Compositions and methods of use for stabilizing and delivering atomic layer deposition coatings of drug-containing lipid emulsion formulations

Abstract

Embodiments of the present disclosure provide novel compositions and methods for making and using thermostable drug-containing lipid emulsion formulations. In certain embodiments, compositions and methods are disclosed for adapting lipid nanoemulsions for drug-containing formulations with improved stability and / or maintaining or enhancing immunogenicity. In other embodiments, drug-containing lipid nanoemulsion formulations can be spray-dried and further embedded in glassy matrices or microparticles to improve stability and compatibility with other drugs by restricting molecular mobility. In other embodiments, these microparticles carrying essentially dry, stabilized drug-containing lipid nanoemulsion formulations can be coated with one or more coating layers to produce stabilized lipid nanoemulsions for drug-containing formulations. In some embodiments, the coating layer is applied by atomic layer deposition (ALD), and the coating layer comprises an organometallic material.

Description

[Technical Field]

[0001]

[0002] Embodiments of the present disclosure provide novel compositions and methods for making and using thermostable lipid emulsion drug formulations. In certain embodiments, compositions and methods are disclosed for adapting lipid nanoemulsions of drug formulations with improved stability and / or maintaining or enhancing immunogenicity. In other embodiments, the lipid nanoemulsions of drug formulations can be spray-dried and further embedded in glassy matrices or microparticles to improve stability and compatibility with other drugs by restricting molecular mobility. In other embodiments, these microparticles carrying the essentially dry stabilized lipid nanoemulsions of the drug formulation can be coated with one or more coating layers to produce stabilized lipid nanoemulsions of the drug formulation. [Background technology]

[0002]

[0003] The therapeutic effectiveness of vaccines or other antigens can be compromised by many challenges. One challenge is the accurate and reliable delivery of the vaccine to the subject. For example, to provide good immunity against a particular pathogen or another health disorder such as cancer, multiple administrations are often required, which can reduce the likelihood that the subject will undergo or be able to undergo the necessary steps to obtain the required two or more administrations. In addition, vaccines and vaccine formulations often experience increased instability during storage, transportation, and handling, and the vaccine supply chain can require expensive and logistically complex refrigeration (e.g., cold chain requirements to reduce the adverse effects of elevated temperatures). Furthermore, certain vaccines against specific pathogens must be administered to subjects simultaneously or separately at different times to avoid ineffectiveness when combined with a different, incompatible vaccine against another pathogen, resulting in additional costs and the need for different administrations to avoid adverse effects. Furthermore, vaccines and vaccine formulations are typically associated with high manufacturing, storage, and delivery costs, which can limit their availability to those in need. Summary of the Invention

[0003]

[0004] Embodiments of the present disclosure provide novel compositions and methods for making and using thermostable lipid emulsion therapeutic agent-containing formulations. In certain embodiments, compositions and methods are disclosed for adapting lipid nanoemulsions of therapeutic agent-containing formulations for improved stability and / or retention or enhancement of immunogenicity or therapeutic agent efficacy. In other embodiments, lipid nanoemulsions of therapeutic agent-containing formulations can be spray-dried to embed the therapeutic agent in a glassy matrix or microparticles to improve stability and compatibility with other therapeutic agents. These embodiments can limit molecular mobility, thereby reducing adverse reactions, degradation, and / or therapeutic agent interactions. In other embodiments, these microparticles carrying essentially dry stabilized lipid nanoemulsions of therapeutic agent-containing formulations can be coated with one or more coating layers to produce stabilized lipid nanoemulsions of drug-containing formulations for delayed or timed delivery in a subject. In some embodiments, the coating layer may be applied by atomic layer deposition (ALD), and the coating layer may comprise an organometallic material, such as a metal oxide or metal alkoxide.

[0004]

[0005] In certain embodiments, further to the above paragraph

[0004] , the spray-dried nanoemulsified lipid therapeutic agent-containing formulations disclosed herein can form essentially dry microparticles in powder form, and these microparticles containing the essentially dry nanoemulsified lipid therapeutic agent-containing formulations can be introduced into a fluidized-bed atomic layer deposition (ALD) reactor disclosed herein to apply one or more coating layers. According to these embodiments, each coating layer of the one or more coating layers can comprise one or more of an organometallic material, a metal oxide, a metal alkoxide, and / or an aluminum-based coating layer. In certain embodiments, the coating layer can comprise, but is not limited to, a composition comprising aluminum oxide (Al2O3), aluminum alkoxide, silicon dioxide (SiO2), titanium dioxide (TiO2), zinc dioxide (ZnO2), and silicon nitride (Si3N4). In certain embodiments, the coating layer may comprise a mixture of two or more aluminum oxide (Al2O3), aluminum alkoxide, silicon dioxide (SiO2), titanium dioxide (TiO2), zinc dioxide (ZnO2), and silicon nitride (Si3N4) compositions, layered in the same, alternating, or other patterns, such as 1:2, 1:3, 1:4, 1:5, or other patterns (e.g., alternating layers of aluminum agents and silicon- or titanium-containing compositions). According to these embodiments, the combination of spray-drying a nanoemulsified lipid therapeutic agent-containing formulation and then ALD-coating the essentially dried nanoemulsified lipid therapeutic agent-containing formulation increases thermal stability, reduces therapeutic agent incompatibility, and provides delayed or timed release of the nanoemulsified lipid therapeutic agent-containing formulation. For example, these coated essentially dry nanoemulsified lipid therapeutic agent-containing formulations have improved stability at room temperature or at higher temperatures (e.g., 40°C to about 70°C).

[0005]

[0006] In certain embodiments, further to paragraphs

[0004] -

[0005] above, ALD-coated microparticles bearing or containing lipid emulsion therapeutic-agent-containing formulations (e.g., lipid adjuvant suspensions and / or lipid nanoparticles) may result in increased stability and / or compatibility of the antigen or other agent of the coated stabilized lipid-containing therapeutic-agent-containing nanoemulsion to provide time-release delivery of the therapeutic antigen or therapeutic agent contained within the coated lipid emulsion therapeutic-agent-containing microparticles, increased compatibility between therapeutic agents when two or more therapeutic agents or antigens are contained within the coated microparticles, and reduced concentrations of therapeutic agents and / or antigens needed to treat, reduce the onset of, ameliorate, or prevent the onset of a health condition.

[0006]

[0007] In some embodiments, further to paragraphs

[0004] -

[0006] above, a prime dose and at least one boost dose of a coated lipid emulsion or lipid suspension therapeutic-agent-containing microparticle formulation can be administered to a subject in a single administration of these coated microparticles. In other embodiments, the coated lipid emulsion or lipid suspension therapeutic-agent-containing microparticles disclosed herein may contain antigens (e.g., immunogenic antigens) or drugs against two or more pathogens or other essentially dry lipid emulsion or lipid suspension therapeutic-agent-containing formulations in the same or separate microparticles.

[0007]

[0008] In certain embodiments, in addition to paragraphs

[0004] through

[0007] above, the compositions and methods disclosed herein describe key formulations, parameters, and methods for spray-drying lipid emulsion adjuvant-, antigen-, and / or therapeutic agent-containing formulations using a squalene-based nanoemulsion system to develop heat-stable lipid emulsion adjuvant-, antigen-, and / or therapeutic agent-containing formulations. In certain embodiments, much of the bulk liquid or water in these formulations can be removed to obtain formulations with residual moisture of about 5.0% w / v or less, about 2.5% w / v or less, about 1.0% w / v or less, or about 0.5% w / v or less, thereby reducing or limiting molecular mobility in the nanoemulsion or nanosuspension lipid adjuvant-, antigen-, and / or therapeutic agent-containing compositions. According to these embodiments, emulsified lipid adjuvant-, antigen-, and / or therapeutic agent-containing compositions or formulations with reduced water content have dramatically reduced or limited molecular mobility, resulting in reduced collision frequency between emulsified nanodroplets or suspended nanoparticles and / or a lower rate of nanodroplet coalescence or nanoparticle aggregation. According to these embodiments, reducing the water or moisture content of lipid emulsion adjuvant-, antigen-, and / or therapeutic agent-containing formulations, thereby reducing the collision frequency between emulsified nanodroplets or suspended nanoparticles, can result in stability at temperatures from about 35° C. up to 60° C., or up to 70° C., or even higher. In other embodiments, the reduced water or moisture content of these compositions and their stabilization at high temperatures can enable coating of these reduced-moisture drug- or antigen-containing lipid emulsion compositions.

[0008]

[0009] In some embodiments, further to the above paragraphs

[0004] through

[0008] , to make reduced moisture or reduced water-containing lipid emulsion or lipid suspension adjuvant-containing, antigen-containing, and / or therapeutic agent-containing formulations, the core of these compositions in essentially dry form may be formed by spray drying in a formulation containing one or more salts, one or more glass-forming polysaccharides (e.g., trehalose, sucrose, glycine and mannitol, other disaccharides, or the like), and a high molecular weight glass-forming or smoothing agent (e.g., hydroxyethyl starch, dextran, carboxymethylcellulose, etc.). According to these embodiments, in the liquid formulation prior to spray drying or dehydration, the salt concentration may be about 0.1 mM to about 250.0 mM (or about 10 mM to about 200 mM or about 80 mM or less), the polysaccharide concentration may be about 2.5% to about 30.0% (or about 7.50% to about 25.0% or about 7.50% to about 20.0%), and the high molecular weight glass former, such as additional glass former, concentration may be about 0.1% to about 10.0% or about 1.0% to about 5.0%. In certain embodiments, the pre-spray dried formulations disclosed herein contain about 0.1% to about 10.0%, or about 1.0% to about 5.0% hydroxyethyl starch, about 2.5% to about 30.0% (or about 7.50% to about 25.0%, or about 7.50% to about 20.0%) sucrose, and / or about 2.5% to about 30.0% (or about 7.50% to about 25.0%, or about 7.50% to about 20.0%) trehalose. In some embodiments, it has surprisingly been discovered that even though adjuvant-, antigen-, and / or therapeutic-agent-containing lipid emulsions may be or were unstable in liquid solution, when formulated with the same high salt concentrations found during spray drying (e.g., about 10 mM to about 80 mM, or about 50 mM to about 1000 mM, or about 150 mM to about 640 mM), the salt-induced instability of these emulsions is reduced or eliminated by formulating the lipid emulsion with at least one glass-forming polysaccharide (e.g., hydroxystarch, sucrose, trehalose, or other comparable polysaccharide).In yet other embodiments, instability can be further reduced or eliminated by combining one or more polysaccharide agents with the lipid emulsion adjuvant-, antigen-, and / or therapeutic agent-containing formulations prior to spray drying in the presence of a high concentration (e.g., 0.5 wt % to about 25.0 wt %, or about 2.0 wt % to about 15.0 wt %) of a polysaccharide-containing solution; the combination of high polysaccharide concentration with rapid (e.g., timing of about a few seconds to less than one second) spray drying times reduces both the frequency of droplet-droplet collisions and the time available for these collisions to result in undesired coalescence or aggregation of the adjuvant-, antigen-, and / or therapeutic agent-containing lipid emulsion or suspension, resulting in a stabilized spray-dried formulation. In certain embodiments, rapid spray drying while incorporating elevated polysaccharide concentrations (e.g., about 0.5 wt% to about 25.0 wt% or about 2.0 wt% to about 15.0 wt%) into these formulations creates microparticle cores (e.g., powders) that can be coated by ALD or similar systems to coat these stabilized microparticles (e.g., applying organometallic, metal oxide agents, metal alkoxides, etc.).

[0009]

[0010] In some embodiments, in addition to the above paragraphs

[0004] to

[0009] , the stabilized lipid emulsion adjuvant-containing, antigen-containing, and / or therapeutic agent-containing powder or glassy matrix or microparticles may be coated with one or more coating layers, each of which may comprise one or more of organometallic materials, metal oxides, metal alkoxides, and / or aluminum-based coating layers. As disclosed herein, the terms particle(s), microparticles, nanoparticles, and the like may be referred to simply as particles and may be used interchangeably without regard to size, and the size of a particle is contemplated herein to be a size that can be administered by syringe or be part of a sprayable, inhalable, or topically applicable form, as appropriate, taking into account the health condition being treated or prevented. In certain embodiments, coating materials for layering on the stabilized particles disclosed herein may include, but are not limited to, aluminum oxide (Al2O3), aluminum alkoxides, silicon dioxide (SiO2), titanium dioxide (TiO2), zinc dioxide (ZnO2), and silicon nitride (Si3N4). According to these embodiments, the combination of spray drying of nanoemulsified or nanosuspended lipid formulations and ALD coating of essentially dry nanoemulsified or nanosuspended lipid microparticles increases the thermal stability of the nanoemulsified or nanosuspended adjuvant-, antigen-, and / or therapeutic-agent-containing lipid microparticles. For example, these coated essentially dry nanoemulsified or nanosuspended microparticles have improved stability upon storage at room temperature or at elevated temperatures. Surprisingly, it has been observed that spray-dried lipid emulsion adjuvant-, antigen-, and / or therapeutic agent-containing formulations can be introduced into a fluidized-bed ALD reactor and stabilized to form microparticle surface coatings without altering the size distribution of the embedded lipid nanoemulsion or nanosuspension adjuvant-, antigen-, and / or therapeutic agent-containing microparticles contained with the powdered or essentially dry formulation.In certain embodiments, the coated lipid emulsion adjuvant-, antigen-, and / or therapeutic-agent-containing powder or glassy matrix can be stored, transported, and reconstituted for use in treating, reducing the occurrence of, or preventing a medical condition. According to these embodiments, the reconstituted coated microparticles are injectable, nebulizable, and / or inhalable, and maintain a size distribution that allows them to be introduced into a subject by any delivery method known in the art.

[0010]

[0011] In certain embodiments, further to the above paragraphs

[0004] through

[0010] , the antibody and / or therapeutic agent is a portion of a central or innermost coated microparticle comprising at least one immunogenic agent and at least one glass-forming agent, and the at least one immunogenic agent forms one or more outer coating layers that coat or encase the central glassy microparticle. According to these embodiments, 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 100, up to 150, up to 200, up to 250, or more coating layers may surround the stabilized lipid emulsion antigen-containing and / or therapeutic agent-containing antigen-containing powder or glassy matrix or microparticle, where the coating layers are readily dissolvable in the subject upon administration to the subject and expose the subject to one or more therapeutic agents or antigens by immediate exposure, delayed release, and / or timed release. In other embodiments, the at least one immunogenic agent or immunogenic antigen may be stabilized in powder form as disclosed herein and encased in another layer of coating agent that forms a prime dose of the at least one immunogenic agent or immunogenic antigen to be exposed before the microparticle dissolves to expose the inner core. In still other embodiments, the at least one immunogenic agent or immunogenic antigen may comprise a mixture of immunogenic agents or antigens or distinct immunogenic agents or antibodies in the inner core from the same or different immunogenic agents intermixed with a coating layer that forms at least one outer layer of the coated microparticle for differential exposure of the at least one immunogenic agent or antigen.

[0011]

[0012] In certain embodiments, in addition to the above paragraphs

[0004] through

[0011] , the present disclosure provides a method for making stabilized lipid emulsion adjuvant-, antigen-, and / or therapeutic agent-containing coated microparticles, stabilized powders, or glassy matrices, comprising combining at least one lipid emulsion of at least one adjuvant, antigen, and / or therapeutic agent with at least one glass-forming agent to form a primary liquid composition, rapidly spray-drying the composition to form lipid emulsion adjuvant-, antigen-, and / or therapeutic agent-containing glassy microparticles, and coating the essentially dry lipid emulsion adjuvant-, antigen-, and / or therapeutic agent-containing glassy microparticles with one or more outer coating layers. In some embodiments, the primary liquid composition may further comprise a second glass-forming agent or a polymeric glass-forming agent. According to these embodiments, the second glass-forming agent may comprise hydroxyethyl starch or a similar agent. Furthermore, according to these embodiments, the hydroxyethyl starch precipitates as a film on the surface of the composition droplets as they dry during spray drying, reducing microparticle aggregation or collision, thereby maintaining particle integrity as they dry to a more stable state in preparation for coatings by ALD or other organometallic layer-forming application systems.

[0012]

[0013] According to these embodiments, in addition to paragraphs

[0004] through

[0012] above, the at least one adjuvant, antigen, and / or therapeutic agent may include one or more adjuvants, antigens, and / or therapeutic agents, including, but not limited to, viral antigens, bacterial antigens, toxins, prions, yeast, fragments or subunits thereof, chemical agents, small molecules, anti-cancer agents, anti-inflammatory agents, anti-autoimmune agents, peptides, polynucleotides, or proteins thereof, or combinations thereof. In some embodiments, the at least one agent or antigen may include one or more agents or antigens, including, but not limited to, recombinant peptides, recombinant proteins, peptides derived from target proteins or pathogens, polysaccharides derived from target pathogens, synthetic peptides or proteins, virus-like particles, live viruses, live attenuated viruses, inactivated viruses, antigens attached to, associated with, or expressed on the surface of viruses or bacteriophages, or combinations thereof. In still other embodiments, the at least one agent or antigen may include one or more agents or antigens represented by or encoded by polynucleotides, etc. According to these embodiments, the polynucleotide may include, but is not limited to, DNA, RNA, mRNA, siRNA, or chimeric molecules thereof. In certain embodiments, the chimera may include a combination of at least one polynucleotide segment and at least one polypeptide segment, or a mixture of multiple polynucleotide segments and / or multiple polypeptide segments. In some embodiments, the polynucleotide may be an mRNA encoding the full length or peptide fragment of a target agent (e.g., a virus or bacterium).

[0013]

[0014] In certain embodiments, in addition to the above paragraphs

[0004] -

[0013] , the at least one agent is an agent that inhibits or inhibits at least one pathogen or antibody derived therefrom, such as, for example, a human papillomavirus (e.g., HPV16, HPV18, or other serotype or type) or other mammalian papillomavirus, ricin toxin, Bacillus anthracis, Clostridium botulinum, or the like. Antibodies may include, but are not limited to, antibodies derived from Salmonella botulinum, Ebola virus, influenza virus, coronavirus (SARS-CoV-2) or other variants or mutants thereof, SARS or other variants, poliovirus, norovirus, rotavirus, hepatitis C, chickenpox, herpes simplex, cytomegalovirus, Japanese encephalitis, dengue virus, West Nile virus, Zika virus or other flavivirus, chikungunya, equine encephalitis virus (EEV) or other alphavirus, pneumonia virus species, Yersinia, Pneumococcus, Salmonella, Clostridium difficile, or combinations thereof. In certain embodiments, at least one antigen or agent may be a multimeric complex. In other embodiments, the at least one antigen or agent may comprise a pathogen or an antigen derived therefrom that can infect humans, companion animals, livestock, wild animals, zoo animals, birds, fish, or reptiles.

[0014]

[0015] In some embodiments, in addition to the above paragraphs

[0004] -

[0014] , the at least one glass-forming agent or polysaccharide or disaccharide disclosed herein may include at least one of trehalose, sucrose, ficoll, dextran, sucrose, maltotriose, lactose, mannitol and sucrose, hydroxyethyl starch, glycine, cyclodextrin, povidone, etc. In certain embodiments, the at least one glass-forming agent or polysaccharide disclosed herein may include trehalose or sucrose. In certain embodiments, the at least one glass-forming agent or polysaccharide disclosed herein may include hydroxyethyl starch, alone or in combination with a second polysaccharide. According to these embodiments, at least one glass-forming agent or polysaccharide disclosed herein may be present in the primary liquid immunogenic composition at a weight-to-volume (w / v) concentration of about 0.1% to about 40.0%, about 1.0% to about 30.0%, about 5.0% to about 20.0%, or about 8.0% to about 15.0%. In certain embodiments, at least one glass-forming agent or polysaccharide disclosed herein may be combined with a lipid emulsion drug or antigen, further spray-dried, and further used in a formulation or preparation for application of one or more coatings to the essentially dried microparticles formed by this process for subsequent formulation and use as disclosed herein.

[0015]

[0016] In other embodiments, in addition to the above paragraphs

[0004] through

[0015] , the glass-forming agent or polysaccharide disclosed herein may include at least one additional polysaccharide. According to these embodiments, the at least one additional polysaccharide may be hydroxyethyl starch or another pharmaceutically acceptable plasma expander, such as human serum albumin (HAS), other serum albumins, dextran, hetastarch, plasma protein factors, or a combination thereof. According to these embodiments, the at least 0.1 additional polysaccharide may be present in the primary formulation prior to spray drying at a weight-to-volume (w / v) concentration of about 0.1% to about 40.0%, about 1.0% to about 30.0%, about 5.0% to about 20.0%, or about 8.0% to about 15.0%. In certain embodiments, the at least 0.1 additional polysaccharide may be different from the primary polysaccharide agent, and the at least one additional polysaccharide may be present in the pre-spray-dried formulation at a weight to volume (w / v) concentration of about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 2.5%, or about 0.1% to about 0.5%. In certain embodiments, the glass-forming agent present in the pre-spray-dried formulation is sucrose or trehalose, and the at least one additional polysaccharide for the formulation is hydroxyethyl starch.

[0016]

[0017] In some embodiments, in addition to the above paragraphs

[0004] through

[0016] , each layer of the one or more outer coating layers may comprise one or more of an organometallic material, a metal oxide, a metal alkoxide, and / or an aluminum-based coating layer. In certain embodiments, each layer of the one or more outer coating layers may comprise one or more of aluminum oxide, aluminum alkoxide (e.g., Alcon), silicon dioxide (SiO2), titanium dioxide (TiO2), zinc dioxide (ZnO2), or silicon nitride (Si3N4), alone or in a suitable combination composition. According to these embodiments, each coating layer may be about 0.1 nm to about 30.0 nm or about 0.1 nm to about 20.0 nm thick. In certain embodiments, the essentially dry microparticles (e.g., after spray drying) disclosed herein may comprise a sufficient number of outer coating layers to delay or provide a timed release of at least one antigen or agent contained in one or more layers of the coated microparticle or from the center or innermost core of the coated microparticle.

[0017]

[0018] In certain embodiments, in addition to the above paragraphs

[0004] to

[0017] , one or more coating layers disclosed herein can function as an adjuvant to enhance an immune response in a subject to one or more drugs or antigens of the drug- or antigen-containing coated microparticle. In some embodiments, the one or more coating layers may include a coating layer(s) capable of inducing a rapid immune response in a subject, and / or an essentially dry coated lipid emulsion having one or more drugs or antigens may be contained in selected one or more layers and / or the core of the coated microparticle that induces a rapid immune response in a subject to one or more drugs or antigens upon dissolution of the coating layer and exposure of the one or more antigens or drugs.

[0018]

[0019] In some embodiments, further to paragraphs

[0004] through

[0018] above, the lipid emulsion drug-containing, lipid nanosuspension drug-containing, or antigen-containing coated microparticles described herein can be stored for extended periods of time without refrigeration at room temperature, at temperatures up to about 50°C, or at temperatures up to about 60°C, or at temperatures up to about 70°C. In certain embodiments, the lipid nanoemulsion drug-containing, lipid nanosuspension drug-containing, or antigen-containing coated microparticles described herein can be stored without refrigeration at up to room temperature, up to about 50°C, or up to about 60°C, or up to about 70°C for up to about 1 day, about 2 days or more, about 1 week, about 2 weeks, about 2-3 weeks, 1 month, about 2 months, about 3 months, up to about 4 months, up to about 6 months, up to about 9 months, up to about 12 months, up to about 15 months, up to about 18 months, up to about 24 months or more without negative effects on the coated microparticles (e.g., degradation, loss of efficacy, loss of immunogenicity, reduced delivery of one or more drugs or antigens) or their encased nanoemulsions or nanosuspensions (e.g., degradation, coalescence of the nanoemulsion, aggregation of the nanosuspension).

[0019]

[0020] In addition to the above paragraphs

[0004] through

[0019] , other embodiments provide combination compositions or formulations comprising a plurality of coated lipid emulsion drug- or antigen-containing microparticles described herein. According to these embodiments, these combination compositions or formulations may comprise a mixture of different coated microparticles containing one or more drugs for treating or preventing a single health condition or multiple health conditions (e.g., pathogen infection or prevention of pathogen infection), and may further comprise at least one pharmaceutically acceptable excipient to create a pharmaceutically acceptable composition or formulation. In other embodiments, the combination composition may comprise at least one representative microparticle-containing pharmaceutical composition mixed with a standard or known composition or formulation to treat, reduce the occurrence of, or prevent a health condition (e.g., infectious disease, cancer, or other condition).

[0020]

[0021] In some embodiments, in addition to paragraphs

[0004] through

[0020] above, the formulations disclosed herein may be part of a single-dose formulation comprising a prime dose of at least one drug or at least one antigen and at least one boost dose. According to these embodiments, the prime dose of at least one antigen or at least one drug and the at least one boost dose may be present in the same coated microparticle or in separate coated microparticles. If present in separate particles, the prime dose of at least one drug or at least one antigen may be sequestered in the microparticle, while the at least one boost dose may be present in separate microparticles. In certain embodiments, release of the prime dose after delivery to a subject may be immediate or delayed, depending on the number of coating layers (if not on the outermost or near-outermost coating layer) coating the lipid emulsion-coated drug- or antigen-containing microparticle prime dose, and release of the at least one boost may occur shortly, several days, or even several months after initial administration of the drug- or antigen-containing coated microparticles to the subject.

[0021]

[0022] In some embodiments, further to the above paragraphs

[0004] through

[0021] , an ALD-coated formulation containing lipid emulsion drug- or antigen-containing coated microparticles may be part of a single-dose formulation having at least two different drugs or two different antigens capable of inducing an immune response or other therapeutic response to two or more different antigens or drugs. According to these embodiments, the two or more different antigens or different drugs may be contained in the same or separate coated microparticles.

[0022]

[0023] In other embodiments, further to the above paragraphs

[0004] -

[0022] , the ALD-coated lipid emulsion drug- or antigen-containing microparticle composition may further comprise a standard vaccine composition (e.g., mRNA, live virus, attenuated virus) and a plurality of the ALD-coated lipid emulsion drug- or antigen-containing microparticles described, wherein at least one of the at least one drug or at least one antigen induces a boost immune response to the standard vaccine composition.

[0023]

[0024] Other embodiments, in addition to the above paragraphs

[0004] to

[0023] , provide methods for inducing a response in a subject, which may include administering to the subject an ALD-coated microparticle-containing formulation described herein. According to these embodiments, the formulation may be administered by any method known in the art. In other embodiments, the formulation may induce a response (e.g., an immune response) in the subject. The immune response induced by the composition may be prophylactic or therapeutic, depending on the at least one antigen or at least one agent.

[0024]

[0025] Still other embodiments provide kits that can include, in addition to paragraphs

[0004] through

[0024] above, at least one ALD-coated lipid emulsion drug- or antigen-containing microparticle or components for making the microparticles described herein. In certain embodiments, the kits can further include at least one container and / or instructions for making or using the coated formulations disclosed herein. [Brief explanation of the drawings]

[0025]

[0026] The accompanying drawings are incorporated into and form a non-limiting part of this specification to illustrate several examples of the present disclosure. [Figure 1]

[0027] FIG. 1 is a representative table showing the moisture content of spray-dried samples having various polysaccharide concentrations, according to some embodiments of the present disclosure. [Figure 2]

[0028] FIG. 2 is a plot of spray drying versus freezing of various samples at increasing concentrations of a representative salt to analyze emulsion droplet size under various conditions, according to some embodiments of the present disclosure. [Figure 3]

[0029] 3A-3B are a representative graph of tested salt concentrations versus emulsion droplet size diameter distribution (a) and a representative plot of tested salt concentrations versus hydrodynamic diameter of microparticles disclosed herein (b), according to some embodiments of the present disclosure. [Figure 4]

[0030] FIG. 4 is a representative plot showing coated versus uncoated lipid emulsion microparticles assessing the range of hydrodynamic radii of entrapped lipid nanodroplets over time, according to some embodiments of the present disclosure. [Figure 5]

[0031] FIG. 5 is a representative plot evaluating the effect of various disaccharide concentrations of spray-dried versus non-spray-dried microparticles on maintaining emulsified nanodroplet size below 300 nm after reconstitution, according to some embodiments of the present disclosure. [Figure 6]

[0032] FIG. 6 is a representative plot evaluating the effect of various disaccharide concentrations, in the presence or absence of additional disaccharide (e.g., hydroxyethyl starch, HES), of spray-dried versus non-spray-dried microparticles on maintaining emulsified nanodroplet size below 300 nm after reconstitution, according to some embodiments of the present disclosure. [Figure 7]

[0033] FIG. 7 is a representative plot evaluating the effect of various disaccharide concentrations of spray-dried versus non-spray-dried microparticles on maintaining emulsified nanodroplet size below 300 nm after reconstitution, and evaluating the effect of ionic strength on size distribution, according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0026]

[0034] In the following sections, various exemplary compositions and methods are described to detail various embodiments. It will be apparent to those skilled in the art that practicing various embodiments does not require the use of all or even some of the specific details outlined herein; rather, concentrations, times, and other specific details can be modified through routine experimentation. In some embodiments, well-known methods or components are not included in the description.

[0027]

[0035] Vaccine formulations containing lipid nanoemulsions can be thermally unstable and typically must be refrigerated to reduce degradation, maintain the immunogenic activity or integrity of the drug or antigen, and make them reliably deliverable. These issues can complicate the distribution of vaccines and therapeutics during pandemics, extreme catastrophic events, for example, to underserved and remote areas where electricity may be unreliable to keep thermally unstable drugs, antigens, and / or vaccines at continuously cool or chilled temperatures. The embodiments disclosed herein solve these problems by spray-drying nanoemulsified lipids to embed these drugs, antigens, and / or vaccines within a glassy matrix that restricts molecular mobility.

[0028]

[0036] In addition to the above paragraph

[0035] , embodiments disclosed herein provide novel compositions and methods for making and using thermostable drugs, antigens, and / or vaccines in nanoemulsified lipid-containing glassy microparticle formulations. In certain embodiments, compositions and methods are disclosed for adapting nanoemulsions of drug- or antigen-containing lipid formulations with improved stability for long-term storage, transportation, and / or retention of drug, antigen, and / or immunogenic efficacy. In other embodiments, nanoemulsions of antigen- or drug-containing lipid formulations can be spray-dried and embedded within glassy matrices or microparticles to restrict molecular mobility and thereby improve stability and compatibility alone or with other drugs. In other embodiments, these essentially dry stabilized nanoemulsions of antigen- or drug-containing lipid formulations can be coated with one or more coating layers to produce stabilized nanoemulsions of lipid-containing drugs. According to these embodiments, the coating may include, but is not limited to, atomic layer deposition (ALD) coatings.

[0029]

[0037] In certain embodiments, in addition to the above paragraphs

[0035] and

[0036] , the spray-dried nanoemulsified antigen- or drug-containing lipid formulations disclosed herein can form essentially dry microparticle powders. These microparticles, including the essentially dry nanoemulsified antigen- or drug-containing lipid formulations, can be introduced into a fluidized-bed atomic layer deposition (ALD) reactor to apply one or more coating layers, each of which may comprise one or more of organometallic materials, metal oxides, metal alkoxides, and / or aluminum-based coating layers. In certain embodiments, the coating layers may include, but are not limited to, aluminum oxide (Al2O3), aluminum alkoxides, silicon dioxide (SiO2), titanium dioxide (TiO2), and silicon nitride (Si3N4). It is noted herein that these processes are solvent-free and are designed to apply metal-containing formulations to coat heat-stabilized antigens or drugs with precision for timed release of targeted antigens or drugs to treat conditions in a subject. According to these embodiments, the combination of spray drying and ALD coating of nanoemulsified antigen- or drug-containing lipid formulations can increase the thermal stability of the nanoemulsified lipid formulations, resulting in timed release of the drug or antigen. For example, these coated, essentially dry nanoemulsified lipid formulations have improved stability upon storage at room temperature or at higher temperatures up to about 60-70°C.

[0030]

[0038] In certain embodiments, further to paragraphs

[0035] to

[0037] above, the coated adjuvant-, antigen-, or drug-containing lipid formulation (e.g., coated lipid nanoparticles) may result in increased stability and / or compatibility of the antigen or other agent in the coated stabilized antigen- or drug-containing lipid nanoemulsion, providing time-release delivery of the antigen or agent contained within the coated antigen- or drug-containing lipid microparticles, increased compatibility between agents (when two or more agents or antigens are contained within the coated particle that would not normally be compatible to combine), and reduced concentrations of agents and / or antigens needed to treat, reduce the occurrence of, or prevent a health condition.

[0031]

[0039] In some embodiments, further to paragraphs

[0035] through

[0038] above, a prime dose and at least one boost dose of ALD-coated antigen- or drug-containing lipid glass microparticles (e.g., coated lipid nanoparticles) can be administered to a subject in a single administration of these coated particles. In other embodiments, the ALD-coated antigen- or drug-containing lipid glass microparticles (e.g., coated lipid nanoparticles) disclosed herein can contain antigens (e.g., immunogenic antigens) or drugs against two or more pathogens, or other essentially dry drug- or antigen-containing nanoemulsion formulations, in the same or separate coated microparticles.

[0032]

[0040] In certain embodiments, in addition to paragraphs

[0035] through

[0039] above, the compositions and methods disclosed herein describe important formulation parameters and methods for spray-drying adjuvants, antigens, or drugs emulsified using an oil-based nanoemulsion system (e.g., a triterpenoid such as squalene) to develop heat-stable emulsified adjuvant-, antigen-, or drug-containing formulations for further stabilization of the adjuvant, antigen, or drug. In certain embodiments, much or essentially all of the bulk liquid or water can be removed from these formulations to obtain formulations with residual moisture of less than about 5.0% w / v, less than about 2.5% w / v, less than about 1.0% w / v, or less than about 0.5% w / v, thereby reducing or limiting molecular mobility in the emulsified lipid adjuvant-, antigen-, or drug-containing compositions disclosed herein. According to these embodiments, emulsified adjuvant-, antigen-, or drug-containing compositions with reduced water content have dramatically reduced or limited molecular mobility, resulting in reduced collision frequency and / or lower droplet coalescence rates. In certain embodiments, reducing collision frequency by reducing the water or moisture content of adjuvant-, drug-, or antigen-containing lipid emulsion compositions or formulations can result in stability at typically unfavorable temperatures, e.g., room temperature, up to about 60°C, 70°C, or higher. In other embodiments, the reduced water or moisture content and stabilization of these compositions at elevated temperatures can enable ALD coating of these reduced-water adjuvant-, drug-, or antigen-containing lipid emulsion compositions without degradation of the adjuvants, drugs, and / or antigens embedded within these formulations.

[0033]

[0041] In some embodiments, in addition to the above paragraphs

[0035] to

[0040] , compositions used herein for making reduced moisture or reduced water adjuvant-, antigen-, or drug-containing lipid emulsion formulations are disclosed, and stabilization of these compositions by spray drying is also described. According to these embodiments, these compositions may contain one or more glass-forming agents, such as one or more salts, one or more polysaccharides or disaccharides (e.g., trehalose, sucrose, or the like), and high molecular weight glass-forming agents, such as, but not limited to, hydroxyethyl starch, polyvinylpyrrolidone, dextran, carboxymethylcellulose, human serum albumin, bovine serum albumin, other serum albumins, etc. According to these embodiments, in the liquid formulation prior to spray drying, the salt concentration may be about 0.1 mM to about 250.0 mM (or about 10.0 mM to about 200.0 mM), the glass former / polysaccharide concentration may be about 2.5% to about 30.0% (or about 7.50% to about 25.0% or about 7.50% to about 20.0%), and the concentration of the high molecular weight agent, such as an additional glass former, may be about 0.1% to about 10.0% or about 1.0% to about 5.0%. In certain embodiments, the pre-spray dried formulations disclosed herein contain about 0.1% to about 10.0%, or about 1.0% to about 5.0% hydroxyethyl starch, about 2.5% to about 30.0% (or about 7.50% to about 25.0%, or about 7.50% to about 20.0%) sucrose, and / or about 2.5% to about 30.0% (or about 7.50% to about 25.0%, or about 7.50% to about 20.0%) trehalose.In some embodiments, it has surprisingly been discovered that even though adjuvant-, drug-, or antigen-containing lipid emulsions may be or were unstable in liquid solution when formulated with the same high salt concentrations found during spray drying (e.g., about 50.0 mM to about 1000.0 mM or about 150.0 mM to about 640.0 mM), the salt-induced instability of these emulsions is reduced or eliminated by formulating the adjuvant-, drug-, or antigen-containing lipid emulsions with at least one glass-forming polysaccharide or disaccharide (e.g., hydroxyl starch, sucrose, trehalose, or other comparable polysaccharide). In still other embodiments, when one or more polysaccharide agents are present at high concentrations (e.g., 0.5 wt% to about 25.0 wt%, or about 2.0 wt% to about 15.0 wt%), combining the one or more polysaccharide agents in the presence of a solution with the adjuvant-, drug-, or antigen-containing lipid emulsion formulation prior to spray drying can further reduce or eliminate instability; the combination of high polysaccharide concentrations with rapid (e.g., timing of about a few seconds to less than one second) spray-drying times reduces both the frequency of droplet-droplet collisions and the time available for these collisions to result in undesirable coalescence or aggregation of the lipid-containing emulsion or suspension, resulting in a stabilized spray-dried formulation. In certain embodiments, rapid spray drying while incorporating elevated polysaccharide concentrations into these formulations creates microparticle cores (e.g., powders) that can be coated by ALD or similar systems to apply metal-containing coatings (e.g., to apply metal oxide agents, metal alkoxides, organometallic materials, etc.).In some embodiments, it has surprisingly been discovered that even though adjuvant-, drug-, or antigen-containing lipid emulsions may be or were unstable in liquid solution when formulated with the same high salt concentrations (e.g., 50 mM to about 1000 mM or about 100 mM to about 500 mM) found during drying, the salt-induced instability of these emulsions is reduced or eliminated by formulating the lipid emulsion with at least one glass-forming polysaccharide, such as sucrose, trehalose, hydroxyethyl starch, or other comparable polysaccharides. In some embodiments, salts used in the liquid formulations disclosed herein prior to spray drying can include, but are not limited to, sodium citrate, sodium chloride, sodium phosphate, calcium chloride, potassium chloride, potassium phosphate, and the like, or combinations thereof. In some embodiments, the salt comprises sodium citrate.

[0034]

[0042] In yet other embodiments, further to the above paragraphs

[0035] through

[0041] , the instability of the adjuvants, drugs, and / or antigens disclosed herein can be further reduced or eliminated when one or more polysaccharide agents are rapidly introduced and / or combined with a lipid emulsion adjuvant-, drug-, or antigen-containing formulation and then spray-dried in the presence of a solution containing a high concentration (e.g., 2.5% to about 30.0%) of one or more of these polysaccharides. According to these embodiments, the combination of a high polysaccharide concentration and a rapid spray-drying time (e.g., about 100 milliseconds to about 1.0 second) reduces both the frequency of droplet-droplet collisions and the time available for these collisions to result in undesirable coalescence or aggregation of the lipid-containing emulsion, resulting in a stabilized spray-dried formulation. In certain embodiments, rapid spray drying while incorporating high polysaccharide concentrations into these formulations creates coatable microparticle cores (e.g., coatable powders) to which a molecular coating layer can be precisely applied by atomic layer deposition (ALD) or similar systems for coating (e.g., applying organometallic agents, metal oxide agents, etc.) to produce time-release coated formulations.

[0035]

[0043] As will be understood by those skilled in the art, in addition to paragraphs

[0035] through

[0042] above, salt concentration can determine the ionic strength of the formulation. At high ionic strength, charges on the surface of emulsion droplets can be shielded, reducing repulsive inter-droplet forces and resulting in a decrease in the distance between emulsion droplets, which promotes droplet-droplet interactions that can lead to droplet coalescence. During spray drying, an increase in ionic strength can compress the electrical double layer surrounding the droplets, further reducing droplet distance. Non-reducing polysaccharides, such as sucrose and trehalose, form glassy matrices during spray drying. The high viscosity within these glassy matrices reduces or prevents translational movement of any lipid nanodroplets of nanoparticles contained within the matrix. In certain embodiments, as disclosed herein, the lipid-to-polysaccharide weight ratio is an important parameter for emulsion stability during freezing, freeze-drying, and spray-drying of these formulations. According to these embodiments, lipid to polysaccharide weight ratios are evaluated and improved condition parameters for spray drying lipid emulsion drug-containing formulations are disclosed herein, followed by further stability of these essentially dry lipid emulsion adjuvant-containing, antigen-containing, and / or drug-containing microparticle spray-dried or essentially dry microparticle coatings contemplated herein.

[0036]

[0044] In certain embodiments, in addition to the above paragraphs

[0035] -

[0043] , the drying rate during spray drying can be adjusted by adding a high molecular weight drying agent, such as hydroxyethyl starch, carboxymethyl cellulose, or a similar agent. In some embodiments, these high molecular weight drying agents (e.g., hydroxyethyl starch) may precipitate as a film on the surface of droplets from the lipid emulsions disclosed herein as they dry during spray drying, e.g., slowing the drying rate. According to these embodiments, this results in a reduced Peclet number for any emulsified nanodroplets or nanoparticles (e.g., lipid emulsions) contained within the droplets, effectively allowing the nanodroplets sufficient time to diffuse away from the receding drying region, reducing their concentration at the drying front, reducing their tendency to collide and coalesce, and reducing particle breakage. In other embodiments, lipid-based emulsions stabilized by spray drying to form glassy powders can be further protected from instability by coating the powder with a metal oxide or other drug disclosed herein, for example, using a nanoscale layer (e.g., molecular layer) of a metal drug coating by atomic layer deposition (ALD). The ALD coating further increases the thermal stability of the emulsion formulation for long-term storage, but the coating can be dissolved either in vivo or in a salt buffer. These observations that the drug within the lipid-based emulsion was stabilized and further stabilized to allow ALD coating were surprising. Additionally, it was surprisingly discovered and disclosed herein that lipid emulsions in polysaccharide-containing formulations that were spray-dried to form essentially dry microparticles can be further introduced into a fluidized-bed atomic layer deposition reactor to coat the microparticle surface without altering the size distribution of the embedded lipid emulsion contained with the powder. Thus, when the coated microparticles disclosed herein are reconstituted, the microparticle size is maintained, and their injectability preserves their internal nanoemulsion droplet size distribution for subsequent delivery.

[0037]

[0045] In some embodiments, in addition to the above paragraphs

[0035] through

[0044] , the stabilized lipid emulsion drug-, adjuvant-, or antigen-containing powders, microparticles, or glassy matrices disclosed herein may be coated with one or more coating layers using ALD, each of which may comprise one or more of organometallic materials, metal oxides, metal alkoxides, and / or aluminum-based coating layers. In certain embodiments, the coating layers may include, but are not limited to, aluminum oxide (Al2O3), aluminum alkoxides, silicon dioxide (SiO2), titanium dioxide (TiO2), and silicon nitride (Si3N4). According to these embodiments, the combination of spray drying and ALD coating of nanoemulsified lipid formulations increases the thermal stability of the nanoemulsified lipid formulations. For example, these coated, essentially dry nanoemulsified lipid formulations have improved stability upon storage at room temperature or at elevated temperatures.

[0038]

[0046] In certain embodiments, further to the above paragraphs

[0035] -

[0045] , ALD-coated lipid emulsion drug-containing, lipid emulsion adjuvant-containing, or lipid emulsion antigen-containing powder, microparticle, or glassy matrix can be stored, transported, and reconstituted for use in treating, reducing the occurrence of, or preventing a medical condition. According to these embodiments, the reconstituted coated microparticles maintain a size distribution that is injectable and can be introduced into a subject by any delivery method known in the art.

[0039]

[0047] In certain embodiments, in addition to paragraphs

[0035] -

[0046] above, one or more agents disclosed herein may include at least one immunogenic agent in a lipid emulsion that forms part of a central or innermost microparticle. According to these embodiments, the central or innermost microparticle may include, but is not limited to, at least one essentially dry lipid emulsion immunogenic agent-containing and at least one glass-forming agent or polysaccharide-containing microparticle, and further, one or more outer coating layers cover or encase the central or innermost lipid emulsion immunogenic agent-containing glassy microparticle. In accordance with these and the above-referenced embodiments, 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 100, up to 150, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, or more coating layers may surround a stabilized lipid emulsion drug-containing, lipid emulsion adjuvant-containing, or antigen-containing powder or glassy matrix, where the coating layers, upon administration to a subject, are readily dissolvable in the subject and expose the subject to one or more drugs or antigens by immediate or timed release. In other embodiments, the at least one agent, the at least one adjuvant, and / or the at least one antigen may be stabilized in powder form as disclosed herein and encased in another or outer layer of coating that forms a prime dose of the at least one agent to be exposed prior to dissolving the microparticles to expose the antigen, adjuvant, and / or agent in the outer layer or to expose at least one of the antigens or agents in the inner core.In yet other embodiments, at least one drug or at least one antigen may comprise a mixture of drugs or separate drugs in an inner core from the same or different species, or from the same or different drug or compound family, and then layered onto the coated microparticle, or mixed and coated onto the coated microparticle, or mixed with at least one outer layer of coating to create a coated microparticle for differential exposure of at least one drug or antigen, etc. The outer layer of coating with the same or different antigen, drug, and / or adjuvant may be further coated with an additional metal-containing composition using ALD.

[0040]

[0048] In certain embodiments, in addition to the above paragraphs

[0035] through

[0047] , a method for making stabilized lipid emulsion drug-, adjuvant-, and / or antigen-containing coated microparticles, stabilized powder, or glassy matrix includes combining at least one lipid emulsion drug / adjuvant / antigen with at least one glass-forming agent or polysaccharide to form a first liquid composition, rapidly spray-drying the composition to form lipid emulsion drug-containing glassy microparticles, and coating the essentially dry lipid emulsion drug-containing glassy microparticles with one or more outer coating layers. In some embodiments, the first liquid composition (before spray-drying) may further include the rapid introduction of a second glass-forming agent. According to these embodiments, the second glass-forming agent or polysaccharide may include hydroxyethyl starch or a similar agent. Furthermore, according to these embodiments, hydroxyethyl starch precipitates as a film on the surface of the composition droplets as they dry during spray drying, slowing the drying rate and reducing particle collisions, thereby maintaining particle integrity as they dry to a more stable state in the preparation of coatings. Rapid spray drying as disclosed herein can refer to spray drying in milliseconds to seconds. According to these embodiments, the particles and / or essentially dry microparticles disclosed herein are formulated and spray-dried so that the therapeutic agent in the lipid nanoemulsion of the therapeutic agent-containing formulation is not primarily located on the surface of the spray-dried particles (reducing exposure). It is known that high molecular weight and low solubility compounds are more likely to end up on the surface of spray-dried particles. Therefore, the embodiments disclosed herein address this commonly observed problem by specific additions to the formulation to form an outer shell-like layer that can shield, for example, polynucleotides, rather than primarily target therapeutic agents, making them more stable under certain conditions.According to these embodiments, the particles and / or essentially dry microparticles disclosed herein can be introduced into an ALD reaction chamber where the particles and / or essentially dry microparticles flow freely within the chamber to reduce clumping and / or aggregation of the particles and / or essentially dry microparticles. It is noted herein that directly introducing a lipid emulsion drug-containing formulation into an ALD reaction chamber without producing thermostable particles or essentially dry glassy microparticles as disclosed herein is an unsuccessful coating protocol. For example, due to the lack of availability of the linking groups required to anchor the coatings produced herein using polymer-containing formulations to produce the essentially dry microparticles or particles disclosed herein for long-term storage or further coating, high temperatures may degrade the formulation and therapeutic agent, or the therapeutic agent may stick to each other, or the chamber and / or applied metal material may not form a layer over the free therapeutic agent (not encased in a protective shell or glassy particles) and / or may not fully encapsulate the therapeutic agent. Until the present disclosure, it was unknown that the formulations disclosed herein could create such a barrier or shell to protect polynucleotides from such conditions. Those skilled in the art will appreciate that the formulations and processes disclosed herein are scalable and readily available for manufacturing to generate large quantities of microparticles for coating, or storage and later coating.

[0041]

[0049] According to these embodiments, in addition to paragraphs

[0035] through

[0048] above, the at least one drug, at least one adjuvant, and / or at least one antigen may include, for example, one or more drugs or antigens and / or adjuvants derived from or part of a viral antigen, a bacterial antigen, a toxin, a prion, yeast, a fragment or subunit thereof, a chemical agent, a small molecule, an anti-cancer agent, an anti-inflammatory agent, an anti-autoimmune agent, a peptide, a polynucleotide, or a protein thereof, or a combination thereof. In some embodiments, the at least one drug, adjuvant, and / or antigen may include one or more drugs, adjuvants, and / or antigens, including, but not limited to, a recombinant peptide, a recombinant protein, a peptide derived from a target protein or pathogen, a synthetic peptide or protein, a polynucleotide encoding a polypeptide, a polynucleotide, a virus-like particle, a live virus, a live attenuated virus, an inactivated virus, or one expressed on the surface of a virus or bacteriophage, or a combination thereof. In yet other embodiments, the at least one agent and / or antigen may comprise one or more agents and / or antigens, such as those represented by or encoded by a polynucleotide. According to these embodiments, the polynucleotide may include, but is not limited to, single-stranded (ss) or double-stranded (ds) DNA, e.g., linear or circular DNA, RNA, mRNA, siRNA, or chimeric molecules thereof, or other polynucleotides thereof. In certain embodiments, the chimera may comprise a combination of at least one polynucleotide segment and at least one polypeptide segment, or a combination of multiple polynucleotide segments and / or multiple polypeptide segments. In some embodiments, the polynucleotide may be, for example, an mRNA encoding the full length or a fragment of a target agent or antibody or its receptor molecule (e.g., a virus or bacterium).

[0042]

[0050] In certain embodiments, in addition to the above paragraphs

[0035] to

[0049] , the at least one agent or antibody is an agent or antibody derived therefrom, such as a human papillomavirus (e.g., HPV16, HPV18) or other mammalian papillomavirus, ricin toxin, Bacillus anthracis, Clostridium botulinum, Antibodies may include, but are not limited to, antibodies derived from viruses such as B. botulinum, Ebola virus, influenza virus, coronavirus (COVID-19 or other variants or mutants thereof), poliovirus, norovirus, rotavirus, hepatitis C, chickenpox, herpes simplex, cytomegalovirus, Japanese encephalitis, dengue virus, West Nile virus, Zika virus or other flaviviruses, alphaviruses such as chikungunya, EEEV, WEEV, VEEV or other alphaviruses, pneumonia virus species, Yersinia, Pneumococcus, Salmonella, Clostridium difficile, or combinations thereof. In certain embodiments, at least one antigen or agent may be a multimeric complex. In other embodiments, at least one antigen or agent may comprise a pathogen or an antigen derived therefrom that can infect humans (e.g., adults, children, infants, or fetuses), companion animals, livestock, wild animals, zoo animals, birds, or reptiles.

[0043]

[0051] In some embodiments, in addition to the above paragraphs

[0035] -

[0050] , the at least one glass former or polysaccharide disclosed herein may include at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol, and glycine, hydroxyethyl starch, glycine, cyclodextrin, povidone, etc. In certain embodiments, the at least one glass former or polysaccharide disclosed herein may include trehalose or sucrose or a combination thereof. In certain embodiments, the at least one glass former or polysaccharide disclosed herein may include hydroxyethyl starch, alone or in combination with a second polysaccharide. According to these embodiments, at least one glass-forming agent or polysaccharide disclosed herein may be present in the primary liquid composition prior to spray drying at a weight-to-volume (w / v) concentration of about 0.1% to about 40%, about 1.0% to about 30%, about 5.0% to about 30%, about 5.0% to about 25.0%, about 5.0% to about 20.0%, about 5.0% to about 15.0%, or about 20%. In certain embodiments, at least one glass-forming agent or polysaccharide disclosed herein may be rapidly incorporated into a lipid emulsion drug or antigen and / or adjuvant, which may then be further spray-dried into an essentially dry formulation; optionally, in preparation for application of one or more coating layers to the essentially dried microparticles formed using the ALD processes described herein. In still other embodiments, the glass former or polysaccharide comprises sucrose at a concentration of about 1.0% to about 40.0%, or about 5.0% to about 30.0%, or about 10.0% to about 30.0%, or about 15.0% to about 30.0%, or about 20.0% to about 40.0%, or about 20.0%. In certain embodiments, drying of microdroplets containing nanoemulsified lipid emulsions by spray drying as disclosed herein can occur over a rapid timescale of about 50 milliseconds to about 3 seconds, or about 100 milliseconds to about 1 second, or about 300 milliseconds.

[0044]

[0052] In other embodiments, in addition to the above paragraphs

[0035] through

[0051] , the glass-forming agent or polysaccharide disclosed herein may include at least one additional polysaccharide agent (or disaccharide agent). According to these embodiments, the at least one additional polysaccharide agent may include, but is not limited to, hydroxyethyl starch (HES), dextran, hetastarch, carboxymethylcellulose, and the like, or combinations thereof. According to these embodiments, the at least one additional polysaccharide may be present in the primary formulation prior to spray drying at a weight-to-volume (w / v) concentration of about 0.1% to about 40%, about 1% to about 30%, about 5% to about 20%, or about 8% to about 20%. In certain embodiments, the at least one additional polysaccharide agent may be different from the primary or first polysaccharide agent, and the additional polysaccharide agent may be present in the liquid or aqueous pre-spray-dried formulation at a weight to volume (w / v) concentration of about 0.1% to about 10.0%, about 0.1% to about 5.0%, about 0.1% to about 2.5%, or about 0.1% to about 0.5%. In certain embodiments, the first or primary glass-forming agent present in the pre-spray-dried formulation may be sucrose or trehalose, and the at least one additional polysaccharide or disaccharide may be hydroxyethyl starch (HES).

[0045]

[0053] In some embodiments, in addition to paragraphs

[0035] through

[0052] above, each layer of the one or more ALD coating layers may comprise one or more of organometallic materials, metal oxides, metal alkoxides, and / or aluminum-based coating layers applied to the essentially dry lipid emulsion drug-, adjuvant-, and / or antigen-containing microparticles. In certain embodiments, each layer of the one or more coating layers may comprise one or more of aluminum oxide, aluminum alkoxide (e.g., Alcon), silicon dioxide (SiO2), titanium dioxide (TiO2), zinc dioxide (ZnO2), or silicon nitride (Si3N4), either alone or in a suitable combination or pattern of layer-by-layer compositions. According to these embodiments, each coating layer may be about 0.1 nm to about 20.0 nm thick. In certain embodiments, the essentially dry microparticles disclosed herein may comprise multiple outer coating layers sufficient to delay or provide a timed release of at least one antigen, at least one adjuvant, and / or drug contained in one or more layers of the coated microparticle or from the center or innermost core of the coated microparticle. In certain embodiments, 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 100, up to 150, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, or more coating layers may surround the stabilized lipid emulsion drug-, adjuvant-, and / or antigen-containing powder or glassy matrix or microparticle disclosed herein, where the coating layers are readily dissolvable in a subject upon administration to the subject and expose the subject to one or more drugs, adjuvants, and / or antigens by immediate or timed release.

[0046]

[0054] In certain embodiments, in addition to paragraphs

[0035] through

[0053] above, one or more ALD coating layers disclosed herein can function as an adjuvant to enhance an immune response in a subject to one or more drugs or antigens of a drug- or antigen-containing ALD-coated microparticle. In some embodiments, the one or more coating layers can include one or more coating layers capable of inducing a rapid immune response in a subject upon exposure. In other embodiments, a coated essentially dry lipid emulsion having one or more drugs or antigens can be contained in selected one or more layers and / or the core of the coated microparticle such that, upon dissolution of the coating layer and exposure of the one or more antigens and / or drugs, a rapid immune response to the one or more drugs or antigens in the subject can be induced in the subject. According to these embodiments, the immune response is enhanced compared to delivery of antigens and / or drugs without spray drying and / or coating of the stabilized microparticles disclosed herein.

[0047]

[0055] In some embodiments, further to paragraphs

[0035] to

[0054] above, the lipid emulsion drug-containing and / or antigen-containing coated microparticles described herein can be stored for extended periods of time at room temperature, at temperatures up to about 50°C, or up to about 60°C, or up to about 70°C, without refrigeration. In certain embodiments, the lipid emulsion drug-containing and / or antigen-containing coated microparticles described herein can be stored without refrigeration at up to room temperature, up to about 50°C, or up to about 60°C, or up to about 70°C for up to about 1 day, about 2 days or more, about 1 week, about 2 weeks, about 2-3 weeks, about several weeks, about 1 month, about 2 months, about 3 months, up to about 4 months, up to about 6 months, up to about 9 months, up to about 12 months, up to about 15 months, up to about 18 months, up to about 24 months or more without negative effects on the coated microparticles or stabilized but uncoated microparticles (e.g., degradation, loss of efficacy, loss of immunogenicity, reduced delivery of one or more drugs or antigens).

[0048]

[0056] In addition to the above paragraphs

[0035] -

[0055] , other embodiments provide combination compositions or formulations comprising a plurality of coated lipid emulsion drug-, adjuvant-, and / or antigen-containing microparticles described herein. According to these embodiments, these combination compositions or formulations may comprise a mixture of different coated microparticles containing one or more drugs or antigens for treating, reducing the onset of, or preventing a single health condition or multiple health conditions (e.g., pathogenic infection, or prevention of pathogenic infection, cancer, inflammation, etc.), and may further comprise at least one pharmaceutically acceptable excipient to create a pharmaceutically acceptable composition or formulation. In other embodiments, the combination composition may comprise at least one representative microparticle-containing pharmaceutical composition as disclosed herein mixed with a standard or known composition or formulation for treating, reducing the onset of, or preventing a health condition. In certain embodiments, the standard formulation may comprise a standard vaccine against a pathogen or anti-cancer agent, etc. In some embodiments, the ALD-coated microparticles disclosed herein can be used as one or more boost doses to standard therapy aimed at treating, preventing, or reducing the onset of a health condition.

[0049]

[0057] In some embodiments, further to paragraphs

[0035] -

[0056] above, the formulations disclosed herein are part of a single-dose formulation comprising a prime dose and at least one boost dose of at least one agent or at least one antigen sequestered within the coated microparticles disclosed herein. According to these embodiments, the prime dose and at least one boost dose of at least one antigen or at least one agent may be present in the same coated microparticle or in separate coated microparticles. If in separate particles, the prime dose of at least one agent or at least one antigen may be sequestered within the microparticle, while the at least one boost dose may be in separate microparticles. In certain embodiments, release of the prime dose after delivery to a subject may be immediate or delayed, depending on the number of coating layers (if not on the outermost or near-outermost coating layer) coating the lipid emulsion-coated microparticle prime dose, and the at least one boost may be exposed to the subject a short time, minutes, hours, days, weeks, or months later. In certain embodiments, compositions can be mixed where one composition contains stabilized coated or uncoated stabilized lipid emulsion drug- or antigen-containing microparticles alone or in combination with different drugs or antigens targeting the same or different pathogens or conditions, or in admixture with other known drugs or formulations.

[0050]

[0058] In some embodiments, further to the above paragraphs

[0035] through

[0057] , formulations containing lipid emulsion drug- or antigen-containing coated microparticles may be part of a single-dose formulation with at least two different drugs or two different antigens capable of inducing an immune response to two or more different antigens or drugs. According to these embodiments, the two or more different antigens or drugs may be contained in the same or separate coated microparticles (e.g., in the core and at least one outer layer) in the same or different coated particle layers of the microparticles. Precision coating layers may be applied by ALD to provide a timed release of one or more drugs over a predetermined period of time upon administration to a subject.

[0051]

[0059] In other embodiments, further to the above paragraphs

[0035] to

[0058] , the thermostable lipid emulsion drug- or antigen-containing microparticle composition may comprise a standard vaccine composition (e.g., mRNA, live virus, attenuated virus) and a plurality of coated or uncoated lipid emulsion drug- or antigen-containing microparticles as described, wherein at least one of the at least one drug induces a boost immune response to the standard vaccine composition.

[0052]

[0060] Other embodiments, in addition to the above paragraphs

[0035] through

[0059] , provide methods for inducing a response in a subject, which may include administering to a subject coated microparticles of lipid emulsion containing a drug, adjuvant, and / or antigen formulation described herein to treat, prevent, or improve a health condition. According to these embodiments, the formulation may be administered by any method known in the art. In other embodiments, the formulation may induce a response in a subject (e.g., an immune response, an anti-inflammatory response, an anti-cancer response, or other response). According to these embodiments, the immune response induced by the formulation or composition may be prophylactic or therapeutic, depending on the at least one antigen and / or at least one drug sequestered in one or more coated or uncoated microparticles of lipid emulsion containing a drug or antigen formulation.

[0053]

[0061] In certain embodiments, further to the above paragraphs

[0035] through

[0060] , the coated or uncoated lipid emulsion drug- or antigen-containing microparticles and formulations or compositions thereof disclosed herein can use less antigen or drug than is used to formulate current vaccines against immunogens or other therapeutic agents (e.g., reducing costs) and can provide enhanced efficacy after a single administration. In other embodiments, the coated or uncoated lipid emulsion drug- or antigen-containing microparticle compositions or formulations provide thermostable formulations that eliminate and / or reduce refrigeration requirements (e.g., cold chain refrigeration requirements), limit the concentration of adverse effects of the drug administered to a subject (e.g., aluminum, excess therapeutic agent), and increase lipid emulsion drug or antigen compatibility. In certain embodiments, the compositions and methods disclosed herein are applicable to a variety of potential agents or antigens, including, but not limited to, polynucleotides, chimeras, carbohydrates, polypeptides, recombinant peptides, or protein immunogens, virus-like particles (VLPs), and inactivated or attenuated pathogens (e.g., viruses), bacteriophages conjugated or associated with various antigens and / or agents.

[0054]

[0062] In addition to the above paragraphs

[0035] through

[0061] , some embodiments disclosed herein relate to dehydration or drying method and formulation parameters that can be adjusted to control the nucleation rate, glass transition temperature, and other material properties of lipid emulsion drug- or antigen-containing microparticles. In certain embodiments, dehydration or drying of the drug, antigen, or other agent can occur, for example, by spray drying and / or spray freeze drying or other equivalent procedures, essentially drying the lipid emulsion drug- or antigen-containing microparticles and facilitating coating as disclosed herein. According to these embodiments, lipid emulsion drug-, adjuvant-, and / or antigen-containing microparticles can be enveloped with one or more coating layers to produce coated lipid emulsion drug-, adjuvant-, or antigen-containing microparticles. In other embodiments, lipid emulsion drug- or antigen-containing microparticles containing one or more antigens or drugs directed against one or more pathogens, one or more target molecules, or one or more small molecules, either in the same microparticle or in separate microparticles, can be combined into an aqueous solution and reconstituted.These lipid emulsion drug- or antigen-containing microparticle formulations described herein are heat-stable and can be produced against any pathogenic agent or for any therapeutic agent for the treatment, improvement, or prevention of a health condition.In certain embodiments, lipid emulsion drug- or antigen-containing microparticles can be produced for use against any pathogenic organism or as a therapeutic agent for the treatment of any condition.

[0055]

[0063] In certain embodiments, in addition to the above paragraphs

[0035] to

[0062] , the pathogenic virus may be, for example, any pathogenic virus. According to these embodiments, the pathogenic virus may be a papovavirus (e.g., a papillomavirus, including human papillomavirus (HPV)), a herpesvirus (e.g., herpes simplex virus, varicella-zoster virus, bovine herpesvirus-1, cytomegalovirus), a poxvirus (e.g., smallpox virus), a reovirus (e.g., rotavirus), a parvovirus (e.g., parvovirus B19, canine parvovirus), a picornavirus (e.g., poliovirus, hepatitis A), a togavirus (e.g., rubella virus), a hepadnavirus (e.g., hepatitis B virus), a flavivirus (e.g., dengue virus, hepatitis C virus, West Nile virus, yellow fever virus, Zika virus), or a virulent virus. The virus may include, but is not limited to, an avian influenza virus (e.g., influenza A virus, influenza B virus, influenza C virus), a paramyxovirus (e.g., measles virus, mumps virus, respiratory syncytial virus, canine distemper virus, parainfluenza virus), a rhabdovirus (e.g., rabies virus), a filovirus (e.g., Ebola virus), an alphavirus (e.g., chikungunya or WEEV, EEEV or VEEV, or other alphaviruses), or a coronavirus, SARS, or the like, or a variant thereof, or a combination thereof. According to these embodiments, the agent or antigen derived therefrom may be part of the microparticles disclosed herein, which may further comprise a pharmaceutically acceptable agent.

[0056]

[0064] In another embodiment, further to the above paragraphs

[0035] to

[0062] , the pathogenic agent or antigen derived from a pathogenic agent may be a bacterium or a bacterial toxin or toxoid agent. According to these embodiments, the virulence agent or antigen derived from a virulence agent may be selected from the group consisting of Pasteurella haemolytica, Clostridium difficile, Clostridium haemolyticum, Clostridium tetani, Corynebacterium diphtheria, Neorickettsia resticii, Streptococcus equi equi, Streptococcus pneumoniae, Salmonella spp., Chlamydia trachomatis, Bacillus anthracis, Yersinia spp. spp., and Clostridium botulinum, or other pathogenic bacteria, or combinations thereof. In accordance with these embodiments, the agent or antigen derived therefrom may be part of the microparticles disclosed herein, which may further comprise a pharmaceutically acceptable agent.

[0057]

[0065] In certain embodiments, further to paragraphs

[0035] to

[0062] above, the pathogenic agent may be a fungus. According to these embodiments, the pathogenic fungi include Cryptococcus spp. (e.g., neoformans and gattii), Aspergillus spp. (e.g., fumigatus), Blastomyces spp. (e.g., dermatitidis), Candida albicans, Paracoccidioides spp. (e.g., brasiliensis), Sporothrix spp. (e.g., schenkii and brasiliensis), Histoplasma capsulatum, and the like. The agent may include, but is not limited to, Pneumocystis capsulatum, Pneumocystis jirovecii, and Coccidioides immitis, or other pathogenic fungi, or combinations thereof. In accordance with these embodiments, the agent or antigen derived therefrom may be part of the microparticles disclosed herein, which may further comprise a pharmaceutically acceptable agent.

[0058]

[0066] In yet other embodiments, further to the above paragraphs

[0035] -

[0062] , the virulence factor may be a toxin. According to these embodiments, the toxin may include, but is not limited to, anthrax toxin, ricin toxin, or botulinum toxin, or other toxins. According to these embodiments, the agent or antigen derived therefrom may be part of the microparticles disclosed herein, which may further include a pharmaceutically acceptable agent.

[0059]

[0067] In yet another embodiment, further to the above paragraphs

[0035] to

[0066] , the one or more antigens or agents may comprise DNA or mRNA encoding one or more antigens derived from a pathogenic factor, including, but not limited to, DNA or mRNA encoding a viral capsid protein or a subunit thereof, viral DNA or mRNA encoding a virus-like particle, DNA or mRNA encoding a viral spike protein, DNA or mRNA encoding a viral enzyme, and DNA or mRNA encoding a viral structural protein.

[0060]

[0068] In some embodiments, in addition to paragraphs

[0035] through

[0067] above, the lipid emulsion drug-containing particles described herein can be used to produce one or more microparticle-containing formulations for use as vaccines for any animal. In certain embodiments, the animal may be a household pet or other companion animal. In some embodiments, the animal may include livestock or other farm animals, wild animals, or zoo animals. In certain embodiments, the animal may include a non-human mammal, reptile, or bird. According to these embodiments, the immunogenic composition may be administered to animals including, but not limited to, dogs (canines), cats (felines), horses (equines), cows (bovines), goats (caprines), sheep (goats), pigs (swine), or poultry (e.g., chickens, turkeys, ducks, geese).

[0061]

[0069] In certain embodiments, further to paragraphs

[0035] through

[0062] above, the lipid emulsion drug-containing coated microparticles described herein can be used to produce one or more compositions contemplated herein or other drug-containing compositions for administration to canines to reduce the incidence of or prevent infectious diseases, or to treat conditions such as cancer, inflammatory conditions, diabetes, or other conditions. According to these embodiments, the infectious diseases may include, but are not limited to, infections associated with canine parvovirus (CPV), canine distemper virus (CDV), canine adenovirus (CAV), rabies, canine parainfluenza virus (CPiV), canine influenza virus, canine coronavirus, measles virus, Bordetella bronchiseptica, Leptospira spp., and Borrelia burgdorferi, or combinations thereof.

[0062]

[0070] In certain embodiments, further to paragraphs

[0035] through

[0062] above, the lipid emulsion drug-containing coated microparticles described herein may be used to produce one or more compositions contemplated herein or other drug-containing compositions for administration to felines to reduce the incidence of or prevent infectious diseases, or to treat conditions such as cancer or inflammatory conditions or diabetes or other conditions. In some embodiments, the lipid emulsion drug-containing coated microparticles described herein can be used to produce formulations containing coated microparticles useful for treating, reducing the risk of developing, or preventing infections in cats, including, but not limited to, immunogenic compositions against feline herpesvirus type 1 (FHV1), feline calicivirus (FCV), feline panleukopenia virus (FPV), rabies, feline leukemia virus (FeLV), feline immunodeficiency virus, virulent systemic feline calicivirus, Chlamydophila felis, Pasteurella haemolytica, and Bordetella bronchiseptica, or combinations thereof.

[0071] In certain embodiments, further to paragraphs

[0035] through

[0062] above, the lipid emulsion drug-containing coated microparticles described herein may be used to produce one or more compositions contemplated herein or other drug-containing compositions for administration to horses to reduce the incidence of or prevent infectious diseases, or to treat conditions such as cancer or an inflammatory condition or diabetes or other conditions. In other embodiments, the lipid emulsion drug-containing coated microparticles described herein may be used to produce formulations containing coated microparticles useful for treating, reducing the risk of developing, or preventing or treating an infection in horses, including, but not limited to, immunogenic compositions directed against Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, Venezuelan Equine Encephalitis Virus, Bovine Papillomavirus, Rabies Virus, Clostridium tetani, West Nile Virus, Equine Influenza Virus, Potomac Fever (Neorickettsia risticii), Streptococcus equie equie, and Rhinopneumonitis (Equine Herpesvirus Type 1), or combinations thereof.

[0063]

[0072] In certain embodiments, further to paragraphs

[0035] through

[0062] above, the lipid emulsion drug-containing coated microparticles described herein may be used to produce one or more compositions contemplated herein or other drug-containing compositions for administration to bovines to reduce the incidence of or prevent infectious diseases, or to treat conditions such as cancer or an inflammatory condition or diabetes or other conditions. In other embodiments, the lipid emulsion drug-containing coated microparticles described herein are effective against bovine rhinotracheitis (IBR), parainfluenza type 3 (PI3), bovine viral diarrhea (BVD), bovine respiratory syncytial virus (BRSV), blackleg disease (Clostridium chauvoei), malignant edema (Clostridium septicum), infectious necrotizing hepatitis (Clostridium novyi), enterotoxemia (Clostridium perfringens types C and D), Pasteurella haemolytica, and red urine (Clostridium haemolyticum). The present invention can be used to treat, reduce the risk of developing, or prevent infections in cattle, or to produce coated microparticle-containing formulations useful for treating infections, including, but not limited to, immunogenic compositions against Bacillus subtilis, Bacillus anguilliformis, Bacillus subtilis, Bacillus haemolyticum, or combinations thereof.

[0064]

[0073] In certain embodiments, further to paragraphs

[0035] through

[0062] above, the lipid emulsion drug-containing coated microparticles described herein may be used to produce one or more compositions contemplated herein or other drug-containing compositions for administration to poultry to reduce the incidence of or prevent infectious diseases, or to treat conditions such as cancer or an inflammatory condition or diabetes or other conditions. In other embodiments, the lipid emulsion drug-containing coated microparticles described herein may be used to produce formulations containing coated microparticles useful for treating, reducing the risk of developing, or preventing infection in poultry, including, but not limited to, immunogenic compositions directed against Marek's disease (Marek's disease virus), tenosynovitis (reovirus), encephalomyelitis (avian encephalomyelitis virus), fowl pox (avipox virus), chicken infectious anemia (chicken anemia virus), fowl cholera (Pasteurella multocida), Newcastle disease / infectious bronchitis (Newcastle disease virus), Riemerella anatipestifer, duck viral hepatitis (duck hepatitis virus), and duck viral enteritis (duck herpesvirus 1), or combinations thereof.

[0065]

[0074] In certain embodiments, further to the above paragraphs

[0035] through

[0062] , the lipid emulsion drug-containing coated microparticles described herein can be used to produce one or more of the compositions contemplated herein or other drug-containing compositions for administration to humans to reduce the incidence of, treat, and / or prevent an infectious disease, or to treat a condition such as cancer or an inflammatory condition or diabetes or other health condition. In other embodiments, the lipid emulsion drug-containing coated microparticles described herein can be used to produce a formulation containing the coated microparticles useful for treating, reducing the risk of developing, or preventing an infectious disease in humans. In certain embodiments, the lipid emulsion drug-containing coated particles described herein can be used to deliver one or more formulations disclosed herein to a human, e.g., an infant or child, or an adolescent, young adult, adult, or elderly human subject, including, but not limited to, antigens or agents derived therefrom for varicella zoster (chickenpox), diphtheria, Haemophilus influenzae type b (Hib), hepatitis A, hepatitis B, influenza, coronavirus, SARS, Ebola virus, measles, mumps, whooping cough, polio, pneumococcal disease, rotavirus, rubella, and tetanus. In other embodiments, the immunogenic agent-containing particles described herein may be used to deliver one or more immunogenic compositions to a human pre-teen or teen, including, but not limited to, vaccines for influenza, tetanus, diphtheria, pertussis, human papillomavirus, meningococcal disease, hepatitis B, hepatitis A, polio, measles, mumps, rubella, and varicella-zoster.In still other embodiments, the lipid emulsion drug-containing coated microparticles described herein may be used to deliver one or more immunogenic compositions to adult humans, including, but not limited to, immunogenic compositions against influenza (e.g., A, B, or C), tetanus, diphtheria, pertussis, shingles, pneumococcal disease, meningococcal disease, measles, mumps, rubella, chickenpox, hepatitis A, hepatitis B, and Haemophilus influenzae type b.

[0066]

[0075] In other embodiments, further to the above paragraphs

[0035] through

[0062] , the lipid emulsion drug-containing coated particles described herein can be used to produce compositions useful for administration to humans. According to these embodiments, treatment of human subjects can include, but is not limited to, immunogenic compositions for travel-related infectious diseases, including, but not limited to, hepatitis A, hepatitis B, typhoid, paratyphoid, meningococcal disease, yellow fever, dengue fever, rabies, Zika virus-related conditions, West Nile virus infection, chikungunya, and Japanese encephalitis, COVID-19 or coronavirus infection, or other infectious diseases, or combinations thereof.

[0067]

[0076] In yet another embodiment, further to the above paragraphs

[0035] to

[0062] , the lipid emulsion drug-containing coated microparticles described herein can be used to produce compositions useful for administration to humans, including, but not limited to, drugs or antigens. According to these embodiments, the agent or antigen may be derived from, and may include, but is not limited to, human papillomavirus (e.g., HPV16, HPV18, HPV31, HPV45, or HPV6 or HPV11, or any other HPV type or serotype), herpes simplex virus, smallpox virus, rotavirus, parvovirus B19 vaccine, chikungunya virus, dengue virus (e.g., dengue 1, dengue 2, dengue 3, or dengue 4, or any new dengue strain or variant), norovirus, hepatitis C virus, West Nile virus, Zika virus, respiratory syncytial virus, rabies virus, and Ebola virus, SARS, COVID-19, or other strains or variants, and the like.

[0068]

[0077] In certain embodiments, in addition to the above paragraphs

[0035] through

[0076] , the lipid emulsion drug-containing coated microparticles described herein can contain a single drug dose or two or more doses of a particular drug (e.g., a prime dose and a boost dose, or just the dose of drug for extended release and timed release). In some embodiments, the lipid emulsion drug-containing coated microparticles can contain doses of two or more different immunogenic agents. In still other embodiments, lipid emulsion drug-containing coated microparticles containing different drug doses can be combined into a mixture of lipid emulsion drug-containing coated microparticles. A mixture of lipid emulsion drug-containing coated microparticles can be combined into a single administration. This can result in fewer administrations and less need to return to the clinic for subsequent administrations.

[0069]

[0078] In certain embodiments, in addition to paragraphs

[0035] through

[0077] above, the lipid emulsion drug- or antigen-containing coated particles (e.g., microparticles) described herein include the methods disclosed herein for controlled ultra-rapid freezing rates in combination with glass-forming agents at various concentrations as set forth herein. According to these embodiments, glass-forming agents can include, but are not limited to, trehalose and sucrose, or similar polysaccharides. These agents can be used to generate glass-like or scaffold-like matrices during these rapid spray-drying procedures. In certain embodiments, when glass-forming agents are dried during the dehydration process (e.g., spray-drying) in the presence of one or more drugs, antigens, or therapeutic agents, they form powders (lipid emulsion drug- or antigen-containing microparticles) containing embedded drugs and / or antigens. In this dehydrated state, physical and chemical degradation pathways of proteins that require molecular movement, as well as other degradation pathways, can be inhibited, thereby stabilizing one or more drugs or antigens from degradation. Some advantages of these procedures for making stabilized coated microparticles include, but are not limited to, reduced cold chain storage requirements, reduced or eliminated need for separate adjuvants when inducing an immune response to one or more drugs and / or antigens, reduced concentration of target drugs and / or antigens, and / or long-term stability at elevated temperatures.

[0070]

[0079] In other embodiments, in addition to paragraphs

[0035] through

[0078] above, one or more adjuvants and / or immunostimulatory agents may be incorporated into or layered on the microparticles. According to these embodiments, the adjuvant or coadjuvant may be combined with at least the lipid emulsion agent, antigen or drug, and at least one glass-forming agent in a formulation prior to spray drying or dehydration, and / or may be applied to the outer layer of the coated ALD microparticles disclosed herein.

[0071]

[0080] In some embodiments, in addition to paragraphs

[0035] through

[0079] above, one or more antigens or drugs contained within a lipid emulsion can be combined with a glass-forming agent to produce an aqueous composition. For example, the composition can then be dehydrated to form drug- or antigen-containing glass microparticles. In other embodiments, adjuvants and / or coadjuvants can be included in the composition in preparation for dehydration or spray drying of the combination.

[0072]

[0081] In yet other embodiments, in addition to paragraphs

[0035] through

[0080] above, an inactivated or attenuated pathogen (e.g., a live attenuated virus) can be part of a lipid emulsion-containing composition along with at least one polysaccharide and rapidly spray-dried to form glassy microparticles. According to these embodiments, inactivated (or killed) viruses or virus particles, bacteria, fungi, toxins, or other pathogens can be inactivated by any means, for example, chemically or mechanically by heat, and incorporated into the thermostable glassy microparticles disclosed herein. Non-limiting examples of inactivated pathogens that can be incorporated into thermostable lipid emulsion-containing glassy microparticles include inactivated whole-cell pertussis (inactivated Bordetella pertussis), Salmonella typhi, and inactivated poliovirus. Live attenuated viruses or bacteria can also be incorporated into microparticles. Non-limiting examples of attenuated viruses and bacteria that can be incorporated into the thermostable glassy microparticles include measles, mumps, rubella, influenza, chickenpox, smallpox, polio, rotavirus, flaviviruses (e.g., dengue, yellow fever), alphaviruses, filoviruses, rabies, typhoid, Mycobacterium bovis, Salmonella typhi, and Rickettsia spp., or other known pathogens.

[0073]

[0082] In some embodiments, in addition to the above paragraphs

[0035] through

[0081] , one or more drug or antigen-supplemented formulations disclosed herein may be included in thermostable lipid emulsion drug-containing glass microparticles containing at least one antigen or drug. According to these embodiments, supplements contemplated herein may include, but are not limited to, one or more aluminum salt adjuvants, one or more buffering agents, one or more volatile salts, one or more immunologically relevant agents, one or more lubricating excipients, and costimulatory agents (e.g., co-immunostimulatory agents). In other embodiments, the formulations disclosed herein may include some or all of an immunogenic agent, such as a pathogenic factor, e.g., an antigen, a glass-forming agent, one or more adjuvants, a buffer, an immunologically relevant agent, and a costimulatory agent prior to spray drying to produce drug-containing glass microparticles useful for making the ALD-coated thermostable lipid emulsion drug-containing glass microparticles described herein. In some embodiments, the formulations disclosed herein can be dehydrated by, for example, freeze drying, vacuum drying, spray drying, or spray freeze drying, or other systems for dehydrating samples.

[0074]

[0083] In some embodiments, further to the above paragraphs

[0035] through

[0082] , aluminum salts useful for producing immunogenic thermostable lipid emulsion drug-containing microparticles may include one or more of aluminum hydroxide, aluminum phosphate, and aluminum sulfate, or a combination thereof. According to these embodiments, the aluminum salt may be in the form of an aluminum hydroxide gel (e.g., Alhydrogel®).

[0075]

[0084] In some embodiments, in addition to the above paragraphs

[0035] through

[0083] , buffers useful for spray-drying or reconstituting the microparticles disclosed herein may include, but are not limited to, acetate buffer, succinate buffer, citrate buffer, prolamin buffer, histidine buffer, borate buffer, carbonate buffer, or phosphate buffer, or combinations thereof. In certain embodiments, the buffer may include one or more salts useful for forming lipid emulsion drug-containing glassy microparticles, including, but not limited to, one or more salts including, but not limited to, sodium acetate, sodium succinate, potassium succinate, sodium citrate, sodium phosphate, potassium phosphate, etc., or combinations thereof. In certain embodiments, the buffer may include histidine, e.g., histidine-HCl. In other embodiments, the one or more volatile salts can include, but are not limited to, ammonium acetate, ammonium formate, ammonium carbonate, ammonium bicarbonate, triethylammonium acetate, triethylammonium formate, triethylammonium carbonate, trimethylamine acetate, trimethylamine formate, trimethylamine carbonate, pyridinal acetate, and pyridinal formate, or combinations thereof. In some embodiments, the volatile salt can include ammonium acetate.

[0076]

[0085] In some embodiments, in addition to the above paragraphs

[0035] through

[0084] , glass-forming agents or polysaccharide agents used herein may include, but are not limited to, one or more of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol, hydroxyethyl starch, glycine, cyclodextrin, glycine and mannitol, trehalose and sucrose, and povidone, or combinations thereof. In certain embodiments, the glass-forming agent may be sucrose or trehalose. In other embodiments, trehalose may be present at a weight-to-volume (w / v) concentration of about 0.1% to about 40% in the immunogenic composition before dehydration; about 1% to about 30% w / v; about 5% to about 20%; or about 10% to about 20% w / v or about 20% w / v in the composition before spray drying. In another embodiment, the glass-forming agent can be sucrose or trehalose at a concentration of about 10% to about 25%; or about 15% w / v to about 25% w / v in the immunogenic composition before dehydration.

[0077]

[0086] In certain embodiments, in addition to the above paragraphs

[0035] through

[0085] , the compositions disclosed herein may include a co-stimulatory agent to further boost the immune response to one or more target antigens or one or more agents used to treat a condition (e.g., cancer) as needed. According to these embodiments, the co-immunostimulatory agent may include, but is not limited to, one or more of: lipid A, lipid A derivatives, monophosphoryl lipid A, chemical analogs of monophosphoryl lipid A, CpG-containing oligonucleotides, TLR-4 agonists, flagellin, flagellin derived from Gram-negative bacteria, TLR-5 agonists, fragments of flagellin capable of binding to the TLR-5 receptor, saponin, saponin analogs, QS-21, purified saponin fractions, ISCOMS, and combinations of saponin with sterols and lipids, or any known co-immunostimulatory agent, or combinations thereof.

[0078]

[0087] It is understood in the art that as the complexity of a vaccine, antigenic, or immunogenic composition increases, the long-term stability of the antigenic agent typically decreases, for example, when the temperature increases, such as during storage or transportation. In certain embodiments, the formulations disclosed herein that possess multiple subunits (e.g., multimers) may be more complex and less stable than a single protein or fragment. For example, a formulation disclosed herein that has an antigen based on multiple capsomere subunit types may be a less stable immunogenic composition than a single subunit type. In certain embodiments, embedding a multimeric unit composition (e.g., capsomers) within a matrix formed during spray drying or spray freeze drying can enhance or increase the thermal stability of the multimeric complex, for example, by stabilizing the tertiary structure of the multimeric units. The embodiments described herein may include methods and compositions for use in stabilizing any complex pathogenic or other construct for use in forming thermostable microparticles, including, but not limited to, recombinant peptide or protein immunogens, and inactivated or attenuated pathogens, or other complex structures that have thermolabile properties when introduced into a process that can be stabilized as disclosed herein.

[0079]

[0088] In certain embodiments, in addition to the above paragraphs

[0035] through

[0087] , the drugs used in the thermostable lipid emulsion drug-containing microparticles of the present disclosure may be useful in prophylactic and / or therapeutic immunogenic compositions. The suitability of a drug for use in lipid emulsion drug-containing microparticles can be tested by reaction with antibodies or monoclonal antibodies that react with or recognize conformational epitopes present on the drug's intact target and based on the drug's ability to induce the production of neutralizing antisera. Suitable assays for determining whether neutralizing antibodies are produced are known to those skilled in the art. In this manner, in certain embodiments, it can be verified whether the immunogenic agents of the present disclosure induce the production of neutralizing antibodies.

[0080]

[0089] In certain embodiments, in addition to paragraphs

[0035] through

[0088] above, the drugs stabilized in the microparticles disclosed herein can be coated in one coating layer or sequestered in one or more coating layers. Certain embodiments relate to the use of molecular deposition processes in methods, compositions, and formulations for producing single-dose and multi-dose compositions. In some embodiments, microparticles can be produced in which the coating or sequestering layers not only serve as adjuvants to induce an immune response but also provide separate prime and boost vaccine doses in a single administration to a subject. In some embodiments, thermal stabilization can be achieved by a combination of embedding one or more lipid emulsion drug-containing formulations in a glassy organic matrix to form one or more stabilized glassy microparticles, and using a molecular deposition process that allows for the application of a variety of molecular layers to one or more antigen- or drug-containing glassy microparticles to obtain encapsulated or coated microparticles. According to these embodiments, the thickness of these coating or isolation layers can be controlled to within 1 or 2 angstroms and can range from about 0.1 nm to about 20.0 nm per layer. According to these embodiments, one or more coating layers can be deposited sequentially on top of each other without leaving the ALD reactor or other reactor until fully coated. In certain embodiments, the thickness of these coating or isolation layers can range from about 5.0 nm to about 25.0 nm. Using a series of alternating, self-limiting surface reactions, these coating layer deposition processes are highly scalable. For example, a fluidized-bed reactor can be used to coat large volumes of essentially dry lipid emulsion antigen- or drug-containing microparticles with coating layers without causing the microparticles to aggregate. This process allows for the complete encapsulation of one or more microparticles for immediate or timed release of one or more antigens or drugs.

[0081]

[0090] In some embodiments, in addition to paragraphs

[0035] through

[0089] above, molecular deposition techniques can be used to apply nanometer-thick coatings of inorganic, organic, or metal-organic materials to the surfaces of essentially dry lipid emulsion antigen-, adjuvant-, and / or drug-containing microparticles. In certain embodiments, the coating or isolating layer can be an aluminum-based material, including, for example, aluminum oxide or a metal oxide, or a metal alkoxide and / or aluminum alkoxide (e.g., Alcon), or a mixture thereof. According to these embodiments, the aluminum-containing material is deposited or applied to the surface of one or more microparticles to coat or isolate the one or more microparticles with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, up to 20, up to 30, up to 50, up to 100, up to 150, up to 200, up to 250, up to 300, up to 350, up to 400, or more layers of metal oxide or other metal-based-containing material to form encased or coated microparticles.

[0082]

[0091] In some embodiments, in addition to paragraphs

[0035] through

[0090] above, an aluminum-based coating layer can be used as a coating applied to essentially dry lipid emulsion microparticles carrying at least one antigen, adjuvant, and / or drug, and at least one glass-forming or polysaccharide agent. In certain embodiments, one or more layers of an aluminum-based film (coating) applied by ALD can be formed by coupling trimethylaluminum to the hydroxyl groups of the microparticles, a layer of amine groups can be formed by coupling ethanolamine to a layer of the aluminum-containing material, and a second layer of hydroxyl groups can be formed by coupling maleic anhydride to the available amine groups. ABC-type reactions can be self-limiting, for example, and can be used to deposit molecular layers of alkone or other metal-containing compositions disclosed herein. In addition to these embodiments, hydroxyl groups on a substrate (e.g., microparticles) react with trimethylaluminum, followed by ethanolamine to leave terminal amine groups on the surface, and available maleic anhydride reacts with the terminal amine groups to regenerate the hydroxyl surface for repeated coatings as needed. This ABC-type molecular deposition process can be repeated to provide additional layers as desired (up to 10, up to 20, up to 100, up to 200, up to 300, up to 400 or more coatings, or any desired number of coatings in between), and can be used to deliver, for example, 1, 2, 3, 4, 5, or 6 doses of at least one antigen or agent to a subject in a single administration, depending on the composition or configuration of the coated or sequestered one or more microparticles. In other embodiments, various chemical surrogates (e.g., alternative sources of aluminum, amines, and / or hydroxyl groups) can be used in these coating or sequestering processes, as will be recognized by those of skill in the art based on this disclosure.

[0083]

[0092] In certain embodiments, in addition to paragraphs

[0035] through

[0091] above, a binary reaction sequence can be used to deposit one or more layers of alumina on essentially dry microparticles. The microparticles can be treated with alternating gas streams containing either trimethylaluminum or water vapor. In certain embodiments, the number of cycles can be varied to control the formation of coating or isolation layers on one or more drug- or antigen-containing microparticles. In certain embodiments, it is understood that, because coating of these microparticles by these ALD processes relies on gas, no solvents are used during these coating processes. It is further understood that the essentially dry drug-, antigen-, and / or adjuvant-containing lipid emulsion microparticles disclosed herein introduced into an ALD reactor for coating are introduced for a predetermined time period to receive a predetermined number of coating layers on the microparticles, depending on what is desired. It is understood that the coated microparticles disclosed herein removed from the ALD reactor can be further processed for preparation and use in treating, reducing the onset of, or preventing a health condition. In some embodiments, the coated microparticles comprise one or more coating layers for exposure of antigens, drugs, and / or adjuvants at a predetermined time (e.g., immediately, within one week, within one month, within three months, or more, or any time in between), as desired and preselected by the number of coating layers.

[0084]

[0093] According to these embodiments, and in addition to paragraphs

[0035] through

[0092] above, some advantages of depositing one or more coating layers on essentially dry lipid emulsion microparticles include, but are not limited to, the ability of the coating layers to dissolve slowly or at a predetermined rate when the microparticles are administered to a subject, thereby enabling temporal control of the release of the particle contents (e.g., one or more drugs). The release time can be controlled by adjusting the composition of the coating layer applied to the immunogenic agent-containing glassy microparticles as well as the number and / or thickness of the molecular layers. In some embodiments, about 10, about 20, to about 200 or more coating or sequestering layers can be used to form the coated or sequestered microparticles of the present disclosure. In some embodiments, release of the antigen or drug from the core of the coated or sequestered immunogenic agent-containing particle can occur within a few hours to about 1 day, about 7 days, about 30 days, about 60 days, about 90 days, or about 120 days after administration to a subject. In some embodiments, release of the coated or sequestered antigen or agent from the coated microparticles can occur from about 10 days to about 90 days after administration to a subject. In other embodiments, release of one or more innermost sequestered or coated antigens or agents from the microparticles can occur from about 30 days to about 90 days after administration to a subject. In some embodiments, release of the innermost antigen and / or agent can occur from about 30 days to about 120 days, or from about 30 days to about 90 days, or from about 30 days to about 90 days, or from about 30 days to about 60 days after administration to a subject. In some embodiments, release of the innermost antigen and / or agent can occur from about 10 days to about 21 days after administration to a subject. In some embodiments, release of the innermost antigen and / or agent can occur from about 14 days to about 21 days after administration to a subject. Furthermore, in some embodiments, release of the innermost antigen and / or agent can occur from about 18 days to about 21 days after administration to a subject.

[0085]

[0094] In certain embodiments, in addition to the above paragraphs

[0035] through

[0093] , the particle size (e.g., microparticles, nanoparticles, etc.) of one or more encapsulated lipid emulsion drug- or antigen- or other therapeutic agent-containing particles or stabilized nanoemulsions of lipid-containing agents is about 0.1 μm to about 10 μm, or about 1.0 μm to about 5.0 μm. In other embodiments, encapsulated antigen- and / or drug-containing microparticles having multiple layers are less than about 5.0 μm in size. It is recognized that the elements of the antigen- and / or drug-containing glassy microparticles, coating layer, and any additional layers can be provided at concentrations capable of providing an appropriate dose of antigen and / or drug while maintaining an appropriate particle size for ease and consistency of delivery to a subject.

[0086]

[0095] In certain embodiments, in addition to paragraphs

[0035] through

[0094] above, one advantage of using one or more aluminum-based materials as a coating or isolation layer is that the aluminum-based material can also act as an adjuvant, but can also be used at reduced concentrations to reduce the need for co-adjuvants. According to these embodiments, the aluminum-based coating layer isolating or surrounding the microparticles exposes essentially the same surface chemistry to immune-activated cells as does standard aluminum-based adjuvant particles known in the art. In some embodiments, the nanoscale aluminum-based coating layer deposited on the lipid emulsion drug-containing glassy microparticles disclosed herein can be significantly thinner than that found in conventional vaccine particles, and thus the total amount of aluminum per dose can be essentially negligible, improving the safety of these drugs and reducing side effects. In certain embodiments, the aluminum-based coating layer can be sufficiently thin so that the total aluminum concentration per administration of the composition to a subject is less than about 100 μg, less than about 20 μg, less than about 10 μg, less than about 5 μg, or less than about 1 μg, or even less.

[0087]

[0096] In some embodiments, in addition to the above paragraphs

[0035] through

[0095] , coating layers other than aluminum-based coating layers can be used to coat or sequester antigen- and / or drug-containing microparticles. According to these embodiments, non-aluminum coating layers, including but not limited to silicon dioxide (SiO2), titanium dioxide (TiO2), zinc dioxide (ZnO2), and / or silicon nitride (Si3N4), may be used in combination with or alone to the exclusion of aluminum-based coating layers, or as a mixture or alternating pattern of layers. Layers of different materials can be deposited on lipid emulsion drug-containing glassy microparticles to vary the temporal release of drug from the microparticle core or other layers, with each type of material having a different characteristic dissolution time. In some embodiments, microparticles can be coated with one or more coating layers of one material, followed by one or more layers of a different material. According to these embodiments, the different material may dissolve more slowly than, for example, the aluminum-based coating layer. Using other materials to coat the particles can reduce the number of aluminum-based layers required to provide a given release time, minimizing the amount of aluminum per dose.

[0088]

[0097] In other embodiments, in addition to the above paragraphs

[0035] through

[0096] , one or more coating layers can be deposited on one or more lipid emulsions of lipid-containing drugs, drug-containing, adjuvant-containing, antigen-containing, or other therapeutic-agent-containing particles or stabilized nanoemulsions, for example, by atomic layer deposition (ALD, e.g., any equipment capable of atomic layer deposition can be used). ALD includes thin-film deposition techniques based on the sequential use of gas-phase chemical processes. ALD is considered a type of chemical vapor deposition. In certain methods, most ALD reactions use two chemicals, called precursors. These precursors react with the surface of the material one at a time in a sequential, self-limiting, or directed manner. Thin films can be slowly deposited by repeated exposure to different precursors. The use of ALD to deposit coating layers on immunogenic agent-containing glassy microparticles can be based on sequential, self-limiting reactions, providing angstrom-level layer thickness control and tunable coating layer composition. Examples of ALD procedures useful in the methods disclosed herein for depositing coating or isolation layers on lipid emulsion drug-containing glassy microparticles can now be found in the art to provide stabilized microparticles as discovered and disclosed herein.

[0089]

[0098] In certain embodiments, in addition to the above paragraphs

[0035] through

[0097] , the ALD method may be optimized for specific situations or conditions. For example, one or more lipid emulsion drug-, antigen-, or other therapeutic agent-containing particles or antigens against pathogenic organisms incorporated into stabilized lipid-drug nanoemulsions may have varying thermal stabilities and, therefore, may not be suitable for higher ALD temperatures due to this vulnerability. In certain embodiments, molecular deposition may occur at a temperature at which at least one drug in the microparticles remains stable. In some embodiments, molecular deposition may occur under vacuum conditions. By performing molecular deposition under vacuum conditions, the coating layer can be applied at a lower temperature, thereby reducing the adverse effects of high temperatures on the targeted immunogenic agent. In certain embodiments, the vacuum required for deposition may be minimal. In some embodiments, the ALD process can be performed under a moderate vacuum of approximately 0.1 atmospheres. In other embodiments, ALD may also include the incorporation of a magnetically coupled powder mixing device, which can provide uniform distribution of powders and reactants within the ALD reactor, resulting in shorter cycle times for material deposition.

[0090]

[0099] In addition to the above paragraphs

[0035] to

[0098] , embodiments of the present disclosure provide thermostable antigen-containing and / or drug-containing lipid emulsion-containing microparticles and thermostable compositions, where the thermostable immunogenic compositions can be produced by formulating the immunogenic agent-containing particles into a pharmaceutical composition. In certain embodiments, these compositions can be used as vaccines.

[0091]

[0100] In one embodiment, further to paragraphs

[0035] through

[0099] above, a single-dose composition can be produced, which may include thermostable lipid emulsion drug-containing microparticles that provide both a prime dose and a boost dose of one or more drugs against pathogens or for other health conditions. In some embodiments, the prime dose and the boost dose can be contained in the same one or more lipid emulsion drug-containing, antigen-containing, or other therapeutic agent-containing particles or stabilized nanoemulsions of lipid-containing drug particles. In other embodiments, the prime dose and the boost dose can be contained in separate microparticles. According to these embodiments, the prime dose and the boost dose, whether together or separately, are encased in a coating layer for delivery to a subject. In certain embodiments, the prime and boost doses of antigen or drug may form the core of a drug-containing microparticle, or the prime and boost doses of antigen or drug may form separate layers of one or more lipid emulsion drug-containing or antigen-containing or other therapeutic agent-containing particles or stabilized nanoemulsions of drug-containing lipid emulsions, each layer surrounded by one or more layers of coating or isolating material.

[0092]

[0101] In some embodiments, in addition to the above paragraphs

[0035] to

[0100] , at least a second outer layer having at least a second drug (e.g., in an aqueous or gelatin solution) can be adsorbed or layered on the outermost coating layer surrounding the drug-containing microparticles containing the first drug. In certain embodiments, the drug-containing microparticles can be suspended in a solution of a second glass-forming agent and a second antigen, and optionally a lubricating excipient, and then freeze-dried or spray-dried to produce drug-containing microparticles containing an innermost or central first antigen and an outer second antigen. In certain embodiments, the first and second drugs and the first and second glass-forming agents can be the same drug or different drugs.

[0093]

[0102] In certain embodiments, in addition to the above paragraphs

[0035] through

[0101] , the multilayer lipid emulsion drug-containing microparticles disclosed herein can be reconstituted with water, an aqueous buffer composition, or other pharmaceutically acceptable solution to form, for example, an immunogenic composition or formulation, which can then be administered to a subject. In one embodiment, a second outer layer carrying at least a second immunogenic agent can act as a conventional prime dose. Subsequently, after a predetermined number of days (which can be adjusted by manipulating the number and / or thickness of the applied coating layers), a sufficient amount of the coating dissolves or degrades, allowing the release of a second dose (e.g., boost) from the particle core, which acts to boost or supplement the prime dose response in the subject.

[0094]

[0103] In other embodiments, further to paragraphs

[0035] through

[0102] above, a prime dose (e.g., a first antigen) and a boost dose of a drug against a pathogen or other target may be contained in separate drug-containing microparticles. According to these embodiments, the prime dose of drug may be contained in a first thermostable lipid emulsion drug-containing microparticle (coated with one or more coating layers) or in a drug-containing microparticle (uncoated). The boost dose of drug may be contained in a second thermostable drug-containing microparticle coated with one or more coating layers as described herein.

[0095]

[0104] As described herein, in addition to paragraphs

[0035] through

[0103] above, lipid emulsion drug-containing microparticles having an organometallic-based coating layer reduce incompatibility between two or more different drugs, whether during storage or after administration to a subject. In other embodiments, lipid emulsion drug-containing particles may contain two or more different drugs (e.g., two or more different antigens) within the same microparticle. In these embodiments, the drug-containing microparticles are protected against incompatibility between two or more different drugs due to their stabilization within the coated glassy matrix and, in certain embodiments, due to the physical separation of the drugs by one or more coating layers.

[0096]

[0105] Protective immunity (delayed onset of symptoms or reduced severity of symptoms or complete elimination of infection) that enables mammals or other animals to resist infection can result from exposure to pathogen, disease, or toxin antigens that otherwise occur after contact with the pathogen. Protective immunity is achieved through one or more of the following mechanisms: mucosal immunity, humoral immunity, or cell-mediated immunity. Mucosal immunity is primarily the result of secretory IgA (sIgA) antibodies on mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts. sIgA antibodies are generated after a series of events mediated by antigen-processing cells, B and T lymphocytes, that result in sIgA production by B lymphocytes on the mucosal lining of the body. "Humoral immunity" includes IgG and IgM antibodies in serum. "Cell-mediated immunity" can be achieved through cytotoxic T lymphocytes or delayed-type hypersensitivity involving macrophages and T lymphocytes, as well as other mechanisms involving T cells that do not require antibodies. The primary outcome of protective immunity is the destruction of the pathogen or the inhibition of its ability to replicate.

[0097]

[0106] In addition to the above paragraphs

[0035] through

[0105] , certain embodiments of the present disclosure include methods for eliciting an immune response to a drug in a coated, stabilized microparticle disclosed herein or a combination of drugs in one or more drug-containing microparticles by administering to a subject a composition comprising the microparticles disclosed herein. The composition comprising the microparticles may be administered in a therapeutically effective amount, i.e., an amount sufficient to generate a protective immune response. Generally, immunogenic or vaccine compositions may be administered at active drug dosages ranging from about 0.001 mg to about 20.0 mg of immunogenic drug, or from about 0.01 mg to about 20.0 mg of drug, or from about 0.1 mg to about 10.0 mg of drug. Single or multiple doses may be administered in a single administration composition. When multiple doses of an immunogenic or vaccine composition are administered in a single administration composition, e.g., a prime-boost composition, one of the two doses may be time-controlled for release at a preselected time after administration.

[0098]

[0107] In certain embodiments, in addition to paragraphs

[0035] through

[0106] above, administration of the therapeutic or vaccine compositions disclosed herein can be carried out using any acceptable means (e.g., parenteral, local, or systemic, including, by way of example, oral, intranasal, intravenous, subcutaneous, intradermal, intravaginal, via suppository, intramuscular, and topical administration). In some embodiments, administration of the compositions disclosed herein can be influenced by factors including the natural route of infection by a particular pathogen. The administered dosage(s) can depend on factors including age, health, weight, type of concomitant treatment, if any, exposure, and the nature and type of the specific antigen or agent. The compositions or vaccine compositions disclosed herein can be used in dosage forms such as capsules, solutions, suspensions, or elixirs for oral administration, or in sterile liquid formulations such as solutions or suspensions for parenteral or intranasal use.

[0099] kit

[0108] In addition to paragraphs

[0035] through

[0107] above, other embodiments provide kits for use with the methods (e.g., methods for inducing an immune response in a subject) and compositions described herein. In certain embodiments, the kits may contain one or more microparticles in dehydrated form. In certain embodiments, microparticles may also be provided. Different microparticles and / or immunogenic agent-containing glassy microparticles may be mixed together or provided separately to a subject. In certain embodiments, different microparticles may be mixed together or provided in separate containers for storage and transport. The microparticles may be provided in known or predetermined amounts and / or predetermined ratios such that, when the particles are reconstituted, the result is a composition having a known concentration of agent.

[0100]

[0109] In other embodiments, in addition to paragraphs

[0035] through

[0108] above, kits for use in the compositions and methods described herein are contemplated. The kits may be portable. In certain embodiments, the kits can be used to transport and use in remote locations, such as military installations or remote villages. The thermal stability of the microparticles allows for transportation and storage without the need for a cold chain (e.g., refrigeration). This may also be advantageous in medical facilities, as it reduces costs associated with cryogenic storage.

[0101]

[0110] In other embodiments, in addition to the above paragraphs

[0035] to

[0109] , the kit may include a suitable carrier or diluent suitable for reconstituting the dry thermostable particles. In certain embodiments, the carrier or diluent may be a pharmaceutically acceptable aqueous buffer suitable for injection, including pyrogen-free water, and capable of resisting changes in pH upon addition of inorganic compounds, organic compounds, acids, alkalis, or dilution with solvents or diluents.

[0102]

[0111] In some embodiments, in addition to the above paragraphs

[0035] through

[0110] , the kit may include one or more suitable containers, such as vials, tubes, mini- or microfuge tubes, test tubes, flasks, bottles, syringes, or other containers. If additional components or agents are provided, the kit may contain one or more additional containers into which the agents or components can be placed. The kits herein also typically include a means for containing the microparticles, the pharmaceutically acceptable carrier or diluent, and any other reagent containers in close confinement for commercial sale. Such containers may include injection-molded or blow-molded plastic containers into which the desired vials are retained.

[0103]

[0112] In yet other embodiments, in addition to paragraphs

[0035] through

[0111] above, the kit may include coated or uncoated lipid emulsion drug-containing glass microparticles and compositions, and, optionally, instructions for coating the microparticles. In other embodiments, the kit may include an apparatus for performing such coating on the microparticles. According to these embodiments, the kit enables the production of microparticles useful in immunogenic compositions against one or more pathogens.

[0104]

[0113] It will be appreciated that the embodiments described herein may be applied to pharmaceutical compositions other than immunogenic compositions. For example, small molecule drugs (e.g., anticancer drugs) and biologics may be coated in a manner similar to that disclosed for the microparticles described herein. The coating layer provides a desired level of temporally controlled release for a particular pharmaceutical agent. The coating layer may help reduce exposure to moisture and reduce degradation. These coatings may function to protect water-soluble drug formulations or other moisture-sensitive agents from degradation or dissolution until the desired exposure to the subject after administration. Furthermore, embodiments may be used for non-therapeutic applications. For example, a coating layer may be applied to a diagnostic marker. The coating may allow for delayed release of the marker, allowing sufficient transport / uptake time. This may be beneficial if the marker has a limited half-life. In certain embodiments, the compositions, components, formulations, and immunogenic compositions disclosed herein, or small molecules encapsulated or coated using the layering / coating techniques described herein, may be transported, stored, and administered directly to a subject or to an affected body area of ​​a subject, such as the liver, lymph nodes, stomach, eyes, kidneys, or brain, depending on the ability of the deposited composition to remain in the target area.

[0105] Example

[0114] The materials, methods, and embodiments described herein are further defined in the following examples. Certain embodiments are defined in the examples herein. It is understood that these examples, while illustrating certain specific embodiments, are given by way of illustration only. From the disclosure herein and these examples, one skilled in the art can ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention.

[0106] Example 1 Emulsion Formulation and Manufacturing

[0115] In one illustrative example, an MF59®-like emulsion was prepared and used as a model emulsion for stability testing. MF59® is a representative oil-in-water emulsion adjuvant and has been approved for use in pandemic and seasonal influenza vaccines in many countries. MF59® is safe and well tolerated in humans. Note that the primary component of MF59® is squalene oil, a naturally occurring oil found in humans, plants, and animals. The squalene oil in MF59® is derived from fish oil and is highly purified before use. First, for the aqueous phase, 0.5 g of polysorbate 80 was dissolved in 94 mL of sodium citrate buffer (10 mM, pH 7.0). The oil phase was prepared by dissolving 0.5 g of Span 85 in 5 mL (=4.3 g) of squalene. The oil phase was added to the aqueous phase, and the mixture was pre-emulsified using a high-shear homogenizer (e.g., The VirTis Company) at 5,000 rpm for 5 minutes to obtain a primary oil-in-water emulsion. The primary emulsion was then processed through an Emulsiflex C5 high-pressure homogenizer (e.g., Avestin) for five passes at an operating pressure of 7,000-8,000 psi. After five passes, the emulsion was collected and filter-sterilized using a 0.2 μm polyethersulfone (PES) filter. The filtered emulsion was stored at approximately 2-8°C until use.

[0107] Measurement of emulsion droplet size and charge by dynamic light scattering (DLS) and zeta potential analysis

[0116] In another exemplary method, the hydrodynamic diameter and zeta potential of emulsion droplets were measured using an Anton Paar Litesizer 500 (Graz, Austria). Prior to analysis, emulsion samples were diluted 1:100 with water (e.g., MilliQ®). Dynamic light scattering analysis was performed using backscattering at approximately 175°. The hydrodynamic diameter was the intensity-weighted average hydrodynamic size and was derived from cumulative analysis of the measured correlation curves. The zeta potential was calculated from the electrophoretic mobility using standard equations incorporated into the software used for the analysis.

[0108]

[0117] As described herein, emulsion samples containing approximately 20% w / v polysaccharide (e.g., sucrose) were also frozen with various starting concentrations of a representative salt (e.g., sodium citrate). In this example, at a sodium citrate concentration of 80.0 mM, there was a slight increase in emulsion droplet diameter after freezing, while samples containing 160.0 mM sodium citrate experienced a larger increase in droplet diameter (Figure 2). Because damage is caused by at least partial ice crystallization, an alternative drying technique, spray drying, was performed to isolate the effect of solute concentration during freezing. During spray drying, solutes become increasingly concentrated as the atomized spray-dried particles dry, avoiding the freezing step that typically introduces ice crystallization and side effects. The powders formed by spray drying had a residual moisture content of less than 1% (Figure 1, Table). Higher salt concentrations destabilized emulsions during spray drying, with starting salt concentrations of 80.0 and 160.0 mM resulting in an increase in emulsion droplet diameter as the instability caused droplet coalescence. Spray-dried emulsion samples with 10.0 mM, 20.0 mM, and 40.0 mM sodium citrate all retained emulsion droplet diameters of approximately 250 nm after reconstitution, while samples with 80 mM and 160.0 mM sodium citrate had emulsion droplet diameters of 300 nm and 360 nm, respectively. Surprisingly, in contrast to the dramatic destabilization observed during freeze-drying-based drying with a freezing step, the destabilizing effect of increased salt concentration induced by drying was reduced or avoided when spray drying was used to achieve drying without a freezing step.

[0109]

[0118] Figure 1, Table 1. Represents the moisture content of spray-dried powders formed by spray drying a representative squalene-based lipid adjuvant nanoemulsion to form solutions containing various amounts of sucrose.

[0110]

[0119] Figure 2 is a plot showing the effect of sodium citrate concentration on droplet diameter for emulsion samples that were frozen (black bars) or spray-dried (white bars). The emulsion concentration was 1.25% v / v squalene and 20% w / v sucrose in the presence of increasing salt concentrations (e.g., sodium citrate). Error bars shown are the standard deviation from the mean of three replicates. i = diameter before freeze or spray drying, D f = diameter after freeze or spray drying.

[0111] Example 2 Emulsion destabilization during drying of squalene adjuvant emulsions to powder formation.

[0120] In another exemplary method, another problem during freezing is analyzed, and the effect of spray drying under various conditions on lipid emulsion composition is evaluated.In addition to the damage caused by crystallization, another stress occurs during freezing, and it is solute concentration.One of the solutes in emulsion formulation is salt (for example, sodium citrate salt).The increase of salt, such as sodium citrate concentration during freezing, can shield the charge between emulsion droplets, resulting in a lower zeta potential value, which then promotes particle-particle interaction between droplets, causing adverse effects on drug-containing lipid emulsion.

[0112]

[0121] To determine how increasing salt concentration affects zeta potential values, emulsion samples were prepared using various concentrations of sodium citrate salt at pH 7.0. Because the maximum concentration of a polysaccharide (e.g., sucrose) is approximately 80% w / v at its glass transition temperature, the starting polysaccharide concentration (e.g., sucrose) dictates the degree of concentration reached by the remaining solute. Therefore, the salt concentrations investigated represent the maximum concentrations of salt (e.g., sodium citrate) that will be present in the freeze concentrate for starting polysaccharide (e.g., sucrose) concentrations of 1.25, 2.5, 5, 10, and 20% w / v. Zeta potential values ​​decreased in magnitude (Figure 3A) in correlation with increasing salt (e.g., sodium citrate) concentrations, indicating that charge screening in concentrated salt solutions reduces electrostatic repulsion between emulsified lipid-based adjuvant nanodroplets (e.g., squalene). For samples with salt (e.g., sodium citrate) concentrations up to 320 mM but below 640 mM (Figure 3B), there was no dependence on zeta potential and diameter, which were all approximately 170 nm. At a starting sucrose concentration of 1.25% w / v, the degree of concentration of the system to reach the 80% w / v disaccharide (e.g., sucrose) threshold at the glass transition temperature was approximately 64-fold, thus resulting in a maximum salt concentration of approximately 640 mM salt (e.g., sodium citrate) in the freeze-concentrated liquid. For samples formulated with the maximum tested concentration of 640 mM salt (e.g., sodium citrate), creaming or turbidity of the emulsion was visible almost immediately after formulation, with an increase in hydrodynamic diameter of up to 240 nm (Figure 3B).

[0113]

[0122] Figures 3A-3B show the effect of salt (e.g., sodium citrate) concentration in an aqueous liquid emulsion of an exemplary lipid emulsion of a squalene-based adjuvant on 3A) zeta potential and 3B) emulsion droplet diameter. The emulsion concentration was 1.25% v / v squalene. Error bars shown are the standard deviation from the mean of three replicates.

[0114]

[0123] Figure 4 shows the droplet size distribution of an exemplary lipid emulsion (squalene-based adjuvant emulsion) in uncoated, reconstituted spray-dried powder compared to the droplet size distribution measured in a sample that had been spray-dried, coated with approximately 100 layers of trimethylalumina using ALD, and then suspended in a buffer (e.g., EDTA / His) to remove the coating and resuspend the emulsion droplets. All samples were subjected to brief centrifugation, which pelleted any undissolved alumina coating fragments. Samples taken from the supernatant after centrifugation were analyzed by dynamic light scattering to determine the average droplet size. The droplet size distribution for the emulsion in the powder with 100 layers of alumina applied by atomic layer deposition was indistinguishable from that in the powder without the coating, demonstrating the effectiveness of these methods without altering microparticle droplet size.

[0115] Example 3 Stability of emulsified squalene-based adjuvants in spray-dried powders coated with atomic layer deposited alumina

[0124] In another exemplary method, a lipid emulsion (e.g., a squalene-based lipid droplet emulsion) was spray-dried from a solution containing exemplary concentrations of a representative disaccharide and salt (e.g., 20% sucrose, 20 mM sodium citrate). Analysis of the powder formed by spray drying indicated a water content of less than approximately 1.0%. The sample was divided into two aliquots. One aliquot of the powder was retained and poured into a glass vial, which was evacuated to 60 mTorr, backfilled with dry nitrogen to atmospheric pressure, and sealed. For the "coated" sample, the powder was placed in a custom-built ALD fluidized-bed reactor, and 100 molecular layers of alumina were applied to the surface of the remaining aliquot sample using 100 cycles of ALD with alternating injections of water vapor and trimethylaluminum gas.

[0116]

[0125] To test whether the droplet size within an exemplary MF-59-like emulsion changed during the ALD application of alumina to the surface of the powder, the alumina-coated powder was suspended in 30 mM EDTA, 5 mM His, pH 6, to dissolve a sufficient amount of the alumina coating to allow the interior contents of the coated powder to be released into solution.

[0117]

[0126] A brief centrifugation of the suspension allowed the remaining alumina shell coating the powder to settle. A sample of the supernatant, containing the released inner contents of the powder, was examined by dynamic light scattering to determine the size distribution of the released exemplary MF59®-like emulsion. The size distribution of the emulsion droplets was compared to that of droplets from reconstituted powder that had not been coated by atomic layer deposition. As shown in Figure 4, the droplet size distribution did not change before or after ALD of 100 molecular layers of alumina.

[0118] Example 4 Polysaccharide concentration for stabilizing squalene-based lipid emulsions

[0127] Figure 5 shows the size distribution of emulsified squalene after reconstitution of the spray-dried powder. Maintaining a droplet size of less than 300 nm requires that the polysaccharide (e.g., sucrose) concentration in the sprayed liquid be at least about 10%, or at least about 20% or more, but less than 40% w / v.

[0119]

[0128] FIG. 6 shows optimal retention of the initial lipid emulsion size distribution after spray drying and reconstitution, demonstrated to be enhanced by the addition of an additional glass former (e.g., hydroxyethyl starch) to the solution being spray dried.

[0120]

[0129] FIG. 7 shows that optimal retention of the initial lipid emulsion size distribution after spray drying and reconstitution requires an ionic strength of less than about 100 mmole / L (e.g., 20 mM sodium citrate) to avoid coalescence during spray drying.

[0121] All compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods have been described with reference to embodiments, it will be apparent to those skilled in the art that modifications can be made to the compositions and methods described herein and to the steps or sequence of steps of the methods without departing from the concept, spirit, and scope of the invention. More specifically, certain agents that are both chemically and physiologically related can be substituted for the agents described herein while achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept as defined by the appended claims.

Claims

1. a central or innermost antigen- or drug-containing lipid emulsion-containing glassy microparticle comprising at least one drug, at least one antigen, or a combination thereof in a lipid emulsion, and at least one glass-forming agent; and one or more coating layers comprising one or more of an organometallic material, a metal oxide, a metal alkoxide, and / or an aluminum-based coating layer coating the central or innermost antigen- or drug-containing lipid emulsion-containing glassy microparticle.

2. The microparticle of claim 1 , wherein the glass former comprises at least one polysaccharide.

3. 3. The microparticle of claim 2, wherein the at least one polysaccharide to lipid concentration of the lipid emulsion comprises a ratio sufficient to stabilize the lipid emulsion during spray drying, optionally a ratio of greater than 5:1, greater than 6:1, greater than 7:1, or at least 8:1 polysaccharide to lipid.

4. The microparticle of any one of claims 1 to 3, further comprising at least one additional glass former or polysaccharide.

5. 5. The microparticle of claim 4, wherein the at least one additional glass former or polysaccharide comprises at least one of hydroxyethyl starch, dextran, carboxymethyl cellulose, or viastarch, or the like.

6. The microparticle of any one of claims 1 to 5, wherein the at least one drug or at least one antigen comprises a drug or antigen derived from a pathogenic organism.

7. 7. The microparticle of claim 6, wherein the at least one agent or the at least one antigen comprises a polynucleotide, a polypeptide, a recombinant protein; a virus-like particle; a live virus; a live attenuated virus; an inactivated virus; a toxoid; or a combination thereof.

8. 8. The microparticle of claim 1, wherein the at least one agent or the at least one antigen comprises one or more antigens derived from a pathogenic virus, a pathogenic bacterium, a pathogenic prion, a pathogenic fungus, a pathogenic protozoan, a helminth, a pathogenic yeast, or other pathogen.

9. 2. The microparticle of claim 1, wherein the at least one agent or the at least one antigen comprises at least one antigen or at least one agent derived from a pathogen in Category A, Category B, or Category C of the National Institute of Allergy and Infectious Diseases (NIAID) list.

10. The at least one agent or the at least one antigen may be selected from the group consisting of Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague), Variola major (smallpox), and other related poxviruses, Francisella tularensis (Francisella tularensis), tularensis; tularensis), viral hemorrhagic fever, arenavirus, Junin, Machupo, Guanarito, Chafa, Lassa, Lujo, Bunyavirus, Hantavirus causing Hantaan pulmonary syndrome, Rift Valley fever, Crimean-Congo hemorrhagic fever, flavivirus alphavirus, and filoviruses such as Ebola virus, Sudan virus, and Marburg virus, or other filoviruses, or combinations thereof.

11. The at least one antigen is derived from at least one pathogen, and the at least one pathogen is selected from the group consisting of human papillomavirus (HPV), Ebola virus, Marburg virus, poliovirus, norovirus, rotavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, varicella-zoster virus, herpes simplex virus, cytomegalovirus, dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, Zika virus, Haemophilus influenzae type b virus, measles virus, mumps virus, rubella virus, respiratory syncytial virus, influenza virus, rabies virus, smallpox virus, parvovirus, chikungunya virus, Corynebacterium diptheriae, Clostridium tetani, and the like. tetani, Clostridium botulinum, Bordetella pertussis, Streptococcus pneumoniae, Neisseria meningitides, Salmonella spp., Bacillus anthracis, Yersinia spp., or a combination thereof.

12. The at least one antigen is derived from at least one pathogen, including a pathogen that infects companion animals or other non-human animals, such as canine parvovirus, canine distemper virus, canine adenovirus, rabies virus, canine parainfluenza virus, canine influenza virus, canine coronavirus, West Nile virus, Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), chikungunya virus, equine influenza virus, equine herpesvirus, Streptococcus equi equi, Clostridium tetani, Neorickettsia risticii, risticii), Clostridium tetani, measles virus, Bordetella bronchiseptica, Leptospira spp., Borrelia burgdorferi, feline herpesvirus type 1, feline calicivirus, feline panleukopenia virus, rabies virus, feline leukemia virus, feline immunodeficiency virus, virulent systemic feline calicivirus, Chlamydia felis, Pasteurella haemolytica, Bordetella bronchiseptica 10. The microparticle of claim 8, comprising at least one of: a hydroxybenzoate; ...

13. The at least one antigen is derived from at least one pathogen, and the at least one pathogen is selected from the group consisting of bovine herpesvirus, parainfluenza virus type 3, bovine viral diarrhea virus, bovine respiratory syncytial virus, Clostridium chauvoei, Clostridium septicum, Clostridium novyi, Clostridium perfringens type C, Clostridium perfringens type D, Pasteurella haemolytica, and Clostridium haemolyticum.

10. The microparticle of claim 8, comprising a pathogen that infects livestock, comprising at least one of: S. haemolyticum; Chikungunya virus; or a combination thereof.

14. 9. The microparticle of claim 8, wherein the at least one antigen is derived from at least one pathogen, wherein the at least one pathogen comprises a pathogen that infects birds, including at least one of Marek's disease virus, reovirus, avian encephalomyelitis virus, avian influenza virus, avipoxvirus, chicken anemia virus, Pasteurella multocida, Newcastle disease virus, Riemerella anatipestifer, duck herpesvirus 1, duck hepatitis virus, or a combination thereof.

15. The at least one antigen is derived from at least one pathogen, and the at least one pathogen is selected from the group consisting of Cryptococcus spp., Aspergillus spp., Blastomyces spp., Candida albicans, Paracoccidioides spp., Sporothrix spp., Histoplasma capsulatum, Pneumocystis jirovecii, and the like.

9. The microparticle of claim 8, comprising at least one of: Bacillus subtilis, Bacillus jirovecii, ...

16. The microparticle of any one of claims 1 to 15, wherein the at least one agent comprises a multimeric protein complex.

17. The microparticle of any one of claims 1 to 16, wherein the antigen or agent is derived from a virus, the virus comprising an enveloped or non-enveloped virus.

18. 17. The microparticle of any one of claims 1 to 16, wherein the antigen or drug is derived from a virus, the virus comprising a positive or negative strand RNA virus, a linear or circular double-stranded or single-stranded DNA virus, or an RNA / DNA hybrid virus, or an mRNA encoding a virus or viral antigen, or an mRNA encoding a therapeutic protein, a cellular receptor protein, an enzyme, a structural protein, a cytokine, or a combination thereof.

19. 19. The microparticle of any one of claims 1 to 18, wherein the at least one glass forming agent comprises at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol and glycine, hydroxyethyl starch, polyvinylpyrrolidone, glycine, cyclodextrin, povidone, and combinations thereof.

20. The microparticle of any one of claims 1 to 18, wherein the at least one glass forming agent comprises sucrose or trehalose.

21. 21. The microparticle of any one of claims 1 to 20, further comprising at least one salt, optionally wherein the at least one salt comprises sodium citrate, sodium acetate, sodium succinate, potassium succinate, sodium phosphate, and potassium phosphate.

22. Each of the one or more coating layers is aluminum oxide (Al ２ O ３ ), aluminum alkoxide, silicon dioxide (SiO ２ ), zinc dioxide (ZnO ２ ), titanium dioxide (TiO ２ ), and silicon nitride (Si ３ N ４ 22. The microparticle of any one of claims 1 to 21, comprising one or more of:

23. 23. The microparticle of any one of claims 1 to 22, wherein each layer of the one or more coating layers has a thickness of about 0.1 nm to about 20.0 nm.

24. 24. The microparticle of any one of claims 1 to 23, further comprising at least a second antigen or at least a second drug deposited as a layer on the outer coating layer of the ALD-coated microparticle and separated from the inner core by one or more coating layers.

25. 25. The microparticle of claim 24, wherein the at least second antigen or drug is the same as or different from the at least one drug or at least one antigen in the central or innermost core of the coated microparticle.

26. a) combining at least one drug or at least one antigen or a combination thereof in a lipid emulsion with at least one glass-forming agent; b) spray drying a) to create essentially dry drug- or antigen-containing lipid emulsion-containing microparticles to produce a central or innermost antigen- or drug-containing lipid emulsion; c) coating by ALD the essentially dry drug- or antigen-containing lipid emulsion-containing microparticles of b) with one or more coating layers comprising one or more of organometallic materials, metal oxides, metal alkoxides, and / or aluminum-based coating layers encasing the central or innermost essentially dry drug- or antigen-containing lipid emulsion-containing microparticles.

27. 27. The method of claim 26, wherein the at least one agent or at least one antigen comprises at least one agent or at least one antigen derived from a pathogen.

28. 28. The method of claim 26 or 27, wherein the at least one agent or at least one antigen comprises a recombinant peptide, a polynucleotide, a polypeptide, a recombinant protein, a virus-like particle, a live virus, a live attenuated virus, an inactivated virus, a toxoid, a pathogenic virus, a pathogenic bacterium, a pathogenic prion, a pathogenic fungus, a pathogenic protozoan, or a combination thereof.

29. 29. The method of any one of claims 26 to 28, wherein the at least one glass former comprises an aqueous composition concentration of about 1.0% to about 25.0% (w / v).

30. 30. The method of any one of claims 26 to 29, wherein the ratio of polysaccharide to lipid emulsion agent concentration is at least 5:1, or at least 6:1, or at least 7:1, or at least 8:1, or at least 9:1, or at least 10:

1.

31. 26. The microparticle-containing composition of any one of claims 1 to 25, comprising a plurality of microparticles of any one of claims 1 to 25 or a combination thereof.

32. 32. The microparticle-containing composition of claim 31, further comprising at least one pharmaceutically acceptable excipient.

33. 33. The microparticle-containing composition of claim 31 or claim 32, wherein the microparticle-containing composition is a single-dose composition comprising a prime dose and a boost dose of at least one antigen derived from a pathogen or at least one pathogen-encoding mRNA or fragment thereof.

34. 34. A method for treating, preventing, or reducing the risk of developing a health condition in a subject, comprising administering to the subject a composition of claim 32 or 33.

35. 35. The method of claim 34, wherein preventing or reducing the risk of onset is prophylactic or therapeutic.

36. A kit comprising at least one microparticle of any one of claims 1 to 25 or 31 to 33 and at least one container.

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