

[0056] As used herein, “isolated” means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. A non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, do not require “isolation” to distinguish it from its naturally occurring counterpart.

[0057] As used herein, nicotine unless indicated otherwise, throughout this disclosure includes, without limitation, the terms “nicotine,” “nicotine moiety,” and “nicotine hapten”, all which can be used interchangeably herein, and are intended to include nicotine per se (i.e., (S)- (-)-, (R)-(-)-, or a combination thereof) as well as metabolites, derivatives, analogues, and haptens thereof. Metabolites of nicotine include any compound that is the product of metabolic processing of nicotine, such as cotinine, continine N'-oxide (CNO), 5 '-hydroxy cotinine (5HC), 3 '-hydroxy cotinine (3HC), 5 -hydroxy cotinine (5HC), 5-hydroxycotinine-N-oxide, 3- hydroxy cotinine glucuronide, norcotinine, nornicotine, nicotine-N-oxide (NNO), (S)-nicotine- N — B-glucuronide (Nicotine-Gluc), and Cotinine-glucuronide (Cotinine-Gluc). Derivatives of nicotine include conjugates of nicotine covalently bonded to another species (such as apolymer, oligomer, or small molecule). Analogues include, for example, nicotine wherein the N-methyl group has been replaced with a higher order alkyl group. Similarly, the term “antinicotine antibody” refers to an antibody typically created in a biological organism (such as an animal) that binds to nicotine and / or metabolites, derivatives, or analogues thereof.

[0058] As used herein, “negative control” refers to a “control” that is designed to produce no effect or result, provided that all reagents are functioning properly and that the experiment is properly conducted. Other terms that are interchangeable with “negative control” include “sham,” “placebo,” and “mock.”

[0059] As used herein, “nucleic acid” and “polynucleotide” refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and doublestranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, doublestranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone; artificial nucleic acids may contain other types of backbones but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or “polynucleotide” as that term is intended herein. As used herein, “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined above.

[0060] As used herein, “nicotine addiction” refers to addiction to nicotine and products and other compositions that contain nicotine (nicotine containing products). Example compositions and products containing nicotine include but are not limited to tobacco and tobacco containing products, electronic cigarettes, vegetables belonging to the family Solanacea, and pharmaceutical nicotine replacement products.

[0061] As used herein, a “particle” refers to any entity having a diameter of less than 10 microns (pm). Typically, particles have a longest dimension (e.g., diameter) of 1000 nm or less. In some embodiments, particles have a diameter of 300 nm or less. Particles include microparticles, nanoparticles, and picoparticles. Nanoparticles can have a diameter of 200 nm or less. In some embodiments, nanoparticles have a diameter of 100 nm or less. In some embodiments, nanoparticles have a diameter of 50 nm or less. In some embodiments, nanoparticles have a diameter of 30 nm or less. In some embodiments, nanoparticles have a diameter of 20 nm or less. In some embodiments, nanoparticles have a diameter of 10 nm or less. In some embodiments, particles can be a matrix of polymers. In some embodiments, particles can be a non-polymeric particle (e.g., a metal particle, quantum dot, ceramic, inorganic material, bone, etc.). Particles may also be liposomes and / or micelles. Particles can be homogenous. Particles can be heterogenous. In some embodiments, particles have a core shell structure.

[0062] As used herein, the term “nanoparticle” refers to any particle having a diameter of less than 1000 nm.

[0063] As used herein, “pharmaceutical formulation” refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.

[0064] s used herein, “pharmaceutically acceptable carrier or excipient” refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, nontoxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient.

[0065] As used herein, “pharmaceutically acceptable salt” refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts.

[0066] As used herein, “positive control” refers to a “control” that is designed to produce the desired result, provided that all reagents are functioning properly and that the experiment is properly conducted.

[0067] As used herein, “preventative” and “prevent” refers to hindering or stopping a disease or condition before it occurs, even if undiagnosed, or while the disease or condition is still in the sub-clinical phase.

[0068] As used interchangeably herein, “protein” and “polypeptide” refer to a molecule composed of one or more chains of two or more amino acids in a specific order. Proteins are required for the structure, function, and regulation of the body's cells, tissues, and organs. As used herein, the term polypeptide includes “peptides”, which is a term of art to that is used to refer to short polypeptides, generally ranging in length from 2 to about 50 amino acids. Methods of characterizing polypeptide sequences, secondary structure, tertiary structure, quaternary structure, and interactions with other molecules are generally known in the art. Methods of making polypeptides are generally known in the art.

[0069] As used herein, “purified” or “purify” is used in reference to a nucleic acid sequence, peptide, or polypeptide that has increased purity relative to the natural environment.

[0070] As used herein, “separated” can refer to the state of being physically divided from the original source or population such that the separated compound, agent, particle, or molecule can no longer be considered part of the original source or population.

[0071] As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species contains about 50 percent of all species present. Generally, a substantially pure composition will have more than about 80 percent of all species present in the composition, in some embodiments more than about 85%, 90%, 95%, and 99%. In some embodiments, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species.

[0072] As used herein, the term “specific binding” refers to non-covalent physical association of a first and a second moiety wherein the association between the first and second moi eties is at least 2 times as strong, at least 5 times as strong as, at least 10 times as strong as, at least 50 times as strong as, at least 100 times as strong as, or stronger than the association of either moiety with most or all other moieties present in the environment in which binding occurs. The binding of two or more entities may be considered specific if the equilibrium dissociation constant, Kd, is I O3M or less, 104M or less, I05M or less, 106M or less, 107M or less, 108M or less, 109M or less, 1010M or less, 101 1M or less, or 1012M or less under the conditions employed, e.g., under physiological conditions such as those inside a cell or consistent with cell survival. In some embodiments, specific binding can be accomplished by a plurality of weaker interactions (e.g., a plurality of individual interactions, wherein each individual interaction is characterized by a Kd of greater than 103M). In some embodiments, specific binding, which can be referred to as “molecular recognition,” is a saturable binding interaction between two entities that is dependent on complementary orientation of functional groups on each entity. Examples of specific binding interactions include primer-polynucleotide interaction, aptamer-aptamer target interactions, antibody-antigen interactions, avidin-biotin interactions, ligand-receptor interactions, metal-chelate interactions, hybridization between complementary nucleic acids, etc.

[0073] As used herein, the terms “T cell antigen” refers to any antigen that is recognized by and triggers an immune response in a T cell (e.g., an antigen that is specifically recognized by a T cell receptor on a T cell via presentation of the antigen or portion thereof bound to a major histocompatibility complex molecule (MHC). In some embodiments, an antigen that is a T cell antigen is also a B cell antigen. In other embodiments, the T cell antigen is not also a B cell antigen. T cells antigens generally are proteins or peptides. T cell antigens may be an antigen that stimulates a CD8+ T cell response, a CD4+ T cell response, or both. The nanocarriers, therefore, in some embodiments can effectively stimulate both types of responses.

[0074] As used herein, “vaccine” refers to a compound, molecule, compositions, and formulations that are capable of inducing an immune response in a subject. The term “vaccine” can also be used to refer to a compound, molecule, compositions, and formulations that are capable of providing protective immunity against an organism or compound to be neutralized or cleared from a subject. The vaccine may provide protection or immunization against a compound, such as nicotine, aberrant protein, toxin, or other undesirable compound. Thevaccine can be capable of stimulating a B cell immune response specific to an abused substance, toxin, aberrant protein, or a mutated polynucleotide. The vaccine can be capable of stimulating a T cell immune response specific to or in support of a B cell immune response to an abused substance, toxin, aberrant protein, or a mutated polynucleotide. The vaccine can be capable of stimulating a B cell and a T cell immune response to an abused substance, toxin, aberrant protein, or a mutated polynucleotide.

[0075] As used herein, “therapeutic” refers to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect. A “therapeutically effective amount” can therefore refer to an amount of a compound that can yield a therapeutic effect.

[0076] As used herein, the terms “treating” and “treatment” can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as euphoric feeling acquired from smoking or other drug abuse. The term “treatment” as used herein covers any treatment of infectious or non-infectious disease or disorder or condition in a mammal, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and / or (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. . In some embodiments, the non-infectious disease is addiction The term “treatment” as used herein can refer to therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. Efficacy can be measured using objective or subjective techniques. For example, efficacy can be measured via determining antibody titers and comparing them to a standard and / or control, mitigation of symptoms, etc. In some embodiments, efficacy can be measured by measuring the occurrence of substance abuse and comparing the amount of use to a standard, control, and / or over a period of time. Efficacy can be measured by querying the subject and determining if cravings for abused substance product have been reduced, remained the same, or increased. Efficacy can be measured by querying the subject and determining any changes in euphoric feeling attained after abused substance consumption. Efficacy can be determined by measuring a metabolite or other molecule (e.g. aneurotransmitter, hormone, protein, etc. that indicates a particular disease state) in the subject and comparing the amount measured to a standard and / or a control. Other methods of determining efficacy will be appreciated by those of skill in the art. Other diseases and conditions that can be treated by the nanoparticles of the present description are described elsewhere herein and will be apparent to those of ordinary skill in the art in view of this description.

[0077] As used herein “greatest dimension” can refer to the largest dimension of a nanoparticle herein as measured along any axis of the nanoparticle.

[0078] As used herein “minimum dimensions” can refer to the smallest dimension of a nanoparticle herein as measured along any axis of the nanoparticle.

[0079] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the primary active agent, and optionally present secondary active ingredient, and / or a pharmaceutical formulation thereof calculated to produce the desired response or responses in association with its administration.

[0080] As used herein, “infectious disease” refers to illness caused by pathogenic microorganisms — such as bacteria, viruses, fungi, parasites (e.g., protozoans or helminths), or prions — that invade and replicate within a host and can be transmitted to other hosts (directly, indirectly, or via vectors), resulting in pathological effects.

[0081] As used herein, “non-infectious disease” refers to is a condition or disorder not caused by transmissible pathogens, arising instead from genetic factors, environmental exposures, lifestyle / behavioral influences, degenerative processes, or immune dysregulation (e.g., cancer, cardiovascular disease, diabetes, autoimmune disorders, neurodegeneration, addiction, autism, depression, etc.).

[0082] As used herein, “non-disease antigens” are antigens that are used to induce an immune response to modify anything that is not considered a disease, such as a microbiome.

[0083] As used herein “T-cell helper epitopes” (also known in the art as “T-helper epitopes”) are CD4+T-cell (“helper T-cell”) peptide epitopes (typically about 12-20 amino acids) that bind MHC Class II molecules on antigen presenting cells and help activate T cells and further potentiate a humoral immune response.

[0084] As used herein, a “target molecule” refers to any molecule or composition capable of recognizing, binding, attaching to, or otherwise interacting with a binding partner that canbe present on the surface of a target cell. Binding partners include, but are not limited to nucleic acids, proteins, peptides, sugars, fats, or any combination thereof or any other molecule or molecules that are present on the surface of a target cell. In some embodiments, the binding partner is unique to a cell type or cell state or to a group of related cell types or cell states.

[0085] As used herein, “cell state” is used to describe transient elements of a cell’s identity. Cell state can be thought of as the transient characteristic profile or phenotype of a cell. Cell states arise transiently during time-dependent processes, either in a temporal progression that is unidirectional (e.g., during differentiation, or following an environmental stimulus) or in a state vacillation that is not necessarily unidirectional and in which the cell may return to the origin state. Vacillating processes can be oscillatory (e.g., cell-cycle or circadian rhythm) or can transition between states with no predefined order (e.g., due to stochastic, or environmentally controlled, molecular events). These time-dependent processes may occur transiently within a stable cell type (as in a transient environmental response), or may lead to a new, distinct type (as in differentiation). See e.g., Wagner et al., 2016. Nat Biotechnol. 34(11): 1145-1160.

[0086] The term “small molecule” as used herein refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, peptides, nucleic acids, etc.).

[0087] As used herein, “least effective” amount refers to the lowest amount of the nanoparticles of the present description and / or optional auxiliary agent that achieves the one or more therapeutic or other desired effects.

[0088] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.Overview

[0089] Nanoparticle vaccines can drive robust, T cell-dependent B-cell activation and immunological memory, but the magnitude and quality of these responses critically depend on how efficiently the platform presents protein antigens at the particle interface. Among widely used carriers, PLGA nanoparticles suffer from material-specific liabilities — including poor encapsulation of certain cargos (notably lipophilic agents), burst release, phagocytic uptakewith short systemic half-life, immune reactivity, and uncontrolled biodistribution — whereas liposomes are prone to hypersensitivity reactions, accelerated blood clearance, and limited stability. Hybrid PLGA-lipid core-shell systems mitigate some of these issues and permit adjuvant incorporation and surface functionalization; however, they have not, in general, solved precise, concurrent surface presentation of multiple antigens with controlled ratios on a single particle.

[0090] Concurrently, many vaccine problems (e.g., variant-rich viruses or concurrent pathogens) require multivalent responses. In practice, “multivalency” is commonly achieved by mixing separate monovalent nanoparticle formulations or co-encapsulating several antigens in one carrier — approaches that can introduce antigen competition and immune interference, complicate dose optimization, and increase formulation complexity.

[0091] With that said, embodiments disclosed herein describe a nanoparticle that contains a polymer core; and a lipid shell, wherein the lipid shell includes a first lipid operably coupled with a first orthogonal reaction handle; a second lipid coupled with a second orthogonal reaction handle; and a third lipid coupled with a third orthogonal reaction handle, wherein the first orthogonal reaction handle, the second orthogonal reaction handle, and the third orthogonal reaction handle are different from each other. In some embodiments, the lipid shell has a programmable stochiometric ratio of a quantity of first lipid, the second lipid, and the third lipid. In this way, the ratio of molecules, such as antigenic polypeptides, adjuvants, and the like, that can be attached to the lipid shell can be controlled to achieve effective amounts and / or ratios of molecules incorporated within the lipid shell, such as to elicit an immune response in a precise and programmable manner. Also described herein are exemplary formulations, such as vaccine formulations, of the nanoparticles described herein. Also described herein are exemplary uses of the nanoparticles and formulations thereof described herein, such as to stimulate an immune response in a subject to which they are administered.

[0092] Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.Nanoparticles

[0093] Described in certain example embodiments herein are nanoparticles (also referred to herein as hybrid nanoparticles (hNPs)) comprising a polymer core and a lipid shell. The lipid shell can encapsulate the polymer core. The lipid shell includes lipids where some of the lipids are operably coupled with or include orthogonal reaction handles. The orthogonal reaction handles can then bind with their partner orthogonal reaction handles, which can be attached to molecules of interest (MOIs) thereby covalently attaching the MOIs to the lipid shell of the nanoparticle. By utilizing different orthogonal reaction handle pairs and controlling the amount of lipids containing the different orthogonal reaction handles, different MOIs can be covalently attached to the lipid shell in a controlled manner to achieve a programmed stochiometric ratio of the quantity of the different MOIs attached to the lipid shell. Thus, in some embodiments, the nanoparticles of the present description can have a programmable stochiometric ratio of the quantity of different MOIs attached to the lipid shell. See also e.g., FIG. 1A and 3A which depict a general schematic of covalently attaching different MOIs to a lipid shell of a nanoparticle of the present description. In some embodiments, the shell includes a first lipid that is coupled with or includes a first orthogonal reaction handle, a second lipid that is coupled with or includes a second orthogonal reaction handle; and a third lipid that is coupled with or includes a third orthogonal reaction handle, where the first orthogonal reaction handle, the second orthogonal reaction handle, and the third orthogonal reaction handle are different from each other.

[0094] The nanoparticles of the present description can have a greatest dimension (e.g., diameter) of less than 100, 10, 5, or 1 microns (pm). The nicotine lipid-polymeric nanoparticles can have a greatest dimension (e.g., diameter) of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. The nanoparticles of the present description can have a greatest dimension (e.g., diameter) of 300 nm or less. The nanoparticles of the present description can have a greatest dimension (e.g., diameter) of 250 nm or less. The nanoparticles of the present description can have a greatest dimension (e.g., diameter) of 200 nm or less, nanoparticles of the present description can have a greatest dimension (e.g., diameter) of 150 nm or less, nanoparticles of the present description can have a greatest dimension (e.g., diameter) of 100 nm or less. The nanoparticles of the present description can have a greatest dimension (e.g. diameter) ranging between 20 nm and 200 nm.

[0095] A population of nanoparticles of the present description can have a mean geometric diameter that is less than 500 nm. A population of the nanoparticles of the present description can have a mean geometric diameter that is greater than 20 nm but less than 500 nm. The nanoparticles of the present description can have a mean geometric diameter of a population of nanocarriers is about 20 nm, 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm. In some embodiments, the mean geometric diameter can be between 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm. In some embodiments, the mean geometric diameter can be between 20-400 nm, 20-350 nm, 20-300 nm, 20-250 nm, or 20-200 nm. In some embodiments, the mean geometric diameter can be between 20-200 nm.

[0096] In certain embodiments, the nanoparticles of the present description are greater in size than the renal excretion limit (e.g., nanoparticles having diameters of greater than 6 nm). In certain embodiments, the nanoparticles of the present description are small enough to avoid clearance of nanocarriers from the bloodstream by the liver (e.g., nanoparticles having diameters of less than 1000 nm). In general, physiochemical features of nanocarriers, such as the nanoparticles of the present description, can allow a nanocarrier to circulate longer in plasma by decreasing renal excretion and liver clearance.

[0097] A population of the nanoparticles of the present description can be generally uniform in terms of size, shape, and / or composition so that each nanoparticle has similar properties. For example, at least 80%, at least 90%, or at least 95% of the nanoparticles of the present description in a nanoparticle population can have a diameter or greatest dimension that falls within 5%, 10%, or 20% of the average diameter or greatest dimension. In some embodiments, a population of nanoparticles of the present description can be heterogeneous with respect to size, shape, and / or composition. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanoparticles of the present description of a population of nanoparticles can have a diameter that is less than 500, 300, 200, 100, 50, or 25 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanoparticles of the present description in the population of nanoparticles have a diameter that is greater than 20 nm but less than 200 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanoparticles of the present description of a population of nanoparticles have a diameter of about 20 nm, 50, nm, 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm,425 nm, 450 nm, or 475 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanoparticles of the present description of a population of nanoparticles can have a diameter that is between 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanoparticles of the present description of a population of nanoparticles have a diameter that is between 20-400 nm, 20-350 nm, 20-300 nm, 20-250 nm, or 20-200 nm.

[0098] As previously discussed, the nanoparticles of the present description can have a core / shell structure, wherein the core is one layer (e.g. a polymeric core) and the shell is a second layer (e.g. a lipid bilayer or monolayer). The nanoparticles of the present description can have any shape. The nanoparticles of the present description can be spheres or spheroids. The nanoparticles of the present description can be flat or plate-shaped. The nanoparticles of the present description can be cubes or cuboids. The nicotine nanoparticles of the present description can be ovals or ellipses. The nanoparticles of the present description can be cylinders, cones, or pyramids. The nanoparticles of the present description can have one or more inner and outer surfaces (e.g. the core can have a surface and the shell can have an inner and an outer surface), and at least one of the one or more surfaces comprises an immunofeature surface (i.e., a surface that contains one or more molecules capable or configured to modulate an immune response and / or interact with an immune molecule).

[0099] Zeta potential is a measurement of surface potential of a particle. The nanoparticles of the present description can have a positive zeta potential. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between -50 mV and +50 mV. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between -25 mV and +25 mV. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between -10 mV and +10 mV. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between -5 mV and +5 mV. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between 0 mV and +50 mV. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between 0 mV and +25 mV. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between 0 mV and +10 mV. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between 0 mV and +5 mV. In some embodiments, the nanoparticles of the present description can have a zetapotential ranging between -50 mV and 0 mV. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between -25 mV and 0 mV. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between -10 mV and 0 mV. In some embodiments, the nanoparticles of the present description can have a zeta potential ranging between -5 mV and 0 mV. In some embodiments, the nanoparticles of the present description can have a substantially neutral zeta potential (i.e. approximately 0 mV). In some embodiments, nanoparticles of the present description can have a negative charge. In some embodiments, nanoparticles of the present description can have a positive charge. In some embodiments, nanoparticles of the present description can be electrically neutral. The overall zeta potential of the nanoparticles of the present description can range from about -100 mV to about 100 mV.

[0100] The nanoparticles of the present description, or any component thereof, can be biodegradable and / or biocompatible. In general, a biocompatible substance is not toxic to cells. A substance can be considered to be biocompatible if its addition to cells results in less than a certain threshold of cell death (e.g. less than 50%, 20%, 10%, 5%, or less cell death). A substance can be considered to be biocompatible if its addition to cells does not induce adverse effects. In general, a biodegradable substance can be one that undergoes breakdown under physiological conditions over the course of a therapeutically relevant time period (e.g., weeks, months, or years). A biodegradable substance can be a substance that can be broken down by cellular machinery. A biodegradable substance is a substance that can be broken down by chemical processes. The nanoparticles of the present description or a component thereof can be both biocompatible and biodegradable. The nanoparticles of the present description or a component thereof can be biocompatible, but not biodegradable. The nanoparticles of the present description or a component thereof can be that is biodegradable, but not biocompatible.

[0101] The nanoparticles of the present description can be prepared using any method known in the art. For example, formulations of particulate nanoparticles of the present description can be formed by methods such as nanoprecipitation, flow focusing fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, nanoprinting, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, as well as other methods well known to those of ordinary skill in the art. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconductor, conductive, magnetic, organic, andother nanoparticles may be utilized. In some embodiments, nanoparticles of the present description can be made by self-assembly. As an example, lipids are mixed with a lipophilic component that can contain a nicotine and then formed into thin films on a solid surface. A hydrophilic component dissolved in an aqueous solution, which can be added to the lipid films to hydrolyze lipids under vortex. Liposomes with lipophilic components and hydrophilic components inside the liposome lumen can be spontaneously assembled. In certain embodiments, pre-formulated polymeric nanoparticles (e.g. polymeric core nanoparticles) can be mixed with small liposomes under gentle vortex to induce liposome fusion onto polymeric nanoparticle surface. The nanoparticles of the present description can be manufactured under sterile conditions. Other assembly methods will be appreciated by those of ordinary skill in the art in view of the present disclosure.Polymer Core

[0102] The nanoparticles of the present description can have a polymer core. A wide variety of polymers and methods for forming polymeric matrices therefrom are known in the art of drug delivery. In general, a polymeric matrix can be composed of one or more polymers. Any suitable polymer can be used in the polymer core. In some embodiments, one, more than one, or all of the polymer(s) present in the polymer core can be biodegradable. The polymers can be natural or unnatural (synthetic) polymers. The polymers can be homopolymers or copolymers composed of two or more monomers. In terms of sequence, copolymers can be random, block, or comprise a combination of random and block sequences. The polymers can be organic polymers. The polymers can be synthetic polymers. The polymers can be dendritic polymers or blends of polymers. Non-limiting examples of suitable core polymers include polyethylenes, polycarbonates (e.g. poly(l,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)), polyhydroxyacids (e.g. poly(P-hydroxyalkanoate)), polypropylfumerates, polycaprolactones, polyamides (e.g. polycaprolactam), polyacetals, polyethers, polyesters (e.g. polylactide, polyglycolide), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, and polyamines.

[0103] Suitable polymers that can be included or make up the polymer core also include those which have been approved for use in humans by the U.S. Food and Drug Administration (FDA) under 21 C.F.R. § 177.2600, including but not limited to polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(l,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.

[0104] In some embodiments, the polymer core contains one or more hydrophilic polymers. For example, a polymer(s) of the polymer core can include anionic groups (e.g., phosphate group, sulphate group, carboxylate group); cationic groups (e.g., quaternary amine group); or polar groups (e.g., hydroxyl group, thiol group, amine group). The polymer core can be a hydrophilic polymeric matrix which generates a hydrophilic environment within the nicotine nanoparticle. In some embodiments, additional hydrophilic immunomodulatory agents and / or immunostimulatory agents may be associated with hydrophilic polymeric matrix core.

[0105] In some embodiments, the polymer core contains one or more hydrophobic polymers. A hydrophobic polymeric matrix can generate a hydrophobic environment within the core of the nanoparticle of the present description. In some embodiments, hydrophobic immunomodulatory agents and / or immunostimulatory agents can be associated with hydrophobic polymeric matrix core.

[0106] The core polymer(s) can be modified with one or more moieties and / or functional groups. Any moiety or functional group can be used. In some embodiments, polymers can be modified with polyethylene glycol (PEG), with a carbohydrate, and / or with acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301; incorporated herein by reference).

[0107] The core polymer(s) can be modified with a lipid or fatty acid group, properties of which are described in further detail below. In some embodiments, a fatty acid group may be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid. In some embodiments, a fatty acid group may be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.

[0108] The core polymer(s) can be or include polyesters, including copolymers including lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide- co-glycolide), collectively referred to herein as “PLGA”; and homopolymers including glycolic acid units, referred to herein as “PGA,” and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L- lactide, collectively referred to herein as “PLA.” In some embodiments, exemplary polyesters include, for example, polyhydroxyacids; PEG copolymers and copolymers of lactide andglycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG copolymers, and derivatives thereof. In some embodiments, polyesters include, for example, polyanhydrides, poly(ortho ester), poly(ortho ester)-PEG copolymers, poly(caprolactone), poly(caprolactone)- PEG copolymers, polylysine, polylysine-PEG copolymers, poly(ethylene imine), poly(ethylene imine)-PEG copolymers, poly(L-lactide-co-L-lysine), poly(serine ester), poly(4- hydroxy-L-proline ester), poly[a-(4-aminobutyl)-L-glycolic acid], and derivatives thereof. In some embodiments, polymer core contains or is Poly(lactic-co-glycolic acid) (PLGA).

[0109] The core polymer(s) can be or include one or more acrylic polymers. Acrylic polymers can include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate, poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations comprising one or more of the foregoing polymers. The acrylic polymer may comprise fully-polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.

[0110] The core polymer(s) can be or include cationic polymers. In general, cationic polymers can condense and / or protect negatively charged strands of nucleic acids (e.g. DNA, RNA, or derivatives thereof). Amine-containing polymers such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7; both of which are incorporated herein by reference), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297; incorporated herein by reference), and poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372; all of which are incorporated herein by reference) are positively-charged at physiological pH, form ion pairs with nucleic acids, and mediate transfection in a variety of cell lines.[OHl] The core polymer(s) can be or include degradable polyesters bearing cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115: 11010; Kwon et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121 :5633; and Zhou et al., 1990, Macromolecules, 23:3399; all of which are incorporatedherein by reference). Examples of these polyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem. Soc., 115: 11010; incorporated herein by reference), poly(serine ester) (Zhou et al., 1990, Macromolecules, 23:3399; incorporated herein by reference), poly(4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121 :5633; both of which are incorporated herein by reference), and poly(4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121 :5633; both of which are incorporated herein by reference).

[0112] The core polymer(s) can be or include carbohydrates, properties of which are described in further detail below. In some embodiments, a carbohydrate may be a polysaccharide comprising simple sugars (or their derivatives) connected by glycosidic bonds, as known in the art. In some embodiments, a carbohydrate may be one or more of pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxycellulose, methylcellulose, dextran, cyclodextran, glycogen, starch, hydroxy ethyl starch, carageenan, glycon, amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch, chitin, heparin, konjac, glucommannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan.

[0113] The core polymer(s) can be or include a protein or peptide, properties of which are described in further detail below. Exemplary proteins that may be used in accordance with the present invention include, but are not limited to, albumin, collagen, a poly(amino acid) (e.g., polylysine), an antibody, etc.

[0114] The core polymer can be a nucleic acid (i.e., polynucleotide), properties of which are described in further detail below. Exemplary polynucleotides that may be used in accordance with the present invention include, but are not limited to, DNA, RNA, etc.

[0115] The polymer core can have a greatest dimension between 1 nm and 1000 nm. The polymer core can have a greatest dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. The polymer core can have a greatest dimension (e.g., diameter) of 300 nm or less. The polymer core can have a greatest dimension (e.g., diameter) of 250 nm or less. The polymer core can have a greatest dimension (e.g., diameter) of 200 nm or less. The polymer core can have a greatest dimension (e.g., diameter) of 150 nm or less. The polymer core can have a greatest dimension (e.g., diameter) of 100 nm or less. The polymer core can have a greatest dimension (e.g., diameter) of 50 nm or less. The polymer core can have a greatest dimension ranging between 20 nm and 200 nm.

[0116] The ratio of the quantity or amount of polymer(s) in the polymer core can range from 0: 100 to 100:0 w / w or v / v for the first two polymers. Each additional polymer can be included such that the first polymer can be present at about 0 to about 100% w / w or v / v, the second polymer can be present at about 0 to about 100% w / w or v / v, and each additional polymer can be present at about 0 to about 100% w / w or v / v. The ratio of each polymer present can be determined from the amount present. For example. If there are three polymers present in the polymer core and the first polymer is present at 25% w / w, the second polymer is present at 25% w / w, and the third polymer is present at 50% w / w, the ratio can be said to be 25:25:50 or 1 : 1 :2.

[0117] In some embodiments, the polymer can be PLGA. PLGA is a biocompatible and biodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid:gly colic acid. Lactic acid can be L-lactic acid, D-lactic acid, or D, L-lactic acid. The degradation rate of PLGA can be adjusted by altering the lactic acid:glycolic acid ratio. In some embodiments, PLGA to be used in accordance with the present invention is characterized by a lactic acid:glycolic acid ratio of any value, such as approximately 100:0, approximately 85: 15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, approximately 15:85, or approximately 0: 100.Lipid Shell

[0118] The nanoparticles of the present description can have a shell that can be composed of one or more lipids. The lipid shell can be such that a nanoparticle of the present description is a liposome. As previously discussed, the density or quantity of different MOIs that are displayed on the lipid shell surface is controlled by the amount of different orthogonal reaction handles that are incorporated with individual lipid molecules that form the surface of the shell (see e.g., FIGs. 1A and 3A). When the nanoparticle of the present disclosure includes three different orthogonal reaction handles, the first lipid (including or coupled with the first orthogonal reaction handle), the second lipid (including or coupled with the second orthogonal reaction handle), and the third lipid (including or coupled with the third orthogonal reaction handle) can each contain or can be the same lipid. When the nanoparticle of the present disclosure includes three different orthogonal reaction handles, the first lipid (including or coupled with the first orthogonal reaction handle), the second lipid (including or coupled withthe second orthogonal reaction handle), and the third lipid (including or coupled with the third orthogonal reaction handle) can each contain or can each be a different lipid.

[0119] When the nanoparticle of the present disclosure includes three different orthogonal reaction handles, at least two of the the first lipid (including or coupled with the first orthogonal reaction handle), the second lipid (including or coupled with the second orthogonal reaction handle), and the third lipid (including or coupled with the third orthogonal reaction handle) can each contain or can be same lipid. When the nanoparticle of the present disclosure includes three different orthogonal reaction handles, at least two of the first lipid (including or coupled with the first orthogonal reaction handle), the second lipid (including or coupled with the second orthogonal reaction handle), and the third lipid (including or coupled with the third orthogonal reaction handle) can each contain or can be a different lipid.

[0120] In some embodiments, the first lipid, the second lipid, the third lipid, or any combination thereof contains or is a pegylated lipid. In some embodiments, the first lipid, the second lipid, the third lipid, or any combination thereof includes or is 1,2-Distearoyl-sn- glycero-3 -phosphoethanolamine (DSPE). In some embodiments, the first lipid, the second lipid, the third lipid, or any combination thereof contains or is pegylated DSPE.

[0121] The lipid shell can be a lipid monolayer, a lipid bilayer, and / or multiple lipid bilayers. For example, a lipid bilayer may form the exterior surface of a nanoparticle of the present description, in which case a nanoparticle of the present description having a lipid bilayer shell can be referred to as a liposome. The nanoparticles of the present description can have relatively moldable surfaces, and the nanoparticles can take on a variety of shapes (e.g., spherical, oblong, cylindrical, etc.) depending on environmental factors. It will be appreciated, therefore, that the maximum diameter of such nanocarriers may change in different environments. The lipid shell can contain one or more types of phospholipids. In some embodiments, the lipid shell can be a lipid monolayer. In some embodiments the nicotine lipidpolymeric nanoparticles can be refer to as a micelle. The lipid shell can be composed of one or more amphiphilic lipids (i.e., lipids that possess both hydrophilic and hydrophobic properties). In some embodiments, an amphiphilic lipid can promote the production of the nicotine lipidpolymeric nanoparticles with increased stability, improved uniformity, and / or increased viscosity.

[0122] When the lipid shell includes a lipid bilayer, the lipid bilayer can be oriented such that the interior and the exterior of the nanoparticles of the present description are hydrophilicand the lumen of the nanoparticles of the present description are hydrophobic. In other embodiments, the lipid bilayer can be oriented such that the interior and the exterior of the nanoparticles of the present description are hydrophobic and the lumen of the nicotine nanoparticles are hydrophilic. One of skill in the art will appreciate the general nature of the compositions of the lipid shell and core that would facilitate such orientations of a lipid bilayer shell

[0123] The percent of lipid in nanoparticles of the present description (when considered as a whole) can range from 0.0001% to 99% by weight, from 10% to 99% by weight, from 25% to 99% by weight, from 50% to 99% by weight, or from 75% to 99% by weight. In some embodiments, the percent of lipid in nanoparticles of the present description can range from 0.0001% to 75% by weight, from 0% to 50% by weight, from 0.0001% to 25% by weight, or from 0.0001% to 10% by weight. In some embodiments, the percent of lipid nanoparticles of the present description can be approximately 1% by weight, approximately 2% by weight, approximately 3% by weight, approximately 4% by weight, approximately 5% by weight, approximately 10% by weight, approximately 15% by weight, approximately 20% by weight, approximately 25% by weight, approximately 40% by weight, approximately 50% by weight, or approximately 60% by weight.

[0124] The lipid shell can include one or more oils. In general, any oil known in the art can be included in the lipid shell. In some embodiments, an oil can be composed of one or more fatty acid groups or salts thereof. A fatty acid group can include digestible, long chain (e.g., C8-C50), substituted or unsubstituted hydrocarbons. A fatty acid group can be a C10-C20 fatty acid or salt thereof. In some embodiments, a fatty acid group can be a C15-C20 fatty acid or salt thereof. A fatty acid group may be a C15-C25 fatty acid or salt thereof. In some embodiments, a fatty acid group may be unsaturated. A fatty acid group can be monounsaturated. A fatty acid group can be polyunsaturated. A double bond of an unsaturated fatty acid group can be in the cis conformation. A double bond of an unsaturated fatty acid can be in the trans conformation. A fatty acid group can be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid. A fatty acid group can be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linolenic, gammalinoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid. The oil can be a liquid triglyceride.

[0125] Suitable oils that can be used in the lipid shell include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, jojoba, kukui nut, lavandin, lavender, lemon, Litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and combinations thereof.

[0126] The lipid shell can include a hormone (e.g. estrogen, testosterone), steroid (e.g., cholesterol, bile acid), vitamin (e.g. vitamin E), phospholipid (e.g. phosphatidyl choline), sphingolipid (e.g. ceramides), lipopolysaccharide (e.g. monophosphoryl lipid A), or lipoprotein (e.g. apolipoprotein). The lipid shell can include any molecular adjuvants, such as toll-like receptor (TLR) agonists. Exemplary toll-like receptor agonists include, but are not limited to, triacylated lipopetides, peptidoglycans, bacterial lipoproteins, lipoteichoic acid, lipopolysaccharides, GPI-anchor proteins, neisserial porins, hemagglutinin, pospholipomannan, LAM, viral ssRNA, viral dsRNA, F-protein, mannan, glycoinositolphospholipids, viral envelope proteins, flagellin, pheno-soluble modulin, diacylated lipopeptides, LTA, zymosan, hemozoin, and unmethylated CpG DNA.

[0127] The lipid shell can include one or more amphiphilic molecules (also refered to herein as “amphiphilic entities”). Any amphiphilic entity known in the art is suitable for use in making nanocarriers in accordance with the present invention. Such amphiphilic entities include, but are not limited to, phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine (DOPE); di ol eyl oxy propyltri ethyl ammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid, such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; sorbitan trioleate (Span®85)glycocholate; sorbitan monolaurate (Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60); polysorbate 65 (Tween® 65); polysorbate 80 (Tween® 80); polysorbate 85 (Tween® 85); polyoxyethylene monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid; cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol; poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethylene glycol)400-monostearate; phospholipids; synthetic and / or natural detergents having high surfactant properties; deoxycholates; cyclodextrins; chaotropic salts; ion pairing agents; and combinations thereof. An amphiphilic entity component may be a mixture of different amphiphilic entities. These amphiphilic entities may be extracted and purified from a natural source or may be prepared synthetically in a laboratory. In certain specific embodiments, amphiphilic entities are commercially available.

[0128] The lipid(s) of the lipid shell can be a lipid-polymer conjugate (i.e. a conjugate molecule having a lipid component and a polymer component). The lipids provided above can be conjugated to a suitable polymer to form a lipid-polymer conjugate. Suitable polymers for a lipid-polymer conjugated include polyethylene glycol (PEG), polynucleotides, polypeptides, polysaccharides, or any kind of polymer. The molecular weight of the PEG can rage from 300 to 10,000,000 g / mol. The molecular weight of the PEG can be indicated herein as a number following “PEG”. For example, a PEG having a molecular weight of about 2000 can be abbreviated as PEG2000.

[0129] The lipid shell can be PEGlayted, in addition to the inclusion of a lipid-PEG conjugate. Methods of PEGylating lipid shelled-nanoparticles are generally known in the art.

[0130] The lipid shell can be positively charged, negatively charged, or electrically neutral. The lipid shell can include one ore more molecules. The lipid shell can include one or more compounds that can affect the surface charge of the lipid shell. These can include, but are not limited to, -palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-dioleoyl-3- trimethylammonium-propane, chloride salt (DOTAP), monosial oganglioside GM3, 1,2-di hexadecanoyl-sn-glycero-3-phospho-L-serine, sodium salt (DPPS), monophosphoryl Lipid A (MPLA), cholesterol (CHOL), and N-4-nitrobenzo-2-oxa-l,3-diazole phosphatidylethanolamine (NBD-PE).

[0131] In some embodiments the lipid shell can include DOTAP, cholesterol, DSPE-PEG- maleimide, and DSPE-PEG-amine, or DSPE-PEG with any reactive terminal groups. The PEG in the DSPE-PEG-maleimide and / or the DSPE-PEG-amine can be PEG2000. The weight percent of DOTAP in the lipid shell can range from 1% w / w to about 99% w / w. The weight percent of DSPE-PEG-maleimide in the lipid shell can range from 1% w / w to about 100% w / w. The weight percent of cholesterol in the lipid shell can range from 5% w / w to about 20% w / w.

[0132] The molar ratio of DOTAP:cholesterol can range from 3.5 to 18. The ratio of DOTAP:DSPE-PEG-maleimide can range from 14 to 18. The molarratio of DSPE-PEG- maleimide:cholesterol can range from 0.25 to 3.Molecules of Interest (MOIs)

[0133] In some embodiments, the lipid shell includes a plurality of molecules of interest (MOI) attached to the lipid shell. In some embodiments, the attachment is via a covalent bond between an orthogonal reaction handle pair with one orthogonal reaction handle of the orthogonal reaction handle pair being coupled to or part of a lipid within the lipid shell and the other orthogonal reaction handle of the orthogonal reaction handle pair being coupled to or being part of the MOI. In some embodiments, the lipid shell includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different MOIs. Molecules of interest include, but are not limited to, antigens, immunostimulatory molecules, reporter molecules, barcoding molecules, targeting molecules, therapeutic molecules, any combination thereof, and the like.

[0134] In some embodiments, the lipid shell is covalently attached via orthogonal reaction handles to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different antigens. In some embodiments, the lipid shell is covalently attached via orthogonal reaction handles to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different non-antigenic molecules (e.g., additional immunostimulatory agents, reporter molecules, barcoding molecules, targeting molecules, therapeutic molecules, etc.,). In some embodiments, the lipid shell is covalently attached via orthogonal reaction handles one antigen, and two non-antigenic molecules (e.g., additional immunostimulatory agents, reporter molecules, barcoding molecules, targeting molecules, therapeutic molecules, etc.,). In some embodiments, the lipid shell is covalently attached via orthogonal reaction handles to two different antigens, and one non-antigenic molecule (e.g., additional immunostimulatory agents, reporter molecules, barcoding molecules, targeting molecules, therapeutic molecules, etc.,). In some embodiments, the lipid shell is covalently attached via orthogonal reaction handles to three different antigens.

[0135] The MOI(s) can be peptides, polypeptides, polynucleotides and analogs, carbohydrates, lipids and lipopeptides, and any combination thereof. In some embodiments, the MOI(s) are peptides, polypeptides, or include a peptide or polypeptide. The MOI can be small chemical molecule

[0136] In some embodiments, the MOIs include any antigen, additional immunostimulatory agent, or other molecule involved inducing an immune response described in U.S. Pat. No.: 11,278,608.Exemplary MOIsAntigens

[0137] Exemplary antigens that can be an MOI in the lipid shell of the nanoparticles of the present disclosure can be disease or non-disease antigens. Disease antigens include antigens for infectious diseases and antigens for non-infectious diseases. It will be appreciated that some types of diseases can be considered an infectious disease or a non-infectious disease depending on the initiating trigger. For example, some cancers have been linked to viral origins as well as non-infectious organism origins.

[0138] In some embodiments, the MOI(s) include one or more antigens for a microorganism. The microorganism can be a pathogenic microorganism. The microorganism can be a commensal microorganism. In some embodiments, the microorganism is a virus, phage, bacterium, fungus, yeast, or a protozoan. The virus can be an RNA virus or a DNA virus. In some embodiments, the RNA virus is from the Birnaviridae, Arteriviridae, Bornaviridae, Nodaviridae, Picobirnaviridae, Reoviridae, Coronaviridae, Astroviridaee, Caliciviridae, Flaviviridae, Hepeviridae, Matonaviridae, Picomaviridae, Togaviridae, Filoviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Arenaviridae, Hantaviridae, Nairoviridae, Peribunyaviridae, Phenuiviridae, or Orthomyxoviridae family. In some embodiments, the RNA virus is from the Aquabimavirus, Avibimavirus, Blosnavirus, Picobirnavirus, Aquareovirus, Coltivirus, Orthoreovirus, Orbivirus, Rotavirus, Seadornavirus, Orthohepevirus, Piscihepevirus, Alphaartervirus, Lambdaartervirus, Deltavirus, Etaaterivirus, Epsilonaterivirus, lotaarterivirus, Thetaartereivirus, Zetaartervirius, Betaarteri virus, Gammaatervirus, Kappaarterivirus, Alphacoronavirus, Betacoronavirus, Gammacoronavirus, Deltacoronavirus, Torovirus, Bafinivirus, Ailurivirus, Ampivirus, Aphtovirus, Aquamavirus, Avihepatovirus, Avisivirus, Cardiovirus, Cosavirus, Crohivirus, Dicipivirus, Enterovirus, Erbovirus, Gallivirus, Harkavirus, Hepatovirus, Hunnivirus, Kobuvirus, Kunsagivirus,Limnipivirus, Megrivirus, Mosavirus, Oscivirus, Parechovirus, Pasivirus, Passerivirus, Potamipvirus, Rabovirus, Rosavirus, Sakobuvirus, Salivirus, Sapelovirus, Senecavirus, Sicinivirus, Teschovirus, Torchivirus, Tremovirus, Avastrovirus, Mamastrovirus, Lagovirus, Nebovirus, Norovirus, Sapovirus, Vesivirus, Flavivirus, Hepacivirus, Pegivirus, Pestivirus, Rubivirus, Alphanodavirus, Betanodivirus, Alphavirus, Orthobornavirus, Carbovirus, Nyavirus, Ephemerovirus, Ephemerovirus, Hapavirus, Ledantevirus, Perhabdovirus, Sprivivirus, Tibrovirus, Tupavirus, Vesiculovirus, Cuevavirus, Ebolavirus, Marburgvirus, Aquaparamyxovirus, Avulavirus, Ferlavirus, Henipavirus, Morbillivirus, Respirovirus, Rubulavirus, Metapneumonvirus, Orthopneumonvirus, Hartmanivirus, Mammarenvirus, Reptarenavirus, Orthohantavirus, Orthonairovirus, Phlebovirus, Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus, Deltainfluenzavirus, Thogotovirus, Isavirus, Quaranjavirus, Orthobunyavirus, Sunshinevirus, Tilapinevirus, or Deltavirus genus.

[0139] In some embodiments, the DNA virus is from the erpesviridae, Alloherpesviridae, Adenoviridae, Papillomaviridae, Polomaviridae, Asfarviridae, Iridoviridae, Poxviridae, Anelloviridae, Circoviridae, Genomoviridae, or Parvoviridae family. In some embodiments, the DNA virus is from the Simplexvirus, Varicellovirus, Mardivirus, Scutavirus, Iltovirus, Cytomegalovirus, Muromegalovirus, Roseolivirus, Proboscivirus, Lymphocrypto-virus, Rhadinovirus, Macavirus, Percavirus, Batrachovirus, Cyprinivirus, Ictalurivirus, Salmonivirus, Mastadenovirus, Aviadenovirus, Atadenovirus, Ichtadenovirus, Siadenovirus,Alphapapillomavirus, Betapapillomavirus, Chipapillomavirus, DeltapapillomavirusDyochipapillomavirus, Dyodeltapapillomavirus, DyoepsilonpapillomavirusDyoetapapillomavirus, Dyiotapapillomavirus, DyokappapapillomavirusDyolambdapapillomavirus, Dyomupapillomavirus, DyonupapillomavirusDyoomegapapillomavirus, Dyoomikronpapillomavirus, DyophipapillomavirusDyopipapillomavirus, Dyopsipapillomavirus, DyorhopapillomavirusDyosigmapapillomavirus, Dyotaupapillomavirus, Dyothetapapillomavirus Dyoupsilonpapillomavirus, Dyoxipapillomavirus, DyozetapapillomavirusEpsilonpapillomavirus, Etapapillomavirus, Gammapapillomavirus, lotapapillomavirus, Kappapapillomavirus, Lambdapapillomavirus, Mupapillomavirus, Nupapillomavirus, Omegapapillomavirus, Omikronpapillomavirus, Phipapillomavirus, Psipapillomavirus, Rhopapillomavirus, Sigmapapillomavirus, Taupapillomavirus, Thetapapillomavirus, Treisdeltapapillomavirus, Treisiotapapillomavirus, Treisepsilonpapilomavirus,Treiskappapapillomavirus, Treisthetapapillomavirus, Treiszetapapillomavirus, Treiszetapapillomavirus, Upsilonpapillomavirus, Xipapillomavirus, Zetapapillomavirus, Alefpapillomavirus, Alpha-polyomavirus, Beta-polyomavirus, Gamma-polyomavirus, Deltapolyomavirus, Asfivirus, Lymphocystivirus, Megalocytivirus, Ranavirus, Avipoxvirus, Capripoxvirus, Cervidopoxvirus, Crocodylidpoxvirus, Leporipoxvirus, Molluscopoxvirus, Orthopoxvirus, Parpoxvirus, Suipoxvirus, Yatapoxvirus, Alphatorquevirus, Betatorquevirus, Gammatorquevirus, Deltatorquevirus, Epsilontorquevirus, Lambdatorquevirus, Kappatorquevirus, Zetatorquevirus, Etatorquevirus, Thetatorquevirus, lotatorquevirus, Gyrovirus, Circovirus, Cyclovirus, Gemycicular-virus, Gemygorvirus, Gemykibivirus, Gemykolovirus, Gemykrogvirus, Gemykroznavirus, Gemytondvirus, Gemyvongvirus, Amdoparvovirus, Aveparvovirus, Protoparvovirus, Copiparvoirus, Erythroparvovirus, Dependoparvovirus, Tetraparvovirus, or Bocaparvovirus genus.

[0140] In some embodiments, the virus is a retrovirus. In some embodiments, the retrovirus is from the Alpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Epsilonretrovirus, Lentivirus, Spumavirus, or the Family Metaviridae, Pseudoviridae, and Retroviridae (including HIV), Hepadnaviridae (including Hepatitis B virus), or Caulimoviridae (including Cauliflower mosaic virus) genus.

[0141] In some embodiments, the virus is an influenza A or B virus. The influenza A or B virus antigen can be M2e (SLLTEVETPIRNEWGCRCNDSS) or a fragment thereof, a polypeptide of the HA stem (HA2 region), a polypeptide of the full-length HA protein, or a polypeptide of the NA protein. In some embodiments, the virus is SARS-CoV-2. The SARS- CoV-2 antigen can be a polypeptide of the spike RBD protein, a polypeptide of the spike S1 / S2 protein, a polypeptide of the pre-fusion spike protein (e.g., 2P / 6P polypeptide). In some embodiments, the virus is Respiratory syncytial virus (RSV). The RSV antigen can be a polypeptide of the prefusion F (pre-F) protein or a G ectodomain peptide. In some embodiments, the virus is a human papillomavirus (HPV) (e.g., HPV 6, 11, 16, 18, etc.). The HPV antigen can be a polypeptide of the capsid protein (e.g., LI protein) or an L2 N-terminus region (e.g., an L2 N-terminal peptide, such as amino acids 17-36). In some embodiments, the virus is Hepatitis B (HBV) or C (HBC). The HBV antigen can be a polypeptide of the hBsAG protein (“S” protein), or a polypeptide of the determinant loop (e.g., aa 124-127). The HBC antigen can be a polypeptide of E2 (CD81-binding region); E1ZE2 heterodomains, or a core peptide. In some embodiments, the virus is a human cytomegalovirus (HCVM). The HCVMantigen can be a polypeptide of the gB ectodomain, a pentamer complex peptide (gH / gL / UL- 128-131). In some embodiments, the virus is an Epstein-Barr virus (EBV). The EBV antigen can be a polypeptide of the gp350 protein or an Ebna3 -derived T-cell peptide. In some embodiments the virus is a human immunodeficiency virus (HIV) (e.g., HIV-1). The HIV antigen can be a polypeptide of the protein Env gpl20 / gpl40; a polypeptide of the protein Gag pl7 / p24 CTL epitopes (e.g., pl7 SL9 SLYNTVATL); or a conserved MPER peptides. In some embodiments, the virus is a flavivirus (e.g., a flavivirus causing Dengue 1-4, Zika, West Nile, or Yellow fever). The flavivirus antigen can be a polypeptide of the envelope EDIII domain or a fusion loop peptide in protein E. In some embodiments, the virus is a poliovirus or an enterovirus. The poliovirus or enterovirus antigen can be a polypeptide of the VP1 or VP3 surface loop of the virus. In some embodiments, the virus is the virus causing measles, mumps, or rubella. The antigen for the measles causing virus can be a polypeptide of the H and / or F glycoprotein. The antigen for the mumps causing virus can be a polypeptide of the HN and / or F proteins. The antigen for the mumps causing virus can be a polypeptide of the El or E2 proteins.

[0142] In some embodiments, the fungus is an Aspergillus, Blastomyces, Candidiasis, Coccidiodomycosis, Cryptococcus neoformans, Cryptococcus gatti, Histoplasma, Mucroymcosis, Pneumocystis, Sporothrix, Exserohilum, or Cladosporiu species fungus.

[0143] In some embodiments, the pathogenic bacterium is Acinetobacter baumanii. Actinobacillus sp., Aclinomyceles. Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii . Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocy tophilum, Anaplasma marginale, Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. ( such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp. (such as Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus), Capnocytophaga sp., Cardiobacteriumhominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enter oinvasive E. coli, enter opathogenic E. coli, enter ohemorrhagic E. coli, enteroaggregative E. coli and uropathogenic E. coli) Enterococcus sp. (such as Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae , Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacter fennelliae), Kingella kingii, Klebsiella sp. ( such as Klebsiella pneumoniae, Klebsiella granulomatis and Klebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Mannheimia hemolytica, Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp. , Mycobacterium sp. (such as Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium paratuberculosis, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm sp. (such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica and Nocardia brasiliensis). Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides. Prevotella sp., Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium), Serratia sp.(such as Serratia marcesans and Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri. Shigella boydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis. Staphylococcus hemolylicus. Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcus pneumoniae (for example chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin- resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin- resistant serotype 9V Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F Streptococcus pneumoniae), Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus pyogenes, Group B streptococci, Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus, Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group F streptococci, and Streptococcus anginosus Group G streptococci), Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela and Vibrio furnish), Yersinia sp. ( such as Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis) or a Xanthomonas maltophilia bacteria.

[0144] In some embodiments, the commensal bacterium is a gut commensal bacterium, mouth commensal bacterium, a vaginal commensal bacterium, or a skin commensal bacterium. In some embodiments, the gut commensal bacterium is within the phyla Firmicutes, Bacteroidetes, Actinobacteria, or Proteobacteria. In some embodiments, the gut commensal bacterium is within the genera Bacteroides, Clostridium, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia, orLactobacillus. In some embodiments, the mouth commensal bacterium is Actinomyces viscosus or A. naeslundii. In some embodiments, the vaginal commensal bacterium is from the genus Lactobacillus, including but not limited to, L. iners, L. crispatus, L. jensenii, L. delbruekii and L. gasseri).

[0145] In some embodiments, the pathogenic yeast is an Aspergillus species, a Geotrichum species, a Saccharomyces species, a Hansenula species, a Candida species, a Kluyveromyces species, a Debaryomyces species, or a Pichia species yeast. In some embodiments, the pathogenic fungus is a mold. In some embodiments, the mold is a Penicillium species, a Cladosporium species, or a Byssochlamys species mold.

[0146] In some embodiments, the pathogenic protozoan is from the Euglenozoa, Heterolobosea, Diplomonadida, Amoebozoa, Blastocystic, or Apicomplexa family. Example Euglenoza include, but are not limited to, Trypanosoma cruzi (Chagas disease), T. brucei gambiense, T. brucei rhodesiense, Leishmania braziliensis, L. infantum, L. mexicana, L. major, L. tropica, and L. donovani. Example Heterolobosea include, but are not limited to, Naegleria fowleri. Example Diplomonadid include, but are not limited to, Giardia intestinalis (G. lamblia, G. duodenalisj Example Amoebozoa include, but are not limited to, Acanthamoeba castellanii, Balamuthia madrillaris, Entamoeba histolytica. Example Blastocystis include, but are not limited to, Blastocystic hominis. Example Apicomplexa include, but are not limited to, Babesia microti, Cryptosporidium parvum, Cyclospora cayetanensis, Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and Toxoplasma gondii.

[0147] In some embodiments, the bacterium is Clostridium tetani. The antigen for Clostridium tetani can be tetanus toxoid or the universal Th epitope TT830-843 (QYIKANSKFIGITE (SEQ ID NO: 1)). In some embodiments, the bacterium is Corynebacterium diphtheriae. The antigen for Corynebacterium diphtheriae can be diphtheria toxoid or a polypeptide of CRM197 (carrier polypeptide). In some embodiments, the bacterium is Bacillus anthracis. The Bacillus anthracis antigen can be a polypeptide of the protective antigen PA83 (e.g., PA83 or fragments thereof). In some embodiments, the bacterium is Neisseria meningitidis (e.g. a bacterium causing meningitis A, B, C, W, or Y). The Neisseria meningitidis antigen can be a polypeptide of NadA, NHBA; or a PorA variable loop. In some embodiments, the bacterium is Streptococcus pneumoniae. The Streptococcus pneumoniae antigen is a polypeptide of the PspA; PsaA; or a pneumolysin toxoid peptides. In some embodiments, the bacterium is a Group A Streptococcus (e.g., S. pyogenes). The Group AStreptococcus (e.g., S. pyogenes) antigen can be a polypeptide of conserved M protein (e.g., QAEDKVKQSREAKKQVEKAL (SEQ ID NO: 2)). In some embodiments, the bacterium is Mycobacterium tuberculosis. The Mycobacterium tuberculosis antigen can be a polypeptide of ESAT-6 (e.g., (1-20: MTEQQWNFAGIEAAASAIQG (SEQ ID NO: 3)); a polypeptide of Ag85B (e.g., FQDAYNAAGGHNAVF (SEQ ID NO: 4)); or a CFP-10 peptide. In some embodiments, the bacterium is Bordetella pertussis. The Bordetella pertussis antigen can be a Pertussis toxoid; an FHA peptide; or a polypeptide of a pertactin loop. In some embodiments, the bacterium is Salmonella, Shigella, or an E. coli. The Salmonella, Shigella, or an E. coli antigen can be a polypeptide of an outer membrane protein, or a type-3 secretion needle tip peptide. In some embodiments, the bacterium is Helicobacter pylori. The Helicobacter pylori antigen can be a polypeptide of the urease subunit, or a CagA or VacA peptide motif. In some embodiments, the fungus is Candida albicans. The Candida albicans antigen can be a polypeptide of the Als3p N-terminal region (recombinant N-domain); or a Sap peptide. In some embodiments, the fungus is Aspergillus fumigatus. The Aspergillus fumigatus antigen can be an Asp fl or f3 peptide. In some embodiments, the protozoan is Plasmodium falciparum or vivax. In some embodiments, Plasmodium falciparum or vivax antigen is a CSP repeat polypeptide (e.g., NANP.... repeats), a CSP junctional peptide, a polypeptide of AMA1, or a MSP1 peptide. In some embodiments, the protozoan is Toxoplasma gondii. The Toxoplasma gondii antigen can be a KMP-11 peptide. In some embodiments, the helminth is a Schistosoma a Onchocerca or a hookworm. The Schistosoma a Onchocerca or a hookworm antigen is a polypeptide of a surface tegument protein or of the protease protein.

[0148] In some embodiments, the antigen is for eliciting an immune response against a cancer cell. Exemplary cancers to which the antigen can be for include, without limitation, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, Kaposi sarcoma (HHV-8 / KSHV), AIDS-related lymphoma (often EBV-positive), primary central nervous system (CNS) lymphoma (often EBV-positive in immunosuppressed patients), anal cancer (HPV), appendix cancer, astrocytomas, atypical teratoid / rhabdoid tumors, basal cell carcinoma of the skin, bile duct cancer (including intrahepatic and extrahepatic cholangiocarcinoma), bladder cancer (urothelial carcinoma), bone cancer (including Ewing sarcoma, osteosarcomas, and malignant fibrous histiocytoma / undifferentiated pleomorphic sarcoma), brain tumors (including glioblastoma, oligodendroglioma, meningioma, medulloblastoma), breast cancer, bronchial tumors, Burkitt lymphoma (endemic formclassically EBV-associated), carcinoid tumor / neuroendocrine tumors, cardiac tumors, germ cell tumors (gonadal and extragonadal), embryonal tumors, cervical cancer (HPV), cholangiocarcinoma, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasms (including polycythemia vera, essential thrombocythemia, primary myelofibrosis), colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, eye cancer (including intraocular melanoma and retinoblastoma), fallopian tube cancer, gallbladder cancer, gastric (stomach) cancer (subset EBV-positive gastric carcinoma), gastrointestinal stromal tumors, CNS germ cell tumors, ovarian germ cell tumors, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancers (including oropharyngeal / tonsillar / base-of-tongue squamous cell carcinomas that are HPV-positive, and nasopharyngeal carcinoma, EBV), hepatocellular (liver) cancer (HBV and / or HCV), Langerhans cell histiocytosis, Hodgkin lymphoma (subsets EBV-positive), hypopharyngeal cancer, islet cell tumors / pancreatic neuroendocrine tumors, kidney (renal cell) cancers (including clear cell, papillary, chromophobe, collecting duct), laryngeal cancer, leukemia, lip and oral cavity cancers (including HPV-associated oropharyngeal SCC), lung cancer (non-small cell and small cell), lymphoma (including diffuse large B-cell, follicular, mantle cell, marginal zone, peripheral T- cell, anaplastic large cell), melanoma, Merkel cell carcinoma (Merkel cell polyomavirus), mesothelioma, metastatic squamous cell neck cancer, midline tract carcinoma with and without NUT gene changes (NUT carcinoma), multiple endocrine neoplasia syndromes, multiple myeloma and plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus cancers (including HPV-associated sinonasal SCC), non-Hodgkin lymphoma (including HCV-associated marginal zone / DLBCL subsets), pancreatic ductal adenocarcinoma, paraganglioma, parathyroid cancer, penile cancer (HPV), pharyngeal cancers (oropharyngeal, nasopharyngeal [EBV], hypopharyngeal), pheochromocytoma, pituitary cancer, peritoneal cancer (including primary peritoneal carcinoma), prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, uterine sarcoma, Sezary syndrome, skin cancers (including cutaneous squamous cell carcinoma and melanoma), small intestine cancer, large intestine (colon) cancer, soft tissue sarcomas (including leiomyosarcoma, liposarcoma, synovial sarcoma, angiosarcoma, dermatofibrosarcoma protuberans), T-cell lymphomas (including extranodal NK / T-celllymphoma, nasal type — EBV), throat cancers, thymoma, thymic carcinoma, thyroid cancers (papillary, follicular, medullary, anaplastic), transitional / urothelial cell cancer of the renal pelvis and ureter, urethral cancer, uterine cancer (including endometrial and uterine serous carcinoma), vaginal cancer (HPV), cervical cancer (HPV), vascular tumors and cancers, vulvar cancer (HPV), Wilms tumor, **adult T-cell leukemia / lymphoma (HTLV-1), primary effusion lymphoma (HHV-8 / KSHV, often EBV co-positive), andKSHV-associated plasmablastic / large B-cell lymphomas arising in multicentric Castleman disease.

[0149] In some embodiments, the cancer antigen is a polypeptide that is shared or overexpressed by a cancer cell. Such antigens include, but are not limited to, HER2 / neu (ERBB2) E75 (KIFGSLAFL (SEQ ID NO: 5) (HLA-A2)); NY-ESO-1 157-165 (SLLMWITQC (SEQ ID NO: 6) (HLA-A2)); gplOO 209-217 (ITDQVPFSV (WT) (SEQ ID NO: 7) or IMDQVPFSV (210M) (SEQ ID NO: 8)); MART- 1 / Mel an- A 26-35 (EAAGIGILTV (SEQ ID NO: 9) or analog ELAGIGILTV)); MAGE- A3 271-279 (FLWGPRALV (SEQ ID NO: 10)); WT1 126-134 (RMFPNAPYL (SEQ ID NO: 11)); Survivin 96-104 (ELTLGEFLK (SEQ ID NO: 12)); hTERT 540-548 (ILAKFLHWL; (SEQ ID NO: 13) EGFRvIII (glioblastoma) (PEPvIII (LEEKKGNYVVTDH (SEQ ID NO: 14)); KRAS (e.g., G12D / G12V) — short neoepitope peptides (variant-specific); mutation-defined CTL peptides; polypeptides of gplOO, MART-1, TYRP2, NY-ESO-1 (melanoma); HER2 ECD peptides (e.g., E75), p95HER2 fragments (HER2+ cancers, such as breast cancer); WT1, NY- ESO-1; patient-specific neoantigens (lung cancer, e.g., NSCLC); CEA peptides; KRAS neoepitopes (colorectal cancer); WT1 peptides; PR1 (from proteinase 3) (hematologic cancers, e.g., a leukemia (e.g., acute myeloid leukemia or MDS); a PSA polypeptide (e.g., PSA-3A peptide) or a PAP fragment (prostate cancer); or a polypeptide of NY-ESO-1; MUC1 VNTR (ovarian cancer).

[0150] In some embodiments, the MOI(s) include one or more antigens for a non-infectious disease. In some embodiments, the non-infectious disease is a genetic disease or disorder, a neurodegenerative disease or disorder, an endocrine disease or disorder, a metabolic disease or disorder, a cardiac disease or disorder, a reproductive disease or disorder, a pulmonary disease or disorder, a muscle disease or disorder, a bone disease or disorder, a blood disease or disorder, a skin disease or disorder, a kidney disease or disorder, a pancreas disease or disorder, a liver disease or disorder, an immune system disease or disorder, a splenic disease or disorder, an inflammatory disease or disorder, an autoimmune disease or disorder, a brain disease ordisorder, a nerve disease or disorder, an intestinal disease or disorder, an addiction or substance abuse disorder, a behavioral disease or disorder, an allergy, a neuropathy, pain (acute or chronic), a venom, a tick borne protein, a prion disease, or any combination thereof.

[0151] In some embodiments, the non-infectious disease is Alzheimer’s disease. The Alzheimer’s disease antigen can be Api-42(DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO: 15)), an A 1-6 or 1-15 short peptide, or a tau phospho-epitope peptide. In some embodiments, the non- infectious disease is hypertension. The hypertension antigen can be Angiotensin II (DRVYIHPFHL (SEQ ID NO: 16)). In some embodiments, the non-infectious disease is hypercholesterolemia. The hypercholesterolemia antigen is a PCSK9 peptide mimotopes (e.g., an AFFITOPE-class short peptide). In some embodiments, the non-infectious disease is obesity. The obesity antigen can be a polypeptide of ghrelin or an active fragment thereof (e.g., an peptide of the octanoylated N-terminus region of ghrelin). In some embodiments, the non- infectious disease is diabetes. The diabetes antigen can be a polypeptide of DPP -4 or a glucagon peptidomimetic. In some embodiments, the non-infectious disease is an allergy (e.g. an allergy to a plant, animal, food, etc.). In some embodiments, the non-infectious disease is a drug or alcohol addiction (e.g., nicotine, cocaine, opioid (oxycodone, fentanyl, heroin, etc.), methamphetamine, MDMA, PCP, THC, etc. addiction).Additional Immunostimulatory Molecules

[0152] In addition to an antigen, the nanoparticles can include one or more additional immunostimulatory molecules (also referred to interchangeably herein as “immunostimulatory agents”). The additional immunostimulatory agent(s) can be capable of stimulating B cells and / or T cells. Assays to determine T cell, B cell, or other immune system component are generally known in the art. Where two or more species of immunostimulatory agents are present, the two or more species can be segregated to different locations (e.g. species one can be attached to the surface of the lipid shell and species two can be attached to the core) or can be non-discrimatorily dispersed on various structures (e.g. all species present can be attached to the core and / or outer surface of the lipid shell). One of ordinary skill in the art will recognize that the preceding examples are only representative of the many different ways in which the optional additional immunostimulatory agent(s) can be associated with different locales of the nanoparticles of the present description.

[0153] Suitable additional immunostimulatory agents can include, without limitation, adjuvants, haptens (antigen and non-antigen haptens), carrier proteins, natural or synthetic Toll-like receptor (TLR) agonists, dendritic cell surface molecule agonists, NOD-like receptor agonists, cytokines, proinflammatory stimulating molecules, complement cascade moleclues, activated components of immue complexes, antigen presenting cell agonists, T-cell receptor agonists, glcyoproteins, glycopolypeptides, proteins, peptides, small molecules, toxins and / or combinstions thereof. Specifc non-limiting examples of suitable immunostimulatory agents can include, without limitation, CpG oligodeoxynucleotides, bacterial lipopolyaacharides, VSV-G viral protein, HMGB-1, additional TLR agonists (e.g. TLR-1TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, TLR-9, and TLR- 10 agonists, urate crystals, CD21 and its agonists, CD35 and its agonists, CD40 agonists, KLH, tetanus toxoid, alum, and any combination thereof.

[0154] In some embodiments, the additional immunostimulatory agent is a hapten. Haptens are generally known in the art and are small non-protein antigens that can be used with a carrier or Th-cell helper epitope. In some embodiments, the hapten is for a drug, such as a drug of abuse. In these embodiments, the hapten can be as follows for e.g., nicotine (e.g., 3'- aminomethylnicotine, 6-carboxynicotine, nicotine-butyric / aminobutyl haptens), cotinine haptens; cocaine (e.g., succinyl-norcocaine, GNE cocaine haptens); heroin / morphine / oxycodone / oxymorphone (e.g., morphine-6-hemi succinate, 6-glutaryl- morphinan, oxy codone-6-hemi succinate); fentanyl / carfentanil and other synthetic opioids (p- aminophenethyl-fentanyl, carfentanil analog haptens); methamphetamine / amphetamine haptens; phencyclidine (PCP) haptens; cannabinoids including A9-THC (THC-COOH and linkerized THC analogs), synthetic cannabinoids (e.g., JWH-018 haptens); benzodiazepines (diazepam / oxazepam temazepam hemi succinates); or alcohol use disorder targets (acetaldehyde-mimetic haptens and malondialdehyde-protein adduct mimetics). In some embodiments, the hapten is for toxins, environmental & food contaminants. In these embodiments, the hapten can be a flatoxin Bi hapten; a microcystin-LR hapten; a saxitoxin / anatoxin-a hapten; a digoxin / lanatoside and ouabain hapten; a paraquat and diquat hapten; a organophosphates (e.g., chlorpyrifos, parathion) hapten; a pyrethroid (e.g., deltamethrin) hapten; a bisphenol A, phthalates (DEHP), per- / polyfluoroalkyl substances (PFOA / PFOS) hapten; a nicotine-derived nitrosamines (NNK) hapten; a polychlorinated biphenyl (PCB) hapten; or any combination thereof. In some embodiments, the hapten is forsmall-molecule hormones and metabolites. In these embodiments, the hapten can be estradiol, progesterone, testosterone, dihydrotestosterone, cortisol / corticosterone haptens; bile acid haptens (e.g., chenodeoxy cholate analogs); oxidized LDL mimetic haptens (e.g., phosphocholine headgroup mimics). In some embodiments, a peptide hormones antigen is present as hapten-style conjugates. For example, Angiotensin II (DRVYIHPFHL (SEQ ID NO: 17)), GnRH / LHRH (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NFE), and ghrelin (GSSFLSPEHQRVQQRKESKKPPAKLQPR (SEQ ID NO: 18), where Ser3is typically O- octanoylated.

[0155] In some embodiments, the additional immunostimulatory agent is a T-cell helper epitope. Exemplary T-cell helper epitopes that can be included in the nanoparticles of the present disclosure are Pan-DR epitope (PARDE) (e.g., AKFVAAWTLKAAA (SEQ ID NO: 19)); tetanus toxoid TT830-843 (e.g., QYIKANSKFIGITE (SEQ ID NO: 20)), and influenza HA306-318 (e.g., PKYVKQNTLKLAT (SEQ ID NO: 21).Targeting Molecules

[0156] In some embodiments, a MOI can be a targeting molecule. In some embodiments, the binding partner is a receptor, channel, or other complex present on the surface of a target cell. In some embodiments, the targeting molecule targets a receptor that is present on all cell types. In some embodiments, the targeting molecule targets a receptor that is present on multiple cell types. In some embodiments, the targeting molecule targets a receptor that is present on a single cell type. In some embodiments, the targeting molecule targets a specific cell or tissue type and / or cell state.

[0157] In some embodiments, the targeting molecule is or includes a peptide or a polypeptide. In some embodiments, the targeting molecule is or includes an antibody or fragment thereof. In some embodiments, the targeting molecule is or includes an aptamer (see e.g., . In some embodiments, the targeting molecule is or includes a small molecule. In some embodiments, the targeting molecule is or includes a nucleic acid (e.g., DNA or RNA). In some embodiments, the targeting molecule is or includes a receptor. In some embodiments, the targeting molecule is or includes a receptor ligand. In some embodiments, the targeting molecule is or includes a carbohydrate (e.g., a sugar). In some embodiments, the targeting molecule is or includes a lipid. In some embodiments, the targeting molecule is an engineered protein scaffold (see e.g., Nat Biotechnol 2005, 23: 1257-1268; Gebauer and Skerra.Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol. 2009,13:245-55; Gill and Damle. Biopharmaceutical drug discovery using novel protein scaffolds. Curr Opin Biotechnol 2006, 17:653-658; Skerra. Engineered protein scaffolds for molecular recognition. J Mol Recognit 2000, 13:167-187; and Skerra.Alternative non-antibody scaffolds for molecular recognition. Curr Opin Biotechnol 2007, 18:295-304). In some embodiments, the targeting molecule is an affibody. In some embodiments, the targeting molecule is an antibody mimetic. In some embodiments, the targeting molecule is an engineered binding protein, such as a designed ankyrin repeat proteins (DARPins) (see e.g., Pliickthun et al., Annu. Rev. Pharmacol. Toxicol. (2015) 55(1): 489-511), avimers (Silverman et al., Nat. Biotechnol. (2005) 23 (12): 1556-1561 and Jeong et al. Nat. Biotechnol. (2005), 23(12): 1493-1494), or affibodies (see e.g., Nord et al., Nat. Biotechnol. (1997) 15(8):772-777). In some embodiments, the targeting molecule is a receptor ligand or binding protein.

[0158] In some embodiments, the targeting moiety is a chemical small molecule, such as a small molecule receptor ligand. Exemplary small organic molecules range in size up to about 5000 Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500 Da. In certain embodiments, the small molecule may act as an antagonist or agonist (e.g., blocking an enzyme active site or activating a receptor by binding to a ligand binding site).

[0159] Suitable specific targeting molecules that can be used in context of the nanoparticles of the present description are generally known to one of skill in the art.

[0160] Exemplary target cells include, but are not limited to liver cells, pancreatic cells, muscle cells (e.g., skeletal, cardiac, and / or smooth muscle cells), brain cells, neurons, nerve support cells (e.g., glial cells, Schwann cells, astrocytes, dendrites, etc.), immune cells (T-cells, B-cells, monocytes, macrophages, dendritic cells, NK cells, neutrophils, plasma cells, etc.), kidney cells, thyroid cells, bone cells, gastrointestinal tract cells, auditory cells (e.g., hair cells), eye cells (e.g., retinal cells, corneal cells, etc.), skin cells, lung cells, adipocytes, bladder cells, olfactory cells, vasculature cells, cancer cells, tumor cells, cancer stem cells, infectious microorganisms (bacteria, phages, viruses, and the like), microbiome cells, and / or the like. In some embodiments, the target cells are diseased. In some embodiments, the target cells are normal (non-diseased). In some embodiments, the target cells are progenitor cells. In some embodiments, the target cells are differentiated cells.Reporter and Barcoding Molecules

[0161] The MOI can be a reporter molecule or a barcode molecule. These allow for selection, monitoring, and identification of the nanoparticles during production and / or during and / or after delivery or use. Suitable reporter ad barcoding molecules include, but are not limited to, affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag; solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP, and GST; chromatography tags such as those consisting of polyanionic amino acids, such as FLAG-tag; epitope tags such as V5-tag, Myc-tag, HA-tag and NE-tag; protein tags that can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging), DNA and / or RNA segments that contain restriction enzyme or other enzyme cleavage sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT)) and the like; DNA and / or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and / or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as P-galactosidase, GUS; optically active molecules, including but not limited to fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), mCherry, dtTomato; luciferase, quantum dots, and cell surface proteins); polynucleotides that can generate one or more new primer sites for PCR (e.g., the juxtaposition of two DNA sequences not previously juxtaposed), DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g. GFP, FLAG- and His-tags), and, polynucleotide or polypeptide sequences that make a molecular barcode or unique molecular identifier (UMI), DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art.Therapeutic Molecules

[0162] The MOI can be a therapeutic molecule, i.e., a molecule that when delivered has a therapeutic effect. Exemplary therapeutic compounds include but are not limited to, polynucleotides (e.g., DNA, RNA, combinations thereof), polypeptides, nucleoproteins, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics,psychoactive compounds, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, radiation sensitizers, chemotherapeutics.

[0163] Suitable hormones include, but are not limited to, amino-acid derived hormones (e.g. melatonin and thyroxine), small peptide hormones and protein hormones (e.g. thyrotropinreleasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle- stimulating hormone, and thyroid-stimulating hormone), eicosanoids (e.g. arachidonic acid, lipoxins, and prostaglandins), and steroid hormones (e.g. estradiol, testosterone, tetrahydro testosterone, cortisol).

[0164] Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g. IL-2, IL-7, and IL-12) , cytokines (e.g. interferons (e.g. IFN-a, IFN-P, IFN-s, IFN-K, IFN-co, and IFN-y), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g. CCL3, CCL26 and CXCL7) , cytosine phosphate-guanosine, oligodeoxynucleotides, glucans, antibodies, affibodies, and aptamers).

[0165] Suitable antipyretics include, but are not limited to, non-steroidal antiinflammatories (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate), paracetamol / acetaminophen, metamizole, nabumetone, phenazone, and quinine.

[0166] Suitable anxiolytics include, but are not limited to, benzodiazepines (e.g., alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotonergic antidepressants (e.g., selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), temgicoluril, fabomotizole, selank, bromantane, emoxypine, azapirones, barbiturates, hydroxyzine, pregabalin, isovaleric acid, and beta blockers.

[0167] Suitable antipsychotics include, but are not limited to, benperidol, bromperidol, droperidol, haloperidol, moperone, pipamperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dixyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupentixol, tiotixene, zuclopenthixol, clotiapine, loxapine, prothipendyl, carpipramine, clocapramine, molindone, mosapramine, sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine,clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride, olanzapine, paliperidone, perospirone, quetiapine, remoxipride, risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie, bifeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetad methionil, vabicaserin, xanomeline, and zicronapine.

[0168] Suitable psychoactive compounds include, but are not limited to caffeine; theobromine (in cocoa); nicotine (in tobacco products and some NRTs); alcohol (ethanol); L- theanine; melatonin (OTC in some countries, Rx in others); kava / kavalactones (legal in many places with restrictions); valerian; chamomile constituents (apigenin); prescription stimulants such as methylphenidate and amphetamine salts; prescription wake-promoters such as modafinil and armodafmil; prescription sedative-hypnotics such as zolpidem, eszopiclone, and benzodiazepines (e.g., diazepam, lorazepam, clonazepam); prescription antidepressants with psychoactive effects such as SSRIs / SNRIs (e.g., fluoxetine, sertraline, venlafaxine) and atypicals (e.g., bupropion, mirtazapine); prescription antipsychotics with sedative or mood effects (e.g., quetiapine, olanzapine); cannabis / cannabinoids where legal (THC products in legal jurisdictions; CBD hemp products where permitted); nonprescription antihistamines with sedating / anticholinergic effects (diphenhydramine, doxylamine); nitrous oxide; heroin / diacetylmorphine; illicit fentanyl and many fentanyl analogs; cocaine; crack cocaine; methamphetamine (note: medical Desoxyn exists but nonmedical possession is typically illegal); MDMA (ecstasy, molly); MDA; MDEA; LSD; psilocybin / psilocin (illegal federally in the U.S. but decriminalized or permitted under some local / regulatory frameworks); mescaline (peyote cacti protected in some religious contexts); DMT and 5-MeO-DMT; ibogaine; PCP; ketamine (medical use legal, nonmedical possession often illegal); designer dissociatives such as MXE and various arylcyclohexylamines; synthetic cannabinoids (“spice,” K2; many specific analogs scheduled); synthetic cathinones (“bath salts,” e.g., mephedrone, MDPV, a-PVP); GHB (medical sodium oxybate is tightly regulated; otherwise often illegal); salvinorin A / salvia divinorum (legal in some places, restricted or banned in others); deliriant anticholinergics such as scopolamine and high-dose diphenhydramine misuse; NBOMe series (e.g., 251-NBOMe) and other potent psychedelic phenethylamines; various tryptamine and phenethylamine research chemicals.

[0169] Suitable analgesics include, but are not limited to, paracetamol / acetaminophen, nonsteroidal anti-inflammantories (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g. morphine, codeine,oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupirtine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g. choline salicylate, magnesium salicylate, and sodium salicylate).

[0170] Suitable antispasmodics include, but are not limited to, mebeverine, papaverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methocarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene. Suitable antiinflammatories include, but are not limited to, prednisone, non-steroidal anti-inflammantories (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immune selective anti-inflammatory derivatives (e.g., submandibular gland peptide-T and its derivatives).

[0171] Suitable anti-histamines include, but are not limited to, Hl -receptor antagonists (e.g., acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine, hydroxyzine, levocetirizine, loratadine, meclizine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2-receptor antagonists (e.g., cimetidine, famotidine, lafutidine, nizatidine, ranitidine, and roxatidine), tritoqualine, catechin, cromoglicate, nedocromil, and p2-adrenergic agonists.

[0172] Suitable anti-infectives include, but are not limited to, amebicides (e.g., nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine, amphotericin b, and iodoquinol), aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g., pyrantel, mebendazole, ivermectin, praziquantel, albendazole, thiabendazole, oxamniquine), antifungals (e.g., azole antifungals (e.g., itraconazole, fluconazole, posaconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g., caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g., nystatin, and amphotericin b), antimalarial agents (e.g., pyrimethamine / sulfadoxine, artemether / lumefantrine, atovaquone / proquanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), antituberculosis agents (e.g., aminosalicylates (e.g., amino salicylic acid),isoniazid / rifampin, isoniazid / pyrazinamide / rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antivirals (e.g., amantadine, rimantadine, abacavir / lamivudine, emtricitabine / tenofovir, cobicistat / elvitegravir / emtricitabine / tenofovir, efavirenz / emtricitabine / tenofovir, abacavir / lamivudine / zidovudine, lamivudine / zidovudine, emtricitabine / tenofovir, emtricitabine / lopinavir / ritonavir / tenofovir, interferon alfa-2v / ribavirin, peginterferon alfa-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscamet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delavirdine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, abacavir, zidovudine, stavudine, emtricitabine, zalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir / ritonavir, fosamprenavir, darunavir, ritonavir, tipranavir, atazanavir, nelfinavir, amprenavir, indinavir, saquinavir, ribavirin, valacyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g., doripenem, meropenem, ertapenem, and cilastatin / imipenem), cephalosporins (e.g., cefadroxil, cephradine, cefazolin, cephalexin, cefepime, ceflaroline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, and ceftazidime), glycopeptide antibiotics (e.g., vancomycin, dalbavancin, oritavancin, and telavancin), glycylcyclines (e.g. tigecycline), leprostatics (e.g. clofazimine and thalidomide), lincomycin and derivatives thereof (e.g. clindamycin and lincomycin), macrolides and derivatives thereof (e.g. telithromycin, fidaxomicin, erythromycin, azithromycin, clarithromycin, dirithromycin, and troleandomycin), linezolid, sulfamethoxazole / trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, penicillins (amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin / clavulanate, ampicillin / sulbactam, piperacillin / tazobactam, clavulanate / ticarcillin, penicillin, procaine penicillin, oxacillin, dicloxacillin, and nafcillin), quinolones (e.g., lomefloxacin, norfloxacin, ofloxacin, moxifloxacin, ciprofloxacin, levofloxacin, Gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin, and sparfloxacin), sulfonamides (e.g., sulfamethoxazole / trimethoprim, sulfasalazine, and sulfasoxazole), tetracyclines (e.g., doxycycline, demeclocy cline, minocycline, doxycycline / salicylic acid, doxycycline / omega-3 polyunsaturated fatty acids, and tetracycline), and urinary anti-infectives (e.g., nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue).

[0173] Suitable chemotherapeutics include, but are not limited to, paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, Cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, dacarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, vismodegib, asparaginase Erwinia chrysanthemin, amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylate, carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium-89 chloride, mechlorethamine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octreotide, dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine, thioguanine (tioguanine), dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustine hydrochloride, triptorelin, arsenic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafluposide, azathioprine, doxifluridine, vindesine, and all-trans retinoic acid.

[0174] Suitable radiation sensitizers include, but are not limited to, 5 -fluorouracil, platinum analogs (e.g., cisplatin, carboplatin, and oxaliplatin), gemcitabine, DNA topoisomerase I- targeting drugs (e.g., camptothecin derivatives (e.g., topotecan and irinotecan)), epidermal growth factor receptor blockade family agents (e.g., cetuximab, gefitinib), farnesyltransferase inhibitors (e.g., L-778-123), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), bFGF and VEGF targeting agents (e.g., bevazucimab and thalidomide), NBTXR3, Nimoral, trans sodium crocetinate, NVX-108, and combinations thereof. See also e.g., Kvols, L.K.., J Nucl Med 2005; 46: 187S— 190S.Programmable Stochiometric Ratios

[0175] In some embodiments, lipid shell comprises a programmable stochiometric ratio of a quantity of first lipid, the second lipid, and the third lipid. The programmable stochiometric ratio is such that the MOIs are incorporated into the surface of the nanoparticles of the present description at specific quantities which is not otherwise achievable by prior approaches, such as linkers, that offer no control of the amounts in which the surface molecules are incorporated.

[0176] In some embodiments the ratio of amounts (e.g., number of molecules / number of molecules / number of molecules; w / w / w, or v / v / v) of MOI 1: MOI 2: MOI 3 is 0.0:0.0:0.0,0.0:0.0:0.1, 0.0:0.0:0.2, 0.0:0.0:0.3, 0.0:0.0:0.4, 0.0:0.0:0.5, 0.0:0.0:0.6, 0.0:0.0:0.7,0.0:0.0:0.8, 0.0:0.0:0.9, 0.0:0.0:1.0, 0.0:0.1:0.0, 0.0:0.1:0.1, 0.0:0.1:0.2, 0.0:0.1:0.3,0.0:0.1:0.4, 0.0:0.1:0.5, 0.0:0.1:0.6, 0.0:0.1:0.7, 0.0:0.1:0.8, 0.0:0.1:0.9, 0.0:0.1:1.0,0.0:0.2:0.0, 0.0:0.2:0.1, 0.0:0.2:0.2, 0.0:0.2:0.3, 0.0:0.2:0.4, 0.0:0.2:0.5, 0.0:0.2:0.6,0.0:0.2:0.7, 0.0:0.2:0.8, 0.0:0.2:0.9, 0.0:0.2:1.0, 0.0:0.3:0.0, 0.0:0.3:0.1, 0.0:0.3:0.2,0.0:0.3:0.3, 0.0:0.3:0.4, 0.0:0.3:0.5, 0.0:0.3:0.6, 0.0:0.3:0.7, 0.0:0.3:0.8, 0.0:0.3:0.9,0.0:0.3:1.0, 0.0:0.4:0.0, 0.0:0.4:0.1, 0.0:0.4:0.2, 0.0:0.4:0.3, 0.0:0.4:0.4, 0.0:0.4:0.5,0.0:0.4:0.6, 0.0:0.4:0.7, 0.0:0.4:0.8, 0.0:0.4:0.9, 0.0:0.4:1.0, 0.0:0.5:0.0, 0.0:0.5:0.1,0.0:0.5:0.2, 0.0:0.5:0.3, 0.0:0.5:0.4, 0.0:0.5:0.5, 0.0:0.5:0.6, 0.0:0.5:0.7, 0.0:0.5:0.8,0.0:0.5:0.9, 0.0:0.5:1.0, 0.0:0.6:0.0, 0.0:0.6:0.1, 0.0:0.6:0.2, 0.0:0.6:0.3, 0.0:0.6:0.4,0.0:0.6:0.5, 0.0:0.6:0.6, 0.0:0.6:0.7, 0.0:0.6:0.8, 0.0:0.6:0.9, 0.0:0.6:1.0, 0.0:0.7:0.0,0.0:0.7:0.1, 0.0:0.7:0.2, 0.0:0.7:0.3, 0.0:0.7:0.4, 0.0:0.7:0.5, 0.0:0.7:0.6, 0.0:0.7:0.7,0.0:0.7:0.8, 0.0:0.7:0.9, 0.0:0.7:1.0, 0.0:0.8:0.0, 0.0:0.8:0.1, 0.0:0.8:0.2, 0.0:0.8:0.3,0.0:0.8:0.4, 0.0:0.8:0.5, 0.0:0.8:0.6, 0.0:0.8:0.7, 0.0:0.8:0.8, 0.0:0.8:0.9, 0.0:0.8:1.0,0.0:0.9:0.0, 0.0:0.9:0.1, 0.0:0.9:0.2, 0.0:0.9:0.3, 0.0:0.9:0.4, 0.0:0.9:0.5, 0.0:0.9:0.6,0.0:0.9:0.7, 0.0:0.9:0.8, 0.0:0.9:0.9, 0.0:0.9:1.0, 0.0:1.0:0.0, 0.0:1.0:0.1, 0.0:1.0:0.2,0.0:1.0:0.3, 0.0:1.0:0.4, 0.0:1.0:0.5, 0.0:1.0:0.6, 0.0:1.0:0.7, 0.0:1.0:0.8, 0.0:1.0:0.9,0.0:1.0:1.0, 0.1:0.0:0.0, 0.1:0.0:0.1, 0.1:0.0:0.2, 0.1:0.0:0.3, 0.1:0.0:0.4, 0.1:0.0:0.5,0.1:0.0:0.6, 0.1:0.0:0.7, 0.1:0.0:0.8, 0.1:0.0:0.9, 0.1:0.0:1.0, 0.1:0.1:0.0, 0.1:0.1:01,0.1:0.1:0.2, 0.1:0.1:0.3, 0.1:0.1:0.4, 0.1:0.1:0.5, 0.1:0.1:0.6, 0.1:0.1:0.7, 0.1:0.1:0.8,0.1:0.1:0.9, 0.1:0.1:10, 0.1:0.2:0.0, 0.1:0.2:0.1, 0.1:0.2:0.2, 0.1:0.2:0.3, 0.1:0.2:0.4,0.1:0.2:0.5, 0.1:0.2:0.6, 0.1:0.2:0.7, 0.1:0.2:0.8, 0.1:0.2:0.9, 0.1:0.2:1.0, 0.1:0.3:0.0,0.1:0.3:0.1, 0.1:0.3:0.2, 0.1:0.3:0.3, 0.1:0.3:0.4, 0.1:0.3:0.5, 0.1:0.3:0.6, 0.1:0.3:0.7,0.1:0.3:0.8, 0.1:0.3:0.9, 0.1:0.3:1.0, 0.1:0.4:0.0, 0.1:0.4:0.1, 0.1:0.4:0.2, 0.1:0.4:0.3,1.0:0.7:0.2, 1.0:0.7:03, 1.0:0.7:0.4, 1.0:0.7:03, 1.0:0.7:0.6, 1.0:0.7:0.7, 1.0:0.7:0.8,1.0:0.7:0.9, 1.0:0.7:1.0, 1.0:0.8:0.0, 1.0:0.8:0.1, 1.0:0.8:0.2, 1.0:0.8:03, 1.0:0.8:0.4,1.0:0.8:03, 1.0:0.8:0.6, 1.0:0.8:0.7, 1.0:0.8:0.8, 1.0:0.8:0.9, 1.0:0.8:1.0, 1.0:0.9:0.0,1.0:0.9:0.1, 1.0:0.9:0.2, 1.0:0.9:03, 1.0:0.9:0.4, 1.0:0.9:03, 1.0:0.9:0.6, 1.0:0.9:0.7,1.0:0.9:0.8, 1.0:0.9:0.9, 1.0:0.9:1.0, 1.0:1.0:0.0, 1.0: 1.0:0.1, 1.0: 1.0:0.2, 1.0: 1.0:03,1.0:1.0:0.4, 1.0: 1.0:03, 1.0:1.0:0.6, 1.0: 1.0:0.7, 1.0: 1.0:0.8, 1.0:1.0:0.9, or 1.0: 1.0: 1.0.Orthogonal Reaction Handles

[0177] As previously discussed, the nanoparticle can include orthogonal reaction handle pairs, which are unique to different MOIs to be attached to the lipid shell, (see e.g., FIGs 1A and 3A). In embodiments, one handle of the orthogonal reaction pair is part of or attached to a lipid of the lipid shell of the nanoparticle of the present description and the other handle of the orthogonal reaction pair is attached to a MOI so as to facilitate covalent attachment of an MOI to the lipid shell. By controlling the amount of a lipid with an orthogonal reaction handle present in the shell, the amount of a specific MOI can be controlled. Thus, this facilitates a programmable stochiometric ratio of multiple MOIs on the surface of the lipid shell. In some embodiments, the orthogonal reaction handles are biorthogonal reaction handles.

[0178] In some embodiments, the first orthogonal reaction handle (that is included in or coupled to a first lipid), the second orthogonal reaction handle (that is included in or coupled to a second lipid), the third reaction handle (that is included in or coupled to a third lipid) are each individually selected from the group consisting of: a maleimide, a tetrazine, an azide, a terminal alkyne, a strained alkyne, trans-cyclooctene, a norbornene, cyclopropane, an aldehyde, a ketone, a vinyl sulfone, an aryl boronic acid, a boronate ester, a sulfonyl fluoride, an aryl fluorosulfate, triarylphosphine, a thiol, or any combination thereof. In these embodiments, the lipid shell can further include a first MOI comprising a fourth orthogonal reaction handle, a second MOI including or coupled to a fifth orthogonal reaction handle, and a third MOI including or coupled with a sixth orthogonal reaction handle, and wherein the first MOI is covalently attached the first lipid via covalent binding between the first orthogonal reaction handle and the forth reaction handle, wherein the second MOI is covalently attached to the second lipid via covalent binding between the second orthogonal reaction handle and the fifth orthogonal reaction handle, and wherein the third MOI is covalently attached to the third lipid via covalent binding between the third orthogonal reaction handle and the sixth orthogonal reaction handle. In some embodiments, the fourth orthogonal reaction handle (that is includedin or coupled to first MOI), the fifth orthogonal reaction handle (that is included in or coupled to a second MOI), the sixth reaction handle (that is included in or coupled to a third MOI) are each individually selected from the group consisting of a maleimide, a tetrazine, an azide, a terminal alkyne, a strained alkyne, trans-cyclooctene, a norbornene, cyclopropane, an aldehyde, a ketone, a vinyl sulfone, an aryl boronic acid, a boronate ester, a sulfonyl fluoride, an aryl fluorosulfate, triarylphosphine, a thiol, or any combination thereof.

[0179] In embodiments, the orthogonal reaction handles are choses so as to form orthogonal reaction pairs. Exemplary orthogonal reaction handle pairs include, but are not limited to, Azide Cyclooctyne (DBCO / BCN / DIBAC); Azide Terminal alkyne; Tetrazine Trans-cyclooctene (TCO); Tetrazine Norbomene; Tetrazine Cyclopropene; Nitrone - Cyclooctyne (SPANC); Photo-nitrile imine Alkene (tetrazole photo-click); Aldehyde / ketone Aminooxy; Aldehyde / ketone Hydrazide; Aldehyde 1,2- Aminothiol(N-terminal cysteine); Aldehyde 2-Aminophenyl boronic acid; Maleimide Thio; Haloacetamide Thiol; Vinyl sulfone Thiol; Acryloyl (Michael acceptor) - Thiol; Sulfonyl fluoride (Ar-SCEF) Phenolate / amine / alcohol (with base / fluoride); Thioester N- terminal cysteine; Disulfide Thiol; Boronic acid Catechol / diol; Phenylboronic acid 1,2- or 1,3-diols (e.g., sugars); LPXTG motif Oligoglycine; SNAP -tag (O6-alkyl-G transferase) Benzylguanine; HaloTag (dehalogenase) Chloroalkane; SpyTag SpyCatcher; malemide«-> Thiol; Maleimide selenol, maleimide furan, maleimide amine; maleimide phosphine / radicals; Sortase A: LPXTG Glyn; HaloTag Chloroalkane; His-tag Ni-NTA; azide Terminal alkyne; azide a Strained cyclooctynes; azide Triarylphosphine; azide nitrene; tetrazine Trans-cyclooctene (TCO / sTCO); tetrazine substituted cyclopropenes; tetrazine Norbornene (exo > endo); tetrazine a vinyl ethers; a strained alkyne tetrazine; and tetrazine an isonitrile. Orthogonal reactions and additional specific handles and pairs are generally known and encompassed by the scope of this disclosure. See also e.g., Oprea and Smith. 2024. ACS Chem Biol. 2024 Dec 27;20(l): 19-32.Pharmaceutical Formulations

[0180] Also within the scope of this disclosure are pharmaceutical formulations (which include vaccine formulations) that can contain an amount of the nanoparticles of the present description as described in greater detail elsewhere herein. The nanoparticles of the present description can be provided to a subject in need thereof alone or as such as an active ingredient,in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulations contain an effective amount of the nanoparticles of the present description. The pharmaceutical formulations described herein can be administered to a subject in need thereof. The subject in need thereof can have a drug addiction. In some embodiments, the subject can be a human. In other embodiments, nanoparticles of the present description can be used in the manufacture of a medicament for the treatment or prevention of nicotine addiction in a subject. The term pharmaceutical formulation also encompasses pharmaceutically acceptable salts of the pharmaceutical formulations and / or active ingredients provided herein.Pharmaceutically Acceptable Carriers and Auxiliary Ingredients and Agents

[0181] The pharmaceutical formulations containing an effective amount of nanoparticles described herein can further include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.

[0182] The pharmaceutical formulations can be sterilized, and if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and / or aromatic substances, and the like which do not deleteriously react with the active composition.

[0183] In addition to the effective amount of nanoparticles described herein, the pharmaceutical formulation can also include an effective amount of an auxiliary active agent, including but not limited to, DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, chemotherapeutics, antihypertensives, anticoagulants, and antiarrhythmics.

[0184] The pharmaceutical formulations can include one or more suitable adjuvants. Suitable adjuvants are generally known in the art and can include but are not limited to aluminum salts (e.g., aluminum phosphate and aluminum hydroxide), organic adjuvants (e.g. squalene), and oil-based (e.g., MF59), CpG oligodeoxynucleotides, resiquimod, flagellin, gardiquimod, imiquimod, monophosphoryl lipid A, poly(I:C), and chitosan.Effective Amounts

[0185] The pharmaceutical formulations can contain an effective amount of the nanoparticles of the present description, and optionally, a therapeutically effective amount of an auxiliary agent. In some embodiments, the amount of the nanoparticles of the present description and / or optional auxiliary agent can be an effective amount, least effective amount, and / or therapeutically effective amount.

[0186] The effective amount, least effective amount, and / or therapeutically effective amount of the nanoparticles of the present description and / or optional auxiliary active agent contained in the pharmaceutical formulation can range from about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 pg, ng, pg, mg, or g or be any numerical value with any of these ranges.

[0187] In some embodiments, the effective amount, least effective amount, and / or therapeutically effective amount of the nanoparticles of the present description and / or optional auxiliary active agent can be an effective concentration, least effective concentration, and / or therapeutically effective concentration, which can each range from about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810,820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 pM, nM, pM, mM, or M or be any numerical value with any of these ranges.

[0188] In some embodiments, the effective amount, least effective amount, and / or therapeutically effective amount of the nanoparticles of the present description and / or optional auxiliary active agent can range from about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 IU or be any numerical value with any of these ranges.

[0189] In some embodiments, the effective amount, least effective amount, and / or therapeutically effective amount of the nanoparticles of the present description and / or optional auxiliary active agent present in the pharmaceutical formulation can range from about 0 to 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23,0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4,0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74,0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91,0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.9, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 % w / w, v / v, or w / v of the pharmaceutical formulation.

[0190] In some embodiments where a cell population is present in the pharmaceutical formulation (e.g., as a primary and / or auxiliary active agent), the effective amount of cells can range from about 2 cells to 1X101cells / mL, 1X1O20cells / mL or more, such as about 1X101cells / mL, 1X102cells / mL, 1X103cells / mL, 1X104cells / mL, 1X105cells / mL, 1X106cells / mL, lX107cells / mL, 1X108cells / mL, 1X109cells / mL, 1X1010cells / mL, 1X1011cells / mL, 1X101cells / mL, lX1013 / mL, 1X1014cells / mL, 1X1015cells / mL, 1X1016cells / mL, 1X1017cells / mL, 1X1018cells / mL, 1X1019cells / mL, to / or about 1X1O20cells / mL.

[0191] In some embodiments, the amount or effective amount of the one or more of the nanoparticles of the present description contained in the pharmaceutical formulation can range from about 1 pg / kg to about 10 mg / kg based upon the body weight of the subject in need thereof or average bodyweight of the specific patient population to which the pharmaceutical formulation can be administered. In some embodiments, the effective amount of the nanoparticles of the present description when administered can range from about 0.3 mg / kg body weight to about 30 mg / kg.

[0192] In embodiments where there is an auxiliary agent contained in the pharmaceutical formulation, the effective amount of the secondary active agent will vary depending on the auxiliary agent, the nanoparticles of the present description, the administration route, subject age, disease, stage of disease, among other things, which will be one of ordinary skill in the art.

[0193] In some embodiments, the nanoparticles of the present description can be included in the pharmaceutical formulation at a value greater than zero but less than 1, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 %, any number between 99.9% up to 100 %, and / or 100% w / w, v / v, or w / v of the total pharmaceutical formulation.

[0194] When optionally present in the pharmaceutical formulation, the auxiliary active agent can be included in the pharmaceutical formulation or can exist as a stand-alone compound or pharmaceutical formulation that can be administered contemporaneously or sequentially with the nanoparticles of the present description or pharmaceutical formulation thereof.

[0195] In some embodiments, the effective amount of the auxiliary active agent can range from about O to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 % w / w, v / v, or w / v of the total auxiliary active agents in the pharmaceutical formulation. In additional embodiments, the effective amount of the auxiliary active agent can range from about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8,99.9 % w / w, v / v, or w / v of the total pharmaceutical formulation.Dosage Forms

[0196] In some embodiments, the pharmaceutical formulations described herein can be provided in a dosage form. The dosage form can be administered to a subject in need thereof. The dosage form can be effective generate specific concentration, such as an effective concentration, at a given site in the subject in need thereof. In some embodiments, the given site is proximal to the administration site. In some embodiments, the given site is distal to the administration site. In some cases, the dosage form contains a greater amount of one or more of the active ingredients present in the pharmaceutical formulation than the final intended amount needed to reach a specific region or location within the subject to account for loss of the active components such as via first and second pass metabolism.

[0197] The dosage forms can be adapted for administration by any appropriate route. Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, parenteral, subcutaneous, intramuscular, intravenous, internasal, and intradermal. Other appropriate routes are described elsewhere herein. Such formulations can be prepared by any method known in the art.

[0198] Dosage forms adapted for oral administration can discrete dosage units such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or nonaqueous liquids; edible foams or whips, or in oil-in-water liquid emulsions or water-in-oil liquid emulsions. In some embodiments, the pharmaceutical formulations adapted for oral administration also include one or more agents which flavor, preserve, color, or help disperse the pharmaceutical formulation. Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as a foam, spray, or liquid solution. The oral dosage form can be administered to a subject in need thereof. Where appropriate, the dosage forms described herein can be microencapsulated.

[0199] The dosage form can also be prepared to prolong or sustain the release of any ingredient. In some embodiments, compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof described herein can be the ingredient whose release is delayed. In some embodiments the primary active agent is the ingredient whose release is delayed. In some embodiments, an optional secondary agent can be the ingredient whose release is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and thelike. Delayed release dosage formulations can be prepared as described in standard references such as "Pharmaceutical dosage form tablets," eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wlkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al., (Media, PA: Wiliams and Wlkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules and delayed release dosage forms of tablets and pellets, capsules, and granules. The delayed release can be anywhere from about an hour to about 3 months or more.

[0200] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0201] Coatings may be formed with a different ratio of water-soluble polymer, water insoluble polymers, and / or pH dependent polymers, with or without water insoluble / water soluble non-polymeric excipient, to produce the desired release profile. The coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, "ingredient as is" formulated as, but not limited to, suspension form or as a sprinkle dosage form.

[0202] Where appropriate, the dosage forms described herein can be a liposome. In these embodiments, primary active ingredient(s), and / or optional secondary active ingredient(s), and / or pharmaceutically acceptable salt thereof where appropriate are incorporated into a liposome. In embodiments where the dosage form is a liposome, the pharmaceutical formulation is thus a liposomal formulation. The liposomal formulation can be administered to a subject in need thereof.

[0203] Dosage forms adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for treatments of the eye or other external tissues, for example the mouth or the skin, the pharmaceutical formulations are applied as a topical ointment or cream. Whenformulated in an ointment, a primary active ingredient, optional secondary active ingredient, and / or pharmaceutically acceptable salt thereof where appropriate can be formulated with a paraffinic or water-miscible ointment base. In other embodiments, the primary and / or secondary active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Dosage forms adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.

[0204] Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders. In some embodiments, a primary active ingredient, optional secondary active ingredient, and / or pharmaceutically acceptable salt thereof where appropriate can be in a dosage form adapted for inhalation is in a particle-size- reduced form that is obtained or obtainable by micronization. In some embodiments, the particle size of the size reduced (e.g. micronized) compound or salt or solvate thereof, is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art. Dosage forms adapted for administration by inhalation also include particle dusts or mists. Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions / suspensions of an active (primary and / or secondary) ingredient, which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators. The nasal / inhalation formulations can be administered to a subject in need thereof.

[0205] In some embodiments, the dosage forms are aerosol formulations suitable for administration by inhalation. In some of these embodiments, the aerosol formulation contains a solution or fine suspension of a primary active ingredient, secondary active ingredient, and / or pharmaceutically acceptable salt thereof where appropriate and a pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol formulations can be presented in single or multi-dose quantities in sterile form in a sealed container. For some of these embodiments, the sealed container is a single dose or multi-dose nasal or an aerosol dispenser fitted with a metering valve (e.g. metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted.

[0206] Where the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon. The aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer. The pressurized aerosolformulation can also contain a solution or a suspension of a primary active ingredient, optional secondary active ingredient, and / or pharmaceutically acceptable salt thereof. In further embodiments, the aerosol formulation also contains co-solvents and / or modifiers incorporated to improve, for example, the stability and / or taste and / or fine particle mass characteristics (amount and / or profile) of the formulation. Administration of the aerosol formulation can be once daily or several times daily, for example 2, 3, 4, or 8 times daily, in which 1, 2, 3 or more doses are delivered each time. The aerosol formulations can be administered to a subject in need thereof.

[0207] For some dosage forms suitable and / or adapted for inhaled administration, the pharmaceutical formulation is a dry powder inhalable-formulations. In addition to a primary active agent, optional secondary active ingredient, and / or pharmaceutically acceptable salt thereof where appropriate, such a dosage form can contain a powder base such as lactose, glucose, trehalose, mannitol, and / or starch. In some of these embodiments, a primary active agent, secondary active ingredient, and / or pharmaceutically acceptable salt thereof where appropriate is in a particle-size reduced form. In further embodiments, a performance modifier, such as L-leucine or another amino acid, cellobiose octaacetate, and / or metals salts of stearic acid, such as magnesium or calcium stearate. In some embodiments, the aerosol formulations are arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the compositions, compounds, vector(s), molecules, cells, and combinations thereof described herein.

[0208] Dosage forms adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Dosage forms adapted for rectal administration include suppositories or enemas. The vaginal formulations can be administered to a subject in need thereof.

[0209] Dosage forms adapted for parenteral administration and / or adapted for inj ection can include aqueous and / or non-aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. The dosage forms adapted for parenteral administration can be presented in a single-unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The doses can be lyophilized and re-suspended in a sterile carrier to reconstitute the dose prior to administration. Extemporaneous injection solutions and suspensions can beprepared in some embodiments, from sterile powders, granules, and tablets. The parenteral formulations can be administered to a subject in need thereof.

[0210] For some embodiments, the dosage form contains a predetermined amount of a primary active agent, secondary active ingredient, and / or pharmaceutically acceptable salt thereof where appropriate per unit dose. In an embodiment, the predetermined amount of primary active agent, secondary active ingredient, and / or pharmaceutically acceptable salt thereof where appropriate can be an effective amount, a least effect amount, and / or a therapeutically effective amount. In other embodiments, the predetermined amount of a primary active agent, secondary active agent, and / or pharmaceutically acceptable salt thereof where appropriate, can be an appropriate fraction of the effective amount of the active ingredient.Co-Therapies and Combination Therapies

[0211] In some embodiments, the pharmaceutical formulation(s) described herein can be part of a combination treatment or combination therapy. The combination treatment can include the pharmaceutical formulation described herein and an additional treatment modality. The additional treatment modality can be a therapeutic drug (e.g., a chemotherapeutic or a biological therapeutic), surgery, radiation, diet modulation, environmental modulation, a physical activity modulation, mental health or behavioral therapy or counseling, and combinations thereof.

[0212] In some embodiments, the co-therapy or combination therapy can additionally include but not limited to, polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, chemotherapeutics, and combinations thereof.Administration of the Pharmaceutical Formulations

[0213] The pharmaceutical formulations or dosage forms thereof described herein can be administered one or more times hourly, daily, monthly, or yearly (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times hourly, daily, monthly, or yearly). In some embodiments, the pharmaceutical formulations or dosage forms thereof described herein can be administered continuously over a period of time ranging from minutes to hours to days. Devices and dosages forms are known in the art and described herein that are effective to provide continuous administration of the pharmaceutical formulations described herein. Insome embodiments, the first one or a few initial amount(s) administered can be a higher dose than subsequent doses. This is typically referred to in the art as a loading dose or doses and a maintenance dose, respectively. In some embodiments, the pharmaceutical formulations can be administered such that the doses over time are tapered (increased or decreased) overtime so as to wean a subject gradually off of a pharmaceutical formulation or gradually introduce a subject to the pharmaceutical formulation.

[0214] As previously discussed, the pharmaceutical formulation can contain a predetermined amount of a primary active agent, secondary active agent, and / or pharmaceutically acceptable salt thereof where appropriate. In some of these embodiments, the predetermined amount can be an appropriate fraction of the effective amount of the active ingredient. Such unit doses may therefore be administered once or more than once a day, month, or year (e.g. 1, 2, 3, 4, 5, 6, or more times per day, month, or year). Such pharmaceutical formulations may be prepared by any of the methods well known in the art.

[0215] Where co-therapies or multiple pharmaceutical formulations are to be delivered to a subject, the different therapies or formulations can be administered sequentially or simultaneously. Sequential administration is administration where an appreciable amount of time occurs between administrations, such as more than about 15, 20, 30, 45, 60 minutes or more. The time between administrations in sequential administration can be on the order of hours, days, months, or even years, depending on the active agent present in each administration. Simultaneous administration refers to administration of two or more formulations at the same time or substantially at the same time (e.g. within seconds or just a few minutes apart), where the intent is that the formulations be administered together at the same time.

[0216] Where the pharmaceutical formulations are vaccines, an initial dose can be followed by 1 or more doses given at varying intervals after the initial dose. In some embodiments, an initial dose can be administered followed with an administration given at about 1-4 weeks after the first initial dose and optionally one or more doses following the second dose about 1-4 weeks after the second dose. This can then be followed up by subsequent doses at larger intervals such as months or years after second dose.Methods of Use

[0217] The nanoparticles and / or pharmaceutical formulations thereof of the present description can be used to deliver an active agent, induce an immune response, particularly aB cell and / or T response in a subject (such as one specific to antigen(s) present on the surface of the nanoparticles of the present description). The nanoparticles and / or pharmaceutical formulations thereof of the present description prevent and / or treat a disease or disorder in a subject.

[0218] The method can include the step of administering an amount, such as an effective amount, of the nanoparticles and / or pharmaceutical formulations thereof of the present description to a subject. The subject can be suffering from a disease or condition. In some embodiments, the subject or a healthy subject (one who is not currently suffering from a disease or condition but where it is desirable to prevent or mitigate a disease or condition). In some embodiments, the method includes administering an amount, such as an effective amount of the nanoparticles or a pharmaceutical formulation thereof of the present disclosure to a subject whereby a therapeutic agent is delivered and thereby treats a disease or a symptom thereof in the subject.

[0219] In some embodiments, the method can include the step of administering an amount, such as an effective amount, to a subject such that a humoral and / or an innate immune response in the subject is stimulated. In some embodiments, the method can include the step of administering an amount, such as an effective amount, to a subject such that a B-cell and / or T- cell response is stimulated in the subject. In some embodiments, the B cell response is the generation of antibodies that can specifically bind an antigen that is an MOI on the surface of the nanoparticles of the present description. In some embodiments, the B-cell response includes memory B cell production. In some embodiments, the T-cell response include memory T-cell production. In some embodiments, antibodies produced can interact with the antigen in a subject and result in neutralization and / or clearance of the antigen from the body. In this way, the amount of antigen in the subject can be reduced and / or eliminated and thus treat the disease or disorder. The amount of the nicotine lipid-polymeric nanoparticles administered can be effective to decrease the amount of an antigen in the brain as compared to a control and / or before administration of the nicotine lipid-polymeric nanoparticles. The amount of the nicotine lipid-polymeric nanoparticles administered can be effective to increase the amount of the antigen in the serum as compared to a control and / or before administration of the nanoparticles of the present description.

[0220] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLES

[0221] Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.Example 1

[0222] Vaccination is the most effective way of protecting humans from various pathogens. Nanoparticle based vaccines (nanovaccines) have emerged as an area of research emphasis in vaccine development because of their ability to elicit strong immune responses.1Nanovaccines are commonly comprised of a nanoparticle conjugated with an antigenic protein, and they could initiate T cell-dependent activation of B cells that elicit a more robust immune response, affinity maturation, and immunological memory.2Different nano-delivery systems offers different efficiency for protein conjugation and plays an important role in the success of a nanovaccine.3

[0223] Among all nano delivery systems, nanoparticles made of poly(lactic-co-glycolic acid) (PLGA), which has been approved for human use by the US Food and Drug Administration (FDA), has been exploited widely in various biomedical applications because of its biodegradability, biosafety, and biocompatibility.3-7However, some constraints associated with the use of PLGA nanoparticles, such as poor drug encapsulation, particularly for lipophilic therapeutic agents, high burst release, phagocytic uptake, short half-life, immune reaction, and uncontrolled tissue distribution, have hampered their biomedical applications.5,7Liposome nanoparticles, on the other hand, have also been used in drug delivery and vaccine development,8 10but the utility of these nanoparticles is plagued by problems such as hypersensitivity, accelerated blood clearance, and limited stability.11 13To overcome the respective limitations of PLGA and liposome nanoparticles, hybrid nanoparticles that are comprised of a PLGA core and a liposome shell have been developed.5,7 14 17The biodegradable PLGA core can encapsulate desired therapeutic agents or adjuvants, is and the lipid layer could reduce the degradation rate of the PLGA core by limiting water diffusion inside of the particle, offering a controllable release kinetics. The lipid layer also allows the seamless incorporation of a widely used adjuvant, monophosphoryl lipid A (MPLA), for vaccine development.18,19Furthermore, by incorporating functionalized lipids, the surface of the hybrid nanoparticles could be chemically modified with targeting ligands or antigens for targeted delivery or eliciting a desired immune response.18,20

[0224] Recently, PLGA / lipid hybrid NPs have been extensively explored as potent nanocarriers for various antigens. For example, recent research results have demonstrated that the OVAencapsulated / adsorbed hNPs were capable of eliciting an extraordinary antigenspecific immune response.21,22Moreover, the versatile functionalized lipids offer the opportunity to display proteins on the surface of the hNPs which could increase the immunogenicity of a protein antigen.23Applicant has develop ed a library of protein-haptens conjugated PLGA / lipid hNPs as nicotine vaccines.15,18,24'31Through these vaccines, Applicant has demonstrated that different adjuvants, such as CpG oligodeoxynucleotides (CpG ODNs) and Resiquimod (R848), can be encapsulated in the PLGA core, while MPLA can be incorporated in the lipid layer.18Different proteins, including KLH (KLH), cross-reactive material 197 (CRM 197), tetanus toxoid (TT), and bovine serum albumin (BSA), were successfully conjugated to the surface of hNPs for enhanced immunological efficacy.15,24,25,28However, it is often necessary to develop multivalent vaccines for a pathogen that has distinctive variants, such as different serotypes of dengue viruses32and SARS-CoV-2,33or different pathogens that may emerge concurrently.34,35Multivalent vaccines have attracted more attention because they can elicit multiple antibodies simultaneously against different virus variants (SARS-CoV-2, influenza, etc.)36,37or opioid substitutes (fentanyl, oxycodone, heroin, morphine, methamphetamine, etc.).38'40Current preparation of multivalent vaccines was limited to simply mixing several monovalent nanovaccines or co-encapsulating several antigens in the formulation.36'40However, these manufacturing processes result multivalentvaccines with issues such as antigen competition and immune interference, difficult dose optimization, and formulation complexity. Therefore, there is an urgent need for a versatile strategy to display multiple antigenic proteins on the surface of the same nanoparticle as an “all-in-one” platform. This platform would make it much easier to control the relative dosage of each protein of the vaccine and minimize the concerns of batch-to-batch variations. In this Example, Applicant developed a strategy to conjugate three different proteins to the surface of a single hNP. Lipids with different functional groups were assembled into the lipid layer. Fluorescent dyes with different colors were used to label the proteins for easy identification, visualization, and quantification. The dye-labeled proteins were conjugated to the PLGA / lipid hNPs step-by-step through the maleimide-thiol Michael addition, Aizdo-DBCO, and TCO- Tetrazine click chemistry, respectively.Materials and MethodsMaterials

[0225] Lactel® (50:50 poly(lactic-co-glycolic acid) (PLGA)) and Poly(vinyl alcohol) (PVA, MW: 38K), was purchased from Durect Corporation (Cupertino, CA, USA). Keyhole limpet hemocyanin subunit (sKLH) was purchased from Biosyn Corporation (Carlsbad, CA, USA). Fluorescein isothiocyanate (FITC), NHS-Rhodamine (5 / 6-carboxy-tetramethyl- rhodamine succinimidyl ester: Rd-NHS), Imject™ Ovalbumin, Traut's Reagent (2- iminothiolane), Micro BCA™ Protein Assay Kits were purchased from Thermos fisher Scientific. Cyanine5 NHS ester (Cy5-NHS) was purchased from Lumiprobe. l,2-Dioleoyl-3- trimethylammonium-propane (DOTAP), cholesterol (CHOL), 1,2- distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide(polye-thylene glycol)-2000] (ammonium salt) (DSPE- PEG2K-Maleimide),l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(poly ethylene glycol)-2000] (ammonium salt) (DSPE-PEG2K Azide) and 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[amino(- polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2K- Amine) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The linker methyltetrazine-NHS ester, trans-cycloctene-NHS (TCO-NHS) and DBCO-NHS ester were purchased from BroadPharm (San Diego, CA, USA). Dialysis tubing with molecular weight cut-off (MWCO) IKDa was purchased from Repligen (Waltham, MA, USA). AmiconEJUltra centrifugal filters MWCO 3K were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade.Preparation of PLGA / lipid hybrid nanoparticles (hNPs)

[0226] Firstly, the PLGA nanoparticles were prepared by using a double emulsion solvent evaporation method with minor modification based on Applicant’s previous report.18,24’25Briefly, 20 mg of PLGA (Lactel 50:50) was dissolved in 2 mL of di chloromethane (oil phase). 200 pL of ultrapure water was added dropwise to the oil phase under vigorous stir, then the mixture was emulsified by sonication for 1 min in a Branson M2800H Ultrasonic Bath sonicator (Danbury, CT, USA). The resultant emulsion was added dropwise to 12 mL of 0.5% w / v poly(vinyl alcohol) solution under continuous stirring (1200 RPM). The suspension was emulsified by sonicating on ice for 40 s at 70% amplitude using a probe sonic dismembrator (Model 500; Fisher Scientific, Pittsburg, PA, USA). The resultant secondary emulsion was stirred overnight to allow complete dichloromethane evaporation. PLGA NPs were collected by centrifugation at 10,000 g, 4 °C for 30 min (Beckman Coulter Avanti J-251, Brea, CA, USA). Pellets were washed three times using ultrapure water, and final PLGA NPs were dispersed in 1 *PBS buffer.

[0227] The PLGA / lipid hNPs were assembled using a film-hydration-sonication method as described previously.18,24,25 The DSPE-PEG2000-Tetrazine lipid were synthesized by mixing DSPEPEG2000- Amine with methyltetrazine-NHS ester (molar ratio of 1 :30) in 0.1M PBS pH7.0 buffer for 1 hour, and the reaction product was dialyzed (MWCO IKDa) against deionized (DI) water at 4 °C in dark overnight. The final product was lyophilized to powder. 2.0 mg of lipids mixture dissolved in chloroform consisting of DOTAP, DSPE-PEG2000- maleimide, DSPE-PEG2000-Tetrazine, DSPE-PEG20000-Azide and CHOL (30:20:20:20: 10 in moles ratio) was evaporated to form a lipid film. 4 mg of PLGA NPs suspended in 1 *PBS buffer was added to the lipid film. The mixture was vortexed for 1 minute and sonicated in an ice-water bath using a bath sonicator for 30 min. The products were collected by centrifugation at 10,000 g for 30 min at 4 °C, washed by DI water three times to remove empty liposome particles. The final PLGA / lipid hNPs were kept in 1 *PBS buffer at 4 °C for short-term storage or -20 °C for long-term storage.Synthesis of fluorescent dye and linker tagged carrier proteins

[0228] Three different color of dyes, red Rhodamine-NHS, green FITC and cyan Cy5-NHS were tagged onto different proteins, CRM, OVA and KLH, respectively, through N- hydroxysulfosuccinimide (NHS) ester chemistry. Briefly, 2 mg of a protein (approximately 34 nmol CRM, 44 nmol of OVA, and 5 nmol of KLH) was dissolved in 500 pL 0. IM PBS buffer(pH7.0), and 5 molar equivalents (Eq) of corresponding fluorescent dye was added to the solution at room temperature and shook for 3 hours, followed by dialysis using 3K MWCO tube against 1 *PBS pH7.0 buffer overnight up to 24 hours to remove the free dyes. The dialysis buffer was changed very 3~4 hours. The absorption of the dialysis buffer was measured after each buffer change on the second day till no changes in the observed absorption. Then, the Traut’s reagent was used to convert the primary amines on the protein, FITC-OVA, to thiol groups. 50 Eq of Traut’s reagent was added into 500 pL of a solution containing 2 mg of FITC- OVA in 0.1M PBS buffer (pH7.0). After 1 hour, the reaction was stop by using a centrifugal filter (3K MWCO) and washed by 1 *PBS buffer 6 times to remove the free linkers. 30 Eq of Trans-cycloctene-NHS (TCO-NHS) or DBCO-NHS ester was added to 2 mg of Rhodamine- CRM and Cy5-KLH in 500 pL 0.1M PBS buffer (pH7.0), respectively. The products were purified by a centrifugal filter (3K MWCO) and washed by 1 *PBS buffer 6 times to remove the excessive linkers.Conjugation of dye-proteins to PLGA / lipid hybrid nanoparticles (hNPs)

[0229] FITC-OVA-SH was first conjugated onto hNPs via maleimide groups because of the relative instability of the maleimide and thiol groups. 0.25 mg of 1.27 mg / mL fresh prepared FITC-OVASH was added to 4 mg of 10 mg / mL freshly prepared hNPs in 1 *PBS pH7.0 buffer. After 30 min, the nanoparticles were collected by centrifugation at 10,000 g for 45 min at 4 °C, washed by 1 *PBS pH7.0 buffer 3 times to remove the unreacted protein. Then, 0.25 mg of 3.33 mg / mL Rhodamine-CRM-TCO were added to the hNPs:OVA in 1 *PBS pH7.0 buffer, and after 120 min, the nanoparticles were collected by centrifugation at 10,000 g for 45 min at 4 °C, washed by 1 *PBS pH7.0 buffer 3 times to remove the unreacted protein. At the final step, 0.25 mg of 2.76 mg / mL Cy5-KLH-DBCO were added to hNPs:OVA:CRM in 1 *PBS pH7.0 buffer, and after 30 min, the nanoparticles were collected by centrifugation at 10,000 g for 45 min at 4 °C, washed by 1 *PBS pH7.0 buffer 3 times to remove the unreacted protein. The final triple protein conjugated hNPs were storage in 1 *PBS buffer at 4 °C.CharacterizationTransmission Electron Microscopy (TEM)

[0230] The nanoparticles diluted to 1 mg / mL in 20 pL water were deposited onto Formvar carbon film coated copper TEM grids (Electron Microscopy Sciences) for 2 min at room temperature. The grids were then washed twice with DI water and stained twice with 2% (w / v) uranyl acetate (UAc) in DI water. The size and morphology of the nanoparticles werecharacterized by using a transmission electron microscope (TEM) operated at 80 kV. The number of PLGA nanoparticles was calculated using 1.3 g / cm3as the density of 100 nm PLGA nanoparticles.41UV / vis spectroscopy

[0231] The dye load was determined by measuring UV / vis absorbance on a BioTek Synergy HTX multimode reader and using the Beer-Lambert law (A = s ic), where: A = the measured absorbance of the dye (arbitrary unit), e = dye-specific molar extinction coefficient (M ' em '), c = concentration of the dye (mol / L), / = path length of the cuvette (fixed at 1 cm). The dye-specific extinction coefficient is 68,000 cm-iM-i for FITC (at 494 nm), 60,000 cm-iM- i for Rhodamine-NHS (at 570 nm), and 250,000 cm-iM-i for Cy5-NHS (at 646 nm), respectively. By measuring the absorbance value (A) at the dye’s peak wavelength (this varies depending on the dye) and ensuring the sample is diluted appropriately to avoid absorbance values higher than 1.0 (due to the linearity range of Beer-Lambert law), the concentration of the dye can be calculated as c = A / (e-l). The protein concentration in the nanoparticle formulations was determined using the Pierce BCA protein quantitation assay kit (Thermo Fisher Scientific).42The quantity of the hNPs was weighed by METTLER TOLEDO analytical electric balance after lyophilized 3 days. The conjugation of protein per hNPs was calculated by Protein loading (pg / mg) = protein concentration / hNPs concentration.Statistical AnalysisDesign and preparation of PLGA / lipid hNPs

[0232] The PLGA nanoparticles were prepared from Lactel (50:50) by using a double emulsion solvent evaporation method with 0.5% poly(vinyl alcohol) (PVA, MW: 38K Da) solution as surfactant. Then the PLGA nanoparticles were passively loaded into liposomes using thin lipid film hydration method. Three functionalized lipid, DSPE-PEG2000- Maleimide, DSPE-PEG2000-Tetrazine and DSPE-PEG2000-Azido, were incorporated into the lipid film (FIG. 1A) to specifically conjugate with different proteins. The size and morphology of the nanoparticles were characterized using TEM as shown in FIG. 1B-1E. The low (FIG. IB) and high (FIG. 1C) magnification TEM images demonstrate that the naked PLGA NPs are spherical with a clean surface and a mean size of approximately 98.7 ± 3.6nm. The TEM images (FIG. 1D-1E) of PLGA / lipid hNPs clearly show the distinctive core-shell structure with a bright PLGA core and a dark lipid shell. The size of the PLGA / lipid hNPs from TEM isaround 102.6 ± 5.4nm, and the size determined by dynamic light scattering (DLS) is 127.9 ± 8.4 nm, likely influenced by the flexible and extended lipid-PEG chains.Labeling fluorescent dyes and tagging the functional linkers onto the proteins

[0233] In this study, three proteins, OVA, CRM, and sKLH, were chosen to demonstrate the proof-of concept of specific conjugation strategies. OVA (MW: 43kDa), a well- characterized protein, has 20 lysine residues available for conjugation.43CRM (MW: 58.4KDa), a nontoxic variant of diphtheria toxin (DT), is a widely used carrier protein for polysaccharide vaccines44and conjugate vaccines against psychoactive compounds.28,45CRM also has ~20 amino groups exposed on the surface of the protein that is available for further reaction.46KLH is a large molecular (MW: ~4-8MDa) comprising a variable number of subunits (KLH1 (390 kDa) and KLH2 (350 kDa)), and this oxygen transporter protein is found in the hemolymph of the giant keyhole limpet.47,48Subunit KLH (sKLH, ~400 kDa) is often used as a vaccine carrier protein that is coupled to a carbohydrate or other non-immunogenic molecule to boost T-cell priming because of its high immunogenicity and the large number of lysine residues available for modification.47sKLH has between 300 and 600 lysine amines available for coupling.49The fluorescent dyes were used to demonstrate the surface lysine residues could be converted to different functional groups for desired conjugation. Furthermore, the fluorescent dyes can be used as color indicators to directly show the success of conjugation as well as a quantitation tool. FIG. 2A-2C shows the schematic reactions and Uvvis absorption spectra of the corresponding fluorescent dye-labeled and functional group- tagged proteins. As shown in FIG. 2A, OVA was labeled by FITC, and then Traut’s reagent converted the amino groups of the protein to thiol groups. The final protein showed FITC’s specific max absorption at 494 nm. CRM was labeled by Rhodamine B, and then the rest amino groups of the protein were converted to Tetrazine moi eties (FIG. 2B). The final protein showed Rhodamine’s specific max absorption at 570 nm. Finally, KLH was labeled by Cy5 and then tagged with DBCO moieties (FIG. 2C), and the specific max absorption of Cy5 was observed at 646 nm. The number of dyes attached to each protein was calculated based on the Beer- Lambert law and BCA protein quantitative assay (FIG. 2D). On average, 2 FITC, 5 Rhodamine, 45 Cy5 were attached to each OVA, CRM, or sKLH molecule, respectively. The sKLH has significantly more dyes per protein than OVA and CRM because of the large number (300-600) of available lysine residues on the protein.49Preparation and characterization of multiple protein conjugated PLGA / lipid hNPs

[0234] The strategy for conjugation of three proteins to the PLGA / lipid hNPs step-by-step is shown in FIG 3A. Reactions between thiols and maleimides have long been recognized as some of the most efficient Michael-type additions.50However, the disadvantage is that the maleimide moiety is easily hydrolyzed and the thiol groups are readily oxidized in physiological conditions.

[0235] Therefore, both moieties must be freshly prepared or stored in deep freeze.50Hence, the thiol tagged OVA was first conjugated to the hNPs. As shown from the TEM image (FIG. 3B), the hNPs-OVA(FITC) does not show obviously changes on the morphology and size. Based on the DLS analysis, the size of the particles in PBS buffer is 132.6 ± 11.3 nm, which is slightly bigger than that of the hNPs (-127.9 ± 8.4 nm).

[0236] In the second step, Rd-CRM-TCO was conjugated to the hNPs-OVA(FITC) through the TCO-Tetrazine click reaction (FIG. 3A). The TCO click chemistry is the 3rd generation click chemistry that is widely used in labeling and bioconjugation due to its fast speed and high selectivity.51,52The reaction between TCO and Tetrazine moiety is an inverse electron demand Diels Alder (IEDDA) reaction followed by a retro-DA reaction to eliminate nitrogen.52Both moieties are very stable in various physiological conditions. From the TEM image shown in FIG. 3C, there is no observable morphology differences between hNPs- OVA(FITC):CRM(Rd) and hNPs-OVA(FITC). The hydrodynamic diameter of hNPs- OVA(FITC):CRM(Rd) increases to 137.8=1=19.6 nm.

[0237] In the last step, the DBCO tagged Cy5-sKLH-DBCO was conjugated to hNPs- OVA(FITC):CRM(Rd) by a copper-free click reaction. The DBCO-Azido click reaction is the 2nd

[0238] generation of click chemistry eliminating the needs of copper catalyst.53The attraction of this copper-free click chemistry is that both DBCO and azido are highly selective and stable under most conditions.53 sKLH is a large protein, and the large size makes it visible in the TEM images as shown in FIG. 3D, a cylinder shape with 32 nm in diameter and 40 nm in length.47 After it was conjugated to the hNPs through copper free click chemistry, multiple sKLH molecules located on the surface of hNPs can be clearly observed in FIG. 3E (indicated by arrows), confirming the success of conjugation. The hydrodynamic size of the final particles dramatically increased to 156.3 ± 23.1 nm.

[0239] The successful conjugation of different proteins onto the surface of hNPs at each step can be corroborated by the UV-vis spectra due to the loading of different color dyes. After the first conjugation step with FITC-OVA-SH, the characteristic absorption peak of FITC (494 nm) was observed in the hNPs-OVA(FITC) solution (FIG. 4A). After the conjugation of Rd- CRM-TCO, two absorption peaks are evident, with the additional peak at 570 nm that is attributed to the presence of Rhodamine. In the final step of conjugation, Cy5-sKLH-DBCO was conjugated onto hNPs that already carried OVA and CRM. From the UV-vis spectrum (FIG. 4C), additional peak belongs to Cy5’s absorption (646nm) is apparent. The sample was analyzed by BCA protein assay kit to quantitate the protein loading and lyophilized to determine the amount of the final product. As shown in FIG. 4D, the protein loadings of hNPs after each conjugation step are hNPs-OVA(FITC) with 89.8 pg / mg, hNPs- OVA(FITC):CRM(Rd) with 121.8 pg / mg, and hNPs- OVA(FITC):CRM(Rd):sKLH(Cy5) with 184.4 pg / mg, respectively. Therefore, the individual protein loading on hNPs is 89.8 pg / mg of OVA, 32.0 pg / mg of CRM, and 62.6 pg / mg of sKLH, which equals to -816 OVA, -224 CRM, and -64 sKLH per hybrid nanoparticle, respectively. The order to which a protein is conjugated onto the hNPs seems to have an effect on the protein loading.

[0240] Even though the molar amount of each lipid responsible for the conjugation of each protein is the same, the number of a protein conjugated onto the hNPs decreases after each conjugation step.

[0241] Particularly, the number of sKLH molecules loaded onto the hNPs is significantly lower than that of OVA or CRM. The major reasons are likely the steric hindrance by the proteins already conjugated on the surface of the hNPs and the large size of the protein itself. Thus, in order to conjugating multiple proteins onto the surface of a nanoparticle, it is advisable to start with small proteins first.

[0242] This Example at demonstrates that three proteins can be conjugated onto the same nanoparticle by different conjugation chemistry. The amount of each protein conjugated can be adjusted by changing the amount of each lipid included in the assembly of the hNPs.24This presents an interesting possibility in the development of vaccines, particularly multivalent vaccines against different pathogens or psychoactive compounds. For example, different antigenic proteins of a pathogen or different antigenic proteins from different pathogens or different variants of a pathogen can be fabricated into the same vaccine formulation to maximize the possibility of a vaccine in eliciting the production of neutralizing antibodies. Forpsychoactive compounds, a multivalent vaccine can conceivably be produced by conjugating different haptens, such as oxycodone and fentanyl, onto different carrier proteins and then subsequently by conjugating the hapten-protein conjugates onto the same hybrid nanoparticles. Moreover, adjusting the amount of each protein loaded onto the hNP could be significant as it may be necessary to develop a balanced immune response to different antigens in an “all-in- one” vaccine.

[0243] This multivalent vaccine strategy described herein has many potential advantages as the quality of the vaccine can be more rigorously controlled and the possibility of different vaccines up-taken differently by the antigen-presenting cells will be eliminated.Conclusion

[0244] In this Example, Applicant at demonstrates the design and development of multiple protein-conjugated PLGA / lipid hybrid nanoparticles. Three different proteins OVA, CRM and sKLH were successfully conjugated to the surface of hNP step-by-step by different conjugation chemistry with 184.4 pg of total protein per mg of hNPs. The order of conjugation may impact the number of protein molecules conjugated onto the hNP. Although it is yet to be seen how different proteins, amounts, and surface exposure (steric hindrance by larger proteins), may impact the immune response, this work presents an exciting possibility of fabricating multivalent nanovaccine in the same nano-formulation.References for Example 1

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[0299] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

[0300] Further attributes, features, and embodiments of the present invention can be understood by reference to the following numbered aspects of the disclosed invention. Reference to disclosure in any of the preceding aspects is applicable to any preceding numbered aspect and to any combination of any number of preceding aspects, as recognized by appropriate antecedent disclosure in any combination of preceding aspects that can be made. The following numbered aspects are provided:Aspect 1. A nanoparticle comprising: a polymer core; and a lipid shell, wherein the lipid shell comprises a first lipid operably coupled with a first orthogonal reaction handle; a second lipid coupled with a second orthogonal reaction handle; and a third lipid coupled with a third orthogonal reaction handle, wherein the first orthogonal reaction handle, the second orthogonal reaction handle, and the third orthogonal reaction handle are different from each other.Aspect 2. The nanoparticle of aspect 1, wherein the lipid shell comprises a programmable stochiometric ratio of a quantity of first lipid, the second lipid, and the third lipid.Aspect 3. The nanoparticle of any one of aspects 1-2, wherein the polymer core comprises or consists of Poly(lactic-co-gly colic acid) (PLGA).Aspect 4. The nanoparticle of any one of aspects 1-3, wherein the first lipid, the second lipid, and the third lipid each comprise or consist of the same lipid.Aspect 5. The nanoparticle of any one of aspects 1-3, wherein at least two of the first lipid, the second lipid, and the third lipid comprise or consist of a different lipid.Aspect 6. The nanoparticle of any one of aspects 1-3 or 5, wherein the first lipid, the second lipid, and the third lipid each comprises or consist of a different lipid.Aspect 7. The nanoparticle of any one of aspects 1-6, wherein the first lipid, the second lipid, the third lipid, or any combination thereof comprises or consists of a pegylated lipid.Aspect 8. The nanoparticle of aspect 7, wherein the first lipid, the second lipid, the third lipid, or any combination thereof comprises or consists of l,2-Distearoyl-sn-glycero-3- phosphoethanolamine (DSPE).Aspect 9. The nanoparticle of any one of aspects 1-7, wherein the first lipid, the second lipid, the third lipid, or any combination thereof comprises or consists of pegylated DSPE.Aspect 10. The nanoparticle of any one of aspects 1-9, wherein the first orthogonal reaction handle, the second orthogonal reaction handle, the third reaction handle is each individually selected from the group consisting of: a maleimide, a tetrazine, an azide, a terminal alkyne, a strained alkyne, trans-cyclooctene, a norbornene, cyclopropane, an aldehyde, a ketone, a vinyl sulfone, an aryl boronic acid, a boronate ester, a sulfonyl fluoride, an aryl fluorosulfate, triarylphosphine, a thiol, or any combination thereof.Aspect 11. The nanoparticle of any one of aspects 1-10, wherein the lipid shell further comprises a first molecule of interest (MOI) comprising a fourth orthogonal reaction handle, a second MOI comprising a fifth orthogonal reaction handle, and a third molecule comprising a sixth orthogonal reaction handle, and wherein the first MOI is covalently attached the first lipid via covalent binding between the first orthogonal reaction handle and the forth reaction handle, wherein the second MOI is covalently attached to the second lipid via covalent binding between the second orthogonal reaction handle and the fifth orthogonal reaction handle, and wherein the third MOI is covalently attached to the third lipid via covalent binding between the third orthogonal reaction handle and the sixth orthogonal reaction handle.Aspect 12. The nanoparticle of aspect 11, wherein the first MOI the second MOI, the third MOI, or any combination thereof is a polypeptide.Aspect 13. The nanoparticle of any one of aspects 11-12, wherein at least two of the first MOI, the second MOI, or the third MOI are different from each other.Aspect 14. The nanoparticle of aspect 13, wherein the first MOI, the second MOI, and the third MOI are different from each other.Aspect 15. The nanoparticle of any one of aspects 11-14, wherein the first MOI, the second MOI, the third MOI, or any combination thereof is an antigen, optionally wherein the antigen is a polypeptide.Aspect 16. The nanoparticle of any one of aspects 11-15, wherein the first MOI, the second MOI, the third MOI, or any combination thereof is an immunostimulatory agent.Aspect 17. The nanoparticle of any one of aspects 11-16, wherein the nanoparticle comprises a programmable stoichiometric ratio of the quantity of the first MOI, the second MOI, and third MOI.Aspect 18. The nanoparticle of aspect 17, wherein the programmable stochiometric ratio of the quantity of the first MOI, the second MOI, and third molecule is effective to stimulate an immune response in a subject.Aspect 19. The nanoparticle of any one of aspects 1-18, further comprising cholesterol, DOTAP, or both.Aspect 20. A pharmaceutical formulation comprising: a nanoparticle of any one of aspects 1-19; and a pharmaceutically acceptable carrier.Aspect 21. A method of delivering a compound to a subject in need thereof, the method comprising: administering a nanoparticle as in any one of aspects 11-19 or a pharmaceutical formulation thereof to the subject in need thereof, the first MOI, the second MOI, the third MOI, or any combination thereof is the compound.Aspect 22. The method of aspect 21, wherein the subject in need thereof has or is suspected of having a disease or condition.Aspect 23. The method of aspect 22, wherein the disease or condition is an infectious disease, a non-infectious disease, a mental health disease or condition, or a combination thereof.Aspect 24. The method of any one of aspects 21-23, wherein the first MOI, the second MOI, and the third MOI are each individually selected from the group consisting of: antigens, immunostimulatory molecules, reporter molecules, barcoding molecules, targeting molecules, therapeutic molecules.Aspect 25. A method of stimulating an immune response in a subject in need thereof, the method comprising: administering a nanoparticle as in any one of aspects 11-19 or a pharmaceutical formulation thereof to the subject in need thereof.Aspect 26. The method of aspect 25, wherein the immune response comprises B cell production.Aspect 27. The method of aspect 26, wherein the B cell is a memory B cell.Aspect 28. The method of any one of aspects 25-27, wherein the immune response comprises T-cell production.Aspect 29. The method of aspect 28, wherein the T cell is a memory T cell.Aspect 30. The method of any one of aspects 25-29, wherein the immune response comprises an adaptive immune response.Aspect 31. The method of any one of aspects 25-31, wherein the immune response comprises an innate immune response.Aspect 32. The method of any one of aspects 25-31, wherein the nanoparticle comprises one or more antigenic molecules, optionally one or more antigenic polypeptides, and further comprising exposing the subject in need thereof to the one or more antigenic molecules one or more days after administering the nanoparticle.Aspect 33. The method of any one of aspects 25-32, wherein the nanoparticle comprises one or more therapeutic molecules.Aspect 34. The method of aspect 33, wherein the one or more therapeutic molecules comprise a psychoactive compound.

Claims

CLAIMSWhat is claimed is:

1. A nanoparticle comprising: a polymer core; and a lipid shell, wherein the lipid shell comprises a first lipid operably coupled with a first orthogonal reaction handle; a second lipid coupled with a second orthogonal reaction handle; and a third lipid coupled with a third orthogonal reaction handle, wherein the first orthogonal reaction handle, the second orthogonal reaction handle, and the third orthogonal reaction handle are different from each other.

2. The nanoparticle of claim 1, wherein the lipid shell comprises a programmable stochiometric ratio of a quantity of first lipid, the second lipid, and the third lipid.

3. The nanoparticle of any one of claims 1-2, wherein the polymer core comprises or consists of Poly(lactic-co-gly colic acid) (PLGA).

4. The nanoparticle of any one of claims 1-3, wherein the first lipid, the second lipid, and the third lipid each comprise or consist of the same lipid.

5. The nanoparticle of any one of claims 1-3, wherein at least two of the first lipid, the second lipid, and the third lipid comprise or consist of a different lipid.

6. The nanoparticle of any one of claims 1-3 or 5, wherein the first lipid, the second lipid, and the third lipid each comprises or consist of a different lipid.

7. The nanoparticle of any one of claims 1-6, wherein the first lipid, the second lipid, the third lipid, or any combination thereof comprises or consists of a pegylated lipid.

8. The nanoparticle of claim 7, wherein the first lipid, the second lipid, the third lipid, or any combination thereof comprises or consists of l,2-Distearoyl-sn-glycero-3- phosphoethanolamine (DSPE).

9. The nanoparticle of any one of claims 1-7, wherein the first lipid, the second lipid, the third lipid, or any combination thereof comprises or consists of pegylated DSPE.

10. The nanoparticle of any one of claims 1-9, wherein the first orthogonal reaction handle, the second orthogonal reaction handle, the third reaction handle is each individually selected from the group consisting of: a maleimide, a tetrazine, an azide, a terminal alkyne, a strained alkyne, trans-cyclooctene, a norbornene, cyclopropane, an aldehyde, a ketone, a vinyl sulfone, an aryl boronic acid, a boronate ester, a sulfonyl fluoride, an aryl fluorosulfate, triarylphosphine, a thiol, or any combination thereof.

11. The nanoparticle of any one of claims 1-10, wherein the lipid shell further comprises a first molecule of interest (MOI) comprising a fourth orthogonal reaction handle, a second MOI comprising a fifth orthogonal reaction handle, and a third molecule comprising a sixth orthogonal reaction handle, and wherein the first MOI is covalently attached the first lipid via covalent binding between the first orthogonal reaction handle and the forth reaction handle, wherein the second MOI is covalently attached to the second lipid via covalent binding between the second orthogonal reaction handle and the fifth orthogonal reaction handle, and wherein the third MOI is covalently attached to the third lipid via covalent binding between the third orthogonal reaction handle and the sixth orthogonal reaction handle.

12. The nanoparticle of claim 11, wherein the first MOI the second MOI, the third MOI, or any combination thereof is a polypeptide.

13. The nanoparticle of any one of claims 11-12, wherein at least two of the first MOI, the second MOI, or the third MOI are different from each other.

14. The nanoparticle of claim 13, wherein the first MOI, the second MOI, and the third MOI are different from each other.

15. The nanoparticle of any one of claims 11-14, wherein the first MOI, the second MOI, the third MOI, or any combination thereof is an antigen, optionally wherein the antigen is a polypeptide.

16. The nanoparticle of any one of claims 11-15, wherein the first MOI, the second MOI, the third MOI, or any combination thereof is an immunostimulatory agent.

17. The nanoparticle of any one of claims 11-16, wherein the nanoparticle comprises a programmable stoichiometric ratio of the quantity of the first MOI, the second MOI, and third MOI.

18. The nanoparticle of claim 17, wherein the programmable stochiometric ratio of the quantity of the first MOI, the second MOI, and third molecule is effective to stimulate an immune response in a subject.

19. The nanoparticle of any one of claims 1-18, further comprising cholesterol, DOTAP, or both.

20. A pharmaceutical formulation comprising: a nanoparticle of any one of claims 1-19; and a pharmaceutically acceptable carrier.

21. A method of delivering a compound to a subject in need thereof, the method comprising: administering a nanoparticle as in any one of claims 11-19 or a pharmaceutical formulation thereof to the subject in need thereof, the first MOI, the second MOI, the third MOI, or any combination thereof is the compound.

22. The method of claim 21, wherein the subject in need thereof has or is suspected of having a disease or condition.

23. The method of claim 22, wherein the disease or condition is an infectious disease, a non-infectious disease, a mental health disease or condition, or a combination thereof.

24. The method of any one of claims 21-23, wherein the first MOI, the second MOI, and the third MOI are each individually selected from the group consisting of: antigens, immunostimulatory molecules, reporter molecules, barcoding molecules, targeting molecules, therapeutic molecules.

25. A method of stimulating an immune response in a subject in need thereof, the method comprising: administering a nanoparticle as in any one of claims 11-19 or a pharmaceutical formulation thereof to the subject in need thereof.

26. The method of claim 25, wherein the immune response comprises B cell production.

27. The method of claim 26, wherein the B cell is a memory B cell.

28. The method of any one of claims 25-27, wherein the immune response comprisesT-cell production.

29. The method of claim 28, wherein the T cell is a memory T cell.

30. The method of any one of claims 25-29, wherein the immune response comprises an adaptive immune response.

31. The method of any one of claims 25-30, wherein the immune response comprises an innate immune response.

32. The method of any one of claims 25-31, wherein the nanoparticle comprises one or more antigenic molecules, optionally one or more antigenic polypeptides, and further comprising exposing the subject in need thereof to the one or more antigenic molecules one or more days after administering the nanoparticle.

33. The method of any one of claims 25-32, wherein the nanoparticle comprises one or more therapeutic molecules.

34. The method of claim 33, wherein the one or more therapeutic molecules comprise a psychoactive compound.

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