Suspension of atomic layer deposition (ALD) coated therapeutic agent-containing particles in non-aqueous suspension agents

Abstract

Embodiments of the present disclosure provide novel compositions and methods for suspending atomic layer deposition (ALD) coated microparticles. In certain embodiments, compositions disclosed herein include non-aqueous suspension agents for suspending ALD-coated therapeutic agent-containing microparticles or powders. In certain embodiments, therapeutic agent-containing formulations can be spray-dried and further embedded within glassy matrices or microparticles and coated by ALD and these ALD-coated microparticles suspended in non-aqueous suspension agents for improved stability and ease of transfer to suitable packaging and ready delivery including, but not limited to, pre-packaging for improved stability in a ready-to-use formulation, and / or for storage, and / or for stable transport and administration to a subject.

Description

SUSPENSION OF ATOMIC LAYER DEPOSITION (ALD) COATED THERAPEUTIC AGENT-CONTAINING PARTICLES IN NON-AQUEOUS SUSPENSION AGENTSPRIORITY

[0001] This International Application claims priority to U.S. Provisional Application No. 63 / 666,088 filed June 28,2024 and U.S. Provisional Application No. 63 / 666,084 filed June 28, 2024. These applications are incorporated herein by reference in their entireties for all purposes. FIELD

[0002] Embodiments of the present disclosure provide novel compositions and methods for suspending atomic layer deposition (ALD) coated particles. In certain embodiments, compositions disclosed herein include non-aqueous suspension agents for suspending ALD- coated therapeutic agent-containing microparticles, particles or powders. In certain embodiments, therapeutic agent-containing formulations can be spray-dried embedding these therapeutic agents within glassy matrices or microparticles in preparation for ALD coating; these coated microparticles can be suspended in non-aqueous suspension agents for ease and reliability of transfer to suitable packaging including, but not limited to, pre-packaging for improved stability and ready delivery to a subject.BACKGROUND

[0003] ALD-coated or metal agent-coated therapeutic agent-containing dry microparticles or powders offer numerous advantages for vaccine formulations and other therapy deliveries, including thermal stability and controlled release of therapeutic agents in vivo. One drawback of these formulations is that the powders and essentially dry microparticles can be cumbersome to accurately fill in vials, syringes, and other containers where transfer is difficult, and product can be lost in the transfer. In addition, these powders and microparticles conventionally have been suspended in aqueous media prior to their administration by injection. ALD-coated powders can exhibit instabilities if suspended in aqueous media. Therefore, aqueous suspensions of metal oxide-coated therapeutic agent-containing essentially dry microparticles or powders for injection must be prepared immediately prior to injection to reduce these effects. Therefore, it is desirable to find improved formulations and protections for storage and delivery of ALD-coated therapeutic agent-containing microparticles or powders.SUMMARY

[0004] Embodiments of the present disclosure provide novel compositions and methods for suspension, storage, and use of ALD-coated therapeutic agent-containing microparticles orpowders for improved stability, ease of filling into containers, ease of storage and ready -use applications in therapeutic settings. In certain embodiments, compositions and methods are disclosed for suspending ALD-coated therapeutic agent-containing microparticles or powders in one or more non-aqueous liquids suitable for parenteral administration, ease of transfer to storage containers and delivery devices, and suitable for generating pre-filled syringes or other administration devices for ease of administration or storing for later use as a stable suspension. In certain embodiments, these ALD-coated microparticle-containing formulations can be stored for prolonged periods or be reliably transported for immediate or later use without the risk of degradation of coating layers and without the risk of damage or premature release of a sequestered therapeutic agent contained within the coating layers. In other embodiments, these formulations disclosed herein can be stored at temperatures to reduce or eliminate particle or microparticle settling where a temperature for storage is below a nonaqueous suspending agent’s solidification temperature (e.g., a temperature below -6 °C for ultrarefined sesame oil, or a temperature below 18 °C for benzyl benzoate).

[0005] In certain embodiments and further to paragraph

[0004] above, one or more nonaqueous agents or suspending agents can include, but are not limited to, benzyl benzoate, 2- hydroxybenzoate (salicylate) esters, polyethylene glycols, polyoxyl 15 hydroxystearate glycerin, sorbitan monolaurate, refined sesame oil, refined soybean oil, refined corn oil, refined cottonseed oil, refined peanut oil, other refined triglyceride mixtures or oils and the like for suspending ALD-coated therapeutic agent-containing microparticles or powders disclosed herein. In some embodiments, one or more non-aqueous agents or suspending agents can include, but are not limited to, benzyl benzoate, or mixtures thereof for suspending ALD-coated therapeutic agent-containing microparticles or powders disclosed herein. In yet other embodiments, refined triglyceride mixture of use as suspension agents can include, but are not limited to, sorbitan monolaurate.

[0006] In certain embodiments and further to paragraphs

[0004] -

[0005] above, ALD- coated therapeutic agent-containing microparticles disclosed herein can be suspended in nonaqueous suspending agents. In accordance with these embodiments, coating of the one or more coating layers can include one or more of a metal agent, metal oxides, metal alkoxides, aluminum-based and / or other ALD-compatible coating agent or mixtures or alternating layers thereof, for layering over a microparticle disclosed herein. In certain embodiments, the ALD coating layer can include, but is not limited to, a composition including, one or more of aluminum oxide (AI2O3), an aluminum alkoxide, silicon dioxide (SiCL), titanium dioxide (TiCh), gallium oxide (Ga2Os), silicon nitride (SisN^, zinc oxide (ZnO), zirconium dioxide(ZrCL), zirconium silicate or zircon (ZrSiCU) and hafnium oxide (HfCL) and combinations thereof. In certain embodiments, the coating layer can include, but is not limited to mixtures of two or more of aluminum oxide (AI2O3), an aluminum alkoxide, silicon dioxide (SiCL), titanium dioxide (TiCh), gallium oxide (Ga2Os), silicon nitride (SisN^, zinc oxide (ZnO), zirconium dioxide (ZrCh), zirconium silicate or zircon (ZrSiC ) and hafnium oxide (HfCL) and combinations thereof in the same, alternating, or other pattern of layering such as 1 :2, 1 :3, 1 :4, 1 :5 or other pattern (e.g., alternating layers of aluminum agents with silicon- or titanium-containing compositions or vice versa, for example). In accordance with these embodiments, these ALD-coated therapeutic agent microparticles can be suspended in one or more non-aqueous suspending agent contemplated herein for improved reliability for transfer, storage, and ready administration. For example, these ALD-coated therapeutic agent microparticles suspended in one or more non-aqueous suspending agents can be directly loaded into an administration device such as an injection device for immediate delivery or for transport and later use with improved stability at room temperature or higher temperature storage (e.g., 25 °C to about 70 °C; or about 40 °C to about 70 °C) for prolonged periods of a few hours up to a few months or up to several months (e.g., 3 months to about 1 year).

[0007] In certain embodiments and further to paragraphs

[0004] -

[0006] above, ALD- coated therapeutic agent-containing microparticles suspended in non-aqueous agents create a stable composition where the ALD-coated therapeutic agent-containing microparticles are preserved within the suspension in an intact form without pre-mature release or leaking of a sequestered therapeutic agent from the ALD-coated therapeutic agent-containing microparticles. In accordance with these embodiments, these suspension compositions provide advantages for preserving intact microparticles with improved methods for reliable therapeutic agent distribution and provide for a more uniformly distributed active agent suspension. In other embodiments, these suspension compositions provide advantages for more reliable delivery of the ALD-coated therapeutic agent-containing microparticles to a subject with reduced product loss and reduced unpredictability of delivery.

[0008] In some embodiments and further to paragraphs

[0004] -

[0007] above, administration-ready formulations of non-aqueous suspending agents with suspended ALD- coated therapeutic agent-containing microparticles disclosed herein can be generated. In accordance with these embodiments, a container, syringe, vial, or other vessel or delivery device can be readily and accurately filled with reliable transfer of the suspended ALD- coated therapeutic agent-containing microparticles to the container, syringe, vial, or other vessel or delivery device. In accordance with these embodiments, these suspensionformulations avoid the issues of transferring a powder or microparticle where product loss from spills or loss due to adherence to a container are reduced or completely avoided; the suspension formulation preserving intact coated particles with improved ease of transfer.

[0009] In certain embodiments and further to paragraphs

[0004] -

[0008] above, nonaqueous suspension agent formulated ALD-coated therapeutic agent-containing microparticles disclosed herein have increased stability at elevated temperatures from about 4 °C up to about 35 °C, up to 40 °C, up to 50 °C, up to 60 °C or up to 70 °C or higher for prolonged periods. In other embodiments, ready -to-administer syringes or other delivery devices can be pre-filled at one location and then delivered to a second location for administration without pre-mature loss or leaking of the therapeutic agent from the ALD coated particles or microparticles.

[0010] In some embodiments and further to paragraphs

[0004] -

[0009] above, the at least one therapeutic agent can include, but is not limited to, at least one antigen, other therapeutic agent, or combination thereof. In accordance with these embodiments, the at least one antigen, other therapeutic agent can include, but is not limited to, a protein or polypeptide, or fragment thereof; a polynucleotide, a nucleic acid sequence or fragment thereof, a lipid assembly-free polynucleotide, nucleic acid sequence or fragment thereof, a small molecule and a recombinant protein. In other embodiments, the at least one antigen, other therapeutic agent can include, but is not limited to, a virus-like particle (VLP); a live virus; a live, attenuated virus; an inactivated virus; a bacterium, a bacteria-derived antigen, a fungus, a prion, a protozoan, a toxoid; and the like. In certain embodiments, the antigen-and / or therapeutic agent includes at least one immunogenic agent.

[0011] In some embodiments and further to paragraphs

[0004] -

[0010] above, the antigen- and / or therapeutic agent and / or immunogenic agent form part of a central or innermost ALD coated microparticle including, but not limited to, at least one therapeutic agent, antigen or immunogenic agent and at least one glass-forming agent, where one or more ALD-applied outer coating layers covering or encasing the central glassy microparticle are added using ALD. In accordance with 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, up to 500, up to 1,000 or more coating layers can encase the central or innermost therapeutic agent. In accordance with these embodiments, the ALD coated therapeutic agent-containing microparticles, powders or glassy matrices’ ALD coating layers are readily dissolvable when administered to a subject, exposing the one or more therapeutic agent or antigen or immunogenic agent to the subject by immediate dissolution of coating layers, delayed release by sequential dissolution of coating layersand / or timed-release depending on type and number of coating layers added.

[0012] Certain embodiments and further to paragraphs

[0004] -

[0011] above, the present disclosure provides methods for making stable administration-ready ALD-coated therapeutic agent-containing microparticle. In accordance with these embodiments, these methods include, but are not limited to, combining ALD-coated therapeutic agent-containing microparticles with at least one non-aqueous suspension agent; and forming a suspension composition.

[0013] In some embodiments and further to paragraphs

[0004] -

[0012] above, the at least one antigen, and / or therapeutic agent can include one or more adjuvant, antigen, and / or other therapeutic agent. In accordance with these embodiments, the at least one antigen can include, but not limited to, 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 antiinflammatory agent, an anti -autoimmune agent, a peptide, polynucleotide, or protein thereof or a combination thereof. In some embodiments, the at least one agent or antigen can include one or more agent(s) or antigen(s) including, but is not limited to, a recombinant peptide, a recombinant protein, a peptide derived from a target protein or pathogen, a polysaccharide derived from a target pathogen, a synthetic peptide or protein, a virus-like particle (VLP), a live virus, a live, attenuated virus, an inactivated virus, an antigen attached to, associated with, or expressed on the surface of a virus or a bacteriophage or a combination thereof. In yet other embodiments, the at least one agent or antigen can include one or more agent(s) or antigen(s) including, but not limited to, a protein, a polypeptide or fragment thereof, polynucleotide, a polypeptide encoded by a polynucleotide or the like. In accordance with these embodiments, a polynucleotide can include, but is not limited to, DNA, RNA, mRNA, siRNA, or a chimeric molecule thereof. In certain embodiments, a chimera can include a combination of at least one polynucleotide segment and at least one polypeptide segment or a mixture of polynucleotide segments and / or polypeptide segments. In some embodiments, the polynucleotide can be mRNA encoding a full length or peptide fragment of a targeted agent (e.g., pathogenic agent such as a virus or bacterium).

[0014] In some embodiments and further to paragraphs

[0004] -

[0013] above provide for single, combination compositions or formulations including a plurality of non-aqueous liquid- suspended ALD coated therapeutic agent-containing microparticles described herein. In accordance with these embodiments, these combination compositions or formulations can include a pharmaceutical composition. In some embodiments, the pharmaceutical composition can further include a pharmaceutically acceptable carrier or excipient. In otherembodiments, these combination compositions or formulations can include mixtures of different ALD coated microparticles containing one or more therapeutic agent for treating or preventing a single health condition, or mixtures of different ALD coated microparticles containing one or more therapeutic agent for treating or preventing multiple health conditions (e.g., pathogenic agent infections or prevention of pathogenic agent infections).

[0015] In some embodiments and further to paragraphs

[0004] -

[0014] above, formulations disclosed herein can be part of a single-administration formulation including a prime dose and at least one boost dose of at least one agent or at least one therapeutic agent sequestered within the ALD-coated microparticles. In accordance with these embodiments, the prime and at least one boost dose of the at least one antigen or at least one therapeutic agent can be in the same coated microparticle, or in separate coated microparticles. When in separate particles, the priming dose of the at least one therapeutic agent or at least one antigen can be sequestered within a population of microparticle while the at least one boost dose can be sequestered in separate microparticles. In certain embodiments, release of the priming dose after delivery to a subject can be immediate or delayed depending on the number of coating layers coating the therapeutic agent-containing or antigen-containing microparticle priming dose (if not on the outermost or near outermost coating layer) and release of the at least one boost can be a short time, days, or several months after the initial dose of agent or antigen containing coated microparticles in the subject. In accordance with these embodiments, these coated microparticles suspended in at least one non-aqueous suspension agent of combination prime and at least one boost doses can be used to create pre-filled delivery devices for administering an accurate, stabilized composition to a subject for accurate prime and boost dose delivery.

[0016] In some embodiments and further to paragraphs

[0004] -

[0015] above, non-aqueous suspension agent formulated ALD-coated compositions can be part of a single administration formulation with coated microparticles containing at least two different therapeutic agents or two different antigens capable of eliciting an immune response or other treatment response to two or more different antigens or agents. In accordance with these embodiments, the two or more different antigens or different agents can be included in the same or separate coated microparticles.

[0017] Other embodiments and further to paragraphs

[0004] -

[0016] above, provide for methods for eliciting a response in a subject, where the method can include administering ALD-coated microparticle-containing non-aqueous suspension agent formulations described herein to the subject. In accordance with these embodiments, the formulation can beadministered by any method known in the art. In other embodiments, these formulations can be administered using a pre-filled syringe for intravenous, intradermal or subcutaneous administration, using an inhalation device, nebulizer, or other delivery device for administering to the subject. In accordance with these methods, a response induced by the composition can be prophylactic or therapeutic depending on the at least one antigen, therapeutic agent, or at least one agent.

[0018] Yet other embodiments and further to paragraphs

[0004] -

[0017] above, provide for kits that can include at least one ALD-coated therapeutic agent- or antigen-containing microparticle suspended in or separately contained from the at least one non-aqueous suspension agent. In some embodiments, the at least one ALD-coated therapeutic agent- or antigen-containing microparticle suspended in at least one non-aqueous suspension agent is contained in an administration ready device (e.g., pre-filled syringe). In certain embodiments, kits can further include at least one container and / or instructions for making or using these formulations disclosed herein. In other embodiments, kits containing the at least one ALD- coated therapeutic agent- or antigen-containing microparticle suspended in at least one nonaqueous suspension agent can be stored at elevated temperatures without refrigeration or frozen or can be stored at temperatures below the suspending agent’s solidification temperature for a predetermined period.BRIEF DESCRIPTION OF THE FIGURES

[0019] The accompanying drawings are incorporated into and form a non-limiting part of the specification to illustrate several examples of the present disclosure.

[0020] FIG. 1 represents an exemplary graph illustrating an example of total antibody response post-immunization using formulations created under various ALD-coated therapeutic agent-containing microparticles suspension conditions according to some embodiments of the present disclosure.

[0021] FIG. 2 represents an exemplary graph illustrating total antibody post-immunization using formulations created under various ALD-coated therapeutic agent-containing microparticles suspension conditions according to some embodiments of the present disclosure.

[0022] FIG. 3 represents an exemplary graph illustrating ELISA assay results of spray dried versus ALD coated microparticles over time post-immunization using formulations created under various suspension conditions according to some embodiments of the present disclosure.

[0023] FIG. 4 represents an exemplary plot of antibody reactions against an exemplary antigen using an ELISA assay representing absorbance versus serum dilution for an animal model injected with therapeutic agent-containing particles prepared by spray drying and ALD coated post immunization using formulations created under various suspension conditions according to some embodiments of the present disclosure.DETAILED DESCRIPTION

[0024] In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, time and other specific details may be modified through routine experimentation. In some embodiments, well known methods or components have not been included in the description.

[0025] Atomic layer deposition (ALD) is a technique that can be used to coat microparticles contemplated herein with nanoscopic layers of metal oxides, metal alkoxides and alumina agents. In one application of ALD, microparticles containing at least one therapeutic agent, vaccine antigen, adjuvant, and / or other vaccine or therapeutic agents can be completely coated with nanoscopic layers of metal oxides such as alumina to create controlled release preparations. The ALD process for creating such coated microparticles begins by formulating the antigens, adjuvants, small molecules, or other therapeutic agent in aqueous formulations that contain glass-forming agents such as polysaccharides. These formulations are spray dried or spray-freeze dried, a process where the aqueous formulation is nebulized to form a mist of microdroplets. In accordance with these embodiments, suspended microdroplets are contacted with dry, warm air, causing them to dry and form solid microparticles with embedded vaccine and / or therapeutic agents. As the droplets dry, the glass transition temperature of the drying mixture is increased until it exceeds the temperature in the spray dryer; at this point the excipients surrounding the vaccine, adjuvant and therapeutic agents form a glass that stabilizes the agents due to the limited molecular mobility allowed within highly viscous glasses. As long as the powders are stored at a temperature that is below the glass transition temperature, thermally labile therapeutic agents are protected from thermal damage, allowing storage and transportation of the powders to occur at higher temperatures, increasing the shelf life of the vaccine or therapeutic preparation, and potentially eliminating the need for exacting “cold chain” conditions that add great expense and logistical challenges in delivering vaccines and / or other therapeutics to subject, especially in remote areas. Importantly, glass transition temperatures of polysaccharides aredramatically affected by the presence of water. Water is a strong plasticizer for polysaccharide glasses, and sorption of as little as a few percents of water into polysaccharide glasses can lower the glass transition temperature to the point where the stabilizing properties of the protective glassy state are lost. Therefore, it was noted that it was important that these therapeutic agents formulated in glassy matrices be kept dry during filling a container, or syringe or other device, storage, and transportation operations prior to their administration to a subject.

[0026] In certain embodiments and further to paragraph

[0025] above, after dry, glassy powders are formed by spray drying of the one or more therapeutic agents, nanoscopic coatings of metal oxides (e.g., alumina) can then be applied to the surface of the microparticles using atomic layer deposition (ALD) techniques. These ALD processes involve repeatedly conducting two sequential reactions where gaseous reagents react with the surface of the microparticles. ALD chemistries for application of various metal oxides, metal alkoxides and / or alumina agents to microparticle surfaces have been described. As previously described, a process for depositing layers of aluminum oxides using repeated two- part, gas-phase reaction cycles that each deposit a molecular layer of alumina is used to form coated microparticles. In addition, in another example, particles formed by spray drying can be introduced into a fluidized bed reactor. Under vacuum, precise quantities of trimethylaluminum gas can be first introduced to the reactor. The trimethylaluminum reacts quantitatively with hydroxyl groups present on the surface of the essentially dried particles (e.g., hydroxyls on the polysaccharides that form the particle surface). Subsequent addition of a second reactive gas, water vapor, releases methane and leaves the surface coated with a molecular layer of alumina, which offers a new layer of exposed hydroxyl groups for coating. These cycles can then be repeated as often as desired to produce microparticles with nanoscopic aluminum oxide layers of any arbitrary thickness having pre-determined coating layers.

[0027] In certain embodiments and further to paragraphs

[0025] to

[0026] above, when the ALD-coated microparticles are exposed to water, (e.g., when administered in vivo), the nanoscopic coating erodes over a period depending on layers and releases the antigen or other embedded therapeutic agent. Depending on the thickness of the applied metal oxide coating, this erosion can over the course of days to months, release the content of microparticle core, including vaccine, adjuvant, therapeutic agent or any other cargo contained within the microparticle. A release profile can be tailored by modifying the number of molecular layers of metal oxide added to the microparticles, providing delayed release that can be near zeroorder, sustained release or delayed, pulsatile release, or combinations thereof. For precision- controlled release of the therapeutic agent from the coated microparticles, it is important that the release not be prematurely initiated, e.g., by exposure to aqueous solutions during filling, storage, or transportation. Premature release can lead to inaccurate delivery of the agents and poorer outcomes.

[0028] It is known in the art that parenteral administration of controlled-release vaccine or other therapeutic agent-containing formulation of ALD-coated microparticles have been achieved by suspending the particles in aqueous solutions (e.g., water for injection or aqueous disaccharide solutions) and using conventional needle-and-syringe technology to inject the resulting suspension. Some drawbacks to this approach are that the aqueous suspensions must be prepared immediately before injection to avoid particle settling, particle sintering, and premature release of microparticle cargo (e.g., therapeutic agent) prior to or prematurely after administration. This state-of-the-art requirement of immediate preparation of aqueous suspensions before injection precludes for example, use of advanced preparation such as high-speed liquid filling operations during the preparation of drug product in containers such as vials and prefilled syringes; and instead requires that the ALD-coated microparticles be filled when in a powder state. These requirements increase cost, reduce stability of a resulting aqueous suspension leading to loss of product by degradation as well as loss of powder during difficult transfers to a vial, syringe, or other delivery container.

[0029] As disclosed herein and further to paragraphs

[0025] to

[0028] , certain non-aqueous liquid suspending agents can be used in place of aqueous suspending solutions to avoid the issues compounded by powder transfers and aqueous suspensions. Compositions and methods disclosed herein relate to suspension of coated microparticles in non-aqueous suspending agents or liquids.

[0030] In some embodiments and further to paragraphs

[0025] to

[0029] above, compositions including, but not limited to, a dried atomic layer deposition (ALD) coated microparticle of a central or innermost therapeutic agent-containing microparticle including, but not limited to, at least one therapeutic agent, at least one antigen or combination thereof; and at least one glass-forming agent; one or more coating layers of one or more of metal oxide, metal alkoxide, and aluminum-based coating layer or combination thereof, covering the central or innermost at least one therapeutic agent, at least one antigen or combination thereof containing glassy microparticle; and at least one non-aqueous suspension agent. In accordance with these embodiments, the at least one non-aqueous suspension agent can include, but is not limited to, at least one of benzyl benzoate, 2-hydroxybenzoate (salicylate)esters, polyethylene glycols, polyoxyl 15 hydroxystearate, glycerin, sorbitan monolaurate, refined triglyceride mixtures or oils and the like. In other embodiments, the at least one refined triglyceride mixture or oil includes, but is not limited to, at least one of refined sesame oil, refined soybean oil, refined corn oil, refined cottonseed oil, refined peanut oil or other triglyceride mixture or oil.

[0031] In some embodiments and further to paragraphs

[0025] to

[0030] above, the at least one therapeutic agent, at least one antigen or combination thereof comprises a protein or polypeptide fragment thereof, a polynucleotide or fragment thereof, a lipid assembly-free polynucleotide or fragment thereof, a recombinant protein; a virus-like particle; a live virus; a live, attenuated virus; an inactivated virus; a toxoid; a small molecule, and the like. In certain embodiments, the at least one therapeutic agent, at least one antigen or combination thereof comprises one or more antigens from a pathogenic virus, a pathogenic bacteria, a fungal pathogen; a peptide or polypeptide derived from a pathogenic virus, a pathogenic bacteria or fungal pathogen; a recombinant molecule derived from a pathogenic virus, a bacterial pathogen; an inactivated pathogenic virus, a fungal pathogen, a pathogenic protozoa, a pathogenic worm, a pathogenic yeast, or other pathogen. In yet other embodiments, the at least one therapeutic agent, at least one antigen or combination thereof includes, but is not limited to, an antibody or fragment thereof, a recombinant protein or fragment thereof (e.g., a virus-like particle; a live virus; a live, attenuated virus; an inactivated virus; one or more viral proteins) or a combination thereof. In certain embodiments, the at least one therapeutic agent, the at least one antigen or combination thereof includes a multimeric protein complex.

[0032] In some embodiments and further to paragraphs

[0025] to

[0031] above, the at least one glass-forming agent can be at least one polysaccharide. In accordance with these embodiments, the at least one glass-forming agent comprises at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol and glycine, hydroxyethyl starch, glycine, cyclodextrin, and povidone and the like. In some embodiments, the at least one glassforming agent or polysaccharide disclosed herein can include trehalose, sucrose or combination thereof. In certain embodiments, the at least one glass-forming agent or polysaccharide disclosed herein can include hydroxy ethyl starch alone or in combination with at least a second polysaccharide. In accordance with these embodiments, the at least one glass-forming agent or polysaccharide disclosed herein can be present in a primary liquid composition prior to spray-drying or spray -freeze drying in a weight-to-volume (w / v) concentration of from about 0.1% to about 40%, from about 1.0% to about 30.0%, from about 5.0% to about 30.0%, about 5.0% to about 25.0%, about 5.0% to about 20.0%. about 5.0% toabout 15.0% or about 20%. In certain embodiments, the at least one glass-forming agent or polysaccharide disclosed herein can be rapidly introduced to the one or more therapeutic agents and further spray-dried to a dried formulation in preparation for application of one or more coating layers to the dried microparticles formed using an ALD process as described herein. In yet other embodiments, the glass-forming agent or polysaccharide includes 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 other embodiments, therapeutic agent in presence of at least one glassforming agent can further include at least one additional agent.

[0033] In other embodiments and further to paragraphs

[0025] to

[0032] above, the ALD- coating can include any ALD coating material. In other embodiments, ALD-coating materials can include a metal agent, a metal oxide, a metal alkoxide, and an aluminum-based coating layer for coating therapeutic agent containing microparticles disclosed herein. In some embodiments, the ALD coating material can include one or more of aluminum oxide (AI2O3), an aluminum alkoxide, silicon dioxide (SiCh), titanium dioxide (TiCh), gallium oxide (Ga2Os), silicon nitride (SisN^, zinc oxide (ZnO), zirconium dioxide (ZrCL), zirconium silicate or zircon (ZrSiCh) and hafnium oxide (HfCh) and combinations thereof, other ALD- coating materials and mixtures, alternating materials and combinations thereof.

[0034] In some embodiments and further to paragraphs

[0025] to

[0033] above, the composition comprises a readily injectable formulation. In some embodiments, the at least one therapeutic agent, at least one antigen or combination thereof of the atomic layer deposition (ALD) coated microparticle remain stably sequestered within the coated microparticles in suspension in the at least one non-aqueous suspension agent.

[0035] In some embodiments and further to paragraphs

[0025] to

[0034] above, the at least one therapeutic agent, the at least one antigen or combination thereof can include, but is not limited to, at least one of a therapeutic agent or antigen derived from or used to treat or prevent infection by Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague), Variola major (smallpox) and other related pox viruses, Francisella tularensis (tularemia), viral hemorrhagic fevers, Arenaviruses, Junin, Machupo, Guanarito, Chapare, Lassa, Lujo, bunyaviruses, hantaviruses causing Hanta pulmonary syndrome, Rift Valley Fever, Crimean Congo Hemorrhagic Fever, flaviviruses-related conditions, alphaviruses, and Filoviruses such as Ebola, Sudan, and Marburg viruses or other Filoviruses or a combination thereof. In certain embodiments, the at least one therapeutic agent or the at least one antigen can include at least one antigen or at least one therapeutic agent derivedfrom a category A, category B or category C pathogen from the National Institute of Allergy and Infectious Diseases (NIAID) listings.

[0036] In some embodiments and further to paragraphs

[0025] to

[0035] above, the at least one therapeutic agent, the at least one antigen or combination thereof can be at least one of a therapeutic agent or antigen derived from human papilloma virus (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, Cory neb acterium diptheriae, Clostridium tetani, Clostridium botulinum, Bordetella pertussis, Streptococcus pneumoniae, Neisseria meningitides, Salmonella spp., Bacillus anthracis, Yersinia spp., or a combination thereof.

[0037] In some embodiments and further to paragraphs

[0025] to

[0036] above, where the at least one of the at least one therapeutic agent, the at least one antigen or combination thereof includes, but is not limited to, at least one of a therapeutic agent or antigen derived from at least one pathogen, wherein the at least one pathogen comprises a pathogen that infects a human, a companion animal, a pet, a non-human animal, livestock, a bird, a fish, or a reptile. In certain embodiments, the subject is a human subject including, a neonate, an infant, a toddler, a child, an adolescent, a young adult, an adult, or an older adult subject.

[0038] In some embodiments and further to paragraphs

[0025] to

[0037] above, wherein the at least one therapeutic agent, the at least one antigen or combination thereof is derived from a virus and the virus comprises an enveloped or non-enveloped virus. In other embodiments, the at least one therapeutic agent, the at least one antigen or combination thereof is derived from a virus and the virus includes, but is not limited to, 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 cell receptor protein, an enzyme, a structural protein, a cytokine or a combination thereof.

[0039] In certain embodiments and further to paragraphs

[0025] to

[0038] above, methods of making timed release stable administration-ready ALD-coated therapeutic agentcontaining microparticle compositions are disclosed. In accordance with these embodiments, these methods can include combining dried ALD-coated therapeutic agent-containing microparticles with at least one non-aqueous suspension agent; and forming a suspensioncomposition having improved uniformity. In other embodiments, the dried ALD-coated therapeutic agent-containing microparticles comprises a powder form of the ALD-coated therapeutic agent-containing microparticles. In other embodiments, the at least one nonaqueous suspension agent includes, but is not limited to, at least one of benzyl benzoate, 2- hydroxybenzoate (salicylate) esters, polyethylene glycols, polyoxyl 15 hydroxystearate, glycerin, sorbitan monolaurate, refined triglyceride mixtures, or similar oils and the like. In other embodiments, the at least one refined triglyceride mixture or oil includes, but is not limited to, at least one of refined sesame oil, refined soybean oil, refined corn oil, refined cottonseed oil, refined peanut oil or other refined triglyceride mixture or refined triglyceride oil or combinations thereof. In some embodiments, these resuspended ALD coated microparticle-containing formulations disclosed herein can be stored at temperatures to reduce, avoid, or eliminate particle or microparticle settling for a predetermined period or indefinitely where a temperature for storage can be below the non-aqueous suspending agent’s solidification temperature (e.g., a temperature below -6 °C for ultrarefined sesame oil or other ultrarefined oils, or a temperature below 18 °C for benzyl benzoate or similar agent). In certain embodiments, the temperature can be up to about 20 °C, or up to about 18 °C, or up to about 15 °C, or up to about 10 °C, or up to about 6 °C as a storage temperature for formulations disclosed herein. In certain embodiments, non-aqueous suspending agent suspension formulations containing ALD coated microparticles disclosed herein can be cooled to reduce, avoid, or eliminate particle or microparticle settling to ensure more uniform distribution of the microparticles in a suspended formulation. For example, suspensions of ALD-coated microparticles in ultrarefined sesame oil or other non-aqueous suspending agents disclosed herein can be cooled to a temperature below the suspending agent’s solidification point (e.g., a temperature below -6 °C for ultrarefined sesame oil or other refined oils, or a temperature below 18 °C for benzyl benzoate or similar agent). In other embodiments, when a cooled suspension is warmed or re-warmed to a temperature above the oil’s or other nonaqueous suspending agent’s solidification temperature (e.g., room temperature, body temperature or above) uniform, unsettled ALD coated microparticle suspensions can be essentially or fully recovered. For example, these conditions can suspend ALD coated microparticles where the microparticles are suspended with little to no loss in settling. In accordance with these embodiments, the warmed or re- warmed composition is ready for administration or immediate administration to a subject. In other embodiments, these suspensions maintain syringability. Further, it was observed that suspensions cooled and then warmed maintain microparticle integrity without premature release of the therapeutic agentand are not altered by this process. This observation can be due in part to the fact that the non-aqueous suspending agents contemplated of use herein do not crystallize (e.g., refrigeration of an oil suspension contemplated herein does not crystallize upon cooling but can solidify). In certain embodiments, when suspensions disclosed herein are stored at temperatures below the non-aqueous suspending agent’s solidification temperature, ALD- coated particles are essentially locked down in a solidified state, reducing or eliminating agglomeration of the ALD-coated microparticles and maintaining a more uniform distribution. In contrast, settled aqueous suspension of these ALD-coated particles can agglomerate and form clumps that are very difficult to resuspend resulting in inaccurate delivery, loss of product and other issues.

[0040] In other embodiments and further to paragraphs

[0025] to

[0039] above, the at least one therapeutic agent, at least one antigen or combination thereof includes, but is not limited to, a protein or polypeptide fragment thereof, a polynucleotide or fragment thereof, a lipid assembly-free polynucleotide or fragment thereof, a small molecule, a recombinant protein, a plasmid, or other biologically active or therapeutically active molecule thereof. In some embodiments, the at least one therapeutic agent, at least one antigen or combination thereof includes, but is not limited to, a virus-like particle; a live virus; a live, attenuated virus; an inactivated virus; a toxoid; and the like. In other embodiments, the at least one therapeutic agent, at least one antigen or combination thereof includes, but is not limited to, one or more antigens from a pathogenic virus, a pathogenic bacteria, a toxoid, a prion, a fungal pathogen or other pathogen; a peptide or polypeptide derived from a pathogenic virus, a pathogenic bacteria, toxin, or fungal pathogen or other pathogen; a recombinant molecule derived from a pathogenic virus, a bacterial pathogen; an inactivated pathogenic virus, a fungal pathogen, a pathogenic protozoa, a pathogenic worm or parasite, a pathogenic yeast, or other pathogen.

[0041] In some embodiments and further to paragraphs

[0025] to

[0040] above, methods can further include loading a therapeutic delivery device with the non-aqueous agent suspension composition for immediate or later administration to a subject. In some embodiments, no other composition or agent is added to the non-aqueous agent suspension prior to loading a therapeutic delivery device for immediate or later administration to a subject. In other embodiments, the non-aqueous suspension composition can be stored at temperatures above the non-aqueous agent solidification temperature; for example, above room temperature, or above about 18 °C or greater (e.g., up to 50° C or 60° C) for a few hours, a day, a couple of days, a few days, up to one week up, up to two weeks, up to a month, up to 45 days, up to 2 months, up to 3 months or more prior to delivery to a subject.

[0042] In some embodiments and further to paragraphs

[0025] to

[0041] above, ALD coated microparticles disclosed herein can further include at least a second therapeutic agent, antigen, or at least a second therapeutic agent deposited as a layer on an outer coating layer of the ALD-coated microparticle and separated from an inner core containing one or more sequestered therapeutic-agent containing microparticles by one or more coating layers. In certain embodiments, the at least second therapeutic agent, antigen or agent is the same or different than the at least one therapeutic agent, antigen or agent of a central or innermost core of the ALD-coated microparticle. In accordance with these embodiments, these ALD coated microparticles can be in powder form and resuspended in a non-aqueous suspending agent disclosed herein for improved delivery.

[0043] In some embodiments and further to paragraphs

[0025] to

[0042] above, methods for treating, preventing, or reducing the risk of onset of a health condition in a subject are disclosed. In some embodiments, methods for treating, preventing, or reducing the risk of onset of a health condition in a subject include administering to the subject ALD-coated therapeutic agent-containing microparticles suspended in nonaqueous suspending agent compositions disclosed herein. In certain embodiments, preventing or reducing the risk of onset or treating is prophylactic or therapeutic.

[0044] In some embodiments and further to paragraphs

[0025] to

[0043] above, administering a nonaqueous liquid suspended ALD-coated therapeutic agent-containing microparticle composition disclosed herein can include, but is not limited to, administering the composition from a pre-filled syringe containing the composition. In other embodiments, administering a nonaqueous liquid suspended ALD-coated therapeutic agent-containing microparticle composition disclosed herein can include, but is not limited to, administering the composition from a derma device (e.g., Pharmjet® or the like).

[0045] In some embodiments and further to paragraphs

[0043] to

[0044] above, the health condition includes, but is not limited to, a subject having or suspected of developing an infection from a pathogenic organism. In other embodiments, the health condition is cancer. In yet other embodiments, the health condition is a genetic disorder or autoimmune disorder.

[0046] In some embodiments and further to paragraphs

[0025] to

[0045] above, the subject in need of treatment, preventing, or reducing the risk of onset of a health condition can include a human. In other embodiments, the subject is a human and the human comprises a neonate, an infant, a toddler, a child, an adolescent, a young adult, an adult, or an older adult subject. In other embodiment, the subject in need of treatment, preventing, or reducing the risk of onset of a health condition can include a non-human mammal or animal. In someembodiments, the subject can include a pet, livestock, a horse (e.g., a performance horse), or other domesticated, captive or wild animal. In other embodiments, the subject can include a reptile, fish or bird. For example, compositions disclosed herein can be delivered by bulk administration or by assembly-line administration to herds, for example such as farm animals or wild animal herds.

[0047] In some embodiments and further to paragraphs

[0025] to

[0046] above, nonaqueous liquid suspending agents contemplated herein can include, but are not limited to, triglycerides listed in the US Food and Drug Administration (FDA) IID list of inactive ingredients approved for parenteral administration by injection such as castor oil, com oil, cottonseed oil, peanut oil, and sesame oil, and mixtures of these triglycerides with sorbitan monolaurate. In other embodiments, nonaqueous liquid suspending agents contemplated herein can include, but are not limited to, suspending agents appearing on the US Food and Drug Administration (FDA) IID 1 list of injectable non-aqueous liquid excipients including benzyl benzoate.

[0048] In certain embodiments and further to paragraphs

[0025] to

[0047] above, ALD- coating materials of use herein are insoluble in non-aqueous liquid suspension agents disclosed herein. In certain embodiments, ALD-coating materials of use herein are insoluble in triglyceride and benzyl benzoate suspending agents. In other embodiments, metal agents, metal oxides and metal alkoxides are insoluble in triglyceride and benzyl benzoate suspending agents. In accordance with these embodiments, metal oxides such as alumina are highly insoluble in triglyceride and benzyl benzoate suspending agents. For example, when in solution, these non-aqueous agents do not prematurely dissolve coating layers of ALD coated therapeutic agent containing microparticles disclosed herein. Therefore, therapeutic agents contained in these microparticles are not prematurely leaked out of these ALD coated microparticles, preserving intact microparticles for improved or more accurate delivery to a subject at reduced loss of product. Therefore, ALD-coated therapeutic agent containing powders (e.g., metal oxide) can be stable in these suspending agents, allowing powders to form suspensions amenable to automated liquid vial- and syringe-filling (e.g., for pre-filled syringe presentations) operations. In certain embodiments, suspending agents such as triglycerides and benzyl benzoate exhibit viscosities greater than that of water, allowing suspensions to be handled and injected without undue settling of microparticles. In some embodiments, because of the highly non-polar nature, of certain non-aqueous suspending agents (e.g., triglyceride and benzyl benzoate) they do not act as solvents and do not cause ALD-coatings, metal agents and metal oxides to dissolve, suspending ALD-coatedtherapeutic-agent containing microparticles in non-aqueous, non-polar agents or suspending agents which dramatically reduces or prevents premature dissolution of the ALD-coating layers that coat the particles. In accordance with these embodiments, these conditions avoid unintended or pre-mature release of therapeutic agents or antigens prior to administration of these ALD-coated microparticles (e.g., by injection). In some embodiments, ALD-deposited coating layers (e.g., alumina) are impermeable to triglycerides. In some embodiments, it was discovered that certain vaccines or therapeutic agents within ALD-coated microparticles that typically interact unfavorably if placed in direct contact with the triglycerides (e.g., mRNA- lipid nanoparticles or mRNA lipid complexes, mRNA, polypeptides or assemblies) are protected from directly contacting the suspending agent by the ALD-deposited layers. Further, because viscosity of the suspending agents (e.g., triglycerides) is higher than that of water, particles settle more slowly and with less propensity for agglomeration.

[0049] In certain embodiments and further to paragraph

[0048] above, maintaining low moisture content within microparticles is important to avoid the destabilizing effects of water- induced plasticization and maintaining sustained, accurate, reliable delivery during storage and transportation. Deposited coatings (e.g., metal agents) of ALD-coated microparticles offer a protective barrier against water sorption. This barrier is augmented by the low solubility of water in the preferred non-aqueous suspending agents, which limits the transport of water from the ambient environment to the suspended ALD-coated microparticles.Further, because viscosity of the triglycerides is higher than that of water, particles settle more slowly and with less propensity for agglomeration. Therefore, embodiments disclosed herein provide a solution to issues of dried ALD-coated microparticles or powders enabling therapeutic product containers such as vials, tubes and pre-filled syringes or other delivery device to be filled using standard high-speed liquid filling lines and facilitates the process for creating ready -to-use syringes, inhalation devices or the like, or other containers for administering to subjects for improved stability, storage, transport, and administration to patients.

[0050] In certain embodiments and further to paragraphs

[0025] to

[0049] above, one or more agent disclosed herein can include at least one therapeutic agent that forms part of a central or innermost microparticle. In accordance with these embodiments, the central or innermost microparticle can include, but is not limited to, at least one microparticle including, but not limited to, one therapeutic agent, antigen, or other therapeutic molecule, and at least one glass-forming agent or polysaccharide, and further, one or more outer coating layers covering or encasing the central or innermost microparticle. In accordance with theseembodiments and those referenced above, one, two, three, four, five, 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 can encase stabilized powders or glassy matrices where the coating layers are readily dissolvable in a subject once administered to the subject, to expose the one or more therapeutic agent to the subject by immediate exposure or timed- release.

[0051] Certain embodiments and further to paragraphs

[0025] to

[0050] above, methods for making stabilized coated microparticles, stabilized powders or glassy matrices, the methods including combining at least one therapeutic naked polynucleotide with at least one glass-forming agent or polysaccharide to form a primary liquid composition, rapidly spraydrying the composition to form glassy microparticles, and coating the dried glassy microparticles with one or more outer coating layers. In some embodiments, a primary liquid composition (prior to spray-drying) can further include rapid introduction of a second glassforming agent. In accordance with these embodiments, the second glass-forming agent or polysaccharide can include hydroxyethyl starch or similar agent. Further, in accordance with these embodiments, hydroxyethyl starch can precipitate as a film on the surface of droplets of the composition as they dry during spray drying, decreasing the rate of drying and reducing collision of particles thereby maintaining integrity of the particles as they dry to a more stabilized state in preparation for coating. Rapid spray drying as disclosed herein can mean spray-drying in a few milliseconds up to a few seconds. In accordance with these embodiments, particulates and / or essentially dry microparticles disclosed herein are formulated and spray dried such that formulations are not principally located at the surface of the spray dry particle (reduced exposure). It is known that high molecular weight compounds and low solubility compounds have a higher likelihood of ending up at the surface of a spray dried particle, therefore, embodiments disclosed herein address this commonly observed issue by specific additions to the formulation to form an external shell-like layer that is not principally the target therapeutic agent and can, for example, shield the therapeutic agent making them more stable under certain conditions. In accordance with these embodiments, particulates and / or essentially dry microparticles disclosed herein can be introduced to an ALD reaction chamber where the particulates and / or essentially dry microparticles flow freely within the chamber for reduced agglomeration and / or aggregation of the particulates and / or dry microparticles. It is noted herein that introduction of therapeutic agent-containing formulations directly to an ALD reaction chamber without creating thermostable particulates or essentially dry glassy microparticles as disclosed herein would be an unsuccessful coatingprotocol; for example, the elevated temperature would degrade the formulation and therapeutic agent or the therapeutic agent would likely stick to one another or the chamber and / or the metal material applied would not layer onto free therapeutic agent (not encased in a protective shell or glassy particle) and / or fully encapsulate the therapeutic agent because there would be no availability of binding groups needed to secure the coating which is created herein using polymer-containing formulations to create the essentially dry microparticles or particulates disclosed herein for long term storage or for further coating.

[0052] In certain embodiments and further to paragraphs

[0025] to

[0051] above, the at least one therapeutic agent or antigen can include, but is not limited to, at least one pathogen or antigen or fragment or construct thereof derived from; for example, antigens derived from human papilloma virus (e.g., HPV16, HPV18) or other mammalian papilloma virus, ricin toxin, Bacillus anthracis, Clostridium botulinum, Ebola virus, influenza virus, Corona virus (SARS-CoV2, Covid-19 or other variants, mutants thereof), poliovirus, norovirus, rotavirus, hepatitis C, varicella, herpes simplex, cytomegalovirus, Japanese encephalitis, dengue virus, West Nile virus, Zika virus or other flaviviruses, an alphavirus such as chikungunya, EEEV, WEEV, VEEV or other alphavirus, pneumonia virus spp., Yersinia, Pneumococcus, Salmonella, Clostridium difficile, Burkholderia pseudomallei (Bp) (e.g., glycoconjugate or recombinant protein; against melioidosis and / or glanders) or a combination thereof. In certain embodiments, the at least one therapeutic agent, antigen or agent can be a multimeric complex. In other embodiments, the at least one therapeutic agent or antigen can include a pathogen or antigen derived therefrom capable of infecting a human (e.g., adult, child, toddler, infant or fetus), companion animal, livestock, pet, wild animal, zoo animal, bird, or reptile.

[0053] In certain embodiments and further to paragraphs

[0025] to

[0052] above, a pathogenic virus can be, for example, any pathogenic virus. In accordance with these embodiments, a pathogenic virus can include, but is not limited to, a papovavirus (e.g., papillomaviruses, including human papilloma virus (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 Bl 9, 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, Japanese encephalitis virus or other flavivirus), an orthomyxovirus (e.g., influenza A virus, influenza B virus, influenza C virus), a paramyxovirus (e.g., measles virus, mumps virus, respiratory syncytial virus, caninedistemper virus, parainfluenza viruses), a rhabdovirus (e.g., rabies virus), a filovirus (e.g., Ebola virus), an alphavirus (e.g., chikungunya or WEEV, EEEV or VEEV, or other alphavirus) or a coronavirus, SARS or the like or mutants thereof or combinations thereof. In accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD-coated microparticles and suspended in non-aqueous suspension compositions disclosed herein.

[0054] In other embodiments and further to paragraphs

[0025] to

[0052] above, a pathogenic agent or antigen derived from a pathogenic agent can be a bacterium or a toxin of a bacterium or toxoid agent. In accordance with these embodiments a pathogenic agent or antigen derived from a pathogenic agent can include, but is not limited to, 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., Neisseria gonorrhoeae, Borrelia burgdorferi, Burkholderia pseudomallei, Klebsiella pneumoniae, Acinetobacter baumanii, E. coli, Enteerococcus faecium, Enterococcus feacalis, Pseudomonas aeruginosa, Clostridium botulinum or other pathogenic bacteria or a combination thereof. In accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD-coated microparticles and suspended in nonaqueous suspension compositions disclosed herein.

[0055] In some embodiments and further to paragraphs

[0025] to

[0052] above, a pathogenic agent can be a fungus. In accordance with these embodiments, a pathogenic fungus can include but is not limited to, Cryptococcus spp. (e.g., neoformans and gatti), Aspergillus spp. (e.g., fumigatus), Blastomyces spp. (e.g., dermatitidis), Candida albicans, Paracoccidioides spp. (e.g., brasiliensis), Sporothrix spp. (e.g., schenkii andbrasiliensis), Histoplasma capsulatum, Pneumocystis jirovecii, Coccidioides immitis, or other pathogenic fungus or combinations thereof. In accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD-coated microparticles and suspended in non-aqueous suspension compositions disclosed herein.

[0056] In yet other embodiments and further to paragraphs

[0025] to

[0052] above, a pathogenic agent can be a toxin. In accordance with these embodiments, a toxin can include but is not limited to, anthracis toxin, ricin toxin or botulinum toxin or another toxin. In accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD-coated microparticles and suspended in non-aqueous suspension compositions disclosed herein.

[0057] In some embodiments and further to paragraphs

[0025] to

[0056] above, microparticles described herein can be used to manufacture one or more microparticlecontaining formulation for use as vaccines for any animals. In certain embodiments, the animal can be a household pet or other companion animal. In some embodiments, an animal can include livestock or other farm animal, wild animal, or zoo animal. In certain embodiments, the animal can include a non-human mammal, reptile, or bird. In accordance with these embodiments, the immunogenic composition can be administered, including, but not limited to, to a dog (canine), a cat (feline), a horse (equine), cattle (bovine), a goat (hircine), a sheep (caprine), pig (swine), or poultry (e.g., chicken, turkey, duck, goose).

[0058] In certain embodiments and further to paragraphs

[0025] to

[0057] above, ALD coated microparticles described herein can be used to generate one or more compositions or other agent-containing compositions contemplated herein for administering to a canine to reduce onset of or prevent an infection or treat a condition such as cancer or an inflammatory condition or diabetes or other condition. In accordance with these embodiments, an infection can include, but is not limited to, infections related to canine parvovirus (CPV), canine distemper virus (CDV), canine adenovirus (CAV), rabies, canine parainfluenza virus (CPiV), canine influenza virus, canine corona virus, measles virus, Bordetella bronchiseptica, Leptospira spp., and Borrelia burgdorferi or combinations thereof. In accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD- coated microparticles and suspended in non-aqueous suspension compositions disclosed herein.

[0059] In certain embodiments and further to paragraphs

[0025] to

[0057] above, ALD coated microparticles described herein can be used to generate one or more compositions or other agent-containing compositions contemplated herein for administering to a feline to reduce onset of or prevent an infection or treat a condition such as cancer or an inflammatory condition or diabetes or other condition. In some embodiments, lipid emulsion agentcontaining coated microparticles described herein can be used to generate coated- microparticle containing formulations of use to treat, reduce the risk of onset or prevent an infection or treat an infection in a feline, including but not limited to, immunogenic compositions directed to feline herpesvirus 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. In accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD-coatedmicroparticles and suspended in non-aqueous suspension compositions disclosed herein.

[0060] In certain embodiments and further to paragraphs

[0025] to

[0057] above, ALD coated microparticles described herein can be used to generate one or more compositions or other agent-containing compositions contemplated herein for administering to a horse to reduce onset of or prevent an infection or treat a condition such as cancer or an inflammatory condition or diabetes or other condition. In other embodiments, therapeutic agents directed to reducing onset of or treating an infection caused by and including, but not limited to, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Venezuelan equine encephalomyelitis virus, bovine papillomavirus, rabies virus, Clostridium tetani, West Nile virus, equine influenza virus, Potomac fever (Neorickettsia risticii), Streptococcus equi equi, and rhinopneumonitis (equine herpesevirus type 1) or combinations thereof. In accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD-coated microparticles and suspended in non-aqueous suspension compositions disclosed herein.

[0061] In certain embodiments and further to paragraphs

[0025] to

[0057] above, ALD coated microparticles described herein can be used to generate one or more compositions or other agent-containing compositions contemplated herein for administering to a bovine to reduce onset of or prevent an infection or treat a condition such as cancer or an inflammatory condition or diabetes or other condition. In other embodiments, ALD coated-microparticle containing formulations of use to treat, reduce the risk of onset or prevent an infection or treat an infection in a bovine, including but not limited to, immunogenic compositions directed to bovine rhinotracheitis (IBR), parainfluenza type 3 (PI3), bovine virus diarrhea (BVD), bovine respiratory syncytial virus (BRSV), blackleg (Clostridium chauvoei), malignant edema (Clostridium septicum), infectious necrotic hepatitis (Clostridium novyi), enterotoxemia (Clostridium perfringens type C and D), Pasteurella haemolytica, and redwater (Clostridium haemolyticum) or combinations thereof. In accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD-coated microparticles and suspended in non-aqueous suspension compositions disclosed herein.

[0062] In certain embodiments and further to paragraphs

[0025] to

[0057] above, ALD coated microparticles described herein can be used to generate one or more compositions or other agent-containing compositions contemplated herein for administering to a poultry to reduce onset of or prevent an infection or treat a condition such as cancer or an inflammatory condition or diabetes or other condition. In other embodiments, coated microparticles described herein can be used to generate coated-microparticle containing formulations of useto treat, reduce the risk of onset or prevent an infection or treat an infection in poultry including but not limited to, immunogenic compositions directed to Marek’s disease (Marek’s disease virus), tenosynovitis (reoviruses), encephalomyelitis (avian encephalomyelitis virus), fowlpox (avipoxviruses), chicken infectious anemia (chicken anemia virus), fowl cholera (Pasteurella multocida), Newcastle / infectious bronchitis (Newcastle disease virus), Riemerella anatipestifer, duck viral hepatitis (duck hepatitis virus), and duck viral enteritis (duck herpesvirus 1) or combinations thereof. In accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD-coated microparticles and suspended in non-aqueous suspension compositions disclosed herein.

[0063] In certain embodiments and further to paragraphs

[0025] to

[0057] above, ALD coated microparticles described herein can be used to generate one or more compositions or other agent-containing compositions contemplated herein for administering to a human to reduce onset of, treat, and / or prevent an infection or treat a condition such as cancer or an inflammatory condition or diabetes or other health condition. In other embodiments, ALD coated microparticles described herein can be used to generate coated-microparticle containing formulations of use to treat, reduce the risk of onset or prevent an infection in a human. In certain embodiments, lipid emulsion agent-containing coated particles described herein can be used to deliver one or more formulation disclosed herein to a human such as an infant or child or adolescent, young adult, adult or elderly human subject including but not limited to antigens or agents derived therefor for varicella-zoster (chicken pox), diphtheria, Haemophilus influenzae type b (Hib), hepatitis A, hepatitis B, influenza, corona virus, SARS, Ebola virus, measles, mumps, pertussis, polio, pneumococcal disease, rotavirus, rubella, and tetanus. In other embodiments, coated particles described herein can be used to deliver one or more suspension 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 accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD-coated microparticles and suspended in non-aqueous suspension compositions disclosed herein.

[0064] In certain embodiments and further to paragraphs

[0025] to

[0057] above, ALD coated microparticles described herein can be used to generate compositions of use for administering to a human. In accordance with these embodiments, treatment of a human subject can include, but not limited to, compositions against travel -related infections, including but not limited to, hepatitis A, hepatitis B, typhoid fever, paratyphoid fever,meningococcal disease, yellow fever, dengue fever, rabies, Zika virus-related conditions, West Nile virus infection, Chikungunya disease, and Japanese encephalitis. Covid-19 or corona virus infection, or other infection or combinations thereof. In accordance with these embodiments, therapeutic agents or antigens derived therefrom can be sequestered in ALD- coated microparticles and suspended in non-aqueous suspension compositions disclosed herein.

[0065] In certain embodiments and further to paragraphs

[0025] to

[0057] above, ALD coated microparticles described herein can be used to generate compositions of use for administering to a human, including but not limited to, sequestered therapeutic agents or antigens. In accordance with these embodiments, therapeutic agents or antigens can be derived from and include, but not be limited to, human papillomavirus (e.g. HPV 16, HPV18, HPV31, HPV45, or HPV 6 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 strain or mutant, or the like.

[0066] In some embodiments and further to paragraphs

[0025] to

[0065] above, suspended coated microparticles described herein can be stored at refrigeration temperatures, at room temperature, or up to about 50 °C, or up to about 60 °C, or up to about 70 °C for extended periods. In certain embodiments, suspended coated microparticles described herein can be stored at temperatures above the solidification temperatures of the suspending non-aqueous liquid such as up to room temperature, or up 50 °C, or up to about 60 °C, or up to about 70 °C for up to about a day, about two or more days, about a week, about a couple of weeks, about a few weeks, about several weeks, about 1 month, about 2 months, about 3 months, up to about 4 months, up to about 6 month, up to about 9 months, up to about 12 months or longer without negative effects on the coated microparticles (e.g. settling out, agglomeration, degradation, loss of efficacy, premature leaking, reduced delivery of the one or more agents or antigens).

[0067] Other embodiments and further to paragraphs

[0025] to

[0066] above, provide for combination compositions or formulations including a plurality of suspended coated microparticles described herein. In accordance with these embodiments, these combination compositions or formulations can include mixtures of different suspended ALD coated therapeutic agent-containing microparticles containing one or more therapeutic agent or antigen for treating, reducing onset of, or preventing a single health condition or multiplehealth conditions (e.g., pathogenic agent infections or prevention of pathogenic agent infections, cancer, inflammation, etc.) and optionally, include at least one pharmaceutically acceptable excipient. In other embodiments, combination compositions can include at least one representative therapeutic agent-containing microparticle pharmaceutical composition as disclosed herein mixed with a standard or known composition or formulation to treat, reduce onset of, or prevent a health condition. In certain embodiments, the standard formulation can include a standard vaccine against a pathogen or an anti -cancer agent or the like. In some embodiments, ALD-coated microparticles disclosed herein can be used as one or more boost doses to a standard treatment directed to treat, prevent, or reduce onset of a health condition.

[0068] In some embodiments and further to paragraphs

[0025] to

[0067] above, buffers of use to spray-dry or reconstitute microparticles disclosed herein can include, but are not limited to, acetate, succinate, citrate, prolamine, histidine, borate, carbonate or phosphate buffer, or a combination thereof. In certain embodiments, a buffer can include one or more salts of use in forming glassy microparticles and can include, but is not limited to, one or more of salt including, but not limited to, sodium acetate, sodium succinate, potassium succinate, sodium citrate, sodium phosphate, potassium phosphate and the like or a combination thereof. In certain embodiments, the buffer can include histidine, for example, histidine-HCl. In other embodiments, 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, a volatile salt can include ammonium acetate.

[0069] Certain embodiments and further to paragraphs

[0025] to

[0068] above, include pharmaceutical compositions of the dried ALD coated therapeutic agent containing microparticle and at least one non-aqueous suspension agent. In certain embodiments, the pharmaceutical composition can include a pharmaceutically acceptable carrier or excipient. In some embodiments, acceptable carriers, excipients, or stabilizers can include, but are not limited to, nontoxic to recipients at the dosages and concentrations used, and can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0070] Certain embodiments and further to paragraphs

[0025] to

[0069] above, include methods to elicit a therapeutic response to an agent of non-aqueous suspension of coated stabilized microparticles or combination of agents of one or multiple agent-containing microparticles disclosed herein, by administering to the subject the suspension disclosed herein. These pharmaceutical compositions containing the ALD-coated therapeutic agent containing microparticles can be administered in therapeutically effective amounts. That is, in amounts sufficient to produce a protective immunological response or therapeutic response (e.g., in a single or multiple dosing delivery by dissolution of coating layers). Generally, the suspension compositions can be administered in active agent dosages ranging from about 0.001 mg to about 20.0 mg immunogenic agent or about 0.01 mg to about 20.0 mg agent, or about 0.1 mg to about 10.0 mg agent. Single or multiple dosages can be administered in a single administration suspension composition. Where multiple doses of a suspension composition are administered in a single administration composition, for example, in primeboost compositions, one of the two or more doses can be temporally controlled for release at a pre-selected time after administration using precision ALD coating layers.

[0071] In other embodiments and further to paragraphs

[0025] to

[0070] above, kits are contemplated. In accordance with these embodiments, kits can include at least one nonaqueous suspending agent separate from a powder form of ALD-coated therapeutic agentcontaining microparticles disclosed herein in at least 2 separate containers. In other embodiments, kits can include at least one ALD-coated therapeutic agent-containing microparticle formulation suspended in at least one nonaqueous suspending agent, and at least one container. In certain embodiments, kits can further include at least one delivery device for combining the at least one non-aqueous suspending agent together with a dry or powder form of ALD-coated therapeutic agent-containing microparticles. In certain embodiments, kits can further include at least one delivery device that is the container harboring the at least one ALD-coated therapeutic agent-containing microparticle suspended in a nonaqueous suspension formulation for immediate or later delivery to a subject. In yetother embodiments, delivery devices can include, but are not limited to, at least one syringe, other injection device or non-invasive or derma injection device. In some embodiments, a delivery device or kit can contain more than one dose of the ALD coated particles in the same or different containers. In yet other embodiments, kits can further include instructions for combining or delivering these formulations.DEFINITIONS

[0072] To facilitate an understanding of certain disclosures and concepts disclosed herein, the following definitions are provided. Otherwise, all technical terms have their ordinary, skill of the art-recognized definitions.

[0073] When introducing elements of the embodiments described herein, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0074] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 2 to about 50” should be interpreted to include not only the explicitly recited values of 2 to 50 but also include all individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 2.4, 3, 3.7, 4, 5.5, 10, 10.1, 14, 15, 15.98, 20, 20.13, 23, 25.06, 30, 35.1, 38.0, 40, 44, 44.6, 45, 48, and sub-ranges such as from 1-3, from 2-4, from 5-10, from 5-20, from 5-25, from 5-30, from 5-35, from 5-40, from 5-50, from 2- 10, from 2-20, from 2-30, from 2-40, from 2-50, etc. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0075] The term “about,” as used herein, can refer to variation of in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry outthe methods and the like. The term “about” also encompasses these variations, which can be up to ± 5%, but can also be ± 4%, 3%, 2%, 1%, etc. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

[0076] In this disclosure, “comprises,” “comprising,” “containing,” and “having” and the like can have the meaning ascribed to them in patent laws and can mean “includes,” “including,” and the like, and are generally interpreted to be open ended terms. The terms “consisting of’ or “consists of’ are closed terms, and include only the components, structures, steps, or the like listed in conjunction with such terms, as well as that which is in accordance with patent laws. “Consisting essentially of’ or “consists essentially of’ can have the meaning generally ascribed to them by patent laws. For example, these terms are generally closed terms, with the exception of allowing inclusion of additional items, materials, components, steps, or elements, that do not materially affect the basic and novel characteristics or function of the item(s) used in connection therewith. For example, trace elements present in a composition, but not affecting the composition’s nature or characteristics would be permissible if present under the “consisting essentially of’ language, even though not expressly recited in a list of items following such terminology. In this specification when using an open-ended term, like “comprising” or “including,” it is understood that direct support should be afforded also to “consisting essentially of’ language as well as “consisting of’ language as if stated explicitly and vice versa.EXAMPLES

[0077] 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 indicating certain embodiments, are given by way of illustration only. From the disclosure herein and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.Example 1Non-aqueous suspending agents for suspension and intramuscular administration of an ALD- coated rabies vaccine

[0078] In certain exemplary experiments, rabies virus particles from Rabivax-S, a killed rabies virus vaccine, was diluted to 5 lU / mL reformulated in 9.5% trehalose, 2.5 % HES, 0.2 mM polysorbate 80, 40 mM ammonium acetate, and 10 mM L-Histidine, pH 6.5. The resulting suspension was spray dried in a Buchii pilot-scale spray dryer to produce glassyparticles with embedded rabies virus particles. For example, 150 or 200 coats of aluminum oxide were applied in these studies to the particles in a custom-built fluidized bed reactor by ALD, using alternating injections of trimethyl aluminum vapor and water vapor.

[0079] Aliquots of the resulting ALD-coated particles were suspended in water, benzyl benzoate, or ultra purified triglyceride mix from sesame oil for administration. 50 microliter doses of the benzyl benzoate and sesame oils suspensions were injected intramuscularly though 25-gauge needles into groups of 10 Balb / c mice; similar suspensions in water for injection were administered in 25-, 26-, or 27-gauge needles after vigorous mixing and / or sonication to resuspend and de-agglomerate microparticles. Aqueous suspensions settle quickly and needed to be agitated vigorously immediately before injection to reduce frequency of syringe needle plugging. Non-aqueous suspensions as used herein required much less vigorous mixing to ensure homogeneity. Antibody responses generated in response to suspensions of the 150-coat material in aqueous solution suspensions and in sesame oil suspensions were indistinguishable (Fig 1); responses to injections in benzyl benzoate appeared to be somewhat more robust. Increasing the thickness of the ALD coating on the microspheres to 250 coats delayed the response by approximately two weeks.

[0080] FIG. 1 represents an example of total antibody titers in Balb / c mice following injection of vaccine coated with 150 ALD cycles of alumina on spray-dried RabiVax antigen. ALD-coated particles were suspended in water, benzyl benzoate, or ultra purified sesame oil for administration by injection. Water samples were injected through needles of 25-, 26-, and 28-gauge. ALD-coated therapeutic agent containing microparticle powders suspended in sesame oil or benzyl benzoate were injected through 25-gauge needles. Suspensions in water and sesame oil generated similar antibody titer levels and kinetics of antibody response. It was surprisingly observed that injections in benzyl benzoate resulted in slightly faster responses with higher maximum antibody titer values.

[0081] FIG. 2 is a representative graph illustrating total antibody titers in Balb / c mice following injection of therapeutic agent containing microparticles coated with 250 ALD cycles of alumina on spray-dried Rabivax antigen. ALD-coated microparticles were suspended in water, benzyl benzoate, or ultra purified sesame oil for administration by injection. Suspensions in water, benzyl benzoate or ultra purified sesame oil demonstrated equivalent immune response trajectories. Compared with immune responses demonstrated in FIG. 1 for suspensions of microparticles with 150 ALD-applied coats of alumina, immune responses to microparticles with 250 coats were delayed by approximately two weeks as desired by methods and compositions disclosed herein.Example 2

[0082] In another exemplary experiment, non-aqueous suspending agents for suspension and subcutaneous administration of a protein-based, ALD-coated human immunodeficiency virus vaccine were studied. A solution of a trimeric protein antigen based on the human immunodeficiency virus (HIV) Env protein was prepared in 9.5% trehalose, 2.5 % HES, 0.2mM polysorbate 80, 40mM ammonium acetate, and lOmM L-Histidine, pH 6.5. The resulting suspension was spray dried in a Buchii pilots scale spray dryer to produce glassy particles containing the HIV Env trimer protein. Nanoscopic coatings of 50 or 100 coats of aluminum oxide were applied to aliquots of powder in a custom-built fluidized bed reactor by ALD, using alternating injections of trimethyl aluminum vapor and water vapor.

[0083] These powders were mixed and suspended in super refined sesame oil (Croda Pharma) prior to subcutaneous injection in Balb / c mice as a single injection containing HIV Env trimer (e.g., 10 micrograms). Control groups received 10 micrograms HIV Env trimer as a single bolus dose formed by reconstituting aliquots of spray-dried powder in water for injection.

[0084] It was observed that doses suspended in super refined sesame oil suspensions of ALD-coated microparticles containing an exemplary agent, HIV Env trimer, provoked the anticipated delayed immune response compared to the bolus doses of soluble, uncoated agent (e.g., HIV Env trimer) dissolved in water (FIG. 3).

[0085] FIG. 3 illustrates a representative plot of immune responses in Balb / c mice to injections of a bolus, aqueous suspension solution of HIV Env trimer reconstituted from a spray dried powder, and sesame oil suspension of ALD-coated HIV Env trimer powders designed to provide two separate release pulses. When compared to conventional aqueous solution of HIV Env trimer, the controlled release of HIV Env trimer from oil suspensions of ALD-coated HIV Env trimer powders yielded a delayed response with a significantly higher antibody response, as measured by the area-under-the-curve from Enzyme-Linked Immunosorbent Assays (ELISA).Example 3Non-aqueous suspending agents for suspension and subcutaneous administration of a proteinbased, ALD-coated human immunodeficiency virus vaccine

[0086] In another exemplary method, a suspension of mRNA-LNPs encoding for a trimeric protein antigen based on the human immunodeficiency virus (HIV) Env protein was prepared in 9.5% trehalose, 2.5 % HES, 0.2mM polysorbate 80, 40mM ammonium acetate, and lOmM L-Histidine, pH 6.5. The resulting suspension was spray dried in a Buchii pilot scale spraydryer to produce glassy particles with embedded LNPs. Nanoscopic coatings of 50 coats of aluminum oxide were applied to aliquots of powder in a custom-built fluidized bed reactor by ALD, using alternating injections of trimethyl aluminum vapor and water vapor.

[0087] ALD-coated powders were suspended in super refined sesame oil (Croda Pharma) and injected subcutaneously into C57 / B16 mice. Mice were injected with 1 microgram mRNA embedded in alum ALD-coated microparticles and suspended in ultra purified sesame oil for injection (Croda Pharma).

[0088] FIG. 4 represents an exemplary anti- HIV Env trimer ELISA assay absorbance vs serum dilution for mice injected with 1 microgram free mRNA (or uncomplexed mRNA from lipid complexes, LNPs) encoding for HIV Env trimer in particles prepared by spray drying and coating with 50 ALD cycles of alumina deposition. Responses are shown for each mouse. Numbers in FIG 4. correspond to serum samples taken from individual mice. ELISA results demonstrate that immune responses were generated from injections of free mRNA formulated within microparticles and injected as a non-aqueous suspension.Example 4Suspensions of ALD-coated powders in triglyceride (sesame oil) / sorbitan monolaurate mixtures.

[0089] In another exemplary method, spray dried powders containing rabies virus antigen were coated with 250 coats of alumina by ALD. 10 mg aliquots of the coated powders were suspended in 1 mL volumes of sesame oil triglycerides and various amounts (0, 0.01. 0.1, 1 and 10 %) of sorbitan monolaurate.

[0090] Powders were readily suspended in the non-aqueous suspending agent mixture. Suspensions were filled into 1 mL syringes and allowed to sit quiescently overnight. Particles settled overnight but could be suspended with gentle agitation of the syringes. Suspensions were placed within and then extruded through 27-gauge needles to test for syringeability. In all samples tested, the resulting microparticle suspensions in a non-aqueous agent mixtures of sesame oil and sorbitan monolaurate could easily be injected through a 27- gauge needle.

[0091] These examples using oils as non-solvents for metal oxide-coated microparticles demonstrate wider application. Oils disclosed herein have limited chemical interactions with metal oxides making them an ideal candidate for the claimed formulations. Because of these limited interactions and non-solvent behavior, microparticles combined with any metal oxide coating contemplated herein would be suspendable in these oils and the other non-aqueous agents disclosed. These oils and coated microparticle combinations remain stable wherenothing happens over an extended period. With these limited chemical interactions and negligible solubility, any metal oxide coated microparticle / oil combo (metal oxide coated particle / non-aqueous non-solvent) disclosed herein would work to permit stable storage and delivery (e.g., administration ready formulations) to a subject in need thereof.Example 5

[0092] In another exemplary experiment, microparticle settling was assessed and it was discovered that microparticle settling could be avoided indefinitely by cooling ultrarefined oil suspensions (e.g., sesame) or other non-aqueous suspensions disclosed herein of ALD-coated microparticles to a temperature below the oil’s solidification point or below the non-aqueous solvent’s solidification point (e.g., a temperature below -6 °C for ultrarefined sesame oil, or a temperature below 18 °C for benzyl benzoate). When re-warmed to a temperature above the oil’s solidification temperature, e.g., room temperature or above, uniform, unsettled microparticle suspensions were recovered. These suspensions could be readily extruded through a 27-gauge needle reducing loss of product and increasing delivery storage and distribution efficiency.All the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of embodiments, it is apparent to those of skill in the art that variations maybe applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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

What is claimed is:

1. A composition comprising: a dried atomic layer deposition (ALD) coated microparticle comprising: a central or innermost therapeutic agent-containing microparticle comprising at least one therapeutic agent, at least one antigen or combination thereof and at least one glass-forming agent; and one or more ALD-applied coating layers encasing the central or innermost therapeutic agent-containing microparticle wherein the ALD-applied coating layers comprise at least one ALD-coating agent comprising a metal agent, a metal oxide, a metal alkoxide, an aluminum-based coating layer, other acceptable ALD coating agent, or mixtures or combination thereof; and at least one non-aqueous suspension agent.

2. The composition according to claim 1, wherein the at least one non-aqueous suspension agent comprises at least one of benzyl benzoate, 2-hydroxybenzoate (salicylate) esters, polyethylene glycols, polyoxyl 15 hydroxystearate, glycerin, sorbitan monolaurate, refined triglyceride mixtures, oil derivatives thereof or combinations thereof.

3. The composition according to claim 2, wherein the at least one refined triglyceride mixture or oil comprises at least one of refined sesame oil, refined soybean oil, refined com oil, refined cottonseed oil, refined peanut oil or other refined triglyceride mixture or refined oil.

4. The composition according to any one of claims 1-3, wherein the at least one therapeutic agent, at least one antigen or combination thereof comprises at least one of a protein and a polypeptide fragment thereof, an antibody and an antibody fragment thereof, a polynucleotide and a polynucleotide fragment thereof, a small molecule, a chimera, a recombinant protein and a fragment thereof.

5. The composition according to any one of claims 1-4, wherein the at least one therapeutic agent, at least one antigen or combination thereof comprises at least one of a lipid assembly-free polynucleotide and a fragment thereof, a virus-like particle (VLP); a live vims; a live, attenuated vims; an inactivated vims; a toxoid and a fragment thereof; and a combination thereof.

6. The composition according to any one of claims 1-4, wherein the at least one therapeutic agent, at least one antigen or combination thereof comprises one or more antigens from a pathogenic virus, a pathogenic bacteria, a fungal pathogen; a peptide or polypeptide derived from a pathogenic virus, a pathogenic bacteria or fungal pathogen; a recombinant molecule derived from a pathogenic virus, a bacterial pathogen; an inactivated pathogenic virus, a fungal pathogen, a pathogenic protozoa, a pathogenic worm, a pathogenic yeast, any other pathogen and combinations thereof.

7. The composition according to any one of claims 1-6, wherein the glass-forming agent comprises at least one polysaccharide.

8. The composition according to claim 1, wherein the at least one glass-forming agent comprises at least one of trehalose, sucrose, ficoll, dextran, maltotriose, lactose, mannitol and glycine, hydroxyethyl starch, glycine, cyclodextrin, and povidone and combination thereof.

9. The composition according to any one of claim 1-8, wherein the metal agent, metal oxide, metal alkoxide, and aluminum-based coating layer or combination thereof comprises one or more of aluminum oxide (AI2O3), an aluminum alkoxide, silicon dioxide (SiCh), titanium dioxide (TiCh), gallium oxide (Ga2Os), silicon nitride (SisN^, zinc oxide (ZnO), zirconium dioxide (ZrCh), zirconium silicate or zircon (ZrSiCU) and hafnium oxide (HfCh) and combinations thereof or combinations or mixtures or alternating pattern layering thereof.

10. The composition according to any one of claims 1-9, wherein the central or innermost therapeutic agent-containing microparticle further comprises at least one additional agent.

11. The composition according to any one of claims 1-10, wherein the composition comprises a readily injectable formulation comprising reduced ALD coated microparticle settling compared to a composition not suspended in the at least one non-aqueous suspension agent.

12. The compositions according to any one of claims 1-11, wherein the at least one therapeutic agent, at least one antigen or combination thereof of the ALD coated microparticle remain stably sequestered within the coated microparticles in suspension in the at least one non-aqueous suspension agent.

13. The compositions according to any one of claims 1-12, wherein the at least one therapeutic agent, the at least one antigen or combination thereof comprises at least one of a therapeutic agent or antigen derived from Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague), Variola major (smallpox) and other related pox viruses, Francisella tularensis (tularemia), viral hemorrhagic fevers, Arenaviruses, Junin, Machupo, Guanarito, Chapare, Lassa, Lujo, bunyaviruses, hantaviruses causing Hanta pulmonary syndrome, Rift Valley Fever, Crimean Congo Hemorrhagic Fever, flaviviruses alphaviruses, and Filoviruses such as Ebola, Sudan, and Marburg viruses or other Filoviruses or a combination thereof.

14. The compositions according to any one of claims 1-12, wherein the at least one therapeutic agent, the at least one antigen or combination thereof comprises at least one of a therapeutic agent or antigen derived from human papilloma virus (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, Cory neb acterium diptheriae, Clostridium tetani, Clostridium botulinum, Bordetella pertussis, Streptococcus pneumoniae, Neisseria meningitides, Salmonella spp., Bacillus anthracis, Yersinia spp., or a combination thereof.

15. The compositions according to any one of claims 1-12, wherein the at least one therapeutic agent, the at least one antigen or combination thereof comprises at least one of a therapeutic agent or antigen derived from at least one pathogen, wherein the at least one pathogen comprises a pathogen that infects a human, a companion animal, a pet, a non-human animal, livestock, a wild animal, a bird, a fish, or a reptile.

16. The compositions according to any one of claims 1-15, wherein the at least one therapeutic agent, the at least one antigen or combination thereof comprises a multimeric protein complex.

17. The compositions according to any one of claims 1-16, wherein the at least one therapeutic agent, the at least one antigen or combination thereof is derived from a virus and the virus comprises an enveloped or non-enveloped virus.

18. The compositions according to any one of claims 1-12 or 15-17, wherein the at least one therapeutic agent, the at least one antigen or combination thereof is derived from a virus and the virus comprises a positive or negative strand RNA virus, a linear or circular doublestranded 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 cell receptor protein, an enzyme, a structural protein, a cytokine or a combination thereof.

19. The composition according to any one of claims 1-18, wherein temperature of the composition is below a non-aqueous suspending agent’s solidification temperature to reduce, avoid, or eliminate microparticle settling for a predetermined period.

20. The composition according to claim 19, wherein the temperature comprises up to about 20 °C, or up to about 18 °C, or up to about 15 °C, or up to about 10 °C, or up to about 6 °C.

21. The compositions according to any one of claims 1-20, wherein the composition comprises a pharmaceutical composition; and optionally, further comprises a pharmaceutically acceptable carrier or excipient.

22. A method of making stable administration-ready ALD-coated therapeutic agentcontaining microparticle compositions according to any one of claims 1-21, the method comprising: combining spray or spray freeze dried ALD-coated therapeutic agent-containing microparticles with at least one non-aqueous suspension agent; and forming a suspension composition of ALD-coated therapeutic agent-containing microparticles.

23. The method according to claim 22, wherein the spray or spray freeze dried ALD-coated therapeutic agent-containing microparticles comprises a powder form of the ALD-coated therapeutic agent-containing microparticles prior to combining with the at least one nonaqueous suspension agent.

24. The method according to claim 22 or 23, wherein the at least one non-aqueous suspension agent comprises at least one of benzyl benzoate, 2-hydroxybenzoate (salicylate) esters, polyethylene glycols, polyoxyl 15 hydroxystearate, glycerin, sorbitan monolaurate, refined triglyceride mixtures or other refined oil and combinations thereof.

25. The method according to claim 24, wherein the at least one refined triglyceride mixture or other refined oil comprises at least one of refined sesame oil, refined soybean oil, refined com oil, refined cottonseed oil, refined peanut oil or other triglyceride mixture and combinations thereof.

26. The method according to any one of claims 22-25, wherein the at least one therapeutic agent, at least one antigen or combination thereof comprises at least one of a protein and a polypeptide fragment thereof, an antibody and an antibody fragment thereof, a polynucleotide and a polynucleotide fragment thereof, a small molecule, a chimera, a recombinant protein and a fragment thereof and a combination thereof.

27. The method according to any one of claims 22-26, wherein the at least one therapeutic agent, at least one antigen or combination thereof comprises at least one of a lipid assembly- free polynucleotide and a fragment thereof, a virus-like particle (VLP); a live virus; a live, attenuated virus; an inactivated virus; a toxoid and a fragment thereof; and a combination thereof.

28. The method according to any one of claims 22-26, wherein the at least one therapeutic agent, at least one antigen or combination thereof comprises one or more antigens from a pathogenic virus, a pathogenic bacteria, a fungal pathogen; a peptide or polypeptide derived from a pathogenic virus, a pathogenic bacteria or fungal pathogen; a recombinant molecule derived from a pathogenic virus, a bacterial pathogen; an inactivated pathogenic virus, a fungal pathogen, a pathogenic protozoa, a pathogenic worm, a pathogenic yeast, any other pathogen and combinations thereof.

29. The method according to any one of claims 22-28, further comprising loading a therapeutic delivery device with the suspension composition for immediate or later administration to a subject.

30. The method according to claim 29, wherein no other composition is added to the suspension prior to loading the therapeutic delivery device.

31. The method according to any one of claims 22-30, further comprising storing the suspension composition at a temperature of about 4 °C up to about 50 °C up to an hour, up to a few hours, up to a day, up to a few days, up to one week up, up to two weeks, up to a month or up to several months or more.

32. The method according to any one of claims 22-31, wherein the ALD coating agent, metal agent, metal oxide, metal alkoxide, and aluminum-based coating layer comprises one or more of aluminum oxide (AI2O3), an aluminum alkoxide, silicon dioxide (SiCh), titanium dioxide (TiCh), and silicon nitride (SisN^, zinc oxide (ZnO), zirconia, hafnium oxide and mixtures and alternating layering pattern thereof.

33. The method according to any one of claims 22-32, further comprising at least a second therapeutic agent, antigen or agent deposited as a layer on an outer coating layer of the ALD coated microparticle and separated from the inner core by one or more coating layers.

34. The method according to claim 33, wherein the at least the second therapeutic agent, antigen or agent is the same or different than the at least one therapeutic agent, antigen or agent of the central or innermost core of the coated microparticle.

35. The method according to any one of claims 22-34, further comprising storing the suspension composition at a temperature below a non-aqueous suspending agent’s solidification temperature to reduce, avoid, or eliminate particle or microparticle settling.

36. The method according to claim 35, further comprising removing the suspension composition from the temperature below the non-aqueous suspending agent’s solidification temperature and warming the composition to a temperature above the non-aqueous suspending agent’s solidification temperature.

37. A method for treating, preventing, or reducing risk of onset of a health condition in a subject, the method comprising administering to the subject a composition according to any one of claims 1-20 or 21 to the subject.

38. The method according to claim 37, wherein the preventing or the reducing the risk of onset is prophylactic or therapeutic.

39. The method according to any one of claims 37-38, wherein the administering the composition comprises, administering the composition from a pre-filled syringe containing the composition.

40. The method according to any one of claims 37-39, wherein the health condition comprises having or suspected of developing an infection from a pathogenic organism.

41. The method according to any one of claims 37-39, wherein the health condition comprises cancer.

42. The method according to any one of claims 37-41, wherein the subject comprises a human or other mammal or animal.

43. The method according to any one of claims 37-41, wherein the subject comprises a pet, livestock, or other domesticated animal.

44. The method according any one of claims 37-41, wherein the subject comprises a human and the human comprises a neonate, an infant, a toddler, a child, an adolescent, a young adult, an adult, or an older adult subject.

45. A kit comprising a dried atomic layer deposition (ALD) coated microparticle comprising: a central or innermost therapeutic agent-containing microparticle comprising at least one therapeutic agent, at least one antigen or combination thereof and at least one glassforming agent; one or more ALD-applied coating layers comprising one or more of an ALD- coating agent comprising one or more of a metal agent, a metal oxide, a metal alkoxide, and an aluminum-based coating layer, other acceptable ALD coating agent, or combination thereof, covering the central or innermost at least one therapeutic agent, at least one antigen or combination thereof containing glassy microparticle in a container; and a non-aqueous suspension agent in at least a separate container.

46. The kit according to claim 45, wherein the at least one non-aqueous suspension agent comprises at least one of benzyl benzoate, 2-hydroxybenzoate (salicylate) esters, polyethylene glycols, polyoxyl 15 hydroxystearate, glycerin, sorbitan monolaurate, refined triglyceride mixtures, oil derivatives thereof or combinations thereof.

47. The kit according to claim 46, wherein the at least one refined triglyceride mixture or oil comprises at least one of refined sesame oil, refined soybean oil, refined corn oil, refined cottonseed oil, refined peanut oil or other refined triglyceride mixture or refined oil.

48. A kit comprising at least one composition according to any one of claims 1-21, and at least one container.

49. The kit according to claim 48, wherein the container further comprises at least one delivery device containing a composition according to any one of claims 1-21.

50. The kit according to claim 48 or 49, wherein the kit further comprises at least one syringe, or other injection device.

Images