Biosoluble microneedle arrays for effective skin immunity - metal-organic structures - vaccine biocomposites and their manufacture

Abstract

Aspects of a microneedle array containing a metal-organic structure (MOF)-vaccine biocomposite and a method for using the same are disclosed herein. The microneedle array further comprises a soluble material that dissolves when inserted into the skin of a subject, thereby releasing the MOF-vaccine biocomposite. The MOF may dissolve in an acidic environment, thereby being selected to target vaccine delivery to specific cellular compartments. Methods for preparing the MOF-vaccine biocomposite and the microneedle array are also disclosed.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of the earliest filing date of U.S. Provisional Patent Application No. 63 / 490,670, filed on 16 March 2023 (which is incorporated herein by reference in its entirety).

[0002] field This disclosure relates to a microneedle array containing a metal-organic structure for effective vaccination.

[0003] Acknowledgment of government support This invention was made with government support under grant number R01 AR079233, awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0004] background Vaccines provide a simple, safe, and effective method of achieving protection against disease (including preventing infection or infectious disease or reducing the severity of its symptoms). Vaccines are often administered by injection using subcutaneous needles, which poses significant challenges related to vaccine administration, immunogenicity, safety, and logistics for effective global immunization campaigns. Furthermore, each vaccine composition requires a specific formulation suitable for the specific antigen it contains. For example, solvents, buffers, stabilizers, and adjuvants must be selected so that the antigen does not react or degrade in the formulation before administration and provides potent immunogenicity. During vaccine delivery, certain antigens may adversely affect the extracellular space before the vaccine composition internalizes into the patient's cells to produce the desired immunological effect, and during vaccine storage, vaccine components may adversely affect due to environmental stressors. Therefore, there is a need for alternative vaccine formulation and delivery technologies that can overcome these problems associated with vaccine formulation, delivery, storage, and distribution. [Overview of the project] [Means for solving the problem]

[0005] Abstract A composite microneedle array that addresses issues associated with vaccine formulation, delivery, storage, and distribution is disclosed herein. In some aspects, the microneedle array comprises a base portion and a plurality of biocompatible and soluble microneedles extending from the base portion, wherein the microneedles comprise metal-organic framework (MOF) particles containing the vaccine composition. The MOF may be a zinc-based MOF (e.g., ZIF-8, ZIF-10, ZIF-90), a copper-based MOF (e.g., HKUST-1), an iron-based MOF (e.g., MIL-88), a magnesium-based MOF, a chromium-based MOF, a calcium-based MOF, a europium-based MOF, a bismuth-based MOF, a titanium-based MOF, a cobalt-based MOF, a nickel-based MOF, or a zirconium-based MOF.

[0006] In any given context, the above-mentioned MOF may be surface-activated with a surface coating containing, for example, PEG, PVA, PVP, hyaluronic acid, silk, or a combination thereof. And / or the above-mentioned MOF particles may have an average size distribution of 10 nm to 50,000 nm.

[0007] In some aspects, the base portion of the microneedle array lacks the MOF particles containing the vaccine composition. In other examples, both the microneedles and the base portion contain the MOF particles containing the vaccine composition. In certain aspects, the MOF particles containing the vaccine composition are integrated into the tip portion of the microneedles.

[0008] In some cases, the MOF particles containing the vaccine composition are pre-fabricated and then loaded into a mass production mold. However, in other cases, the MOF particles containing the vaccine composition are synthesized within the mass production mold.

[0009] In some aspects, the vaccine composition comprises antigens selected from proteins, mRNA, self-replicating mRNA, nucleic acid antigens, recombinant viral vector antigens, nucleic acid origami antigens, extracellular vesicle antigens, inactivated viral antigens, liposome-encapsulated antigens, extracellular vesicle-encapsulated antigens, cell lysate antigens, bacterial antigens, or combinations thereof. Furthermore, or alternatively, the vaccine composition comprises adjuvants which may be low-molecular-weight immune enhancers (e.g., saponins); STING pathway agonists; or nucleic acid and nucleic acid origami immune enhancers (e.g., single-stranded and / or double-stranded RNA and / or DNA-based innate immune agonists).

[0010] In some aspects, the microneedle array comprises a first plurality of MOF particles containing a first vaccine composition, and a second plurality of MOF particles containing a second vaccine composition. The first vaccine composition may contain a first antigen, and the second vaccine composition may contain a second antigen. However, in other aspects, the first vaccine composition may contain an antigen, while the second vaccine composition may contain an adjuvant.

[0011] In any configuration, each microneedle may further contain soluble biocompatible materials (such as CMC, CMC and trehalose, CMC and sucrose, CMC and maltodextrin, CMC and lactose, hyaluronic acid, chitosan, alginate, gantlets, PVP, PVA, PVP and PVA, gelatin, polylactic acid, pullulan, silk, polyphosphazene, poly-γ-glutamate, poly(lactic acid-co-glycolic acid), or combinations thereof).

[0012] Aspects of a method for fabricating a microneedle array comprising a metal-organic structure containing vaccine components (antigen and adjuvant) disclosed herein are also disclosed herein. In some aspects, the fabrication method comprises the steps of forming a solution comprising a soluble biocompatible material and a plurality of metal-organic structure (MOF) particles comprising the vaccine composition, and applying the solution to a microneedle array mold. However, in other aspects, the method comprises the step of forming the microneedle array by adding the solution comprising the soluble biocompatible material to a microneedle array mold comprising a plurality of metal-organic structure particles comprising the vaccine composition. The method may further comprise the step of loading the plurality of metal-organic structure particles comprising the vaccine composition into the mold before the step of adding the solution comprising the soluble biocompatible material. Alternatively, the method may comprise the step of loading precursors of the vaccine composition and the metal-organic structure into the mold before the step of adding the solution comprising the soluble biocompatible material to form the plurality of MOF particles comprising the vaccine composition in the mold.

[0013] Furthermore, aspects of a method for vaccinating a subject are disclosed herein. The above method may include the step of applying the microneedle array metal-organic structure vaccine biocomposite disclosed herein to a region of the subject to deliver the vaccine composition to the subject.

[0014] The aforementioned and other purposes, features, and advantages of this disclosure will become more apparent from the following detailed description, which will proceed with reference to the attached drawings. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows a transmission electron microscopy (TEM) image of a zeolitic imidazolate framework-8 (ZIF-8) particle in its initial state.

[0016] [Figure 2] Figure 2 provides the powder X-ray diffraction (PXRD) pattern of as-synthesized ZIF-8 particles.

[0017] [Figure 3] Figure 3 is a graph of weight vs. temperature showing the thermogravimetric analysis of as-synthesized ZIF-8 particles.

[0018] [Figure 4] Figure 4 is a graph of adsorption amount vs. relative pressure showing the nitrogen adsorption / desorption isotherm analysis of as-synthesized ZIF-8 particles.

[0019] [Figure 5] Figure 5 is a graph of cumulative pore volume and pore volume change vs. pore width showing the pore size distribution analysis of as-synthesized ZIF-8 particles, showing one pore measured at approximately 11 Å.

[0020] [Figure 6] Figure 6 is a schematic diagram showing that the pore size distribution conforms to a theoretical pore size of 12 Å.

[0021] [Figure 7A-C] Figures 7A - 7C provide transmission electron microscopy (TEM) images of as-synthesized ZIF-8 particles having sizes of 66 ± 20 nm (Figure 7A), 198 ± 38 nm (Figure 7B), and 763 ± 108 nm (Figure 7C).

[0022] [Figure 8] Figure 8 provides a transmission electron microscopy (TEM) image of ovalbumin (OVA) subunit vaccine-loaded ZIF-8 (OVA@ZIF-8) particles. [[ID=�9]]

[0023] [Figure 9] Figure 9 provides the powder X-ray diffraction (PXRD) pattern of ovalbumin (OVA) subunit vaccine-loaded ZIF-8 (OVA-loaded ZIF-8) particles.

[0024] [Figure 10] Figure 10 is a weight-to-temperature graph illustrating the thermogravimetric analysis of OVA-loaded ZIF-8 and unloaded ZIF-8 particles.

[0025] [Figure 11] Figure 11 is a graph of the amount of adsorption against relative pressure, illustrating the nitrogen adsorption / desorption isotherm analysis of OVA-loaded ZIF-8 and unloaded ZIF-8 particles.

[0026] [Figure 12] Figure 12 provides a digital image illustrating hydrophobic material (poly(dimethylsiloxane)-PDMS) fragments wetted with 5 μl of OVA AF647 (top row) and 7.5 μl of OVA AF647@ZIF-8 (bottom row).

[0027] [Figure 13] Figure 13 provides a digital image illustrating hydrophilic material (agarose) wetted with 5 μl of OVA AF647 (top row) and 7.5 μl of OVA AF647@ZIF-8 (bottom row).

[0028] [Figure 14] Figure 14 shows a transmission electron microscopy (TEM) image of Alexa Fluor 647-labeled ovalbumin vaccine-loaded zeolite-type imidazolate structure-8 (OVA AF647@ZIF-8), which has a size of 441±86 nm, a vaccine loading efficiency of 99.82%, and approximately 8% vaccine loading.

[0029] [Figure 15] Figure 15 is an optical microscopy image of a top view of a mass-production OVA AF647@ZIF-8 loaded MNA manufactured from plasma-treated poly(dimethylsiloxane) (PDMS).

[0030] [Figure 16]Figure 16 is an optical microscopy image of the OVA AF647@ZIF-8 loaded MNA mass production model.

[0031] [Figure 17] Figure 17 is an image obtained by optical stereomicroscopy of an obelisk-shaped carboxymethylcellulose (CMC) / trehalose microneedle (CMC / trehalose MNA-OVA AF647@ZIF-8 biocomposite) loaded with OVA AF647@ZIF-8.

[0032] [Figure 18] Figure 18 is an optical stereomicroscope image of an obelisk-shaped CMC / trehalose microneedle-OVA AF647@ZIF-8 biocomposite.

[0033] [Figure 19] Figure 19 shows a composite of bright-field and fluorescence microscopy images of an obelisk-shaped CMC / trehalose microneedle-OVA AF647@ZIF-8 biocomposite.

[0034] [Figure 20] Figure 20 provides optical stereomicroscopy images at different time points illustrating the obelisk-shaped CMC / trehalose MNA-OVA AF647@ZIF-8 biocomposite attached to 4% agarose.

[0035] [Figure 21] Figure 21 provides optical stereomicroscopy images at different time points illustrating obelisk-shaped OVA AF647-loaded CMC / trehalose MNA imparted to 4% agarose.

[0036] [Figure 22]Figure 22 is an optical stereomicroscope image of the fabricated obelisk-shaped carboxymethylcellulose (CMC) / trehalose microneedle array-zeolite-type imidazolate structure-8-Alexa Fluor 647-labeled ovalbumin biocomposite (OVA AF647@ZIF-8@CMC / trehalose MNA).

[0037] [Figure 23] Figure 23 is an optical stereomicroscope image of an obelisk-shaped carboxymethylcellulose (CMC) / trehalose microneedle array-zeolite-type imidazolate structure-8-Alexa Fluor 647-labeled ovalbumin biocomposite (OVA AF647@ZIF-8@CMC / trehalose MNA) after application to mouse skin for 20 minutes in vivo.

[0038] [Figure 24] Figure 24 shows the in vivo animal fluorescence imaging analysis of mice treated with OVA AF647@ZIF-8@CMC / trehalose MNA biocomposite.

[0039] [Figure 25] Figure 25 is an optical stereomicroscope image of the fabricated obelisk-shaped carboxymethylcellulose (CMC) / trehalose microneedle array-zeolite-type imidazolate structure-8-Alexa Fluor 647-labeled ovalbumin biocomposite (OVA AF647@ZIF-8@CMC / trehalose MNA).

[0040] [Figure 26] Figure 26 is a stereomicroscope image of the apical surface of a human skin explant after 5 minutes of treatment with OVA AF647@ZIF-8@CMC / trehalose MNA biocomposite.

[0041] [Figure 27]Figure 27 is an optical stereomicroscope image of the remaining material of the OVA AF647@ZIF-8@CMC / trehalose MNA biocomposite after 5 minutes of application to a human skin sample.

[0042] [Figure 28] Figure 28 shows the fluorescence imaging analysis of living human skin explants treated with OVA AF647@ZIF-8@CMC / trehalose MNA biocomposite using the IVIS system.

[0043] [Figure 29] Figure 29 shows epifluorescence microscopy images of frozen-sectioned human skin explants treated with OVA AF647@ZIF-8@CMC / trehalose MNA biocomposite. Blue: DAPI and Red: OVA AF647@ZIF-8.

[0044] [Figure 30] Figure 30 is a schematic diagram illustrating the immunization schedule of C57BL / 6 mice that received two doses of either CMC / trehalose MNA-delivered ovalbumin (OVA) or OVA@ZIF-8, two weeks apart. Five days after the booster dose, vaccine-induced humoral and cellular immune responses were evaluated by ELISA and in vivo lysis assays, respectively.

[0045] [Figure 31] Figure 31 is a graph of anti-OVA IgG levels in response to treatment, illustrating serum levels of OVA-specific IgG antibodies (mean ± SEM, N=3 mice / group).

[0046] [Figure 32] Figure 32 is a graph of the percentage of specific cytolysis for each treatment, illustrating the activity of OVA-specific cytotoxic T lymphocytes (CTLs) in different treatment groups.

[0047] [Figure 33A]Figure 33A is a transmission electron microscopy (TEM) image of ovalbumin subunit protein vaccine-loaded ZIF-8 (OVA@ZIF-8: size: 441±86 nm and concentration: 1000 μg / mL, loading efficiency: 99.82%).

[0048] [Figure 33B] Figure 33B is a transmission electron microscopy (TEM) image of ZIF-8 loaded with SARS-CoV-2 spike protein subunit vaccine (spike protein @ ZIF-8: size: 318 ± 66 nm and concentration: 140 μg / mL).

[0049] [Figure 33C] Figure 33C is a transmission electron microscopy (TEM) image of adenovirus 5-loaded ZIF-8 (Ad5@ZIF-8: size: 406±99 nm and concentration: 7.8 × 10¹⁰ vp / mL).

[0050] [Figure 33D] Figure 33D is a transmission electron microscopy (TEM) image of mRNA-loaded ZIF-8 (mRNA@ZIF-8: size: 454±107 nm and concentration: 67 μg / mL, loading efficiency: 97.91%).

[0051] [Figure 34] Figure 34 provides powder X-ray diffraction (PXRD) patterns of ZIF-8 integrating various types of vaccines from Figures 33A-33D.

[0052] [Figure 35] Figure 35 provides a frequency-as-particle-size graph illustrating the size distribution of biocalcified, biodegradable zeolite-type imidazolate structure-8 (ZIF-8) particles along with various biological agents.

[0053] [Figure 36]Figure 36 is a graph comparing the size of biocalcified, biodegradable zeolite-type imidazolate structure-8 (ZIF-8) particles with various biological agents, relative to vaccine-loaded MOFs.

[0054] [Figure 37] Figure 37 is an optical stereomicroscope image of a CMC / trehalose MNA integrating an Alexa Fluor 647-labeled ovalbumin (OVA)-loaded ZIF-8 (OVA AF647@ZIF-8).

[0055] [Figure 38] Figure 38 shows a composite of bright-field and fluorescence microscopy images of CMC / trehalose MNA integrating OVA AF647@ZIF-8.

[0056] [Figure 39] Figure 39 is an optical stereomicroscope image of CMC / trehalose MNA incorporating SARS-CoV-2 spike subunit protein vaccine-loaded ZIF-8 (S protein@ZIF-8).

[0057] [Figure 40] Figure 40 is an integrated image of CMC / trehalose MNA integrating Alexa Fluor 647-labeled SARS-CoV-2 S1 subunit vaccine-loaded ZIF-8 (S1 AF647@ZIF-8) using bright-field and fluorescence microscopy.

[0058] [Figure 41] Figure 41 is an optical microscope image illustrating CMC / trehalose MNA loaded with ZIF-8 (Ad5.eEGP@ZIF-8), which integrates adenovirus 5 encoding enhanced green fluorescent protein.

[0059] [Figure 42]Figure 42 is an optical microscope image illustrating CMC / trehalose MNA loaded with ZIF-8 (mRNA.Luciferase@ZIF-8), which incorporates an mRNA vaccine encoding luciferase.

[0060] [Figure 43] Figure 43 shows the in vivo animal fluorescence imaging analysis of mice treated with SARS-CoV-2 S1 AF647@ZIF-8@CMC / trehalose MNA biocomposite.

[0061] [Figure 44] Figure 44 shows the fluorescence imaging analysis of living human skin explants treated with SARS-CoV-2 S1 AF647@ZIF-8@CMC / trehalose MNA biocomposite using the IVIS system.

[0062] [Figure 45] Figure 45 is a bright-field microscopy image of OVA AF647@ZIF-8-poly(I:C)rhodamine@CMC / trehalose biocomposite on a PMMA stem.

[0063] [Figure 46] Figure 46 shows fluorescence microscopy images of OVA AF647@ZIF-8-Poly(I:C) rhodamine@CMC / trehalose biocomposite on a PMMA stem using a filter corresponding to rhodamine.

[0064] [Figure 47] Figure 47 shows fluorescence microscopy images of OVA AF647@ZIF-8-poly(I:C)rhodamine@CMC / trehalose biocomposite on a PMMA stem using a filter corresponding to AF647.

[0065] [Figure 48] Figure 48 is an integrated image of Figures 45-47 obtained by microscopic examination.

[0066] [Figure 49] Figure 49 is an optical stereomicroscope image of the OVA AF647@ZIF-8-poly(I:C)rhodamine@CMC / trehalose biocomposite on a PMMA stem after skin application.

[0067] [Figure 50] Figure 50 shows a bright-field microscopy image of the OVA AF647@ZIF-8-poly(I:C)rhodamine@CMC / trehalose biocomposite on a PMMA stem after skin application.

[0068] [Figure 51] Figure 51 shows the in vivo animal fluorescence imaging analysis of OVA AF647@ZIF-8-poly(I:C)rhodamine@CMC / trehalose on PMMA stem MNA biocomposite-treated mice using the IVIS system, illustrating rhodamine fluorescence.

[0069] [Figure 52] Figure 52 shows the in vivo animal fluorescence imaging analysis of OVA AF647@ZIF-8-poly(I:C)rhodamine@CMC / trehalose on PMMA stem MNA biocomposite treated mice using the IVIS system, illustrating the fluorescence of AF647.

[0070] [Figure 53] Figure 53 shows fluorescence imaging analysis of OVA AF647@ZIF-8-poly(I:C)rhodamine@CMC / trehalose on PMMA stem MNA biocomposite-treated living human skin explants using the IVIS system, illustrating rhodamine fluorescence.

[0071] [Figure 54]Figure 54 shows fluorescence imaging analysis of OVA AF647@ZIF-8-poly(I:C)rhodamine@CMC / trehalose on PMMA stem MNA biocomposite-treated living human skin explants using the IVIS system, illustrating AF647 fluorescence.

[0072] [Figure 55A] Figure 55A is a fluorescence microscopy image of DC 2.4 cells transfected with mRNA.mCherry recovered from mRNA@ZIF-8.

[0073] [Figure 55B] Figure 55B shows the flow cytometry analysis of DC 2.4 cells transfected with mRNA.mCherry recovered from mRNA@ZIF-8.

[0074] [Figure 56] Figure 56 is a graph of anti-SARS-CoV-2 S1 IgG levels in response to treatment, illustrating serum levels of SARS-CoV-2-S1 specific IgG antibodies (N=5 mice / group).

[0075] [Figure 57A] Figure 57A is a scanning electron microscope (SEM) image of Hendra virus soluble glycoprotein (GP) loaded ZIF-8 (HeV-sG@ZIF-8).

[0076] [Figure 57B] Figure 57B is a scanning electron microscopy (SEM) image of Hendra virus soluble glycoprotein (GP) + poly(I:C) loaded ZIF-8 (HeV-sG + poly(I:C)@ZIF-8).

[0077] [Figure 57C] Figure 57C is a graph of anti-HeV-sG IgG levels in response to treatment, illustrating serum levels of HeV-sG-specific IgG antibody (N=5 mice / group).

[0078] [Figure 58A] Figure 58A shows a series of representative photographs of mouse skin treated with HeVsG + poly(I:C)@ZIF-8 loaded MNA.

[0079] [Figure 58B] Figure 58B shows a series of body weight measurements of mice treated with HeVsG+ poly(I:C)@ZIF-8 loaded MNA (N=5 mice).

[0080] [Figure 58C] Figure 58C shows a series of body temperature measurements in mice treated with HeVsG+ poly(I:C)@ZIF-8 loaded MNA (N=5 mice). [Modes for carrying out the invention]

[0081] Detailed explanation I. Definitions and Terms

[0082] The singular forms “a” (one, a), “an” (one, a), and “the” (the above, this, that) refer to one or more things unless the context otherwise explicitly states. The term “or” refers to a single element or a combination of two or more elements from the alternative elements mentioned unless the context otherwise explicitly states. As used herein, “comprises” means “includes.”

[0083] Unless otherwise indicated, all numbers representing quantities such as components, molecular weights, percentages, temperatures, and times should be understood to be modified by the term “about” when used herein or in the claims. Unless otherwise indicated by context, “about” refers to ±5% of the reference value. For example, “about” 100 refers to 95–105.

[0084] Unless otherwise stated, all technical and scientific terms shall be used in accordance with their conventional usage. Methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure, but suitable methods and materials are described below. These materials, methods, and examples are illustrative and not intended to be limiting.

[0085] The term "adjuvant" refers to any component added to a vaccine that modifies its effects (e.g., enhancing the immune response and / or stabilizing the formulation). Adjuvants are often pharmacological and / or immunological agents. In some contexts, adjuvants are small molecule immune enhancers (e.g., saponins); STING pathway agonists; or nucleic acid and nucleic acid origami immune enhancers (e.g., single-stranded and / or double-stranded RNA and / or DNA-based innate immune agonists).

[0086] The term “effective amount” refers to a sufficient quantity of vaccine composition to provide the desired outcome (e.g., to provide or enhance an immune response and / or provide protection from a pathogen). It is understood that multiple doses of the disclosed vaccine composition, and / or administration of an alternative vaccine composition targeted to the pathogen, may be required to obtain a protective immune response against the pathogen of interest. Accordingly, the vaccine compositions of this disclosure may be used as the “prime” component or the “boost” component (or both) of a prime-boost immune protocol. The amount of vaccine composition constituting the “effective amount” will vary depending on the vaccine composition, the disease, the nature and age of the subject, etc.

[0087] The term "dissolvable microneedle" refers to a microscale needle made from a water-soluble or biodegradable bio-based material, or a microscale needle made from a water-soluble or biodegradable bio-based material integrated with a non-soluble and non-degradable biocompatible stem region.

[0088] The terms "subject" or "patient" refer to mammals and other animals, particularly humans. Therefore, the disclosed technologies are applicable to both human therapeutic and veterinary applications.

[0089] II. Overview

[0090] Dissolvable microneedle arrays enable efficient and safe vaccine delivery to their targeted skin microenvironment and mucosal surfaces. However, vaccines incorporated into dissolvable microneedle arrays can be adversely affected by chemical and thermal, biological, and environmental stressors during manufacturing, skin delivery (e.g., extracellular enzymes), and storage and distribution, respectively. Therefore, each vaccine requires specific formulation to achieve, typically, thermal, chemical, and / or biological stability. This can increase the cost and complexity of vaccine formulation preparation. Aspects of metal-organic frameworks (MOFs)-vaccine biocomposites suitable for use in dissolvable microneedle arrays are disclosed herein. The MOFs provide chemical and thermal stability of the vaccine during manufacturing, storage, and distribution of the dissolvable microneedle array. Furthermore, the MOFs provide protection for the vaccine in the extracellular space but degrade at appropriate cellular locations to release the vaccine. Importantly, the above-mentioned MOF provides vaccine components suitable for use in a wide range of vaccine types (including, but not limited to, protein vaccines, polysaccharide vaccines, mRNA vaccines, self-replicating mRNA vaccines, nucleic acid vaccines, inactivated viral vaccines, cell lysate vaccines, viral vector vaccines, and bacterial vaccines), as well as standardized delivery vehicles for various adjuvant types (including, but not limited to, nucleic acid adjuvants and small molecule adjuvants).

[0091] III. MOF-Vaccine Biocomposites

[0092] The MOF in the above biocomposite may be any MOF suitable for use in a microneedle array and suitable for use with a vaccine composition. Examples of MOFs include, but are not limited to, zeolite-type imidazolate structures (ZIFs) (e.g., ZIF-8, ZIF-10, ZIF-90), iron-based MOFs (e.g., MIL-53 and MIL-88), copper-based MOFs (e.g., HKUST-1), and other MOFs (e.g., MAF-7, Eu / Tb-BDC, magnesium-based MOFs, chromium-based MOFs, calcium-based MOFs, europium-based MOFs, bismuth-based MOFs, titanium-based MOFs, cobalt-based MOFs, nickel-based MOFs, or zirconium-based MOFs).

[0093] In some aspects, the MOF is selected to have reaction synthesis conditions compatible with the vaccine composition contained within the MOF. Suitable reaction conditions include, but are not limited to, reaction temperature, solvent, and / or reagents compatible with the vaccine composition so that the vaccine composition does not substantially decompose during MOF formation.

[0094] In some cases, the above-mentioned MOFs are stable in the extracellular space, but are selected to dissolve once they enter a cell. In some cases, the above-mentioned MOFs dissolve in an acidic pH environment (e.g., pH less than 7 or 6.5 or less), for example, in an environment with a pH between less than 7 and 5, for example, between 6.5 and 5.

[0095] The above MOF-vaccine biocomposites also contain one or more vaccine components. In some cases, the above vaccine components may be antigens, such as proteins, mRNA, self-replicating mRNA, nucleic acid antigens, recombinant viral vectorized antigens, nucleic acid origami antigens, extracellular vesicle antigens, inactivated viral antigens, liposome-encapsulated antigens, extracellular vesicle-encapsulated antigens, cell lysate antigens, bacterial antigens, or combinations thereof. In some cases, multiple antigens may be encapsulated in the same MOF. In some cases, different antigens may be encapsulated in different MOFs.

[0096] In some aspects, the vaccine antigens described above are derived from pathogens (including viruses, parasites, cardiomyocytes, and bacteria). In some contexts, the above pathogens include: viruses (e.g., viruses originating from one of the following families, but not limited to these: retroviridae (e.g., human immunodeficiency virus (HIV); human T-cell leukemia virus (HTLV)); picornaviridae (e.g., poliovirus, hepatitis A virus; hepatitis C virus; enterovirus, human coxsackievirus, rhinovirus, echovirus; foot-and-mouth disease virus); caliciviridae (e.g., gastroenteritis strains); togaviridae (e.g., equine encephalitis virus, rubella virus); flaviviridae (e.g., dengue virus; yellow fever virus; West Nile virus; St. Louis encephalitis virus; Japanese encephalitis virus; and other encephalitis viruses); coronavirusidae (e.g., coronavirus; severe acute respiratory syndrome (SARS) virus); rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); filoviridae (e.g., Ebola virus); paramyxoviridae (e.g., paramyxoviridae) Influenza virus, mumps virus, measles virus, respiratory syncytial virus (RSV); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., Hunter virus; Sin Nombre virus, Rift Valley fever virus; Bunyaviridae virus, Phlebovirus and Nairovirus); Arenaviridae (hemorrhagic fever virus; Machupovirus; Junin virus); Reoviridae (e.g., Reovirus, Orbivirus and Rotavirus); Birnaviridae; Hepadnaviridae (B Hepatitis viruses; Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses; BK viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV)-1 and HSV-2; cytomegalovirus (CMV); Epstein-Barr virus (EBV); varicella-zoster virus (VZV); and other herpesviruses (including HSV-6)); Poxviridae (smallpox virus, vaccinia virus, poxviruses);and Iridoviridae (e.g., African swine fever virus); Filoviridae (e.g., Ebola virus; Marburg virus); Caritiviridae (e.g., Norwalk virus); and unclassified viruses (e.g., causative agents of spongiform encephalopathy, agents of hepatitis delta (considered to be a defective satellite of hepatitis B virus); and astroviruses).

[0097] In other contexts, the above target antigens include, but are not limited to, antigens derived from bacteria such as Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter sp., and Enterococcus. sp., Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, or Actinomyces israelli.

[0098] In a further context, the antigens are derived from fungi (e.g., Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans). In other contexts, the antigens are derived from parasites (e.g., Plasmodium falciparum or Toxoplasma gondii, but not limited to these).

[0099] In some aspects, the above antigen is a cancer antigen. The above cancer may be a solid tumor or a hematogenous cancer. In certain cases, the above solid tumor may be a sarcoma or carcinoma (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, or another sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, These include papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular cancer, bladder cancer, or central nervous system tumors (e.g., glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, or retinoblastoma).

[0100] In some contexts, the hematogenous cancers mentioned above include leukemia, e.g., acute leukemia (e.g., acute lymphoblastic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphoblastic leukemia); polycythemia vera; lymphoma; Hodgkin's disease; non-Hodgkin lymphoma (painless and high-grade forms); multiple myeloma; Waldenström's hypergammaglobulinemia; heavy chain disease; myelodysplastic syndrome; hairy cell leukemia; or myelodysplasia.

[0101] Tumor antigens are known in the field and include, for example, carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), alpha-fetoprotein (AFP), lectin-reactive AFP (AFP-L3), thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase (hTERT), RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, protease, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivorbin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), melanoma-associated antigen (MAGE), ELF2M, neutrophil elastase, ephrin B2 and CD22. CH2 or CH3 domain molecules can also bind to any cancer-related protein (e.g., IGF-I, IGF-II, IGR-IR, or mesoserine).

[0102] In some contexts, the above antigen is an autoantigen. The above antigens may be associated with autoimmune diseases (e.g., rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjögren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus urinaria, psoriasis, vitiligo, type 1 diabetes, non-obesity diabetes, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosus, autoimmune thrombocytopenia purpura, Goodpasture syndrome, Addison's disease, systemic scleroderma, polymyositis, dermatomyositis, autoimmune hemolytic anemia, or pernicious anemia).

[0103] Furthermore, or alternatively, the vaccine components may include an adjuvant. The adjuvant may be single-stranded RNA, double-stranded RNA, single-stranded DNA, synthetic polynucleotides, liposomes, extracellular vesicles, aluminum salts, bacterial lipopolysaccharides, polyphosphazenes, STING, TLR, CLR, and RLR agonists. In some cases, the adjuvant may be encapsulated in a MOF that similarly encapsulates the antigen. In some cases, the adjuvant is adsorbed onto the surface of the MOF that encapsulates the antigen. In some cases, the adjuvant is encapsulated in a MOF different from the MOF that encapsulates the antigen. In some cases, the adjuvant is adsorbed onto a MOF different from the MOF that encapsulates the antigen.

[0104] Typically, the pores in the above-mentioned MOF are too small to allow the vaccine components to enter and exit the MOF structure. Therefore, the above-mentioned MOF is typically synthesized in the presence of the vaccine components in such a way that the MOF surrounds the vaccine components, forming a nucleus, thereby encapsulating the components within the MOF.

[0105] The above MOF-vaccine biocomposites are formed as particles (e.g., nanoparticles or microparticles). In some examples, the above MOF-vaccine biocomposite particles have an average size of 10 nm to 50,000 nm (e.g., 10 nm to 10,000 nm, 10 nm to 5,000 nm, 10 nm to 2,000 nm, 10 nm to 100 nm, 200 nm to 900 nm, or 1,000 nm to 2,000 nm). As used herein, the particle size refers to the longest dimension of the particle. In some cases, MOFs with larger pore sizes (i.e., larger than the size of the vaccine components) are synthesized, and the vaccine components can be loaded after synthesis.

[0106] IV. Microneedle array containing the above-mentioned MOF-vaccine biocomposites

[0107] A section of a microneedle array containing the above-mentioned MOF-vaccine biocomposite particles is disclosed herein. In some sections, the microneedle array comprises a soluble biocompatible material forming the needles and containing the MOF-vaccine biocomposite particles. In some sections, the MOF particles are located within the needles and / or on the surface of the needles. The soluble biocompatible material is non-toxic and dissolves or decomposes under the physiological conditions of the skin. Suitable soluble biocompatible materials include, but are not limited to, carboxymethylcellulose (CMC), CMC and trehalose, CMC and sucrose, CMC and maltodextrin, CMC and lactose, hyaluronic acid, chitosan, alginate, gantlets, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), poly(vinyl alcohol) (PVA), PVP and PVA, gelatin, polylactic acid, pullulan, silk, polyphosphazene, poly-γ-glutamate, poly(lactic acid-co-glycolic acid), or combinations thereof.

[0108] In some cases, the soluble substance is selected to dissolve at least partially in the skin microenvironment or in mucosal tissue, releasing at least a portion of the MOF-vaccine biocomposite.

[0109] In some aspects, the microneedles in the above array state have a size and shape suitable for facilitating vaccine delivery to the subject.

[0110] In some aspects, the microneedle array includes microneedles having a length (height from base layer to tip) of 50 μm to 1000 μm (e.g., 300 μm to 750 μm). In some aspects, the base layer lacks the MOF particles containing the vaccine composition. In some aspects, the microneedles have a base diameter or width of 50 μm to 500 μm and a taper toward the tip. In some aspects, the taper starts at the base, thereby forming a pyramidal or conical needle. However, in other aspects, the taper starts partway along the microneedle (e.g., at about 20%, 30%, 40%, 50%, 60%, or 70% along the microneedle from the base). In such examples, the microneedle includes a support structure extending from the base, and the tapered portion of the microneedle forms a pyramidal or conical structure located at the end of the support structure. See, for example, Figures 15-18, 21, and 37-42. As can be seen from the above, the microneedles may be substantially circular, or they may be rectangular, quadrilateral, triangular, trapezoidal, polygonal, or irregular in shape.

[0111] In some cases, the microneedle array has a sufficient number of needles to deliver a beneficial amount of the vaccine composition to a patient. In some cases, the microneedle array has 5 to 1000 needles / (5mm-30mm)×(5mm-30mm) arrays (e.g., 50-150 needles / 10mm×10mm array, 100-400 needles / 20mm×20mm array, 250-750 needles / 25mm×25mm array, or approximately 1000 needles / 30mm×30mm array).

[0112] In the above array, the microneedles are separated from each other by a distance suitable for facilitating the delivery of an effective amount of the vaccine to the subject. In some aspects, the microneedles are arranged in a regular pattern (e.g., in parallel and perpendicular lines) (Figure 15). In some aspects, the microneedles are arranged such that the gap between two needles is 100 μm to 1000 μm, for example, 250 μm to 750 μm.

[0113] In some cases, the MOF-vaccine biocomposite is located throughout the entire microneedle, while in other cases, the MOF-vaccine biocomposite is substantially located in the tapered portion of the microneedle. In some cases, more than 50% of the MOF-vaccine biocomposite is located in the tapered portion of the microneedle (e.g., more than 60%, 70%, 80%, 90%, 95%, 97%, or 99% of the MOF-vaccine biocomposite) (Figures 17, 18, and 19).

[0114] V. Preparation of Master and Production Molds

[0115] The master model of the above microneedle array-MOF-vaccine biocomposite is prepared using a microfabrication strategy (including, but not limited to, 3D laser printing, photolithography, dry etching, or microfabrication) to define the microneedle and array geometry, as well as the dimensions of the microneedles and their spatial distribution across the array.

[0116] For example, a microneedle array can be manufactured based on a methodology from a master mold (positive) to a production mold (negative) and then back to the array (positive). Micromilling technology can be used to generate various microscale geometries for various materials, including metals, polymers, and ceramic components. Micromilled master molds of various shapes and configurations can be effectively used to produce a large number of identical female production molds. These female production molds can then be used to microcast various microneedle arrays. In some cases, these female production molds can be used to replicate the master mold using UV-curable resin.

[0117] The master mold described above is preferably formed from a reusable material so that a single master mold can be repeatedly used to produce a large number of production molds. Similarly, each production mold can preferably produce a large number of microneedle arrays.

[0118] Master molds can be micro-milled from various materials (e.g., stainless steel, brass, aluminum, PEEK, PMMA, Cirlex® (DuPont, Kapton® polyimide)). The master mold material can preferably be clearly separated from the mass-production mold material and preferably withstand any high curing temperature that may be required to cure the mass-production mold material. For example, in the illustrated plane, SYLGARD® 184 (Dow Corning), a silicone-based compound, is the mass-production mold material, which may require a curing temperature of about 80-90°C.

[0119] Alternatively, the master mold can be prepared using 3D laser printing. The master mold can be fabricated from IP-S photoresist by 3D direct laser writing. IP-S is a special material designed for 3D laser lithography, providing high resolution and mechanical integrity for microstructures and nanostructures. 3D laser lithography based on two-photon polymerization provides an effective means for fabricating microneedle array designs that have smooth edges and sharp tips and are free from any undesirable residues (e.g., machining tips).

[0120] Further information relating to the preparation of the master mold can be found in U.S. Patent Application Nos. 2016 / 0136407 and 2022 / 0241570, both of which are incorporated herein by reference in their entirety.

[0121] Master molds can be used to produce flexible mass-production molds from suitable materials (e.g., EcoFlex (hydrophobic), polydimethylsiloxane (PDMS, hydrophobic), plasma-treated PDMS (more hydrophilic than PDMS), copolymer of PDMS and poly(ethylene glycol) (PEG) (more hydrophilic than PDMS), and agarose (hydrophilic). In some cases, plasma-treated PDMS or PDMS-PEG is used.

[0122] In some aspects, the material for the mass-production type is selected to have surface properties that are compatible with the MOF and / or vaccine composition. These surface properties include, but are not limited to, surface interactions (e.g., hydrophobic and / or hydrophilic interactions) between the type material and the MOF and / or vaccine composition. In some aspects, the mass-production type material is selected to have the desired hydrophobicity or hydrophilicity to localize the vaccine-loaded MOF in the microneedle-shaped wells of a microneedle array type.

[0123] Furthermore, in the process of preparing the MOF-vaccine biocomposite within the mold, the mold material may be selected to reduce the interaction between the vaccine composition and the mold material before the MOF-vaccine biocomposite is formed.

[0124] VI. Manufacturing of Microneedle Array-MOF-Vaccine Biocomposites

[0125] In some aspects, the microneedle array containing the MOF-vaccine biocomposite is prepared by first forming the MOF-vaccine biocomposite, and then forming the microneedle array containing the MOF-vaccine biocomposite. In some aspects, the MOF-vaccine biocomposite is prepared outside the microneedle array mold. In such aspects, a solution containing the MOF starting material and the vaccine composition is formed. The solution is formed in an aqueous solvent (e.g., water). The solution is stirred by stirring, shaking and / or vortexing, and after formation, the MOF-vaccine biocomposite is separated from the solution. The MOF-vaccine is then resuspended in a suitable suspension, and the suspension is added to the mass production mold. The suspension may contain water-soluble hydrophilic bio-derived materials and / or surfactants (e.g., carboxymethylcellulose (CMC), CMC and trehalose, CMC and sucrose, CMC and maltodextrin, CMC and lactose, hyaluronic acid, chitosan, alginate, gantlets, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), PVP and PVA), PEG, PVP, PVA, PVP and PVA, gelatin, pullulan, silk, polyphosphazene, poly-γ-glutamate, and / or nucleic acid adjuvants (poly(I:C) or CpG). In some aspects, the suspension may be selected to coat the MOF-vaccine biocomposite particles with a hydrophilic compound to overcome any hydrophobic / hydrophobic interactions and / or charge interactions with the mass-production type.

[0126] Furthermore, or alternatively, the suspension may contain an organic solvent that is water-soluble (e.g., alcohol (e.g., methanol, ethanol, isopropanol, or a combination thereof), acetonitrile, or a combination thereof).

[0127] After adding the above MOF-vaccine biocomposite to the above mold, the soluble / structural material of the array is added to form the above microneedles and the above backing layer.

[0128] In an alternative scenario, the MOF components and the vaccine composition are added directly to the mass-production mold, and the MOF-vaccine biocomposite is formed in situ. Once the MOF-vaccine biocomposite is formed, the soluble / structural material of the array is added to form the microneedles and the backing layer.

[0129] In some aspects, the microneedle array includes microneedles having a length (height from base layer to tip) of 50 μm to 1000 μm (e.g., 300 μm to 750 μm). In some aspects, the base layer lacks the MOF particles containing the vaccine composition. In some aspects, the microneedles have a base diameter or width of 50 μm to 500 μm and taper toward a certain point to form the needle tip. In some aspects, the taper starts at the base, thereby forming a pyramidal or conical needle. However, in other aspects, the taper starts partway along the microneedle (e.g., at about 20%, 30%, 40%, 50%, 60%, or 70% along the microneedle from the base). In such an example, the microneedle includes a support structure extending from the base, and the tapered portion of the microneedle forms a pyramidal or conical structure located at the end of the support structure. As can be seen from the above, the microneedles may be substantially circular, or they may be rectangular, quadrilateral, triangular, trapezoidal, polygonal, or irregular in shape.

[0130] In some cases, the microneedle array has a sufficient number of needles to provide a beneficial amount of the vaccine composition to a patient. In some cases, the microneedle array has 5 to 1000 needles / (5mm-30mm)×(5mm-30mm) arrays (for example, 50-150 needles / 10mm×10mm array, 100-400 needles / 20mm×20mm array, 250-750 needles / 25mm×25mm array, or approximately 1000 needles / 30mm×30mm array).

[0131] In the above array, the microneedles are separated from each other by a distance suitable for facilitating the delivery of an effective amount of the vaccine to the subject. In some aspects, the microneedles are arranged in a regular pattern (e.g., in parallel and perpendicular lines) (Figure 15). In some aspects, the microneedles are arranged such that the gap between two needles is 100 μm to 1000 μm, for example, 250 μm to 750 μm.

[0132] In some cases, the MOF-vaccine biocomposite is located throughout the entire microneedle, while in other cases, the MOF-vaccine biocomposite is substantially located in the tapered portion of the microneedle. In some cases, more than 50% of the MOF-vaccine biocomposite is located in the tapered portion of the microneedle (for example, more than 60%, 70%, 80%, 90%, 95%, 97%, or 99% of the MOF-vaccine biocomposite).

[0133] VII. Use of the above microneedle array-MOF-vaccine biocomposites

[0134] The microneedle array-MOF-vaccine biocomposites of this disclosure are useful for delivering vaccines to subjects in need. Typically, the microneedle array is applied to a site on the subject's skin, for example, in the form of a patch. Suitable sites on the patient include, but are not limited to, the subject's arms, legs, chest, back, or neck, or mucosal surfaces. The patch is applied for a suitable period of time to facilitate the delivery of an effective amount of the MOF-vaccine biocomposites. In some cases, the patch is applied to the skin for a period longer than zero but up to 24 hours (e.g., 1 to 2 hours, 1 to 30 minutes). [Examples]

[0135] VIII. Examples Example 1: Synthesis of zeolite-type imidazolate structure-8 (ZIF-8)

[0136] A 3M solution of 2-methylimidazole was prepared by sonication of a mixture of 246.3 mg, 3 mmol of 2-methylimidazole in 1 mL of nuclease-free water (H2O). A 1M solution of zinc acetate dihydrate was prepared by sonication of a mixture of 183.5 mg, 1 mmol of zinc acetate dihydrate in 1 mL of nuclease-free water. 853 μL, 2.56 mmol of the 3M 2-methylimidazole solution and 67 μL of nuclease-free water were sequentially added to a 1.5 mL Eppendorf microcentrifuge tube, and the tube was vortexed for 10 seconds. 80 μL, 0.08 mmol of 1M zinc acetate dihydrate was added to the tube, and the tube was vortexed again for 30 seconds. The solution was reacted at room temperature or lower for 1 hour or less to obtain a turbid solution. The suspension was centrifuged at 10,000 rpm for 2 minutes. The precipitate was washed with nuclease-free water (2 mL, 2 ×) and ethanol or methanol (2 mL, 2 ×), dried under vacuum, and the original ZIF-8 crystals were obtained as a white or colored (in the case of fluorescently labeled biocargo) precipitate.

[0137] Transmission electron microscopy (TEM, A JEOL JEM2100F) Analysis by [method / analysis] demonstrated the successful formation of polydisperse ZIF-8 particles with an average size distribution of 468±92 nm (10⁴ ZIF-8 particles were measured) (Figure 1).

[0138] Powder X-ray diffraction (PXRD, A Bruker AXS D8) Discover X-Ray Powder Diffractometer) Analysis suggests that the PXRD pattern of the ZIF-8 particles closely matches the results of the above simulation, indicating that the successfully synthesized ZIF-8 particles are highly crystalline and phase-pure (Figure 2).

[0139] Thermogravimetric analysis (TGA, TA Instruments Q500 Thermal Analysis System The thermal stability and decomposition profile of ZIF-8 were shown, suggesting that the initial ZIF-8 particles are thermally stable up to 550°C (at which point the ZIF-8 particles began to decompose) (Figure 3).

[0140] Nitrogen adsorption / desorption isotherm (Micromeritics 3Flex Gas Absorption Analyzer) Therefore, the initial ZIF-8 particles are highly porous, and are fused to type I isotherms and approximately 2,070 m 2 It was shown to have a Brunauer-Emmett-Teller (BET) surface area of ​​1 / g. Analysis of numerous ZIF-8 samples using the aforementioned technique demonstrated the reproducibility of an easily obtainable, scalable MOF synthesis protocol (Figure 4).

[0141] Pore ​​size distribution analysis From this, a pore size of 11 Å (1.1 nm) was indicated. This fit well with a theoretical pore size of 12 Å (Figures 5 and 6). The relative sizes of the vaccine antigen / adjuvant and the ZIF-8 pores suggested that the vaccine might be large enough to be loaded into the ZIF-8 pore network. However, vaccine components could be loaded into mesopores within the ZIF-8 bulk crystal via biomimetic calcification.

[0142] Transmission electron microscopy (TEM, A JEOL JEM2100F)Analysis by [method / analysis] demonstrated the successful formation of ZIF-8 particles with adjustable sizes (Figure 7A: 66±20 nm, Figure 7B: 198±38 nm, and Figure 7C: 763±108 nm).

[0143] Example 2: Synthesis of vaccine-loaded zeolite-type imidazolate structure-8 (ZIF-8) A 3M solution of 2-methylimidazole was prepared by sonication of a mixture of 246.3 mg, 3 mmol of 2-methylimidazole in 1 mL of nuclease-free water (H2O). A 1M solution of zinc acetate dihydrate was prepared by sonication of a mixture of 183.5 mg, 1 mmol of zinc acetate dihydrate in 1 mL of nuclease-free water. An ovalbumin (OVA) vaccine solution was prepared by mixing 25.6 mg of OVA with 1 mL of nuclease-free water. The 3M 2-methylimidazole solution (853 μL, 2.56 mmol), nuclease-free water (28 μl), and OVA solution (39 μl, 1 mg) were sequentially added to a 1.5 mL Eppendorf microcentrifuge tube, and the tube was vortexed for 10 seconds. Next, 1 M zinc acetate dihydrate (80 μL, 0.08 mmol) was added, and the tube was vortexed again for 30 seconds. The solution was allowed to react at room temperature or lower for 1 hour or less to obtain a turbid solution. The suspension was centrifuged at 10,000 rpm for 2 minutes. The precipitate was washed with nuclease-free water (2 mL, 2 ×) and ethanol or methanol (2 mL, 2 ×), and dried under vacuum to obtain subunit vaccine @ZIF-8 crystals as a precipitate.

[0144] Transmission electron microscopy (TEM, A JEOL JEM2100F) Analysis revealed the successful formation of vaccine-loaded ZIF-8 particles with an average size distribution of 441±86 nm (101 OVA@ZIF-8 particles were measured) (Figure 8). There was no significant difference between the size of the initial ZIF-8 and the size of the OVA@ZIF-8.

[0145] Powder X-ray diffraction (PXRD, A Bruker AXS D8 Discover X-Ray Powder Diffractometer)Analysis showed that the pattern of OVA@ZIF-8 matches the results of the above simulation. This indicates that the OVA@ZIF-8 particles are highly crystalline and phase-pure (Figure 9).

[0146] Thermogravimetric analysis (TGA, TA Instruments Q500 Thermal Analysis System) The thermal stability and degradation profile of OVA@ZIF-8 particles were shown, suggesting that OVA@ZIF-8 particles are thermally stable up to approximately 250°C (where OVA@ZIF-8 particles degrade due to the degradation of OVA) (Figure 10). These results suggest that OVA@ZIF-8 particles degrade at a lower temperature than the original ZIF-8, but the failure temperature is significantly higher than the temperature predicted during vaccine storage and distribution.

[0147] The vaccine loading efficiency (99.82%) of OVA@ZIF-8 particles was calculated as follows: ((Initial mass of vaccine - Residual mass of vaccine in supernatant) / Initial mass of vaccine) × 100).

[0148] The vaccine loading capacity (8%) of OVA@ZIF-8 particles was calculated as follows: (Loading efficiency × Initial vaccine mass) / (Mass of MOF particles) × 100).

[0149] These results were consistent with the weight reduction observed in thermogravimetric analysis of OVA@ZIF-8 at high temperatures, which is attributed to the decomposition of OVA from ZIF-8.

[0150] Nitrogen adsorption / desorption isotherm (Micromeritics 3Flex Gas Absorption Analyzer) Therefore, the OVA@ZIF-8 particles exhibit type I isotherms similar to those of the original ZIF-8 particles, and as predicted, the OVA@ZIF-8 particles have lower porosity than the original ZIF-8 particles, due to vaccine loading, approximately 1,850 m 2 It was shown to have a Brunauer-Emmett-Teller (BET) surface area of ​​1 / g (Figure 11).

[0151] Example 3: Synthesis of a microneedle array-metal-organic structure-vaccine biocomposite Microneedle array-metal-organic structure-vaccine biocomposites were prepared using a three-step manufacturing strategy.

[0152] Production of master molds and mass-production molds

[0153] Master molds for the microneedle array-metal-organic structure-vaccine biocomposites were prepared using microfabrication strategies (including 3D laser printing, photolithography, dry etching, or microfabrication) to define the microneedle and array geometry, as well as the dimensions of the microneedles and their spatial distribution across the array. Production molds for the microneedle array-metal-organic structure-vaccine biocomposites, which have microneedle-shaped wells / cavities, were prepared by replicating the master molds using microfabrication of different hydrophobic or hydrophilic materials to control the surface interactions between the production molds and the metal-organic structure-vaccine biocomposites (Figures 12 and 13). Candidate materials for the production molds include EcoFlex (hydrophobic), polydimethylsiloxane (PDMS, hydrophobic), plasma-treated PDMS (more hydrophilic than PDMS), copolymer of PDMS and poly(ethylene glycol) (PEG) (more hydrophilic than PDMS), and agarose (hydrophilic).

[0154] Manufacturing of Microneedle Arrays - Metal-Organic Structures - Vaccine Biocomposites

[0155] Metal-organic structures—vaccine biocomposites—were loaded into mass-production microneedle arrays using a number of methods, including the following:

[0156] 1) Metal-organic structure-vaccine biocomposites were resuspended in water-soluble / hydrophilic bio-derived materials / surfactants (carboxymethylcellulose (CMC), CMC and trehalose, CMC and sucrose, CMC and maltodextrin, CMC and lactose, hyaluronic acid, chitosan, alginate, gantlets, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), PVP and PVA) or nucleic acid adjuvants (poly(I:C) or CpG) to overcome hydrophobic / hydrophobic interactions and charge interactions with mass-production types of microneedle arrays by coating them with hydrophilic compounds;

[0157] 2) A more hydrophilic material (plasma-treated PDMS or PDMS-PEG) was used as the material for the mass-production type described above;

[0158] 3) The metal-organic structure-vaccine biocomposites were resuspended in low-density organic solvents (ethanol, acetonitrile, or methanol) because they are stable in organic solvents; and

[0159] 4) Individual components of the metal-organic structure-vaccine biocomposite were loaded into a mold for in situ synthesis of the metal-organic structure-vaccine biocomposite.

[0160] Finally, the structural materials (CMC, CMC and trehalose, CMC and sucrose, CMC and maltodextrin, CMC and lactose, hyaluronic acid, chitosan, alginate, gantlets, PVP, PVA, PVP and PVA, poly(lactic acid-co-glycolic acid)) were loaded to form the remainder and backing layer of the microneedles.

[0161] Characterization of microneedle-array-organic-structure-vaccine biocomposites

[0162] Analysis of mass-production and soluble microneedle array-metal-organic structure-vaccine biocomposites using optical stereomicroscopy (ZEISS Stemi 2000-C microscope and Olympus OM-D E-M5 II camera) and fluorescence microscopy (Keyence BZ-X700) revealed the following:

[0163] 1) The effect of the above-mentioned surface modification on the spread of the improved vaccine;

[0164] 2) Successful loading of mass-production type metal-organic structure-vaccine biocomposites; and

[0165] 3) High quality final soluble microneedle array - metal-organic structure - vaccine biocomposite.

[0166] In vitro release tests in 4% agarose demonstrated the stability of the microneedle array-metal-organic structure-vaccine biocomposite, which is soluble in an aqueous environment, compared with a vaccine-loadable, soluble microneedle array (Figures 20 and 21).

[0167] Example 4: The skin-targeted delivery performance of a microneedle array-metal-organic structure vaccine biocomposite was evaluated in vivo in mouse skin and ex vivo in human skin samples. A soluble microneedle array-zeolite-type imidazolate structure-8-Alexa Fluor 647-labeled ovalbumin biocomposite (OVA AF647@ZIF-8@MNA) was prepared from a mixture of two water-soluble materials, carboxymethylcellulose (CMC) and trehalose (both indicated as "GRAS," meaning generally recognized as safe by regulatory authorities), using the three-step fabrication strategy described herein, and conjugated in vivo to mouse skin and ex vivo to freshly excised human skin explants.

[0168] In vivo study in mouse skin

[0169] Analysis of the OVA AF647@ZIF-8@MNA biocomposite before and after in vivo application to mouse skin using optical stereomicroscopy (ZEISS Stemi 2000-C microscope and Olympus OM-D E-M5 II camera) demonstrated that OVA AF647@ZIF-8@MNA integrates a design and bio-derived material with ideal physiological and chemical properties (sufficient mechanical strength for unfailed skin penetration and efficient solubility in an aqueous environment) for effective in vivo skin penetration into mouse skin and subsequent dissolution in the skin microenvironment.

[0170] In vivo animal fluorescence imaging (IVIS Lumina XR) analysis of mice treated with the OVA AF647@ZIF-8@MNA biocomposite showed that the OVA AF647@ZIF-8@MNA biocomposite can deposit OVA AF647@ZIF-8 into the mouse skin (Figures 22-24).

[0171] Exovivo study in freshly excised, living human skin explants

[0172] Analysis of OVA AF647@ZIF-8@CMC / trehalose MNA biocomposites before (Figure 25) and after (Figures 26 and 27) application to freshly excised human skin samples using optical stereomicroscopy (ZEISS Stemi 2000-C microscope and Olympus OM-D E-M5 II camera) demonstrated that OVA AF647@ZIF-8@CMC / trehalose MNA integrates a design and bio-derived material with ideal physiological and chemical properties (sufficient mechanical strength for unfailed skin penetration and efficient solubility in an aqueous environment) for effective human skin penetration and subsequent dissolution.

[0173] Analysis of human skin samples treated with the OVA AF647@ZIF-8@MNA biocomposite using optical stereomicroscopy (ZEISS Stemi 2000-C microscope and Olympus OM-D E-M5 II camera) and fluorescence imaging (IVIS Lumina XR) demonstrated that the OVA AF647@ZIF-8@MNA biocomposite can deposit OVA AF647@ZIF-8 into human skin. IVIS fluorescence imaging further showed that MNA achieves a more effective spatial distribution of OVA AF647@ZIF-8 compared to intradermal injection using conventional subcutaneous needles (Figure 28).

[0174] Frozen sectioning of human skin samples treated with the OVA AF647@ZIF-8@MNA biocomposite, followed by fluorescence microscopy (Keyence BZ-X700) analysis, demonstrated that the OVA AF647@ZIF-8@CMC / trehalose MNA biocomposite penetrates human skin and delivers OVA AF647@ZIF-8 to an immunologically rich human skin microenvironment (Figure 29).

[0175] These results suggest that the soluble microneedle array-metal-organic structure-vaccine biocomposite provided precise targeting of the metal-organic structure-vaccine biocomposite to the immune-capable microenvironment in mouse and human skin.

[0176] Example 5: In vivo evaluation of immunogenicity and safety of a soluble microneedle array-metal-organic structure vaccine biocomposite. A biosoluble microneedle array-zeolite-type imidazolate structure-8-ovalbumin biocomposite (OVA@ZIF-8@MNA) was prepared from a mixture of two water-soluble bio-based materials, carboxymethylcellulose and trehalose (which are indicated as "GRAS" by regulatory authorities as generally recognized as safe) using the three-step manufacturing strategy described herein. A soluble, ovalbumin-integrated microneedle array (OVA@MNA) was also prepared from the same mixture of carboxymethylcellulose and trehalose using the same three-step manufacturing strategy.

[0177] Immunogenicity of biosoluble microneedle arrays, metal-organic structures, and vaccine biocomposites.

[0178] Humoral immune response: C57BL / 6 female mice (N=3 mice / group) received two doses of 1) OVA (OVA IM) via intramuscular injection, 2) OVA@MNA, and 3) OVA@ZIF-8@MNA, two weeks apart. Naive mice were used as unimmunized controls. Five days after the booster dose, vaccine-induced antibody responses were evaluated by ELISA measurement of anti-OVA total IgG antibodies in mouse serum (Figures 30-31).

[0179] Humoral immune response: C57BL / 6 female mice (N=5 mice / group) received primary doses of 1) 2 MNA-VAX(S1) and 2) 2 MNA-ZIF-8-VAX(S1). Naive mice were used as unimmunized controls. Two weeks after primary dose, vaccine-induced antibody response was evaluated by ELISA measurement of anti-SARS-CoV-2-S1 total IgG antibodies in mouse serum (Figure 56).

[0180] Humoral immune response: C57BL / 6 female mice (N = 5 mice / group) received a single dose of 1) HeV-sG MNA, 2) HeV-sG@ZIF-8 MNA, 3) HeV-sG + poly(I:C) MNA, and 4) HeV-sG + poly(I:C)@ZIF-8 MNA. Naïve mice were used as non-immunized controls. Four weeks after the single dose, the vaccine-induced antibody response was evaluated by ELISA measurement of anti-HeV-sG total IgG antibodies in mouse sera (Figure 57C).

[0181] Cellular immune response: C57BL / 6 female mice (N = 3 mice / group) received two doses of 1) OVA@ZIF-8@MNA and 2) OVA@MNA, two weeks apart. Naïve mice were used as non-immunized controls. To determine the activity of OVA-specific cytotoxic T lymphocytes (CTLs), equal numbers of non-pulsed splenocytes (CFSE low "control" cells) and OVA 257-264 peptide-pulsed splenocytes (CFSE high "target" cells) were transferred into naïve and immunized mice five days after the booster dose (2×10 7 total cells / mouse). The spleen was isolated the next day, and the quantification of specific cell lysis (100% lysis corresponds to complete elimination of target cells) was performed (Figures 30 and 32).

[0182] Results from the tests were compared by one-way ANOVA, followed by Tukey's post hoc test. Significant differences were indicated by * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001.

[0183] Safety of Dissolvable Microneedle Array-Metal Organic Framework-Vaccine Biocomposites

[0184] During these immunogenicity studies, no abnormal animal behavior, no detectable weight loss, and no visible signs of obvious skin and systemic reactogenicity were observed (Figures 58A - 58C).

[0185] Due to their advantages of easy synthesis, chemical robustness, thermal stability, and biocompatibility, soluble microneedle array-metal-organic structure-vaccine biocomposites offer enhanced immunogenicity and bypass cold chain storage and distribution, paving the way for the development of next-generation vaccines.

[0186] Example 6: A reproducible and easily obtainable synthesis of vaccine-loaded zeolite-type imidazolate structure-8 (ZIF-8) was achieved using a simple, cost-effective, and scalable protocol and aqueous solution. A 3M solution of 2-methylimidazole was prepared by sonication of a mixture of 246.3 mg, 3 mmol of 2-methylimidazole in 1 mL of nuclease-free water (H2O). A 1M solution of zinc acetate dihydrate was prepared by sonication of a mixture of zinc acetate dihydrate (183.5 mg, 1 mmol) in 1 mL of nuclease-free water. The vaccine solution was prepared to the desired concentration. 3M 2-methylimidazole solution (853 μL, 2.56 mmol) and Ag solution (67 μL) were sequentially added to a 1.5 mL Eppendorf microcentrifuge tube, and the tube was vortexed for 10 seconds. Then, 1M zinc acetate dihydrate (80 μL, 0.08 mmol) was added, and the tube was vortexed again for 30 seconds. The solution was reacted at room temperature or lower for 1 hour to obtain a turbid solution. The suspension was centrifuged at 10,000 rpm for 2 minutes. The precipitate was washed with nuclease-free water (2 mL, 2 ×) and ethanol or methanol (2 mL, 2 ×), and dried under vacuum to obtain subunit vaccine @ZIF-8 crystals as a precipitate.

[0187] Analysis by transmission electron microscopy (TEM, An FEI Morgagni 268) demonstrated the successful formation of vaccine-loaded ZIF-8 particles with an average size distribution of 441±86 nm (OVA@ZIF-8 particles), 318±66 nm (SARS-CoV-2 S1+S2@ZIF-8 particles), 406±99 nm (Adenovirus 5@ZIF-8 particles), and 454±107 nm (mRNA@ZIF-8 particles). There were differences in size between ZIF-8 particles prepared with different types of biomolecules (Figures 33A-33D and 35-36).

[0188] Powder X-ray diffraction (PXRD, A Bruker AXS D8 Discover X-Ray Powder Diffractometer) analysis showed that the pattern of vaccine@ZIF-8 particles matched the simulated results. This indicates that the Vaccube@ZIF-8 particles are highly crystalline and phase-pure (Figure 34). The vaccine loading efficiencies of OVA@ZIF-8 particles (99.82%) and mRNA@ZIF-8 particles (97.91%) were calculated as follows: (Initial mass of vaccine - residual mass of vaccine in supernatant) / (Initial mass of vaccine) × 100).

[0189] Fluorescence microscopy and flow cytometry analysis of DC 2.4 cells transfected with mRNA.mCherry (i.e., an mRNA vector encoding the mCherry gene) recovered from mRNA@ZIF-8 showed mCherry expression (Figure 55A-B). This indicates the preservation of mRNA integrity within the MOF encapsulation.

[0190] Example 7: Fabrication and Characterization of Microneedle Array-Metal-Organic Structure-Vaccine Biocomposites with Various Antigen Constructs Synthesis of soluble microneedle arrays, metal-organic structures, and vaccine biocomposites.

[0191] Microneedle array-metal-organic structure-vaccine biocomposites with various antigen constructs (ovalbumin protein, SARS-CoV-2 subunit protein, adenovirus 5 viral vector, and mRNA nucleic acid) were prepared using the multi-step manufacturing strategy disclosed herein. Dissolvable MNAs having obelisk-shaped microneedles integrating ZIF-8 loaded with different biomolecules were manufactured from two FDA-recognized "generally recognized as safe" (GRAS) biomaterials, a biomaterial combination of carboxymethylcellulose (CMC) and trehalose, using the three-step (master-production-dissolvable MNA) microfabrication technique described above.

[0192] Characterization of Microneedle Array-Organic Framework-Vaccine Biocomposites

[0193] Analysis of soluble microneedle array-metal-organic structure-vaccine biocomposites with different antigen constructs using optical stereomicroscopy (ZEISS Stemi 2000-C microscope and Olympus OM-D E-M5 II camera) and fluorescence microscopy (Keyence BZ-X700) demonstrated the high quality of the soluble microneedle array-metal-organic structure-vaccine biocomposites with different antigen constructs (including ovalbumin protein, SARS-CoV-2 subunit protein, adenovirus 5 viral vector, and mRNA nucleic acid) (Figures 37-42).

[0194] Example 8: Intradermal delivery performance of a soluble microneedle array-metal-organic structure-SARS-CoV-2 vaccine biocomposite. The skin targeting and delivery performance of a microneedle array-metal-organic structure SARS-CoV-2 vaccine biocomposite was evaluated in vivo in mouse skin and ex vivo in freshly excised human skin samples.

[0195] A soluble microneedle array-zeolite-type imidazolate structure-8-Alexa Fluor 647-labeled SARS-CoV-2 S1 subunit vaccine biocomposite (S1 AF647@ZIF-8@MNA) was prepared from a mixture of two water-soluble materials, carboxymethylcellulose (CMC) and trehalose (both indicated as "GRAS," meaning generally recognized as safe by regulatory authorities), using the three-step manufacturing strategy described above.

[0196] In vivo study in mouse skin

[0197] In vivo animal fluorescence imaging (IVIS Lumina XR) analysis of mice treated with the S1@ZIF-8@MNA biocomposite showed that the S1 AF647@ZIF-8@CMC / trehalose MNA biocomposite can deposit SARS-CoV-2 S1 AF647@ZIF-8 into mouse skin (Figure 43).

[0198] Exovivo study in freshly excised, living human skin explants

[0199] Fluorescence imaging (IVIS Lumina XR) analysis of human skin samples treated with S1 AF647@ZIF-8@MNA biocomposite demonstrated that the S1 AF647@ZIF-8@CMC / trehalose MNA biocomposite can deposit SARS-CoV-2 S1 AF647@ZIF-8 into human skin (Figure 44).

[0200] Therefore, these results demonstrate that a soluble microneedle array-metal-organic structure-vaccine biocomposite provides reliable and consistent cutaneous targeted delivery of the metal-organic structure-SARS-CoV-2 vaccine biocomposite to an immune-capable microenvironment in mouse and human skin.

[0201] Example 9: Fabrication and Characterization of Rapidly Separable and Soluble Microneedle Array-Metal-Organic Structure-Vaccine Biocomposite Synthesis of rapidly separable microneedle array-metal-organic structure-vaccine biocomposites

[0202] A separable microneedle array-metal-organic structure-vaccine biocomposite was prepared using the three-step manufacturing strategy disclosed herein.

[0203] Master mold manufacturing and production

[0204] Master and production molds for microneedles were prepared as described herein.

[0205] Manufacturing of separable microneedle arrays - metal-organic structures - vaccine biocomposites

[0206] Separable metal-organic structure-vaccine biocomposites were modified in the final step to prepare soluble microneedle array-metal-organic structure-vaccine biocomposites using the approach described herein. Biocalcification of zeolite-type imidazolate structure-8-Alexa Fluor 647-labeled ovalbumin (OVA AF647@ZIF-8) was achieved as described herein, and rhodamine-loaded, negatively charged polyinosinic acid-polycytidic acid (Poly(I:C)rhodamine) was adsorbed onto OVA AF647@ZIF-8 via electrostatic interaction (OVA AF647@ZIF-8-Poly(I:C)rhodamine). Next, the MNA-MOF-vaccine biocomposite was prepared with water-soluble bio-derived materials (e.g., carboxymethylcellulose (CMC), CMC and trehalose, CMC and sucrose, CMC and lactose, CMC and sucrose, hyaluronic acid (HA), or PVP or PVP and PVA) that are generally recognized as safe by regulatory authorities, and integrated onto a PMMA stem with a water-insoluble and organic solvent-soluble (e.g., ethyl lactate) thermoplastic (e.g., poly(lactic acid-co-glycolic acid), polymethyl methacrylate, or polylactic acid) as part of a scalable manufacturing process.

[0207] Characterization of microneedle array-metal-organic structure-vaccine biocomposites

[0208] Analysis of separable microneedle array-metal-organic structure-vaccine biocomposites using optical stereomicroscopy (ZEISS Stemi 2000-C microscope and Olympus OM-D E-M5 II camera) and fluorescence microscopy (Keyence BZ-X700) demonstrated the successful formation and conferral of separable microneedle array-metal-organic structure-vaccine biocomposites (Figures 45-50).

[0209] Example 10: Skin vaccine delivery using a separable microneedle array-metal-organic structure-vaccine biocomposite. We evaluated skin-targeted vaccine delivery using separable microneedle array-metal-organic structure vaccine biocomposites in mouse skin and in freshly excised human skin samples ex vivo.

[0210] A separable microneedle array-zeolite-type imidazolate structure-vaccine biocomposite was prepared using the multi-step manufacturing strategy described herein.

[0211] In vivo study in mouse skin

[0212] Analysis of mice treated with separable MNA-MOF-vaccine biocomposites (OVA AF647@ZIF-8-poly(I:C)rhodamine@CMC / trehalose biocomposite on PMMA or OVA AF647@ZIF-8-poly(I:C)rhodamine@PVP biocomposite on PMMA) by in vivo live animal fluorescence imaging (IVIS Lumina XR) showed that these separable MNA-MOF-vaccine biocomposites can in vivo deposit OVA AF647@ZIF-8-poly(I:C)rhodamine@water-soluble material biocomposite into mouse skin.

[0213] Exovivo study in freshly excised, living human skin explants

[0214] Fluorescence imaging (IVIS Lumina XR) analysis of human skin samples treated with separable MNA-MOF-vaccine biocomposites on PMMA (OVA AF647@ZIF-8-poly(I:C)rhodamine@CMC / trehalose biocomposite or OVA AF647@ZIF-8-poly(I:C)rhodamine@PVP biocomposite on PMMA) demonstrated that these novel, separable MNA-MOF-vaccine biocomposites can rapidly deposit OVA AF647@ZIF-8-poly(I:C)rhodamine@water-soluble material biocomposite into human skin ex vivo.

[0215] In summary, these results demonstrate that these high-performance, separable MNA-MOF-vaccine biocomposites delivered precise and rapid targeting of MOF-multi-component vaccine-biocomposites to the cutaneous microenvironments of both humans and mice, with improved cutaneous delivery characteristics.

[0216] In light of the many possible situations in which the principles of the art disclosed herein may be applied, it should be recognized that the illustrated situations are merely preferred examples of the art and should not be interpreted as limiting the scope of the art. Rather, the scope of the art is defined by the following claims. Therefore, the inventors claim all of their disclosures that fall within the scope and spirit of these claims.

Claims

1. A microneedle array, base part; Multiple biocompatible and soluble microneedles extending from the base portion A microneedle array comprising a microneedle containing metal-organic structure (MOF) particles containing a vaccine composition.

2. The microneedle array according to claim 1, wherein the MOF is a zinc-based MOF, a copper-based MOF, or an iron-based MOF.

3. The microneedle array according to claim 2, wherein the zinc-based MOF is ZIF-8, ZIF-10, ZIF-90; the copper-based MOF is HKUST-1; or the iron-based MOF is MIL-53, MIL-88.

4. The microneedle array according to claim 1, wherein the MOF is surface-functionalized with a surface coating comprising CMC, CMC / trehalose, PEG, PVA, PVP, or hyaluronic acid, silk, or a combination thereof.

5. The microneedle array according to claim 1, wherein the MOF particles have an average size distribution of 10 nm to 50,000 nm.

6. The microneedle array according to claim 1, wherein the base portion lacks the MOF particles containing the vaccine composition.

7. The microneedle array according to claim 1, wherein both the microneedle and the base portion incorporate the MOF particles containing the vaccine composition.

8. The microneedle array according to any one of claims 1 to 7, wherein the MOF particles containing the vaccine composition are incorporated into the tapered portion of the microneedle.

9. The microneedle array according to claim 1, wherein the vaccine composition comprises an antigen selected from a protein, mRNA, self-replicating mRNA, nucleic acid antigen, recombinant viral vector antigen, nucleic acid origami antigen, extracellular vesicle antigen, inactivated viral antigen, liposome encapsulated antigen, extracellular vesicle encapsulated antigen, cell lysate antigen, bacterial antigen, or a combination thereof.

10. The microneedle array according to claim 1, wherein the vaccine composition comprises an adjuvant.

11. The aforementioned microneedle array is A first plurality of MOF particles comprising a first vaccine composition; and A second plurality of MOF particles containing a second vaccine composition The microneedle array according to claim 1, comprising:

12. The microneedle array according to claim 11, wherein the first vaccine composition comprises a first antigen, and the second vaccine composition comprises a second antigen.

13. The microneedle array according to claim 11, wherein the first vaccine composition comprises an antigen, and the second vaccine composition comprises an adjuvant.

14. The microneedle array according to claim 1, wherein each microneedle further comprises a soluble biocompatible material.

15. The microneedle array according to claim 14, wherein the soluble biocompatible material is CMC, CMC and trehalose, CMC and sucrose, CMC and maltodextrin, CMC and lactose, hyaluronic acid, chitosan, alginate, gantlets, PVP, PVA, PVP and PVA, gelatin, polylactic acid, pullulan, silk, polyphosphazene, poly-γ-glutamate, or poly(lactic acid-co-glycolic acid).

16. A method for fabricating a microneedle array according to claim 1, wherein the method is: A step of forming a solution comprising a plurality of metal-organic structure (MOF) particles containing a soluble biocompatible material and a vaccine composition; and A step of applying the aforementioned solution to a microneedle array, A method of including.

17. A method for producing a microneedle array according to claim 1, the method comprising the step of adding a solution containing a soluble biocompatible material to a microneedle array mold containing a plurality of metal-organic structural particles containing a vaccine composition to form the microneedle array.

18. The method of claim 17, further comprising the step of loading the plurality of metal-organic structure particles comprising the vaccine composition into the mold before adding the solution comprising the soluble biocompatible material.

19. The method of claim 17, further comprising the step of loading the vaccine composition and the precursor of the metal-organic structure into the mold before adding the solution containing the soluble biocompatible material to form the plurality of MOF particles containing the vaccine composition in the mold.

20. A method for vaccinating a subject, comprising the step of applying the microneedle array described in claim 1 to a region of the subject to deliver the vaccine composition to the subject.

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