Manganese doped metal organic frameworks and methods of making and use thereof

Abstract

Disclosed herein are manganese doped metal organic frameworks and methods of making and use thereof. For example, disclosed herein is a manganese doped metal organic framework (Mn-MOF) comprising a metal organic framework (MOF) comprising a zeolitic imidazolate framework (ZIF) comprising metal ions connected by imidazolate linkers, wherein the metal ions comprise Mn and Zn. In some examples, the Mn-MOF comprises from greater than 0% to 50% (mol%) Mn based on the total amount of metal ions. Also disclosed herein are methods of making and methods of use of the Mn-MOFs.

Description

[0001] MANGANESE DOPED METAL ORGANIC FRAMEWORKS AND METHODS OF MAKING AND USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 609,390 filed December 13, 2023, which is hereby incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] The emerging field of metalloimmunology has sought to control immunological responses using metals as adjuvants. An important pathway of the innate immune system is the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. The stimulation of this pathway has been attracting attention because it promotes the release of interferons, which are essential in immune signaling to help eliminate infected or cancerous cells. Current STING agonists suffer from poor uptake and bioavailability. More efficient intracellular delivery strategies promoting STING agonist uptake are required. The compositions and methods discussed herein address these and other needs.

[0006] SUMMARY

[0007] In accordance with the purposes of the disclosed compositions and methods as embodied and broadly described herein, the disclosed subject matter relates to manganese doped metal organic frameworks and methods of making and use thereof.

[0008] For example, disclosed herein is a manganese doped metal organic framework (Mn- MOF) comprising a metal organic framework (MOF) comprising a zeolitic imidazolate framework (ZIF) comprising metal ions connected by imidazolate linkers, wherein the metal ions comprise Mn and Zn. In some examples, the ZIF is ZIF-8.

[0009] In some examples, the Mn-MOF comprises from greater than 0% to 50% (mol%) Mn based on the total amount of metal ions. In some examples, the Mn-MOF comprises from 10% to 50% Mn. In some examples, the Mn-MOF comprises 15% Mn, 30% Mn, or 50% Mn. In some examples, the Mn-MOF comprises 50% Mn.

[0010] In some examples, the Mn-MOF is crystalline.

[0011] In some examples, the Mn-MOF is nanostructured.

[0012] In some examples, the Mn-MOF is in the form of a plurality of particles. In some examples, the plurality of particles have an average particle size of from 100 nanometers to 2 micrometers. In some examples, the plurality of particles have an average particle size of from 500 nanometers to 1 micrometer, or from 400 nanometers to 700 nanometers. In some examples, the plurality of particles have a particle shape that is a rounded cubic structure. In some examples, the plurality of particles have a sodalite topology.

[0013] In some examples, the Mn-MOF further comprises a therapeutic agent loaded on and / or in the Mn-MOF. In some examples, the therapeutic agent is conjugated to the Mn-MOF, for example electrostatically. In some examples, the therapeutic agent comprises an anticancer agent, an anti-inflammatory agent, an antimicrobial agent, an immunotherapy agent, or a combination thereof. In some examples, the therapeutic agent comprises a STING agonist. In some examples, the therapeutic agent comprises a cyclic dinucleotide (CDN). In some examples, the therapeutic agent comprises cyclic di-adenosine monophosphate (CD A).

[0014] Also disclosed herein are methods of making any of the Mn-MOFs disclosed herein.

[0015] In some examples, the method comprises in situ incorporation of Mn.

[0016] In some examples, the method comprises dispersing a Zn salt, a Mn salt, a reducing agent, and an imidazolate in a green solvent. In some examples, the method comprises adding reactants in the following order: the green solvent, the reducing agent, the Mn salt, the Zn salt, and the imidazolate, thereby forming a mixture.

[0017] In some examples, the method further comprises agitating the mixture, such as by vortexing. In some examples, after agitating, the mixture is left static for an amount of time. In some examples, the method further comprises isolating the Mn-MOF. In some examples, the method further comprises washing and / or drying the Mn-MOF.

[0018] In some examples, the Zn salt comprises zinc(II) acetate dihydrate. In some examples, the Mn salt comprises manganese(II) acetate tetrahydrate. In some examples, the imidazolate comprises 2-methylimidazole.

[0019] In some examples, the reducing agent comprises sodium ascorbate, sodium citrate, tris(2- carboxyethyljphosphine (TCEP), or a combination thereof. In some examples, the reducing agent comprises citrate, tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof. In some examples, reducing agent comprises tris(2-carboxyethyl)phosphine (TCEP).

[0020] In some examples, the green solvent comprises water. In some examples, the green solvent consists essentially of water. In some examples, the green solvent consists of water.

[0021] In some examples, the method is substantially free of organic solvents.

[0022] In some examples, the method is substantially free of methanol.

[0023] In some examples, the method is conducted at room temperature.

[0024] In some examples, the Mn-MOF is formed in an amount of time of from 10 minutes to 30 minutes, such as from 15 to 25 minutes.

[0025] In some examples, the method is a one pot method. In some examples, the method is substantially biocompatible.

[0026] In some examples, the method further comprises contacting the Mn-MOF with a therapeutic agent to thereby load the therapeutic agent in and / or on the Mn-MOF. In some examples, the therapeutic agent is conjugated to the Mn-MOF via electrostatic interactions.

[0027] Also disclosed herein are pharmaceutical compositions comprising any of the Mn-MOFs disclosed herein. In some examples, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

[0028] Also disclosed herein are methods of use of any of the Mn-MOFs or any of the pharmaceutical compositions disclosed herein.

[0029] Also disclosed herein are methods of treating, preventing, or ameliorating a disease in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of any of the Mn-MOFs or any of the pharmaceutical compositions disclosed herein.

[0030] In some examples, the disease comprises cancer. In some examples, the disease is an infectious disease, e.g. an infection, such as a microbial infection. In some examples, the disease is tuberculosis.

[0031] In some examples, the method comprises immunotherapy. In some examples, the Mn- MOF or pharmaceutical composition activates the STING pathway to thereby treat, prevent, and / or ameliorate the disease.

[0032] In some examples, the Mn-MOF or pharmaceutical composition activates antigen presenting cells to thereby treat, prevent, and / or ameliorate the disease.

[0033] In some examples, the method comprises cell uptake followed by degradation of the Mn- MOF, thereby providing delivery and release of Mn and the therapeutic agent (when present) to the cell cytosol to thereby treat, prevent, and / or ameliorate the disease.

[0034] In some examples, the method triggers cGAS-STING activation, resulting in proinflammatory cytokine production and / or bone marrow dendritic cell activation.

[0035] Also disclosed herein are vaccines comprising any of the Mn-MOFs or any of the pharmaceutical compositions disclosed herein.

[0036] Also disclosed herein are immunomodulators comprising any of the Mn-MOFs or any of the pharmaceutical compositions disclosed herein.

[0037] Additional advantages of the disclosed compositions and methods will be set forth in part in the description which follows, and in part will be obvious from the description. The advantages of the disclosed compositions and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed devices, systems, and methods, as claimed.

[0038] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0039] BRIEF DESCRIPTION OF THE FIGURES

[0040] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the disclosure, and together with the description, serve to explain the principles of the disclosure.

[0041] Figure lA-Figure 1C: Synthesis of Mn-ZIF and delivery of Mn for immune activation. Figure 1A) Synthetic overview of Mn-ZIF and CDA@Mn-ZIF. Zn. Mn, a reducing agent TCEP, and HMIM were mixed in water and left static at RT to afford Mn-ZIF. CDA was incorporated post-synthetically by incubating with Mn-ZIF suspended in water and methanol at RT on a rotisserie to make CDA@Mn-ZIF. Figure IB) Uptake of the multivariate MOF was measured to demonstrate the delivery and release of Mn and CDA into cells. Figure 1C) The immune- stimulatory effect of Mn and CDA delivery was characterized by flow cytometry, western blot, and ELISA of cell supernatants for proinflammatory cytokines.

[0042] Figure ID: CDA@Mn-ZIF was combined with the Mtb fusion protein CysVac2 and used a vaccine in a pre-clinical mouse Mtb challenge experiment.

[0043] Figure 2A-Figure 2E: Characterization Mn-ZIF Figure 2A) PXRD spectra of different Mn ratios starting with ZIF-8 control with 0% Mn up to 50% Mn to Zn. Figure 2B) SEM of ZIF- 8 control, Figure 2C) SEM 15% Mn-ZIF, Figure 2D) SEM of 30% Mn-ZIF, Figure 2E) SEM of 50% Mn-ZIF.

[0044] Figure 3A-Figure 3D: In vitro cytotoxicity measured by resazurin assay comparing ZIF-8 to 15% Mn-ZIF, 30% Mn-ZIF, and 50% Mn-ZIF in Figure 3A) RAW 264.7 cells, Figure 3B) 4T1 cells, and Figure 3C) HEK 293 cells. Figure 3D) Calculated ICso values of each sample on the three cell lines. In Figure 3D, for each of the three cell lines, the bars represent, from left to right, 0% Mn-ZIF (e.g., ZIF-8), 15% Mn-ZIF, 30% Mn-ZIF, and 50% Mn-ZIF.

[0045] Figure 4A-Figure Figure 4F: Characterization of CDA r / Mn-ZIF and uptake. Figure 4A) Scheme of post synthetic loading of CDA onto Mn-ZIF. Figure 4B) CDA loading after incubating each concentration of CDA with 1 mg / ml of Mn-ZIF. Figure 4C) Surface charge of Mn-ZIF before and after loading with CDA measured by zeta ( -potential. Figure 4D) Epifluorescent micrographs of CDA@Mn-ZIF loaded with rhodamine in RAW cells stained with Hoechst and lysotracker green after 6 h. Figure 4E) Representative histogram of RAW cell uptake of CF encapsulated in Mn-ZIF at 4 h and 8 h. Figure 4F) Average uptake at 4 h and 8 h of CF. CF r / Mn-ZIF. CDA@CF@Mn-ZIF calculated by geometric mean of FITC intensity where n = 5.

[0046] Figure 5A-Figure 5D: Immune activating potential of 50% Mn-ZIF and CDA@Mn-ZIF. Figure 5A) Mouse BMDC activation measured by flow7cytometry. Activation was defined as CDl lc+cells double positive for CD80+and CD86+. Figure 5B) Western blot of cGAS-STING downstream proteins. The first set of proteins were extracted from cells incubated in a T-25 flask and the other two samples from a 6-well dish. Figure 5C) TNF-a and Figure 5D) IL-6 produced by BMDCs after 24 h incubation with each sample measured by ELISA on the cell supernatant. P = 95%

[0047] Figure 6: PXRD showing amorphous nature beyond 50% substitution.

[0048] Figure 7A: Survey XPS spectra of ZIF-8 vs 50% Mn-ZIF.

[0049] Figure 7B: Ols XPS spectra of ZIF-8 vs 50% Mn-ZIF.

[0050] Figure 8: Raw7data from a 4 h LDH test on RAW cells. In this assay dead cells release LDH into the media which generates signal, living cells create minimal signal. Lysed groups were treated with lyse buffer to induce cell death and serve as a positive control where cells treated with Mn prevent the lysed cells from generating signal.

[0051] Figure 9: Controls experiment with the deep blue viability assay. In this assay living cells generate a fluorescent signal and lysed cells will not generate signal. Lysed Mn-ZIF works as proper control here and also doesn't inhibit living cells from generating signal.

[0052] Figure 10: LC traces of CD A and supernatant CDA@Mn-ZIF used to calculate absorption percent.

[0053] Figure 11. Standard curve of CDA concentration based on integrated peak area of LC curve. Supernatants of CDA@Mn-ZIF were collected after loading and run on LC to find concentration of CDA not bound to Mn-ZIF, this was used to calculate loading efficiency. The peak area was 6.57 which equates to 1.7 pg / ml CDA meaning 28.3 pg / ml or 94% of the starting CDA was absorbed to the Mn-ZIF.

[0054] Figure 12: Average CDA released from 2 different samples of post synthetically loaded CDA@Mn-ZIF. samples of CDA@Mn-ZIF were suspended in water and placed on a rotisserie, at each time point the samples w ere centrifuged and 20 pl supernatant was taken. Aliquots w ere run on LC and samples were resuspended by vortex and placed on rotisserie until the next time point. Figure 13A: Epiflourescent image of CF@Mn-ZIF on the GFP channel. Scale bar is 400 pm.

[0055] Figure 13B: Epiflourescent image of Rh@Mn-ZIF on the RFP channel. Scale bar is 400 pm.

[0056] Figure 14A: Amount of relative ROS generated by ZIF-8 and Mn-ZIF compared to untreated control cells for RAW cells.

[0057] Figure 14B: Amount of relative ROS generated by ZIF-8 and Mn-ZIF compared to untreated control cells for 4T1 cells.

[0058] Figure 15. Water based synthesis to incorporate Mn into ZIF-8 and then post- synthetically load CD A, a STING agonist.

[0059] Figure 16. Overview of project.

[0060] Figure 17A-Figure 17G: Adjuvant effect of CDA@Mn-ZIF in-vivo in a pre-clinical Tb mouse model. Figure 17A) Timeline of experiments including vaccination, blood draw, challenge, and sacrifice for lung CFU counting. Percentage of CD4+CD44hlCD62L10T cells secreting Figure 17B) IL-17, Figure 17C) IFN-y, Figure 17D) IL-2, and Figure 17E) TNF from PBMCs after in vitro stimulation with CysVac2. PBMC was taken approximately 2 weeks after the final vaccination. Figure 17F) Example flow cytometry gating strategy for phenotyping of PBMCs. Figure 17G) Mean Logio colony forming units (CFU) in the lungs of C57BL / 6 mice inoculated with CDA@Mn-ZIF formulated with either 10, 3, or 1 pg of CysVac2 via the I.M. route, 3-times at 2-week intervals. Mice were rested and infected with low-dose aerosol (50-100 CFU) of Mtb Erdman KOI, and CFU was determined at day 30 post-infection. N=5 mice per group. Statistical analysis was performed using a one-way ANOVA. *p<0.05, **p<0.01. ***p<0.005, ****p<0.0001 . Data is presented as mean ± standard deviation.

[0061] Figure 18: LC traces of CDA and supernatant CDA@Mn-ZIF used to calculate absorption percent.

[0062] Figure 19: 24-hour cytotoxicity results for CDA@Mn-ZIF in RAW 264.7 cells. IC50 was calculated to be 61 .0 ± 6.3 pg / mL

[0063] Figure 20A-Figure 20C: Protection of CDA by Mn-ZIF. Figure 20A) Structure of CDA before and after hydrolysis degradation by SVPD into pApA. Figure 20B) Reaction scheme followed for the experiment where CDA@Mn-ZIF was mixed with SVPD and then heated for 5 min. to stop the enzyme, centrifuged and pellet collected. The pellet was then treated with acetic acid and left on a rotisserie to break down the Mn-ZIF and release CDA. This was centrifuged again to collect any debris, and then the supernatant ran on LC. Figure 20C) LC traces of pApA, CDA, CDA@Mn-ZIF, and SVPD-treated CDA7 / ,Mn-ZIF. CDA@Mn-ZIF served as a control to ensure the enzyme treatment and CDA recover}' process was not too harsh.

[0064] Figure 21. Uncropped chemiluminescent scans of each western blot used in Figure 5B, and colorimetric scan of the ladder used to confirm molecular weight.

[0065] Figure 22: Graphs of quantified western blot bands.

[0066] Figure 23A-Figure 23B: Cytokine production measured by ELISA in supernatants of BMDC’s incubated for 20 hours with each sample. Figure 23 A) IFN-y production and Figure 23B) IFN-P production. Statistical analysis was performed with Welches t-test. confidence interval of 95%. Data is presented as mean ± standard deviation.

[0067] Figure 24: Amount of relative ROS generated by ZIF-8, Mn-ZIF, and CDA@Mn-ZIF compared to untreated control cells for RAW 264.7 cells. 1 pg / mL LPS was used as a positive control and generated a 186 ± 16 % increase in ROS compared to untreated cells. Data is presented as mean ± standard deviation.

[0068] DETAILED DESCRIPTION

[0069] The compositions and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein.

[0070] Before the present compositions, methods, and devices are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary'. It is also to be understood that the terminology' used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0071] Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0072] General Definitions

[0073] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings.

[0074] Throughout the description and claims of this specification the word ‘’comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.

[0075] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0076] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 5% of the value, e g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0077] Values can be expressed herein as an “average” value. “Average” generally refers to the statistical mean value.

[0078] By “substantially” is meant within 5%. e.g., within 4%, 3%. 2%, or 1%.

[0079] “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.

[0080] It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms.

[0081] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0082] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included. The term '‘or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC. BC, or ABC, and if order is important in a particular context, also BA. CA, CB, CBA. BCA, ACB, BAC. or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that ty pically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0083] As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g, cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0084] The term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80. 90. 100%, or any amount of reduction in between as compared to native or control levels.

[0085] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor grow th" means reducing the rate of grow th of a tumor relative to a standard or a control.

[0086] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. For example, the terms “prevent” or “suppress” can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity' of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms.

[0087] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is. treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0088] The term “therapeutically effective amount” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0089] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0090] The term “anticancer” refers to the ability to treat or control cellular proliferation and / or tumor grow th at any concentration.

[0091] As used herein, “molecular weight” refers to number average molecular weight as measured by NMR spectroscopy, unless indicated otherwise.

[0092] As used herein, the term “delivery” encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and secreted into patient's circulation system (e g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery’). As used herein, the term "‘encapsulation,’’ or grammatical equivalent, refers to the process of confining an individual nucleic acid molecule within a nanoparticle.

[0093] As used herein, “expression’’ of a mRNA refers to translation of an mRNA into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme) and also can include, as indicated by context, the post-translational modification of the peptide, polypeptide or fully assembled protein (e.g., enzyme). In this application, the terms “expression” and “production,” and grammatical equivalent, are used inter-changeably.

[0094] As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one peptide, polypeptide or protein. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g.. 2-aminoadenosine, 2-thiothymidine. inosine, pyrrolo-pyrimidine. 3-methyl adenosine, 5-methylcytidine. C-5 propynyl-cytidine, C-5 propynyl-uridine. 2-aminoadenosine. C5 -bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5- methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N- phosphoramidite linkages).

[0095] As used herein, the term “nucleic acid.” in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodi ester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA and / or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0096] The organic moieties mentioned when defining variable positions within the general formulae described herein (e.g., the term “halogen”) are collective terms for the individual substituents encompassed by the organic moiety. The prefix Cn-Cmpreceding a group or moiety indicates, in each case, the possible number of carbon atoms in the group or moiety that follows.

[0097] The term “ion,” as used herein, refers to any molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom that contains a charge (positive, negative, or both at the same time within one molecule, cluster of molecules, molecular complex, or moiety (e.g., zwitterions)) or that can be made to contain a charge. Methods for producing a charge in a molecule, portion of a molecule, cluster of molecules, molecular complex, moiety', or atom are disclosed herein and can be accomplished by methods known in the art, e.g., protonation, deprotonation, oxidation, reduction, alkylation, acetylation, esterification, de-esterification, hydrolysis, etc.

[0098] The term “anion” is a ty pe of ion and is included within the meaning of the term “ion.” An “anion” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains a net negative charge or that can be made to contain a net negative charge. The term “anion precursor” is used herein to specifically refer to a molecule that can be converted to an anion via a chemical reaction (e.g., deprotonation).

[0099] The term “cation” is a type of ion and is included within the meaning of the term “ion.” A “cation” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom, that contains a net positive charge or that can be made to contain a net positive charge. The term “cation precursor” is used herein to specifically refer to a molecule that can be converted to a cation via a chemical reaction (e.g.. protonation or alkylation).

[0100] Mn-MOFs

[0101] Disclosed herein are manganese doped metal organic frameworks (Mn-MOFs) and methods of making and use thereof.

[0102] Metal-Organic Frameworks (MOFs) are a class of nanostructured porous hybrid materials assembled via coordination bonds between metal-containing centers and organic linkers.

[0103] For example, disclosed herein are manganese doped metal organic frameworks (Mn- MOFs) comprising a metal organic framework (MOF) comprising a zeolitic imidazolate framework (ZIF) comprising metal ions connected by imidazolate linkers, wherein the metal ions comprise Mn and Zn. In some examples, the ZIF is ZIF-8.

[0104] In some examples, the Mn-MOF comprises greater than 0% (mol%) Mn based on the total amount of metal ions (e.g., 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more). In some examples, the Mn-MOF comprises 50% (mol%) or less Mn based on the total amount of metal ions (e.g., 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less). The amount of Mn (mol%) in the Mn-MOF can range from any of the minimum values described above to any of the maximum values described above. For example, the Mn-MOF can comprise from greater than 0% to 50% (mol%) Mn based on the total amount of metal ions (e.g., from greater than 0% to 25%, from 25% to 50%, from greater than 0% to 10%, from 10% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50%, from greater than 0% to 45%, from greater than 0% to 40%, from greater than 0% to 30%, from greater than 0% to 20%, from greater than 0% to 15%, from 1% to 50%, from 5% to 50%, from 10% to 50%, from 15% to 50%, from 20% to 50%, from 30% to 50%, or from 1% to 45%). In some examples, the Mn-MOF comprises from 10% to 50% Mn. In some examples, the Mn-MOF comprises 15% Mn, 30% Mn, or 50% Mn. In some examples, the Mn-MOF comprises 50% Mn.

[0105] In some examples, the Mn-MOF comprises Mn and Zn in a molar ratio (Mn:Zn) of greater than 0: 100 (e.g., 1:99 or more, 2:98 or more, 3:97 or more, 4:96 or more, 5:95 or more, 10:90 or more, 15:85 or more, 20:80 or more, 25:75 or more, 30:70 or more, 35:65 or more, 40:60 or more, or 45:55 or more). In some examples, the Mn-MOF comprises Mn and Zn in a molar ratio (Mn:Zn) of 50:50 or less (e.g., 45:55 or less, 40:60 or less, 35:65 or less, 30:70 or less, 25:75 or less, 20:80 or less, 15:85 or less, 10:90 or less, 5:95 or less, 4:96 or less, 3:97 or less, 2:98 or less, or 1 :99 or less). The molar ratio of Mn to Zn (Mn:Zn) in the Mn-MOF can range from any of the minimum values described above to any of the maximum values described above. For example, the Mn-MOF can comprise Mn and Zn in a molar ratio (Mn:Zn) of from greater than 0: 100 to 50:50 (e.g., from greater than 0: 100 to 25:75, from 25:75 to 50:50, from greater than 0: 100 to 10:90, from 10:90 to 20:80, from 20:80 to 30:70, from 30:70 to 40:60, from 40:60 to 50:50. from greater than 0: 100 to 45:55, from greater than 0: 100 to 40:60, from greater than 0: 100 to 30:70, from greater than 0: 100 to 20:80. from greater than 0: 100 to 15:85, from 1 :99 to 50:50, from 5:95 to 50:50, from 10:90 to 50:50, from 15:85 to 50:50, from 20:80 to 50:50, from 30:70 to 50:50, or from 1:99 to 45:55). In some examples, the Mn-MOF comprises Mn and Zn in a molar ratio (Mn:Zn) of from 10:90 to 50:50. In some examples, the Mn-MOF comprises Mn and Zn in a molar ratio (Mn:Zn) of 15:85, 30:70, or 50:50. In some examples, the Mn-MOF comprises Mn and Zn in a molar ratio (Mn:Zn) of 50:50.

[0106] In some examples, the metal ions further comprise Co, Fe, or a combination thereof.

[0107] In some examples, the metal ions consist essentially of Mn and Zn. In some examples, the metal ions consist of Mn and Zn.

[0108] In some examples, the Mn-MOF is crystalline.

[0109] In some examples, the Mn-MOF is nanostructured. As used herein, “nanostructured” means any structure with one or more nanosized features. A nanosized feature can be any feature with at least one dimension less than 1 pm in size. For example, a nanosized feature can comprise a nanowire, nanotube, nanoparticle, nanopore, and the like, or combinations thereof. As such, the Mn-MOF can comprise, for example, a nanowire, nanotube, nanoparticle, nanopore, or a combination thereof. In some examples, the Mn-MOF can comprise a material that is not nanosized but has been modified with a nanowire, nanotube, nanoparticle, nanopore, or a combination thereof.

[0110] In some examples, the Mn-MOF can comprise a plurality of particles (e.g., the Mn-MOF can be in the form of a plurality of particles) having an average particle size. “Average particle size” and “mean particle size” are used interchangeably herein, and generally refer to the statistical mean particle size of the particles in a population of particles. For example, the average particle size for a plurality7of particles with a substantially spherical shape can comprise the average diameter of the plurality7of particles. For a particle w ith a substantially spherical shape, the diameter of a particle can refer, for example, to the hydrodynamic diameter. As used herein, the hydrodynamic diameter of a particle can refer to the largest linear distance between two points on the surface of the particle. For an anisotropic particle, the average particle size can refer to, for example, the average maximum dimension of the particle (e.g., the length of a rodshaped particle, the diagonal of a cube shape particle, the bisector of a triangular shaped particle, etc.) For an anisotropic particle, the average particle size can refer to, for example, the hydrodynamic size of the particle. Mean particle size can be measured using methods known in the art, such as evaluation by electron microscopy, such as scanning electron microscopy.

[0111] In some examples, the plurality7of particles have an average particle size of 100 nanometers (nm) or more (e.g., 150 nm or more, 200 nm or more. 250 nm or more. 300 nm or more, 350 nm or more. 400 nm or more. 450 nm or more. 500 nm or more. 550 nm or more. 600 nm or more, 650 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, 1 micrometer (micron, pm) or more, 1.25 pm or more, 1.5 pm or more, or 1.75 pm or more). In some examples, the plurality of particles have an average particle size of 2 micrometers (microns, pm) or less (e.g., 1.75 gm or less, 1.5 gm or less, 1.25 gm or less, 1 gm or less, 900 nanometers (nm) or less, 800 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, or 200 nm or less). The average particle size of the plurality of particles can range from any of the minimum values described above to any of the maximum values described above. For example, the plurality of particles can have an average particle size of from 100 nanometers to 2 micrometers (e.g., from 100 nanometers to 750 nanometers, from 750 nanometers to 2 micrometers, from 100 nanometers to 500 nanometers, from 500 nanometers to 1 micrometer, from 1 micrometer to 1.5 micrometers, from 1.5 micrometers to 2 micrometers, from 100 nanometers to 1.5 micrometers, from 100 nanometers to 1 micrometer, from 100 nanometers to 900 nanometers, from 100 nanometers to 800 nanometers, from 100 nanometers to 700 nanometers, from 100 nanometers to 600 nanometers, from 100 nanometers to 400 nanometers, from 200 nanometers to 2 micrometers, from 400 nanometers to 2 micrometers, from 500 nanometers to 2 micrometers, from 600 nanometers to 2 micrometers, from 700 nanometers to 2 micrometers, from 200 nanometers to 1.5 micrometers, from 300 nanometers to 1 micrometer, or from 400 nanometers to 700 nanometers). In some examples, the plurality- of particles have an average particle size of from 500 nanometers to 1 micrometer. In some examples, the plurality of particles have an average particle size of from 400 nanometers to 700 nanometers.

[0112] In some examples, the plurality' of particles can be substantially monodisperse. “Monodisperse” and “homogeneous size distribution,” as used herein, and generally describe a population of particles where all of the particles have the same or nearly the same particle size. As used herein, a monodisperse distribution refers to particle distributions in which 80% of the distribution (e.g., 85% of the distribution, 90% of the distribution, or 95% of the distribution) lies within 25% of the average particle size (e.g., within 20% of the average particle size, within 15% of the average particle size, w ithin 10% of the average particle size, or w ithin 5% of the average particle size).

[0113] In some examples, the plurality' of particles can comprise particles of any shape(s). In some examples, the plurality' of particles can have a shape that is a rounded cubic structure.

[0114] In some examples, the plurality' of particles have a sodalite topology'.

[0115] In some examples, the Mn-MOF further comprises a therapeutic agent loaded on and / or in the Mn-MOF. The therapeutic agent can, for example, be conjugated to the Mn-MOF. for example electrostatically.

[0116] The therapeutic agent can, for example, comprise an anticancer agent, an antiinflammatory agent, an antimicrobial agent, an immunotherapy agent, or a combination thereof. As used herein, antimicrobials include, for example, antibacterials, antifungals, antivirals, and antiparasitics.

[0117] Examples of antibacterials include, but are not limited to, acetoxy cycloheximide, aciduliprofundum, actaplanin, actinorhodin, alazopeptin, albomycin, allicin, allistatin, allyl isothiocyanate, ambazone, aminocoumarin, aminoglycosides, 4-aminosalicylic acid, ampicillin, ansamycin, anthramycin, antimycin A, aphidicolin, aplasmomycin, archaeocin, arenicin, arsphenamine, arylomycin A2, ascofuranone, aspergillic acid, avenanthramide, avibactam, azelaic acid, bafilomycin, bambermycin, beauvericin, benzoyl peroxide, blasticidin S, bottromycin, brilacidin, caprazamycin, carbomycin. cathelicidin, cephalosporins, ceragenin, chartreusin, chromomycin A3, citromycin, clindamycin, clofazimine, clofoctol, clorobiocin, coprinol, coumermycin Al, cyclic lipopeptides, cycloheximide, cycloserine, dalfopristin, dapsone, daptomycin, debromomarinone, 17-dimethylaminoethylamino-17- demethoxygeldanamycin, echinomycin, endiandric acid C, enediyne, enviomycin, eravacycline, erythromycin, esperamicin, etamycin, ethambutol, ethionamide, (6S)-6-fluoroshikimic acid, fosfomycin, fosmidomycin, friulimicin, furazolidone, furonazide, fusidic acid, geldanamycin, gentamycin, gepotidacin, glycy ciclines, glycyrrhizol, gramicidin S, guanacastepene A, hachimycin, halocyamine, hedamycin. helquinoline, herbimycin, hexamethylenetetramine, hitachimycin, hydramacin-1. isoniazid, kanamycin, katanosin, kedarcidin, kendomycin, kettapeptin, kidamycin, lactivicin, lactocillin, landomycin, landomycinone, lasalocid, lenapenem, leptomycin, lincosamides, linopristin, lipiarmycins, macbecin, macrolides, macromomycin B, maduropeptin, mannopeptimycin glycopeptide, marinone, meclocycline, melafix, methylenomycin A, methylenomycin B. monensin, moromycin, mupirocin, mycosubtilin, myriocin, myxopyronin, naphthomycin A, narasin, neocarzinostatin, neopluramycin, neosalvarsan, neothramycin, netropsin, nifuroxazide, nifurquinazol, nigericin, nitrofural, nitrofurantoin, nocathiacin I, novobiocin, omadacycline, oxacephem, oxazolidinones, penicillins, peptaibol, phytoalexin, plantazolicin, platensimycin, plectasin, pluramycin A, polymixins, polyoxins, pristinamycin, pristinamycin IA, promin, prothionamide, pulvinone, puromycin, pyocyanase, pyocyanin, pyrenocine, questiomycin A, quinolones, quinupristin, ramoplanin, raphanin, resistome, reuterin, rifalazil, rifamycins, ristocetin, roseophilin, salinomycin, salinosporamide A, saptomycin, saquayamycin, seraticin, sideromycin, sodium sulfacetamide, solasulfone, solithromycin, sparassol, spectinomycin, staurosporine, streptazolin. streptogramin. streptogramin B, streptolydigin, streptonigrin, styelin A, sulfonamides, surfactin, surotomycin, tachyplesin, taksta, tanespimycin, telavancin, tetracyclines, thioacetazone, thiocarlide, thiolutin, thiostrepton, tobramycin, trichostatin A, triclosan, trimethoprim, trimethoprim, tunicamycin, tyrocidine, urauchimycin, validamycin, viridicatumtoxin B, vulgamycin, xanthomycin A, xibomol, amikacin, amoxicillin, ampicillin, atovaquone, azithromycin, aztreonam, bacitracin, carbenicillin, cefadroxil, cefazolin, cefdinir, cefditoren, cefepime, cefiderocol, cefoperazone, cefotetan, cefoxitin, cefotaxime, cefpodoxime, cefprozil, ceftaroline, ceftazidime, ceftibuten. ceftizoxime, ceftriaxone, chloramphenicol, colistimethate, cefuroxime, cephalexin, cephradine, cilastatin, cinoxacin, ciprofloxacin, clarithromycin, clindamycin, dalbavancin, dalfopristin, daptomycin, demeclocycline, dicloxacillin, doripenem, doxycycline, eravacycline, ertapenem, erythromycin, fidaxomicin, fosfomycin, gatifloxacin, gemifloxacin. gentamicin, imipenem, lefamulin, lincomycin. linezolid. lomefloxacin, loracarbef, meropenem. metronidazole, minocycline, moxifloxacin, nafcillin, nalidixic acid, neomycin, norfloxacin, ofloxacin, omadacycline, oritavancin, oxacillin, oxytetracycline, paromomycin, penicillin, pentamidine, piperacillin, plazomicin, quinupristin, rifaximin, sarecycline, secnidazole, sparfloxacin, spectinomycin. sulfamethoxazole, sulfisoxazole, tedizolid, telavancin, telithromycin. ticarcillin, tigecycline, tobramycin, trimethoprim, trovafloxacin, vancomycin, and combinations thereof.

[0118] Examples of antifungals include, but are not limited to, abafungin, acibenzolar, acibenzolar-S-methyl, acrisorcin, allicin, aminocandin, amorolfme, amphotericin B, anidulafungin, azoxystrobin. bacillomycin, bacillus pumilus, barium borate, benomyl, binapacryl, boric acid, bromine monochloride, bromochlorosalicylanilide, bupinmate, butenafine, candicidin, capr lic acid, captafol, captan, carbendazim, caspofungin, cerulenin, chloranil, chlormidazole, chlorophetanol, chlorothalonil, chloroxylenol, chromated copper arsenate, ciclopirox, cilofungin, cinnamaldehyde, clioquinol, copper(I) cyanide, copper(II) arsenate, cruentaren, cycloheximide, davicil, dehydroacetic acid, dicarboximide fungicides, dichlofluanid, dimazole, diphenylamine, echinocandin, echinocandin B, epoxiconazole, ethonam, falcarindiol, falcarinol, famoxadone, fenamidone, fenarimol, fenpropimorph, fentin acetate, fenticlor, filipin, fluazinam, fluopicolide, flusilazole, fluxapyroxad. fuberidazole, griseofulvin, halicylindramide. haloprogin, hamycin, hexachlorobenzene, hexachlorocyclohexa- 2,5-dien-l-one, 5-hydroxy-2(5H)-furanone, iprodione, lime sulfur, mancozeb, maneb, melafix, metalaxyl, metam sodium, methylisothiazolone, methylparaben, micafungin, miltefosine, monosodium methyl arsenate, mycobacillin, myclobutanil, natamycin, beta-nitrostyrcne. nystatin, paclobutrazol, papulacandin B, parietin, pecilocin, pencycuron, pentamidine, pentachloronitrobenzene, pentachlorophenol, perimycin, 2-phenylphenol. polyene antimycotic, propamocarb, propiconazole, pterulone, ptilomycalin A, pyrazophos, pyrimethanil, pyrrolnitrin, selenium disulfide, sparassol, strobilurin, sulbentine, tavaborole, tebuconazole, terbinafine, theonellamide F, thymol, tiabendazole, ticlatone, tolciclate, tolnaftate, triadimefon, triamiphos, tribromometacresol, 2,4,6-tribromophenol, tributyltin oxide, triclocarban, triclosan, tridemorph, trimetrexate, undecylenic acid, validamycin, venturicidin, vinclozolin, vinyldithiin, vusion, xanthene, zinc borate, zinc pyrithione, zineb, ziram, voriconazole, itraconazole, posaconazole, fluconazole, ketoconazole, clotrimazole, isavuconazonium, miconazole, caspofungin, amdulafungin, micafungin, griseofulvin, terbinafine, flucytosine, terbinafine, nystatin, amphotericin b., and combinations thereof.

[0119] Examples of antivirals include, but are not limited to, afovirsen, alisporivir, angustific acid, angustifodilactone, alovudine, beclabuvir, 2,3-bis(acetylmercaptomethyl)quinoxaline, brincidofovir, dasabuvir. docosanol, fialuridine. ibacitabine, imiquimod, inosine, inosine pranobex, interferon, metisazone, miltefosine, neokadsuranin, neotripterifordin, ombitasvir, oragen, oseltamivir, peg lated interferon, podophyllotoxin, radalbuvir, semapimod, tecovirimat, telbivudine, theaflavin. tilorone, triptofordin C-2, variecolol, ZMapp, abacavir, acyclovir, adefovir, amantadine, amprenavir. atazanavir, balavir. baloxavir marboxil, boceprevir, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docasanol, dolutegravir, doravirine, ecoliever, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, forscamet, fosnonet, famciclovir, favipravir, fomivirsen, foscavir, ganciclovir, ibacitabine, idoxuridine. indinavir, inosine, inosine pranobex, interferon type 1, interferon type II. interferon type III. lamivudine, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nitazoxanide, oseltamivir, peginterferon alfa-2a, peginterferon alfa-2b, penciclovir, peramivir, pleconaril, podophyllotoxin, pyramidine, raltegravir, remdesevir, ribavirin, rilpivirine, rimantadine, rintatolimod, ritonavir, saquinavir, simeprevir. sofosbuvir, stavudine, tarabivirin, telaprevir, telbivudine, tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, umifenovir, valaciclovir, valganciclovir, vidarabine, zalcitabine, zanamivir, zidovudine, and combinations thereof.

[0120] Examples of antiparasitics include, but are not limited to, 5 ’-S-methyl-5 ’-thioadenosine, abamectin, abametapir, albendazole, albendazole oxide, allipurinol riboside, amodiaquine, amphotericin B, amprolium, andrographolide, anisomycin, arsenamide, antimony potassium tartarate, arprinocid, artefenomel, artemether, artemotil, artenimol, artesunate, ascaricide, atovaquone, avermectin, benzimidazole, bephenium, bephenium hydroxy naphthoate. betulinic acid, bithionol, bunamidine, cambendazole, carbadox. carbendazim, chloroquine, chloroxylenol, chlorproquanil, chlortetracycline, ciclobendazole, clopidol, clorsulon, coumaphos, cycloquanil, cymiazole, dapsone, decoquinate, desaspidin, dichlorophen, dichlorvos, diclazuril, diethelcarbamazine, diiodohydroxyquinoline, diloxanide, diloxanide furoate, diminazene, dimetridazole, dithiazanine, doramectin, doxyxyxline, ectoparasiticide, eflomithine, emetine, emodepside, eprinomectin, ethopabate, etofamide, febantel, fenbendazole, fexinidazole, flubendazole, fumagillin, furazolidone, geneticin, hachimycin, halofantrine, halofuginone, hexylresorcinol, homidium, hy canthone, hydroxychloroquine, hygromycine B, imidocarb, isometamidium chloride, ivermectin, lasalocid, levamisole, lonidamine, lotilaner, lucanthone, lumefantrine, lufenuron, malathion, mebendazole, mefloquine, meglumine antimoniate, melarsomine, melarsoprol, mepartricin, metrifonate, metronidazole, milbemycin oxime, miltefosine, misonidazole, mizoribine. monensin, moxidectin, narasin, nicarbazin, niclosamide, nifuratel. nifurtimox. nimorazole, niridazole, nitazoxanide, nitroxinil. nitroxoline, oltipraz. omidazole, oryzalin, oxamniquine, oxantel, oxfendazole, oxibendazole, pafuramidine, paromomycin, pentamidine, permethrin, phenothiazine, piperaquine, piperazine, plumbagin, posaconazole, praziquantel, primaquine, proguanil, propamidine, psoralen, puromucin, pyrantel, pyrimethamine, pyronaridine, pyrvinium, quinacrine, quinapyramine. quinidine, quinine, radicicol, robenidine, roxarsone, salicylhydroxamic acid, salinomycin, secnidazole, selamectin, sinefungin, sodium stibogluconate, spiramycin, stibophen, sulfadiazine, sulfadoxine, sulfametopyrazine, sulfaquinoxaline, suramin, tafenoquine, tetrandrine, tetraphenylporphine sulfonate, tiabendazole, tinidazole. toltrazuril, triclabendazole, zoalene, and combinations thereof.

[0121] In some examples, the therapeutic agent comprises an anticancer agent. In some examples, the therapeutic agent comprises a chemotherapeutic agent, an immunotherapeutic agent, or a combination thereof.

[0122] In some examples, the therapeutic agent can comprise a chemotherapeutic agent. Chemotherapy is the treatment of cancer with one or more cytotoxic anti -neoplastic drugs (e.g., chemotherapeutic agents) as part of a standardized regimen. Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms. In some cases, it can be used in conjunction with other cancer treatments, such as radiation therapy, surgery, hyperthermia therapy, or a combination thereof. Examples of chemotherapeutic agents include, but are not limited to, 13-cis-Retinoic Acid, 2-Amino-6-Mercaptopurine, 2-CdA, 2- Chlorodeoxyadenosine, 5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine, Accutane, Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretinoin, Alkaban-AQ. Alkeran, All-transretinoic acid, Alpha interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron, Anastrozole, Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenic trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib, Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar, Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine, Carmustine wafer, Casodex, CCNU, CDDP. CeeNU, Cerubidine, cetuximab, Chlorambucil, Cisplatin. Citrovorum Factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide. Cytadren, Cytarabine, Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposomal, DaunoXome, Decadron, Delta-Cortef, Deltasone, Denileukin diftitox, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate. Dexasone, Dexrazoxane, DHAD, DIC, Diodex. Docetaxel. Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludara. Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex, Mechlorethamine, -Mechlorethamine Hydrochlorine, Medralone, Medrol, Megace, Megestrol, Megestrol Acetate. Melphalan, Mercaptopurine. Mesna. Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Mylocel, Letrozole. Neosar, Neulasta, Neumega. Neupogen. Nilandron, Nilutamide, Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-asparaginase, Phenylalanine Mustard, Platinol, PlatinoL AQ, Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin, Prolifeprospan 20 with Carmustine implant, Purinethol, Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alfa-2a), Rubex, Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen. Targretin, Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid, Vesanoid, Viadur, Vinblastine. Vinblastine Sulfate, Vincasar Pfs, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP- 16, Vumon, Xeloda, Zanosar, Zevalin. Zinecard, Zoladex, Zoledronic acid, Zometa, Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulating factor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea, Hydrocort Acetate. Hydrocortisone, Hydrocortisone sodium phosphate, Hydrocortisone sodium succinate, Hydrocortone phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin, Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL- 11, Imatinib mesylate, Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG conjugate). Interleukin 2, Interleukin- 11 , Intron A (interferon alfa-2b), Leucovorin, Leukeran, Leukine, Leuprohde, Leurocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L- PAM, L-Sarcolysin, Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan, Isotretinoin, Kidrolase, LanacorL L-asparaginase, LCR, FAM-HYD-1, Marizomib (NPI-0052). Lenalidomide, Carfilzomib, Panobinostat. Quisinostat, Selinexor, Oprozomib, and combinations thereof. The anticancer agent can also include biopharmaceuticals such as, for example, antibodies.

[0123] Examples of suitable immunotherapeutic agents include, but are not limited to, alemtuzumab, cetuximab (ERBITUX), gemtuzumab, iodine 131 tositumomab, rituximab, trastuzamab (HERCEPTIN), and combinations thereof.

[0124] In some examples, the therapeutic agent can comprise an anti -infl ammatory agent, such as steroidal and / or non-steroidal anti-inflammatory agents. Examples of steroidal antiinflammatory agents include, but are not limited to, hydrocortisone, dexamethasone, prednisolone, prednisone, triamcinolone, methylprednisolone, budesonide, betamethasone, cortisone, and deflazacort. Examples of non-steroidal anti-inflammatory drugs include acetaminophen, aspirin, ibuprofen, naproxen, Celebrex, ketoprofen, tolmetin, etodolac, fenoprofen, flurbiprofen, diclofenac, piroxicam, indomethacin, sulindax, meloxicam, nabumetone, oxaprozin, mefenamic acid, and diflunisal.

[0125] In some examples, the therapeutic agent comprises a STING agonist.

[0126] In some examples, the therapeutic agent comprises a cyclic dinucleotide (CDN), such as cyclic di-adenosine monophosphate (CDA).

[0127] Pharmaceutical Compositions

[0128] Also disclosed herein are pharmaceutical compositions comprising any of the Mn-MOFs disclosed herein. In some examples, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

[0129] In some examples, the disclosed compositions optionally further comprise other therapeutic ingredients or adjuvants.

[0130] In some examples, the composition is administered to a subject. In some examples, the subject is a mammal. In some examples, the mammal is a primate. In some examples, the mammal is a human. In some examples, the human is a patient. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0131] Methods of Making

[0132] Also disclosed herein are methods of making any of the compounds or compositions disclosed herein.

[0133] For example, also disclosed herein are methods of making any of the Mn-MOFs disclosed herein. In some examples, the method comprises in situ incorporation of Mn.

[0134] In some examples, the methods comprise dispersing aZn salt, a Mn salt, a reducing agent, and an imidazolate in a green solvent.

[0135] The Zn salt can comprise any suitable salt comprising Zn, such as those known in the art. For example, the Zn salt can comprise a zinc(II) acetate, a zinc(II) nitrate, or a combination thereof. In some examples, the Zn salt can comprise zinc(II) acetate dihydrate, zinc(II) nitrate hexahydrate, or a combination thereof. In some examples, the Zn salt can comprise zinc(II) acetate dihydrate.

[0136] The Mn salt can comprise any suitable salt comprising Mn, such as those know n in the art. For example, the Mn salt can comprise a manganese(II) acetate, a manganese(II) chloride, or a combination thereof. In some examples, the Mn salt can comprise manganese(II) acetate tetrahydrate, manganese(II) chloride tetrahydrate, or a combination thereof. In some examples, the Mn salt can comprise manganese(II) acetate tetrahydrate.

[0137] The imidazolate can comprise any suitable compound, such as those known in the art. For example, the imidazolate can comprise 2-methylimidazole.

[0138] The reducing agent can comprise any suitable reducing agent, such as those known in the art. For example, the reducing agent can comprise sodium ascorbate, sodium citrate, tris(2- carboxyethyl)phosphine (TCEP), or a combination thereof. In some examples, the reducing agent can comprise sodium citrate, tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof. In some examples, the reducing agent comprises tris(2-carboxyethyl)phosphine (TCEP).

[0139] The green solvent can comprise any suitable green solvent, such as those known in the art. In some examples, the green solvent comprises water. In some examples, the green solvent consists essentially of water. In some examples, the green solvent consists of water. In some examples, the method is substantially free of organic solvents. In some examples, the method is substantially free of methanol.

[0140] In some examples, the method comprises adding reactants in the following order: the green solvent, the reducing agent, the Mn salt, the Zn salt, and the imidazolate, thereby forming a mixture.

[0141] In some examples, the method further comprises agitating the mixture. Agitating the can be accomplished, for example, by mechanical stirring, shaking, vortexing, sonication (e.g., bath sonication, probe sonication, ultrasonication), homogenizing (e.g., using a high shear homogenizer), and the like, or combinations thereof.

[0142] In some examples, after agitating, the mixture is left static for an amount of time.

[0143] In some examples, the method further comprises isolating the Mn-MOF, for example by filtering, centrifuging, extraction, or a combination thereof.

[0144] In some examples, the methods can further comprise washing and / or drying the Mn- MOFs.

[0145] In some examples, the method is conducted at room temperature. As used herein, room temperature means at a temperature of from 14°C to 25°C (e.g., from 18°C to 25°C).

[0146] In some examples, the Mn-MOF is formed in an amount of time of 10 minutes or more (e.g.. 11 minutes or more, 12 minutes or more, 13 minutes or more. 14 minutes or more, 15 minutes or more, 16 minutes or more, 17 minutes or more, 18 minutes or more, 19 minutes or more, 20 minutes or more, 21 minutes or more, 22 minutes or more, 23 minutes or more, 24 minutes or more, 25 minutes or more, 26 minutes or more, 27 minutes or more. 28 minutes or more, or 29 minutes or more). In some examples, the Mn-MOF is formed in an amount of time of 30 minutes or less (e.g., 29 minutes or less, 28 minutes or less, 27 minutes or less, 26 minutes or less, 25 minutes or less, 24 minutes or less, 23 minutes or less, 22 minutes or less, 21 minutes or less, 20 minutes or less, 19 minutes or less, 18 minutes or less, 17 minutes or less, 16 minutes or less, 15 minutes or less, 14 minutes or less, 13 minutes or less, 12 minutes or less, or 11 minutes or less). The amount of time in which the Mn-MOF is formed can range from any of the minimum values described above to any of the maximum values described above. For example, the Mn-MOF can be formed in an amount of time of from 10 minutes to 30 minutes (e.g., from 10 to 20 minutes, from 20 to 30 minutes, from 10 to 15 minutes, from 15 to 20 minutes, from 20 to 25 minutes, from 25 to 30 minutes, from 10 to 25 minutes, from 15 to 30 minutes, or from 15 to 25 minutes). In some examples, the Mn-MOF is formed in an amount of time of from 15 to 25 minutes.

[0147] In some examples, the method is a one pot method. In some examples, the method is substantially biocompatible.

[0148] In some examples, the method further comprises contacting the Mn-MOF with the therapeutic agent to thereby load the therapeutic agent in and / or on the Mn-MOF. In some examples, the therapeutic agent is conjugated to the Mn-MOF via electrostatic interactions.

[0149] Methods of Use

[0150] Also disclosed herein are methods of use of any of the Mn-MOFs or pharmaceutical compositions disclosed herein.

[0151] For example, also disclosed herein are methods of treating, preventing, or ameliorating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the Mn-MOFs or any of the pharmaceutical compositions disclosed herein.

[0152] Also disclosed herein are vaccines comprising any of the Mn-MOFs or any of the pharmaceutical compositions disclosed herein, for example for treating, preventing, or ameliorating a disease in a subject.

[0153] Also disclosed herein are immunomodulators comprising any of the Mn-MOFs or any of the pharmaceutical compositions disclosed herein, for example for treating, preventing, or ameliorating a disease in a subject..

[0154] Examples of diseases include, but are not limited to. cancer, inflammatory diseases, infectious diseases, and combinations thereof.

[0155] In some examples, the disease comprises cancer. For example, also disclosed herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the compounds or compositions disclosed herein. For example, the compounds and compositions described herein or pharmaceutically acceptable salts thereof are useful for treating cancer in humans, e.g, pediatric and geriatric populations, and in animals, e.g.. veterinary' applications. The disclosed methods can optionally include identifying a patient who is or may be in need of treatment of a cancer. Examples of cancer types treatable by the compounds and compositions descnbed herein include bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary' cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, and testicular cancer. Further examples include cancer and / or tumors of the anus, bile duct, bone, bone marrow, bowel (including colon and rectum), eye, gall bladder, kidney, mouth, lary nx, esophagus, stomach, testis, cervix, mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid, vagina, vulva, uterus, liver, muscle, blood cells (including lymphocytes and other immune system cells). Further examples of cancers treatable by the compounds and compositions described herein include carcinomas, Karposi’s sarcoma, melanoma, mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, and other), and lymphoma (Hodgkin’s and non-Hodgkin’s). and multiple myeloma.

[0156] The methods of treatment or prevention of cancer described herein can, in some examples, further include treatment with one or more additional agents (e.g., an anti-cancer agent or ionizing radiation). For example, the compounds or compositions or pharmaceutically acceptable salts thereof as described herein can be combined into a pharmaceutical composition with an additional anticancer agent, such as a chemotherapeutic agent, an immunotherapeutic agent, or a combination thereof.

[0157] The additional anti-cancer agent can also include biopharmaceuticals such as, for example, antibodies. Many tumors and cancers have viral genome present in the tumor or cancer cells. For example. Epstein-Barr Virus (EBV) is associated with a number of mammalian malignancies. The compounds disclosed herein can also be used alone or in combination with anticancer or antiviral agents, such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc., to treat patients infected with a virus that can cause cellular transformation and / or to treat patients having a tumor or cancer that is associated with the presence of viral genome in the cells. The compounds disclosed herein can also be used in combination with viral based treatments of oncologic disease.

[0158] Also described herein are methods of suppressing tumor growth in a subject. The method includes contacting at least a portion of the tumor with a therapeutically effective amount of any of the compound or compositions as described herein. In some examples, the methods further include the step of irradiating at least a portion of the tumor with a therapeutically effective amount of ionizing radiation. As used herein, the term ionizing radiation refers to radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization. An example of ionizing radiation is x-radiation. A therapeutically effective amount of ionizing radiation refers to a dose of ionizing radiation that produces an increase in cell damage or death when administered in combination with the compounds described herein. The ionizing radiation can be delivered according to methods as known in the art, including administering radiolabeled antibodies and radioisotopes.

[0159] In some examples, the diseases comprises an inflammatory disease. Inflammatory diseases include, but are not limited to, acne vulgaris, ankylosing spondylitis, asthma, autoimmune diseases, Celiac disease, chronic prostatitis, Crohn's disease, glomerulonephritis, hidradenitis suppurativa, inflammatory bowel diseases, pelvic inflammatory disease, psoriasis, reperfusion injury, rheumatoid arthritis, sarcoidosis, vasculitis, interstitial cystitis, type 1 hypersensitivities, systemic sclerosis, dermatomyositis, polymyositis, and inclusion body myositis.

[0160] In some examples, the disease comprises an infection, such as with an infectious microbe (e.g., bacteria, virus, fungi, protozoa, etc.). In some examples, the disease comprises a microbial infection. In some examples, the disease is tuberculosis. Also disclosed herein are methods for treating, preventing, inhibiting, and / or ameliorating a microbial infection in a subject in need thereof, comprising administering to the subject an effective amount of any of the Mn-MOFs or pharmaceutical compositions described herein.

[0161] In some examples, the method comprises immunotherapy.

[0162] In some examples, the Mn-MOF or pharmaceutical composition activates the STING pathway to thereby treat, prevent, and / or ameliorate the disease.

[0163] In some examples, the Mn-MOF or pharmaceutical composition activates antigen presenting cells to thereby treat, prevent, and / or ameliorate the disease.

[0164] In some examples, the method comprises cell uptake followed by degradation of the Mn- MOF, thereby providing delivery' and release of Mn and the therapeutic agent (when present) to the cell cytosol to thereby treat, prevent, and / or ameliorate the disease. In some examples, the method triggers cGAS-STING activation, resulting in proinflammatory cytokine production and / or bone marrow dendritic cell activation.

[0165] The specific dose level for any particular subject will depend upon a variety' of factors. Such factors include the age. body weight, general health, sex, and diet of the subject. Other factors include the time and route of administration, rate of excretion, drug combination, and the ty pe and severity^ of the particular disease or disorder.

[0166] The methods of treatment of the disease or disorder described herein can further include treatment with one or more additional agents. The one or more additional agents and the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be administered in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart. The methods can also include more than a single administration of the one or more additional agents and / or the compounds and compositions or pharmaceutically acceptable salts thereof as described herein. The administration of the one or more additional agents and the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be by the same or different routes. When treating with one or more additional agents, the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be combined into a pharmaceutical composition that includes the one or more additional agents.

[0167] The methods, compounds, and compositions as described herein are useful for both prophylactic and therapeutic treatment. As used herein the term treating or treatment includes prevention; delay in onset; diminution, eradication, or delay in exacerbation of signs or symptoms after onset; and prevention of relapse. For prophylactic use, a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein are administered to a subject prior to onset (e.g, before obvious signs of the disease or disorder), during early onset (e.g., upon initial signs and symptoms of the disease or disorder), or after an established development of the disease or disorder. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a disease or disorder. Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein after the disease or disorder is diagnosed.

[0168] In certain embodiments, it is desirable to target a nanoparticle using a targeting moiety that is specific to a cell type and / or tissue type. In some embodiments, a nanoparticle may be targeted to a particular cell, tissue, and / or organ using a targeting moiety. Exemplary nonlimiting targeting moieties include ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies (e.g., full-length antibodies, antibody fragments (e.g., Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, or F(ab')2 fragments), single domain antibodies, camelid antibodies and fragments thereof, human antibodies and fragments thereof, monoclonal antibodies, and multispecific antibodies (e.g... bispecific antibodies)). In some embodiments, the targeting moiety may be a polypeptide. The targeting moiety may include the entire polypeptide (e.g., peptide or protein) or fragments thereof. A targeting moiety is typically positioned on the outer surface of the nanoparticle in such a manner that the targeting moiety is available for interaction with the target, for example, a cell surface receptor. A variety of different targeting moieties and methods are known and available in the art, including those described, e.g., in Sapra et al., Prog. Lipid Res. 42(5):439-62, 2003 and Abra et al., J. Liposome Res. 12: 1-3, 2002.

[0169] The targeting moiety can target any known cell type, including, but not limited to, hepatocytes, colon cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells (including primary tumor cells and metastatic tumor cells). In particular embodiments, the targeting moiety targets the lipid nanoparticle to a hepatocyte. In other embodiments, the targeting moiety targets the lipid nanoparticle to a colon cell. In some embodiments, the targeting moiety targets the lipid nanoparticle to a liver cancer cell (e.g., a hepatocellular carcinoma cell) or a colorectal cancer cell (e.g., a primary tumor or a metastasis).

[0170] Compositions, Formulations, Methods of Administration, and Kits

[0171] In vivo application of the disclosed Mn-MOFs, and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed Mn-MOFs can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrastemal administration, such as by injection. Administration of the disclosed Mn-MOFs or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.

[0172] The Mn-MOFs disclosed herein, and compositions comprising them, can also be administered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time. The Mn-MOFs can also be administered in their salt derivative forms or crystalline forms.

[0173] The Mn-MOFs disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well know n and readily available to those skilled in the art. For example, Remington ’s Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the Mn-MOFs disclosed herein can be formulated such that an effective amount of the Mn-MOF is combined with a suitable excipient in order to facilitate effective administration of the Mn-MOF. The compositions used can also be in a variety of forms. These include, for example, solid, semisolid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and application. The compositions can also include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the Mn-MOFs include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired application, compositions disclosed herein can comprise between about 0.1% and 100% by weight of the total of one or more of the subject Mn- MOFs based on the weight of the total composition including earner or diluent.

[0174] The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0175] Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the excipients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.

[0176] Mn-MOFs disclosed herein, and compositions comprising them, can be delivered to a cell either through direct contact with the cell or via a earner means. Carrier means for delivering Mn-MOFs and compositions to cells are known in the art.

[0177] For the treatment of oncological disorders, the Mn-MOFs or compositions disclosed herein can be administered to a patient in need of treatment in combination with other antitumor or anticancer substances and / or with radiation and / or photodynamic therapy and / or with surgical treatment to remove a tumor. These other substances or treatments can be given at the same as or at different times from the Mn-MOFs or compositions disclosed herein. For example, the Mn- MOFs or compositions disclosed herein can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea. DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and / or other anti-cancer drugs or antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or an immunotherapeutic such as ipilimumab and bortezomib.

[0178] In certain examples, Mn-MOFs and compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of unwanted cell growth (such as a tumor site or benign skin growth, e.g., injected or topically applied to the tumor or skin growth), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. Mn-MOFs and compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient’s diet. For oral therapeutic administration, the Mn-MOF can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

[0179] The tablets, troches, pills, capsules, and the like can also contain the following: binders such as gum tragacanth, acacia, com starch or gelatin; diluents such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of Wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the phy sical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir can contain the Mn-MOF, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry’ or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the Mn-MOF can be incorporated into sustained-release preparations and devices.

[0180] Mn-MOFs and compositions disclosed herein, including pharmaceutically acceptable salts thereof, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0181] Pharmaceutical compositions disclosed herein suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some examples, the final injectable form can be sterile and can be effectively fluid for easy syringability. In some examples, the pharmaceutical compositions can be stable under the conditions of manufacture and storage; thus, they can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0182] Sterile injectable solutions are prepared by incorporating a Mn-MOF and / or agent disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0183] Pharmaceutical compositions disclosed herein can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, solution, tincture, and the like. In some examples, the compositions can be in a form suitable for use in transdermal devices. In some examples, it will be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid. Mn-MOFs and agents and compositions disclosed herein can be applied topically to a subject’s skin. These formulations can be prepared, utilizing any of the Mn-MOFs disclosed herein or pharmaceutically acceptable salts thereof, via conventional processing methods.

[0184] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the Mn-MOFs can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.

[0185] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid earners to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0186] Pharmaceutical compositions disclosed herein can be in a form suitable for rectal administration wherein the carrier is a solid. In some examples, the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted earners) follow ed by chilling and shaping in molds.

[0187] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing any of the Mn-MOFs disclosed herein, and / or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

[0188] Useful dosages of the Mn-MOFs and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, body weight, general health, condition, sex, diet, and extent of the disease in the patient and can be determined by one of skill in the art. Other factors include the time and route of administration, rate of excretion, drug combination, and the type and severity' of the particular disease or disorder. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily’, for one or several days.

[0189] Also disclosed are kits that comprise a Mn-MOF or composition disclosed herein in one or more containers. The disclosed kits can optionally include pharmaceutically acceptable carriers and / or diluents. In one embodiment, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit includes instructions or packaging materials that describe how to administer a Mn-MOF or composition of the kit. Containers of the kit can be of any’ suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a Mn-MOF and / or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, a Mn-MOF and / or agent disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing a Mn-MOF and / or agent disclosed herein in liquid or solution form.

[0190] In some examples, the kit further comprises at least one agent, wherein the Mn-MOF and the agent are co-formulated.

[0191] In some examples, the Mn-MOF and the agent are co-packaged.

[0192] The kits can also comprise Mn-MOFs and / or products co-packaged, co-formulated, and / or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed Mn-MOF and / or product and another component for delivery' to a patient.

[0193] It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making, the disclosed methods of using, and / or the disclosed Mn-MOFs and compositions.

[0194] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. The examples below are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims.

[0195] EXAMPLES

[0196] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0197] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by w eight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of measurement conditions, e.g., component concentrations, temperatures, pressures and other measurement ranges and conditions that can be used to optimize the described process.

[0198] Example 1 - Aqueous Synthesis of a Manganese-Doped Multivariate Metal-Organic Framework for Metallo-Immunotherapy

[0199] Introduction. Metals are essential micronutrients and critical components in many cellular and biological processes [1], Expanded knowledge of cellular functions involving metals has led to many being used therapeutically, including zinc, magnesium, calcium, iron, aluminum, platinum, lithium, manganese, and copper [2], The emerging field of metalloimmunology has sought to control immunological responses using metals as adjuvants [3], In particular. Al and Mn have emerged as potent stimulators of cellular processes that promote either innate or adaptive immune responses. The innate immune system involves cells like macrophages, providing immediate, nonspecific defense against a pathogen. In contrast, the adaptive immune system uses T- and / or B-cells to provide targeted, long-term protection against specific pathogens [4], An important pathway of the innate immune system that has gained significant interest is the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway [5], The stimulation of this pathw ay has been attracting attention because it promotes the release of interferons, which are essential in immune signaling to help eliminate infected or cancerous cells. The STING pathway serves as a sensor for cytosolic DNA. DNA should be in the nucleus of a cell, and if it is detected in the cytosol, it may be a sign of pathogen invasion or cancer; consequently, the STING pathway has become a therapeutic and prophylactic target for cancer therapy and vaccine adjuvants [6-8], The canonical agonists for STING are a class of messaging molecules called cyclic dinucleotides (CDNs), of which cyclic di-adenosine monophosphate (CDA) is one of the most commonly used [9], Cyclic dinucleotide variants are in clinical trials (NCT02675439, NCT03010176, NCT03956680) for cancer immunotherapy and vaccine adjuvating, but these anionic small molecules suffer from poor uptake and bioavailability

[0010] , As an alternative route to cGAS-STING activation, Mn has been reported to induce activation of STING on its own [11, 12], For Mn to activate cGAS-STING, it needs to accumulate in the cytosol of cells where the proteins that control the pathways reside; how ever, high concentrations of free Mn salts are required for intracellular accumulation to occur, as cells are notoriously selective in permitting the translocation of charged metal ions through their lipid bilayer

[0013] , Therefore, using Mn salts alone presents significant issues in therapeutic use, and more efficient intracellular delivery strategies promoting STING agonist uptake are required.

[0200] A relatively underexplored method of intracellular metal delivery uses nano-sized metalorganic frameworks (MOFs). MOFs are a crystalline class of porous coordination polymers of metal ions interconnected by organic linkers

[0014] , MOFs have become ubiquitous since their discover}’ owing to a wide range of applications attributed to their tunable size, scalability, porosiN, and post-synthetic functionalization

[0015] , Some of their industrial-focused applications include molecular separation

[0016] and energy storage

[0017] , Concurrently, MOFs have been expanding within the biomedical field with applications such as imaging [18. 19], biocatalysis

[0020] , biosensing

[0021] , and cargo deliver}’

[0022] , These materials stand out in biomedicine because of their tunable qualities like size, surface area, and stable ordered conformation of labile metals. While there are many nanocarriers, like liposomes

[0023] and virus-like particles

[0024] , the robustness and rigidity of MOFs can provide the thermal and mechanical stability most soft biomaterials cannot. They can also serve as an excellent platform to deliberately traffic certain metals into cells if desired. MOFs can be degraded under different physiological or environmental conditions while also resisting damage from external stimuli [25, 26], This kinetic lability and thermodynamic stability has been exploited for therapeutic and prophylactic treatments [27-32], Of the many MOFs, zeolitic-imidazolate framework-8 (ZIF-8) has become a popular candidate for biomedical applications

[0033] , ZIF-8’s popularity is attributable to several factors, including its well-studied biodegradability in biological systems and its friendly synthesis conditions, which alloyv for incorporating other biomaterials like protein or DNA [34, 35], Previous literature has explored promising vaccine adjuvanting effects of ZIF-8. which could expand the library of metal-based adjuvants

[0036] , MOFs — the ZIF family in particular — can be tuned through reticular synthesis, allowing for the modular incorporation of multiple different linkers

[0037] or multiple metals

[0038] to create multivariate MOFs with properties unique or distinct from MOFs that are made of a single metal or ligand

[0039] , Diversifying the composition of metals in such ZIF structures can provide different functions, such as reducing its cytotoxicity, as each metal is metabolized differently in a cell. Moreover, metals reported to have therapeutic value can be strategically chosen and the metal delivery can be increased while providing a synergistic effect. Considering ZIF’s intercellular delivery potential, combining Mn into the framework would allow for efficient delivery of metal and attached cyclic dinucleotide molecules

[0040] , While there are literature reports ofZIFs incorporating Mn, they have extremely low percentages of incorporated Mn and are synthesized in methanol, thus requiring extensive washing and activation before in vivo use and making the synthesis incompatible with biomacromolecule encapsulation [41, 42], In contrast, the reported synthesis of ZIF made with 100% Mn is unstable outside an argon environment [41-43],

[0201] Herein, a green, bio-friendly approach was developed to dope different Mn ratios into zinc-based ZIF, creating Mn-ZIF with varying percentages of Mn in a simple, 20-minute, one- pot reaction (Figure 1 A). Electrostatic interactions between the anionic CDA and the cationic surface of Mn-ZIF allow for the co-delivery of additional STING agonists. It was found that Mn- ZIF exhibits significantly decreased cytotoxicity compared to ZIF-8 at 50% doping. Mn-ZIF- based particles allow for efficient cell uptake, effectively delivering Mn into cells (Figure IB). Degradation of the particle and release of the components into the cytosol tngger cGAS-STING activation, resulting in proinflammatory cytokine production and murine bone marrow dendritic cells (BMDCs) activation (Figure 1C).

[0202] Results and Discussion. An aqueous synthesis incorporating Mn and Zn was developed to make the reaction green and biofriendly. This method allows for easier scalability, more direct use in vitro, and future application in the growing field of biomimetic encapsulation, where an expanded library of MOFs is desirable

[0044] , Various synthetic conditions were tested to achieve a wide range of Mn incorporation in nano and micron sizes. Mn2+oxidizes in water to form various manganese oxides, and it was quickly realized that a mild reducing agent is needed to keep the Mn2+stable as stocks and during the reaction. Three reducing agents, sodium ascorbate, sodium citrate, and tris(2-carboxyethyl)phosphine (TCEP), were tested. It was found that TCEP did not interfere with the formation of the MOF while allowing for the maximum Mn loading possible and was, hence, the reducing agent of choice for future experiments. The results of the optimization experiments are summarized and tabulated in Table 1. Table 1. Mn substitution % vs reducing agent data. Comparison of TCEP and sodium citrate as reducing agents.

[0203] TCEP-assisted Mn-doped ZIF (Mn-ZIF) was prepared by adding DI water, TCEP, manganese(II) acetate tetrahydrate, zinc(II) acetate dihydrate, and 2-methylimidazole (HMIM) — in that order — and left to react statically at room temperature. Synthetic conditions for these reactions have been tabulated in Table 2. It was found that particle sizes could be controlled from 500 nm to 2 pm by varying the metal and ligand ratios, the results of which have been tabulated in Table 3. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the amount of Mn-doped into ZIF. The percent doping was calculated based on the mole percent of Mn from the total moles of Mn and Zn in a sample. More specifically, the Mol% Mn was found by converting ppm of Mn and Zn to mmol and using the following equation: ((X ppm / 1000 mg / ppm)*10ml) / 55mg / mmol. Mol% was found by using: 100*( mmol Mn / mmol Mn + mmol Zn)).

[0204] Table 2. Reaction Conditions at 15 ml final volume for ZIF-8 and Mn-ZIF with different concentration of manganese.

[0205] Table 3. Formulation vs particle size. It was found that the concentration of Mn was highly tunable; 15%, 30%, and 50% were chosen as representative samples whose crystallinity was retained when examined using powder x-ray diffraction (PXRD) (Figure 2A). The PXRD patterns confirmed that the Mn-ZIF formulas and ZIF-8 synthesized with TCEP (0% Mn) were isostructural to simulated sodalite ZIF-8. The diffraction results demonstrate that TCEP does not interfere with the sodalite topology formation of ZIF-8 or Mn-ZIF. Past 50%, the material became amorphous with no distinct peaks in the PXRD pattern. PXRD of a representative sample calculated to have 70% Mn is shown in Figure 6. The morphology of the various Mn-loaded samples was then assessed by scanning electron microscopy (SEM). A less common rounded cubic structure can be seen for each sample compared to the typical rhombic dodecahedron of sodalite ZIF-8 (Figure 2B-Figure 2E). SEM further shows a sub-500 nm size for each formulation, but the size slightly increases as more Mn is incorporated. X-ray photoelectron spectroscopy (XPS) survey and Ols spectrums were collected for 50% Mn-ZIF and ZIF-8 (Figure 7A-Figure 7B). Oxygen was detected in both samples, most likely due to three sources surface metal oxides, metal carbonates, and residual water with respective bond energies of 529-530 eV, 530-532 eV, and 533-534 eV [45, 46], Similar oxidation character was observed with Mn-ZIF having a slightly shifted peak to higher energies, suggesting more water and carbonate bonding than oxide bonding as seen in ZIF-8. Further, the survey spectrum for Mn-ZIF shows the signature Mn peaks along with Zn peaks seen in the ZIF-8 spectrum, supporting the mixed metal results of ICP-MS.

[0206] Before proceeding to immune activation experiments, it is important to understand the cytotoxicity of the material so that the right dosage can be determined for in vitro and in vivo applications. Cell viability assays were used to determine the half maximal inhibitory concentration (ICso) of Mn-ZIF with different doping percentages compared to control ZIF-8. Three cell lines were used: RAW 264.7, 4T1, and HEK 293 (Figure 3A-Figure 3C). These represent various cell types and tissues: murine immune cells, murine breast cancer epithelial cells, and human kidney epithelial cells respectively. Three viability’ assays were tested, including tetrazolium-based lactate dehydrogenase (LDH), MTT assay, and resazurin. Tetrazolium-based dyes that utilize redox-active formazan rings were incompatible with Mn as it interfered with the assay’s color development; consequently, the LDH and MTT assays produced spurious results. An example can be seen in Figure 8 where after lysing cells in the presence of Mn-ZIF or Mn2+salt, LDH still had low signal, indicating living cells similar to media when they should produce signal similar to the lysed control group. Assays using tetrazolium dyes have been previously reported to have issues measuring redox-active compounds [47, 48] . In contrast, the resazurin-based dye appears inert to Mn. Though the resazurin assays are also redox-based, extensive control experiments were performed to confirm that Mn-ZIF did not react with the dye, and the dye's fluorescence eliminated interference from the physical absorption of the MOF (Figure 9). Across all three cell lines, viability increased as Mn substitution increased. While the difference observed in RAW macrophages was small but not statistically significant, cell viability of 50% Mn-ZIF was considerably higher than that of ZIF-8 in 4T1 and HEK cells. 4T1 showed an 84% increase in tolerance (60.3 ± 3.1 pg / mL vs 1 11.3 ± 0.8 pg / mL) and HEK was even larger, with an 87% increase (81.8 ± 9.6 pg / mL vs 153. 1 ± 0.4 pg / mL) when comparing the two groups. The results have been summarized in Figure 3D. It is hypothesized that the increased viability could be attributed to Mn and Zn being metabolized using different biological processes and the reduced total amount of each metal likely reduces the overall toxicity in some cell lines. The ICso values are promising as only a small amount of Mn — in the pM range based on endogenous levels

[0049] — is needed to induce cGAS-STING activation, allowing for flexible dosing with Mn-ZIF

[0011] ,

[0207] With cytotoxicity data for Mn-ZIF, next the ability to exploit it as a delivery method for Mn2+to activate cGAS-STING activation was investigated. For all future experiments, the 50% Mn-ZIF formulation was used. Mn co-delivered with a cyclic dinucleotide can work synergistically to activate STING and create a more robust immune response

[0050] , As a model, the known STING agonist CD A. a molecule belonging to the cyclic dinucleotide family

[0051] , was incorporated. An additional benefit of loading CDA on to Mn-ZIF is nanoparticle-mediated delivery', which can enhance uptake compared to just CDA — a negatively charged small molecule. First, CDA was post-synthetically loaded onto the surface of Mn-ZIF in methanol over 24 hours on rotisserie at room temperature (Figure 4A). Amount of CDA absorbed from solution was above 96% as determined by nanodrop UV-Vis spectroscopy (Figure 4B). Liquid chromatography (LC) supported this with greater than 94% absorption (Figure 10 and Figure

[0208] 11). Notably, a 30 pg / ml concentration of CDA to 1 mg / ml of Mn-ZIF was ideal, as efficiency decreased with increasing CDA concentration. Zeta-potential further confirmed the loading as the surface of the Mn-ZIF became more negatively charged owing to the negative charge of the CDA (Figure 4C). Additionally, the CDA-loaded Mn-ZIF (CDA@Mn-ZIF) retained 94% of the loaded CDA after 24 h, confirming good stability' and CDA retention of the composite (Figure

[0209] 12).

[0210] To demonstrate the uptake of Mn-ZIF and co-dehvery of Mn and CDA. a fluorescent dye was incorporated into the pore structure via coprecipitation during synthesis before loading CDA. 5,6-carboxyfluorescein (CF) and Rhodamine B (Rh) were chosen because of their high quantum yield and loading efficiency. CF and Rh were incorporated into the crystalline structure during the Mn-ZIF reaction by conducting the MOF synthesis in an aqueous solution of concentrated CF and Rh to form fluorescent nanoparticles (Mn-ZIF(CF)) and (Mn-ZIF(Rh)). Mn-ZIF (CF) and Mn-ZIF (Rh) were then coated with CD A post-synthetically with the same method to create CDA@Mn-ZIF(CF) and CDA <7 n-ZIF (Rh) and can be seen in the green or red channels of an epifluorescence microscope respectively (Figure 13A-Figure 13B). Both Mn- ZIF(Rh) and CDA? / Mn-ZIF(Rh) were used to conduct uptake studies on RAW macrophage cells. Uptake was visualized on an epifluorescence microscope at 6 h, and CDA@Mn-ZIF(Rh) is represented in Figure 4D. The Mn-ZIF(Rh) signal can be seen around the nucleus in the red channel, suggesting effective uptake into the cell's cytosol. A quantitative assessment of uptake was performed using flow cytometry on RAW cells. Mn-ZIF was used as a non-fluorescent control to account for scattering as a potential source of signal. Pure CF dye was used to show uptake of a representative, negatively-charged small molecule. Four- and eight-hour time points were assessed for uptake by incubating cells with Mn-ZIF, CF, Mn-ZIF(CF) and CDA@Mn- ZIF(CF) at each time. A representative histogram of 8 h is shown in Figure 4E, and the geometric mean of the CF intensity using the FITC channel is represented in Figure 4F. Free CF's uptake was very limited, but saw a significant increase once encapsulated within Mn-ZIF. The enhanced CDA@Mn-ZIF(CF) uptake compared to Mn-ZIF(CF) was surprising, given that its surface charge was more negative than Mn-ZIF(CF) alone. It was hypothesized that this result could be attributed to protein corona formation from the bovine serum albumin used in the cell media, which has previously been reported to enhance the uptake of negatively charged particles [52, 53],

[0211] Following uptake studies of Mn-ZIF, the ability of antigen presenting cells (APCs) to be activated in-vitro by delivering a combination of Mn and CDA was investigated. Activation of APCs is extremely important for host defense from pathogens and cancer and stimulation of cGAS-STING is one way to achieve activation

[0054] , cGAS-STING responses can be measured in-vitro, by surface marker staining and by quantifying the proinflammatory cytokines produced by the cells. Primary murine bone marrow dendritic cells (BMDCs) were isolated and used as target APCs. MnCh, ZIF-8, CDA, Mn-ZIF, and CDA@Mn-ZIF were normalized to achieve 10 pg / ml Mn and 1.7 pg / ml CDA then tested against the BMDCs. When added to BMDC cultures, Mn-ZIF outperformed a mole-equivalent dose of MnCh while performing similarly to LPS, a positive control which is well known to activate BMDCs. CDA@Mn-ZIF outperformed all the other groups based on dendritic cell activation — CD80+CD86+CD1 lc+cells (Figure 5A). Cytokines released from the BMDCs into the cell supernatants were measured by ELISA. Mn- ZIF and CDA@Mn-ZIF elicited a proinflammatory response, quantified using cytokines TNF-a (Figure 5C) and IL-6 (Figure 5D). Cytokine production from equal doses of ZIF-8 and CDA were below limit of detection, and MnCb alone only produced IL-6. While cytokine production was considerably higher in the LPS control, it is important to note this level is undesirably high; excess IL-6 produced from LPS can lead to toxicity, as seen in sepsis

[0055] , This demonstrates that Mn-ZIF can generate an immune response alone and synergistically increase potency with CDA.

[0212] To confirm that the BMDC activation and cytokine production was a result of STING, western blot was used to see activation of two proteins downstream of STING, TBK-1 and IRF- 3. Upon STING activation, TBK-1 will phosphorylate (P-TBK-1) and induce IRF-3 phosphorylation (P-IRF-3), further leading to proinflammatory gene expression generating cytokines [6], Western blot confirmed that Mn-ZIF and CDA@Mn-ZIF could trigger phosphorylation of both TBK-1 and IRF-3 in RAW 2.647 cells, while the other controls could not (Figure 5B). This result undoubtedly links the cytokine production observed to Mn-ZIF and CDAi a n-ZIFs ability to activate STING.

[0213] In addition to cytokine production, increased radical oxygen species (ROS) generation inside the cells without triggering additional cell death was observed using an intercellular ROS detection kit (Figure 14A-Figure 14B). ROS have been implicated in promoting lymphocyte activation by playing a role in antigen process and presentation, and increased ROS is indicative of activated immune cells

[0056] ,

[0214] Materials and Methods

[0215] Chemicals: TCEP-HC1 tris(2-carboxyethyl) phosphine hydrochloride was purchased from Goldbio. Zinc (II) acetate dihydrate, 2-methylimidazole. and manganese (II) acetate tetrahydrate, L-glutamine solution, sodium hydroxide, 5(6)-carboxyfluorescin and fluorometric intracellular ROS kit (deep red) were purchased from Millipore Sigma. Deep Blue Cell Viability kit, FITC anti-mouse CD11c antibody, Alexa Fluor® 594 anti-mouse CD80 antibody, and Pacific Blue™ anti-mouse CD86 antibody were purchased from Biolegend. CDA was purchased from Invivogen. Nitric acid (trace metal grade) was purchased from Thermo Fisher Scientific. HyClone phosphate buffered saline solution, HyClone Dulbecco's modified eagle's medium (DMEM), HyClone RPMI 1640 medium, and penicillin-streptomycin were purchased from Cytiva. FB Essence was purchased from Avantor.

[0216] Instruments. SEM micrographs were captured on a Zeiss Supra 40. PXRD spectra were collected using Rigaku SmartLab X-ray Diffractometer. DLS measurements for size and zeta potential were carried out using Malvern Analytical Zetasizer Nano ZS. Fluorescence intensity' measurements on 96-well plates were carried out using Biotek Synergy H4 Hybrid microplate reader. Epifluorescence images were taken on EVOS FL digital inverted fluorescence microscope. Cell counting was carried out using Thermo Countess II. Zinc and manganese quantification was done using Agilent 7900 ICP-MS. CDA quantification was done using NanoDrop and Agilent 1100 series LC. X-ray photoelectron spectra was recorded using PHI VersaProbe II Scanning XPS Microprobe. Flow cytometry was performed on the BD Fortessa.

[0217] Cell lines: RAW 264.7 cells were received as a gift from Dr. Rockford Draper (Department of Biological Sciences, UT Dallas). 4T1 cells were received as gifts from Dr. Laurentiu Pop (Department of Radiation Oncology , UT Southwestern). HEK 293 cells were received as a gift from Mateusz Durbacz (Department of Molecular Biology and Hamon Center for Regenerative Science and Medicine, UT Southwestern).

[0218] Mn-ZIF synthesis: Four stocks were prepared in MilliQ water, a 1 M solution of zinc (II) acetate dihydrate, 3 M 2-methylimidazole (HMIM), 0.5 M TCEP-HC1, and 1 M manganese (II) acetate tetrahydrate with lOmM TCEP. For a 15 ml reaction of 50% Mn-ZIF reactants were added in the following order: (1) 550 pl H2O, (2) 450 pl TCEP, (3) 800 pl Mn, (4) 400 pl Zn, and (5) 12.8 ml HMIM. After adding HMIM, the reaction was vortexed and left static at RT for 20 mins, then centrifuged at 4,300 * G for 15 m. Supernatant was discarded, the pellets were washed twice with water, and subsequently dried under high vacuum overnight.

[0219] Sample preparation for in vitro and in vivo experiments: 10 mg of Z1F-8 or 15 / 30 / 50% Mn-ZIF was w eighed on an analytical balance and suspended in a glass vial with endotoxin-free, sterile w ater to make a 10 mg / ml stock. The stock solution w as vortexed and sonicated to ensure proper dispersion.

[0220] In vitro cytotoxicity: Biolegend’s resazurin based “Deep Blue Cell Viability” kit was used for cytotoxicity and IC50 calculations. 25,000 cells per well were seeded in a 96 well plate overnight in 50 pl of media and incubated at 37° C and 5% humidity. The following morning, 50 pl of 2x desired final concentration of sample was added to n = 4 wells. The plate was placed back in the incubator for 24 h. 30 min before the 24 h time point, 10 pl of lyse buffer was added to appropriate wells as a negative control. At 24 h, 10 pl of the resazurin reagent was added to each well and mixed before being returned to the incubator to incubate for 4 h before being read at an excitation of 530 nm, and an emission of 590 nm on a micro plate reader. % viability7was normalized to media only and lysed cell control and data is presented as average ± standard deviation (n = 4 with outlier analysis done in GraphPad Prism with Grubbs’ method). IC50 was determined using linear regression function in Microsoft Excel.

[0221] CDA loading on Mn-ZIF. To prepare CDA@Mn-ZIF, 1 mg of Mn-ZIF w as mixed with 30 pg CDA in methanol. This sample was placed on rotisserie at RT for 24 h. After the 24 h period, the CDA <7,Mn-ZIF was centrifuged at 17,000 * G for 10 m and the supernatant was collected for concentration determination by LC and NanoDrop UV-VIS. CDA@Mn-ZIF was washed once with water and then dried overnight in a high vacuum chamber for storage, or directly resuspended in DI water for use.

[0222] Determination of CDA concentration by LC. A standard curve of CDA was first prepared by diluting a 1 mg / mL stock to 25 pg / mL and making half serial dilutions down to 1.5625 pg / mL. These standards were run through PLRP column on the LC with UV detector set to 260 nm wavelength. Water and acetonitrile with 0.1% formic acid were used as eluting solvents. 5 pL of each sample was injected on the following gradient: 20% acetonitrile for 2 minutes, an increasing gradient from 20% to 60% for 30 minutes, hold at 60% for 3 minutes, and a decrease down to 20% over 2 minutes. Unknown samples were run under the same conditions and concentration detemiined by peak area integration on GraphPad.

[0223] Bone marrow dendritic cell cultures. Femurs and tibias were isolated from naive mice and placed in PBS. Surgical scissors were used to snip both ends of the bone, and a 25 G needle was used to flush the marrow out using RPMI over a 70 pm single cell suspension filter. Cells were centrifuged at 500 x G for 5 m and supernatant removed. RBC lysis buffer was used to removed red blood cells, after which the cells were counted and suspended in 10 mL RPMI with 20 ng / mL of GM-CSF in a T-75 at a concentration of 2 x 106cells / ml. 10 mL of RPMI with GM- CSF was added 3 days later, and media was refreshed on day 6 and 9.

[0224] BMDC activation and cytokine production . Non-adherent cells were collected on day 10 from the previously described cultures and seeded at a concentration of 200,000 cells per w ell in a 96 well plate with final volume being 200 pL with samples. Final concentration of each sample was 1.7 pg / ml of CDA, 36 pg / ml of MnCb • 4 H2O, and 60 pg / ml of ZIF-8, 60 pg / ml Mn-ZIF loaded with 1.7 pg / ml CDA. Samples were incubated for 20 h at which point the plate was spun and supernatants were taken and frozen for cytokine ELISA. BMDC's were then washed with PBS and followed a staining protocol. First samples were incubated in zombie UV in PBS followed by addition of a staining cocktail of CD11c, CD80, and CD86 and left on ice. Samples were w ashed twice with cell staining buffer then resuspended and ran on flow.

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[0281] Example 2

[0282] Main Idea. A water based synthesis was designed to incorporate Mn into ZIF-8 and then post-synthetically load CDA, a STING agonist (Figure 1 A, Figure 15). The synthesized Mn-ZIF was less cytotoxic and works as a carrier to take Mn and CDA into the cytosol of cells (Figure IB). Once in the cytosol. Mn and CDA can trigger the cGAS-STING pathway, activate dendritic cells, and induce proinflammatory cytokine signaling (Figure 1C).

[0283] Mn-ZIF Synthesis at Various Mn Incorporation percentages. Mn was incorporated into ZIF-8 by varying the Mn to Zn metal ratios in a one pot synthesis. The mole ratios of Mn to Zn were determined by ICP-MS and sodalite topology was confirmed through PXRD analysis of samples with 15%. 30%. and 50% Mn (Figure 2A). Size tends to increase with more Mn incorporated, but stays below 500 nm, with the largest being 50% as shown in SEM (Figure 2B- Figure 2E).

[0284] Cytotoxicity Evaluation of Mn-ZIF. Cytotoxicity' of the different Mn-ZIF percentages were determined through a fluorescent assay. Across three different cell lines, increasing concentration of Mn led to increased inhibitory concentration (Figure 3A-Figure 3D). This allows for more flexibility in dosing before the MOF becomes toxic.

[0285] CDA@Mn-ZIF and Uptake. Post-synthetic loading of CDA onto Mn-ZIF was confirmed through UV-VIS and zeta-potential (Figure 4A-Figure 4F). Two dyes were also loaded for visualization and quantification of uptake demonstrating Mn-ZIFs potential for delivery.

[0286] In Vitro Immune Response. Increased Mn and CDA in the cytosol has been shown to stimulate a proinflammatory immune response through activation of the cGAS-STING pathway. Murine BMDCs were isolated and incubated with samples for 24 h. Cell supernatants were collected and tested for TNF-a and IL-6 by ELISA. Confirmation of STING activation was shown through WB of phosphorylated axis proteins. Taken together CDA@Mn-ZIF is a potent immune stimulator (Figure 5A-Figure 5D).

[0287] Example 3 - Biofriendly Synthesis of Manganese-Doped ZIF with Reduced Cytotoxicity and Enhanced Immunogenicity

[0288] Metal-organic frameworks (MOFs) have become ubiquitous since their discovery' due to a wide range of applications attributed to their tunable size, scalability, porosity, and post synthetic functionalization. Some of their original applications include gas separation, storage, and catalysis while there is an expanding field of biomedical applications such as imaging, biocatalysis, and cargo delivery. A reason MOFs stand out in biomedicine is their ability7to be grown nano size. While there are a many nanocarriers for example virus-like particles, liposomes, or polymeric materials. MOFs can protect and / or target delivery while maintaining kinetic lability. These desirable properties have been exploited for both prophylactic and therapeutic treatment with recent focus in immune modulation. Of these MOFs, zeolitic- imidazolate framework-8 (ZIF-8) has become a popular candidate for biomedical applications. The challenge with MOFs in biomedical applications is the lack of clinical relevance while one is in phase one. none have made it through clinical trials and been FDA approved. Part of the challenge is that there are toxicity concerns related to their metal component. Cells regulate materials differently and one way to address toxicity' is by offsetting the concentration byreplacing with an analogue that can be cleared differently. Additionally, the MOF itself does not contribute to therapeutic or immunological effect as they typically function as a carrier or platform. MOFs can be tuned through reticular synthesis such as incorporating two linkers or multiple metals to gain more favorable properties, potentially addressing both concerns.

[0289] ZIF is well investigated in this area with many known polymorphs and isoreticular structures, including mixed-metal versions.

[0290] Some metals can have therapeutic functions on their own but suffer from poor uptake and cell delivery. Recently, Mn has been reported to induce the cGAS-STING pathway, which is an immune stimulating pathway important to both pathogen defense and tumor immunity. Mn needs to be in the cytosol of cells to activate cGAS-STING pathway but high concentrations (mg / mL) of Mn salt are needed for activation to achieve this so a delivery- method is desirable. Considering ZIFs intercellular delivery potential, combining Mn into the framework could allow for efficient delivery7but there is limited development in ZIFs incorporating Mn. Herein, a green, biofriendly synthesis was developed to dope different ratios of manganese into zinc based ZIF creating Mn-ZIF with varying precents of manganese (Figure 16). Interestingly by swapping enough Zn for Mn, Mn-ZIF exhibited decreased cytotoxicity compared to ZIF-8. Mn-ZIF was grown nano sized to allow for efficient cell uptake through the endosome. Degradation of the particle and release of the components into the cytosol potentiate activation in murine BMDC’s.

[0291] Results and Discussion. A water-based synthesis was employed to form ZIF-8 with manganese doped in so the reaction would be green and be compatible with other applications such as protein encapsulation. Since Mn2+oxidizes in water, reducing agents were used to scrub oxygen and keep the Mn2+stable as stocks and during the reaction. Three reducing agents, sodium ascorbate, sodium citrate, and tris(2-carboxyethyl)phosphine (TCEP), were tested where TCEP yielded the most flexibility in substitution precent so it was used in material for future experiments.

[0292] ZIF-8 synthesis protocols that were previously established were modified to achieve a wide range of Mn incorporation with various sizes. Briefly, stocks were made of TCEP, Mn, Zn, and 2-methylimidazole (HMIM) in water and then combined in that order at different ratios and left statically at room temp.

[0293] Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the amount of Mn doped into ZIF-8. The % doping w as calculated based on mol % of Mn from total moles of Mn and Zn in a sample.

[0294] Powder X-ray diffraction (PXRD) was used to confirm topology of ZIF-8 synthesized with TCEP matched simulated sodalite ZIF-8. Spectra correlated well showing no interference from the TCEP. Next PXRD was used to confirm ZIF-8’s topology was maintained at different doping precents of Mn. The concentration of Mn w as highly tunable but 15%, 30%, and 50% were chosen as doping precents to check.

[0295] PXRD of Mn-ZIF up to 50% Mn correlated well with the simulated pattern showing crystallinity is retained even with a large portion of manganese added (Figure 2A). Past 50% the material became amorphous and lost all low angle crystallinity so 50% Mn became the max for future experiments.

[0296] Next SEM was used to visualize samples and interestingly shows that morphology is not the typical rhombic dodecahedron of sodalite ZIF. SEM does demonstrate a sub 500 nm size for each formulation ideal for uptake but as more Mn is incorporated the size slightly increases (Figure 2B-Figure 2E). XPS survey scan was used to show presence of Mn and Zn in sample of 50% Mn-ZIF and just Zn in ZIF-8 (Figure 7A).

[0297] Before proceeding to immune activation experiments it is important to understand the cytotoxicity' of the material so a proper dose can be selected. Cell viability assays were used to determine IC50 of Mn-ZIF with different precents of doping compared to control ZIF-8. Three different cell lines were used, RAW 264.7, 4T1, and HEK 293. These represent a wide range of cell ty pes and tissue including murine immune cell, murine breast cancer epithelial cells, and human kidney epithelial cells. Three different viability’ assays were tested including tetrazolium based LDH-Cytox. MTT assay, and Biolegend’s resazurin based Deep Blue Cell Viability kit (DBCV). Tetrazolium based dyes caused problems where Mn2+can interfere through redox reactions and the undissolved MOF can cause increased background absorption. It is notable that while DBCV kit is also redox based, controls showed the Mn-ZIF did not react with the dye and the fluorescence of the dye eliminated interference from physical absorption of the MOF. Across all three cell lines, viability' increased as Mn substitution increased where the difference between ZIF-8 and 50% Mn-ZIF in 4T1 and HEK cells was significant: 60.3 ± 3.1 pg / ml vs 111.3 ± 0.8 pg / ml for 4T1 and 81.8 ± 9.6 pg / ml vs 153.1 ± 0.4 pg / ml (Figure 3A-Figure 3D). The increased viability could be attributed to manganese and zinc using different metal transporters in the cells. By maintaining total metal concentration between ZIFs but balancing the zinc and manganese concentration the cells can more easily maintain homeostasis.

[0298] With toxicity7data for Mn-ZIF, its ability’ to act as a nano carrier for Mn2+and deliver enough Mn2+into cells to sensitize cGAS-STING activation was investigated. For these tests the 50% Mn-ZIF was used to give the best chance at response. When added to BMDC cultures from mice, Mn-ZIF was able to outperform a mole equivalent dose of MnCb while performing similar to LPS based on activated dendritic cells — those double positive for CD80 and CD86 gated on CD1 lc+. Supernatants were taken at the end of the BMDC incubation time and cytokine production was measured by ELISA. Mn-ZIF was able to elicit the excretion of proinflammatory cytokines TNF-a and IL-6 where equal doses of ZIF-8 and MnCh were unable (Figure 5A- Figure 5D). While cytokine production was less than the LPS control, excess IL-6 produced from LPS can lead to toxicity' such as seen in sepsis. Demonstrating Mn-ZIF can generate a reasonable immune response.

[0299] Western blot was used to confirm that immune activation was a result of STING activation promoted by manganese binding. TBK-1 and IRF-3 are proteins downstream of STING that will phosphorylate because of STING activation and can be used to definitively show activation. Additionally, increased radical oxygen species (ROS) generation was seen inside the cells without triggering additional cell death (Figure 14A-Figure 14B). ROS has been implicated in promoting lymphocyte activation by playing a role in antigen process and presentation.

[0300] To further increase the immune adjuvating potential, a known STING agonist, cyclic diadenosine monophosphate (CD A), from a class of messaging molecules called cyclic dinucleotides (CDNs), was incorporated. Manganese when delivered with CDNs can work synergistically to create a much stronger immune response.

[0301] Materials and Methods

[0302] Chemicals. TCEP tris(2-carboxyethyl) phosphine was purchased from Fisher Scientific. Zinc (II) acetate dihydrate, 2-methylimidazole, and Manganese (II) acetate tetrahydrate were purchased from Millipore Sigma. Deep Blue Cell Viability kit and all antibodies were purchased from Biolegend.

[0303] Mn-ZIF synthesis. In general, four stocks were prepared in milliq DI water, a 1 M solution of zinc (II) acetate dihydrate, 3 M 2-methylimidazole (HMIM), 0.5 M TCEP-HC1, and 1 M manganese (II) acetate tetrahydrate with lOrnM TCEP. For a 15 ml reaction of 50% Mn-ZIF reactants were added in this order: 550 pl FEO first, 450 pl TCEP, 800 pl Mn, 400 pl Zn, and 12.8 ml HMIM. After adding HMIM the reaction was mixed by vortex and left static at RT for 20 mins then centrifuged at 4,300 x G for 15 minutes. Supernatant was discarded and reaction were w ashed twice with water and then dried on high vacuum overnight.

[0304] Sample preparation for in-vitro and in-vivo experiments. Roughly 10 mg of ZIF-8 or x% Mn-ZIF was weighed on an analytical balance and suspended in a glass vial with endotox free sterile water for a concentration of 10 mg / ml. This was vortexed and sonicated to ensure proper dispersion.

[0305] In vitro cytotoxicity. Biolegend’s “Deep Blue Cell Viability ” kit was used for cytotoxicity and LD50 calculations. 25,000 cells per well were seeded in a 96 well plate overnight in 50pl of media and incubated at 37°C and 5% humidity. The following morning. 50 pl of 2x desired final concentration of sample was added to n = 4 wells. The plate was placed back in the cell incubator for 24 h. 30 min before 24 h time point, 10 pl of lyse buffer was added to appropriate wells as a negative control. At 24 h 10 pl of the deep blue dye was added to each well and mixed before being returned to the incubator to incubate for 4 h before being read at 530 exc 590 emm on a micro plate reader. % Viability was normalized to media only and lysed cell control and data is presented as average ± standard deviation (N = 4 with outlier analysis done in GraphPad Prism with Grubbs’ method). ICso was determined using linear regression function in excel. Bone marrow dendritic cell cultures. Femurs and tibias were isolated from naive mice and placed in PBS, scissors were used to crack both ends and a 25 g needle was used to flush the cavity with RPMI over a 70 pm single cell suspension filter. Cells were centrifuged at 500 x G for 5 min and supernatant removed. RBC lysis buffer was used to removed red blood cells and cells were counted and suspended in 10 ml RPMI with 20 ng / ml of GM-CSF in a t75 at a concentration of 2 xl06 cells / ml. 10ml of RPMI with GM-CSF was added 3 days later, and media was refreshed on day 6 and 9.

[0306] BMDC activation and cytokine production . Non adherent cells w ere collected on day 10 for use and seeded at a concentration of 200,000 cells per well in a 96 well plate with final volume being 200 pl with samples. Samples were incubated for 20 h at which point the plate was spun and supernatants were taken and frozen for cytokine ELISA. BMDC’s were then washed with PBS and followed a staining protocol.

[0307] Instruments. SEM images were captured on a Zeiss Supra 40 at 2.0 kV using an SE2 detector. Low beam voltage was used to reduce charging and material decomposition. Samples were prepared by suspending material in water and drop casting by pipetting on a silicon substrate. PXRD spectra was determined using Rigaku SmartLab X-ray diffractometer.

[0308] Example 4

[0309] A Manganese-based metal-organic framework was developed that encapsulates biomacromolecules (including but not limited to proteins, DNA, RNA, and lipids) within its interior. The nanoparticle composition of this material facilitates cellular uptake of the manganese metal and the encapsulated biomacromolecules. This material induces the activation of multiple cells in the immune system and promotes the activation of STING pathways. This material has applications in several immunotherapeutic agents for cancer and infectious disease. Further, the material may be used to treat intracellular bacterial disease, it may promote cellular sensitivity to radiotherapy in both radiation and non-radiation tolerant cancer.

[0310] Described herein is a nanoparticle that is prepared using manganese as a bioavailable metal. Also described herein is a nanomaterial where additional metals may be incorporated, including Zinc, aluminum, copper, magnesium, etc.

[0311] Also described herein is a nanomaterial that coats and contains a therapeutic small molecule or biological drugs or a combination of several therapeutics.

[0312] Also described herein is a nanomaterial where the small molecule or biological therapeutic is protected from degradation by enzymes or chemical agents.

[0313] Also described herein is a manganese nanoparticle that works synergistically with other molecules that stimulate the STING response. Also described herein is a manganese nanoparticle that stimulates the STING response on its own.

[0314] Example 5 - Mn and Zn-Doped Multivariate Metal-Organic Framework as a Metalloimmunological Adjuvant to Promote Protection Against Tuberculosis Infection

[0315] Abstract: A first-in-class vaccine adjuvant delivery system, Mn-ZIF, was developed by incorporating manganese (Mn) into the zinc-containing zeolitic-imidazolate framework-8 (ZIF- 8). The mixed metal approach, which allowed for tunable Mn doping, was made possible by including a mild reducing agent into the reaction mixture. This approach allowed up to 50% Mn. with the remaining 50% Zn within the ZIF. This multivariate approach exhibited significantly decreased cytotoxicity compared to ZIF-8. The porous structure of Mn-ZIF enabled the codelivery of the STING agonist cyclic di-adenosine monophosphate (CDA) through postsynthetic loading, forming CDA@Mn-ZIF. The composite demonstrated enhanced cellular uptake and synergistic activation of the cGAS-STING pathway, producing proinflammatory cytokines and activating antigen-presenting cells (APCs). In a preclinical Mycobacterium tuberculosis (Mtb) model, CDA? / Mn-ZIF formulated with the CysVac2 fusion protein elicited a potent antigen-specific T-cell response and significantly reduced the mycobacterial burden in the lungs of infected mice. These findings highlight the potential of CDA@Mn-ZIF as a promising adjuvant for subunit vaccines, offering a novel approach to enhancing vaccine efficacy and protection against infectious diseases such as tuberculosis.

[0316] Introduction. Modem vaccine technologies have moved away from traditional vaccine formulations such as inactivated whole-cell and live attenuated because of their lower safety profile, incompatibility with immunocompromised patients, and inability to protect against certain diseases [Al], One disease where live pathogen vaccination is still utilized as the primary mode of disease prevention is tuberculosis (TB), the second leading cause of infectious disease- related death in the world [A2], While the Bacille Calmette-Guerin (BCG) vaccine is sufficiently effective in young children, the protection is limited with age. and BCG boosters have limited efficacy [A3], It has been observed that there is a large variance in efficacy observed across populations — ranging from 0% to 80% [A4], Moreover, BCG causes disseminated BCG disease in HIV-positive populations, a common comorbidity factor for pulmonary7TB in the developing world [A5], In short, while BCG is the only fully clinically approved vaccine against TB, its efficacy and safety demand further work to develop new vaccines.

[0317] To overcome this wide array of limitations, there has been a gradual shift toward subunit vaccines — which contain pieces (<?.g., proteins, polysaccharides, toxoids) of the pathogen known as antigens. While this strategy effectively reduces the reactogenic effects that come with whole- cell formulations, using one, two, or even three antigens in the same vaccine formulation often does not stimulate an immune response as strongly as the inactivated or attenuated pathogen. In such cases, adjuvants — highly immunostimulatory materials used to enhance the efficacy of vaccines — are relied on to supplement the antigen [A6], Successful adjuvants work by triggering a specific pathway or pathways that synergistically work with the antigen to amplify the immune response, ultimately generating protection against the pathogen. Currently, there are only eight different FDA-approved vaccine adjuvants, and most vaccines use aluminum salts, approved more than 70 years ago [A7], An issue with the small portfolio of approved adjuvants is that most generate an innate and humoral or antibody -mediated response, leaving few options for triggering cell-mediated immunity. The innate immune system involves cells like macrophages and dendritic cells — known as antigen-presenting cells (APCs) — that provide early, nonspecific defense against pathogens and help potentiate adaptive immunity. In contrast, the adaptive immune system uses T- and / or B-cells to provide targeted, long-term protection against specific pathogens [A8], To create a robust immune response, innate and adaptative immunity must be triggered, including the humoral and cellular components of the adaptive response. Therefore, an adjuvant that activates dendritic cells to prime long-lasting adaptive immunity is desirable. An important pathway of the innate immune system that has gained significant interest is the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway [A9], It is traditionally a cytosolic DNA sensor responsible for host defense against pathogens and cancer. However, as agonists for cGAS-STING have become available, stimulation of this pathway has become a good target for adjuvants because of the potent innate immune response it triggers, which can activate APCs [A10, Al 1], By engaging APCs that interact with and activate T-cells, cGAS-STING can promote a strong adaptative response [A 10], These features have made the STING pathway a therapeutic and prophylactic target for cancer therapy and vaccine adjuvants [A12-A14], The canonical agonists for STING are a class of messaging molecules called cyclic dinucleotides (CDNs), the most studied of which is cyclic di-adenosine monophosphate (CDA, Figure 1A) [Al 5], A report by Van Dis et al. illustrates how subunit vaccine formulations adjuvanted with CDNs can activate the STING pathw ay and provide enhanced protection against a Mycobacterium tuberculosis (Mtb) infection [Al 6], CDN variants are in clinical trials (NCT02675439, NCT03010176. NCT03956680) for cancer immunotherapy and vaccine adjuvants, but these anionic small molecules suffer from poor cellular uptake and sensitivity to degradation by phosphodiesterases [ A 17] . As an alternative route to cGAS-STING activation, the metal manganese has been reported to sensitize and even induce activation of STING on its own [Al 8, A19], For Mn2+to activate cGAS-STING, it must accumulate in the cytosol of cells where the proteins that control the pathways reside. However, high concentrations of free Mn2+salts are required for intracellular accumulation, as cells are notoriously selective in permitting the translocation of charged metal ions through their lipid bilayer via selective and promiscuous transmembrane transporters [A20], Using Mn2+salts alone is limiting and insufficient for therapeutic use and calls for developing more efficient intracellular delivery strategies promoting STING agonist uptake. Ideally, a carrier that can both improve intercellular delivery and protection from degradation is needed, but the requirement of both these properties limits the list of potential carriers.

[0318] A promising option to co-deliver adjuvants and metal intracellularly is metal-organic frameworks (MOFs). MOFs are a crystalline class of porous coordination polymers of metal ions interconnected by organic linkers [A21], MOFs have become ubiquitous since their discovery owing to a wide range of applications attributed to their tunable size, scalability, porosity, and post-synthetic functionalization [A22], Some of their industrial-focused applications include molecular separation [A23] and energy storage [A24], Concurrently, MOFs have been expanding within the biomedical field with applications such as imaging [A25, A26], biocatalysis [A27], biosensing [A28], vaccines [A29], and cargo delivery [A30], MOFs specifically work well as nanocarriers because of their ability to traffic material into a cell and then release cargo via multiple paths [A31], While there are many nanocarriers, such as liposomes [A32] and virus-like particles [A33], the robustness and rigidity of MOFs can provide the thermal and mechanical stability7most soft biomaterials cannot [A34], While providing stability, most MOFs can be easily degraded under different physiological or environmental conditions, as inorganic anions and proteins can strip metals from these frameworks [A35, A36], This kinetic lability and thermodynamic stability have been exploited in preclinical therapeutic and prophylactic treatment research [A37-A42], Of the many MOFs, zeolitic-imidazolate framework-8 (ZIF-8) has become a popular candidate for biomedical applications [A43], ZIF-8’s popularity is attributable to several factors, including its well-studied biodegradability in biological systems and its friendly synthesis conditions, which allow for incorporating sensitive vaccine constituents like protein or DNA [A44, A45], Previous literature has explored promising adjuvanting effects of ZIF-8, which could expand the library of metal-based adjuvants [A46], The presence of zinc in ZIF-8 has recently been shown to traffic to the lymph node and increase T-cell activation and cytokine production [A47], Further, Zn2+has implications in the activation of cGAS; Zn2+is necessary for the enzyme to produce the CDN cGAMP after the detection of DNA [A48], MOFs - -the ZIF family in particular can be tuned through reticular synthesis, allowing for the modular incorporation of multiple different linkers [A49] or multiple metals [A50] to create multivariate MOFs with properties unique or distinct from MOFs made of a single metal or ligand [A51], Diversifying the composition of metals in such ZIF structures can provide different functions, such as reducing its cytotoxicity, as each metal is metabolized differently in a cell. Moreover, metals that have been reported to have therapeutic value can be strategically chosen and MOFs can be used as a delivery vehicle to traffic these metals into the cell. Finally, because MOFs can deliver many types of cargo, adjuvants can be selected that can work synergistically with the metal or linker. Considering ZIF’s intercellular delivery potential, combining Mn into the framework would allow for the efficient delivery of metal and attached CDN molecules [A52], While there are literature reports of ZIFs incorporating Mn, they have extremely low percentages of incorporated Mn or are synthesized in organic solvents, thus requiring extensive washing and activation before in vivo use and making the synthesis incompatible with biomacromolecule encapsulation [A53, A54], In contrast, the only reported synthesis of ZIF made with 100% Mn is unstable outside an argon environment [A53-A55],

[0319] Here, a green, bio-friendly approach has been developed to dope different Mn ratios into zinc-based ZIF, creating Mn-ZIF with varying percentages of Mn in a simple, 20-minute, one- pot reaction (Figure 1A). It was found that Mn-ZIF exhibits significantly decreased cytotoxicity compared to ZIF-8 at 50% Mn-doping. Electrostatic and coordination interactions between the anionic CDA and the cationic surface of Mn-ZIF allow for the co-delivery of additional STING agonists. Mn-ZIF-based particles allow for efficient cell uptake, effectively delivering Mn, Zn, and CDA into cells (Figure IB). Degradation of the particle and release of the components into the cytosol combined for synergistic cGAS-STING activation, resulting in murine bone marrow dendritic cells (BMDCs) activation and proinflammatory cytokine production (Figure 1C). Finally, the CDA@Mn-ZIF was formulated with CysVac2, a fusion protein of two Mtb antigens and used as a vaccine in a pre-clinical animal model to assess the capacity of the formulation to reduce the mycobacterial burden after infection with a virulent strain of Mtb (Figure ID).

[0320] Results and Discussion. An aqueous synthesis incorporating Mn2+and Zn2+was developed to make the reaction green and biofriendly. This method allows for scalability, more direct use in vitro and in vivo, and future application in the growing field of biomimetic encapsulation, where an expanded library of MOFs is desirable [A 6], Various synthetic conditions were tested to achieve a wide range of Mn incorporation in nano- and micrometer sizes of ZIF. Mn2+oxidizes in water to form various manganese oxides, and it was quickly realized that a mild reducing agent is needed to keep the Mn2+stable as stocks and during the reaction. Three reducing agents, sodium ascorbate, sodium citrate, and tris(2- carboxy ethyljphosphine (TCEP), were tested. It was found that TCEP did not interfere with the formation of the MOF while allowing for the maximum Mn loading possible and was, hence, the reducing agent of choice for future experiments. The results of the optimization experiments are summarized and tabulated in Table 1. TCEP-assisted Mn-doped ZIF (Mn-ZIF) was prepared byadding DI water, TCEP, manganese(II) acetate tetrahydrate, zinc(II) acetate dihydrate, and 2- methylimidazole (HMIM) — in that order — and left to react statically at room temperature for 20 min. Synthetic conditions for these reactions have been tabulated in Table 2 and sizes of each formula have been calculated from the SEM using ImageJ and displayed in Table 4.

[0321] Table 4: Calculated sizes of each Mn-ZIF formula.

[0322] Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the amount of Mn doped into ZIF. The percent doping was calculated based on the mole percent of Mn from the total moles of Mn and Zn in a sample (Equation El). it was found that the concentration of Mn was highly tunable; 15%, 30%, and 50% were chosen as representative samples whose crystallinity' was retained when examined using powder x-ray' diffraction (PXRD) (Figure 2A). The PXRD diffractograms confirmed that the Mn-ZIF formulas and ZIF-8 synthesized with TCEP (0% Mn) were isostructural to simulated sodalite ZIF-8. The diffraction results demonstrate that TCEP does not interfere with the sodalite topology formation of ZIF-8 or Mn-ZIF. A loss in crystallinity is observed in the diffraction patterns as Mn doping is increased, and past 50%, the material became amorphous with no distinct peaks in the PXRD diffractograms. The PXRD of a representative sample that was calculated to have 70% Mn is shown in Figure 6. The morphology- of the various Mn-loaded samples was then assessed by scanning electron microscopy (SEM). A less common rounded cubic structure can be seen for each sample compared to the typical rhombic dodecahedron of sodalite ZIF-8 (Figure 2B-Figure 2E). SEM further shows a size between 400-600 nm for each formulation, but the size slightly increases as more Mn is incorporated. X-ray photoelectron spectroscopy (XPS) survey and Ols spectra were collected for 50% Mn-ZIF and ZIF-8 (Figure 7A-Figure 7B). Oxygen was detected in both samples, most likely due to three sources: surface metal oxides, metal hydroxides, and residual water with respective bond energies of 529-530 eV, 530-532 eV, and 533-534 eV [A57, A58], A similar oxidation character was observed with Mn-ZIF having a slightly shifted peak to higher energies, suggesting more water and hydroxide bonding than oxide bonding, as seen in ZIF-8. From this, it can be concluded that most Mn is incorporated into the lattice and not just trapped as metal oxides. Further, the survey spectrum for Mn-ZIF shows the signature Mn peaks along with Zn peaks seen in the ZIF-8 spectrum, supporting the mixed metal results of ICP-MS.

[0323] Before proceeding to immune activation experiments, it is important to understand the cytotoxicity of these materials so the right dosage can be determined for in vitro and in vivo applications. Cell viability assays were used to determine the half-maximal inhibitory concentration (IC50) of Mn-ZIF with different doping percentages compared to control ZIF-8. Three cell lines were used: RAW 264.7, 4T1, and HEK-293. These represent various cell types and tissues: murine immune cells, murine breast cancer epithelial cells, and human kidney epithelial cells, respectively. Three viability assays were tested, including tetrazolium-based lactate dehydrogenase (LDH), MTT assay, and resazurin assay. Tetrazolium-based dyes that utilize redox-active formazan rings were incompatible with Mn as they interfered with the assay’s color development; consequently, the LDH and MTT assays produced spurious results. An example can be seen in Figure 8, where after lysing cells in the presence of Mn-ZIF or Mn2+salt, LDH still had a low signal, indicating living cells similar to media when they should produce a signal similar to the lysed control group. Assays using tetrazolium dyes have been previously reported to have issues measuring redox-active compounds [A59, A60], In contrast, the resazurin-based dye appears inert to Mn. Resazurin assays are also redox-based; however, extensive control experiments were performed to confirm that Mn-ZIF did not react with the dye. Further, as a fluorescent assay, it avoids interference from the MOF’s absorbance (Figure 9). Across all three cell lines, viability increased as Mn substitution increased. While the difference observed in RAW 264.7 was small but not statistically significant (Figure 3 A), cell viability of 50% Mn-ZIF was considerably higher than that of ZIF-8 in 4T1 and HEK-293 cells. 4T 1 showed an 84% increase in tolerance (60.3 ± 3.1 pg / mL vs. 111.3 ± 0.8 pg / mL). and HEK- 293 was even higher, with an 87% increase (81.8 ± 9.6 pg / mL vs. 153.1 ± 0.4 pg / mL) when comparing ZIF-8 to 50% Mn-ZIF (Figure 3B-Figure 3C). These results have been summarized in Figure 3D. It is hypothesized that the increased viability could be attributed to Mn and Zn being metabolized using different biological processes, and the reduced amount of each metal likely reduces the overall toxicity in some cell lines compared to a pure Zn MOF [ A61 ] . The IC50 values are promising as only a small amount of Mn — in the pM range based on endogenous levels [A62] — is needed to induce cGAS-STING activation, allowing for flexible dosing with Mn-ZIF [Al 8], With cytotoxicity data for Mn-ZIF, it was next sought to exploit it as a delivery method for Mn2+to activate cGAS-STING. The 50% Mn-ZIF formulation was used for all future experiments because it had the highest biocompatibility of all formulations tested. Additionally, the ability to boost STING activation by also incorporating an agonist was investigated. Mn codelivered with a CDN can work synergistically to activate STING and create a more robust immune response [A63], As a model, the known STING agonist CDA, a molecule belonging to the CDN family [A64], was incorporated. An additional benefit of loading CDA onto Mn-ZIF is nanoparticle-mediated delivery, which can enhance uptake compared to just CDA — a negatively charged small molecule. First, CDA was post-synthetically loaded onto the surface of Mn-ZIF for 24 h on a rotisserie at room temperature (Figure 4A). Owing to the previously reported higher affinity of CDA to metals in methanol over other solvents, that approach was tried first [A63], As determined by UV-Vis spectroscopy, over 96% of the CDA in the solution was absorbed into the Mn-ZIF (Figure 4B). Liquid chromatography (LC) supported the nanodrop result with greater than 94% absorption (Figure 18 and Figure 11). Loading was also tested in water to eliminate the need for methanol; excitingly, it was found that a comparable 93% was absorbed by Mn-ZIF, as determined by LC. The highest percent loading was observed when adding 30 pg / mL concentration of CDA to 1 mg / mL of Mn-ZIF, and when increasing CDA concentrations were used, the loading percentage decreased. Thus, the most efficient loading route was used for future experiments, giving about 28 pg of CDA per one mg of Mn-ZIF. ,- potential measurements show that the surface of Mn-ZIF became more negatively charged owing to the anionic phosphodiesters of the loaded CDA (Figure 4C). Additionally, the CDA- loaded Mn-ZIF (CDA@Mn-ZIF) retained 94% of the loaded CDA after 24 h, confirming good stability and CDA retention of the composite (Figure 12). Cytotoxicity was performed on the CDA@Mn-ZIF in the same way as in Figure 3A-Figure 3D and ICso was determined to be 61.0 ± 6.3 pg / mL, which was slightly lower than ZIF but not significantly different (Figure 19).

[0324] Mn-ZIF was meant to serve as an adjuvant delivery vehicle for the CDA and was also designed to protect CDA in vitro. Cells contain enzymes known as phosphodiesterases that can hydrolyze the phosphodiester bond of CDNs, converting them to linear dinucleotides that will not activate STING (Figure 20 A). An experiment was adapted from literature using snake venom phosphodiesterase (SVPD) to degrade CDA to linear phosphadenylyl-adenosine (pApA) [A65], The change in structure generated a large shift in elution time, so the degradation could be observed by LC, as seen in the pApA and CDA traces (Figure 20C). CDA@Mn-ZIF was treated with SVPD or buffer as a control, heated to deactivate the enzyme, and finally treated with dilute acetic acid to decompose the MOF and release the CDA into supernatant (Figure 20B). After running on LC, traces overlapping with intact CDA could be seen for the SVPD- treated and untreated samples (Figure 20C). This suggests the Mn-ZIF could preserve the CDA from degradation even with a surface loading process.

[0325] To demonstrate the uptake of Mn-ZIF and co-delivery of Mn and CDA. a fluorescent dye was incorporated into the pore structure via coprecipitation during synthesis before loading CDA. 5,6-carboxyfluorescein (CF) and rhodamine B (Rh) were chosen because of their high quantum yield. CF and Rh were incorporated into the crystalline structure during the Mn-ZIF reaction by conducting the MOF synthesis in an aqueous solution of concentrated CF and Rh to form fluorescent nanoparticles (Mn-ZIF(CF)) and (Mn-ZIF(Rh)). Mn-ZIF(CF) and Mn-ZIF(Rh) were then coated with CDA post-synthetically with the same method to create C D A? / Mn- ZIF(CF) and CDA(ajMn-ZIF(Rh) and can be seen in the green or red channels of an epifluorescence microscope respectively (Figure 13A-Figure 13B). Both Mn-ZIF(Rh) and CDA rt Mn-ZIFfRh) were used to conduct uptake studies on RAW 264.7 cells. Particle-cell association of CDA@Mn-ZIF(Rh) was visualized on an epifluorescence microscope at 6 h and is represented in Figure 4D. The CDA@Mn-ZIF(Rh) signal can be seen around the nucleus in the red channel, associating with the green lysotracker channel and has a Pearson colocalization coefficient of 0.72. This suggests CDA@Mn-ZIF can effectively associate with the cell, potentially through the lysosome. Since 2-D epifluorescence can only confirm association, a quantitative uptake assessment was performed using flow cytometry on RAW 264.7 cells. Mn- ZIF was used as a non-fluorescent control to account for scattering as a potential source of background signal. Pure CF dye was used to show the uptake of a representative, negatively charged small molecule. Samples were washed with both PBS and flow cytometry buffer, which contains the chelating agent EDTA, to remove material only bound to the surface so that signal is from dye internalized in the cell and not bound to the surface. Four- and eight-hour time points were assessed for uptake by incubating cells with Mn-ZIF, CF, Mn-ZIF(CF), and CDA@Mn- ZIF(CF) at each time. A representative histogram of 8 h is shown in Figure 4E. and the geometric mean of the CF intensity using the FITC channel is represented in Figure 4F. Free CF’s uptake was very limited but significantly increased once encapsulated within Mn-ZIF. The enhanced CDA@Mn-ZIF(CF) uptake compared to Mn-ZIF(CF) was surprising, given that its surface charge was more negative than Mn-ZIF(CF) alone. It was hypothesize that this result could be attributed to protein corona formation from the bovine serum albumin used in the cell media, which has previously been reported to enhance the uptake of negatively charged particles [A66, A67], Taken together, these results suggest that Mn-ZIF is a good delivery’ vehicle for both therapeutic metals like manganese and adjuvanting small molecules like CDA. Following uptake studies of Mn-ZIF, it was checked if APCs could be activated in vitro by delivering a combination of Mn and CD A. Activating APCs is extremely important for host defense against pathogens and cancer, and stimulation of cGAS-STING is one way to achieve activation [A68], cGAS-STING responses can be measured in vitro by surface marker staining and by quantifying the pro-inflammatory cytokines produced by the cells. BMDCs were isolated and used as target APCs. MnCh, ZIF-8, CDA, Mn-ZIF, and CDA@Mn-ZIF were normalized to achieve 10 pg / mL Mn and 1.7 pg / mL CDA then tested on BMDCs. Successful dendritic cell (DC) activation was measured by the percent of CD1 lc+cells double positive for CD80+and CD86+. the surface proteins that allow APCs to present and activate T-cells. When added to BMDC cultures, Mn-ZIF provided greater activation than the mole-equivalent dose of MnCh and lipopolysaccharides (LPS), a positive control known to activate BMDCs. Excitingly, CDA@Mn-ZIF outperformed all the groups and offered greater DC activation than Mn-ZIF alone, suggesting a strong synergistic effect (Figure 5A). ELISA was used to measure cytokines released from the BMDCs into the cell supernatants. Mn-ZIF and CDA@Mn-ZIF elicited a proi inflammatory response, quantified using cytokines TNF-a (Figure 5C) and IL-6 (Figure 5D). Cytokine production from equal doses of ZIF-8 and CDA w as below the limit of detection, and MnCh alone only produced IL-6. While cytokine production was significantly higher in the LPS control, it is important to note this level is undesirably high; excess IL-6 produced from LPS can lead to toxicity, as seen in sepsis [A69], This demonstrates that Mn-ZIF can generate an immune response alone and synergistically increase the potency of CDA. To confirm that the BMDC activation and cytokine production resulted from STING, a western blot was used to see the activation of two proteins downstream of STING — TBK-1 and IRF-3. TBK-1 will phosphorylate (P-TBK-1) upon STING activation and induce IRF-3 phosphorylation (P-IRF-3), further leading to proinflammatory gene expression generating cytokines [A12], Western blot confirmed that Mn-ZIF and CDA@Mn-ZIF could trigger phosphorylation of both TBK-1 and IRF-3 in RAW 264.7 cells, while only P-TBK-1 was seen for MnCh and the other controls did not induce phosphorylation of either protein (Figure 5B and Figure 21). The western blot bands were quantified (Figure 22) and normalized to fyactin and results are tabulated in Table 5. This result links the cytokine production observed to Mn-ZIF and CDA@Mn-ZIF’s ability to activate STING. IFN-fy a type-1 interferon, was measured and is reported in Figure 23B where a modest increase in IFN-0 production was observed from CDA@Mn-ZlF. IFN-y was also measured but no sample generated significant production (Figure 23 A). In addition to cytokine production, increased radical oxygen species (ROS) generation was observed inside RAW macrophages for the ZIF-8 and Mn-ZIF containing samples using an intercellular ROS detection kit (Figure 24). ROS have been implicated in promoting lymphocyte activation by playing a role in antigen process and presentation, and increased ROS is indicative of activated immune cells [A70],

[0326] Table 5. Quantification and normalization of the western blot using Bio-Rad Image Lab software. Protein bands were quantified and then normalized to P-actin and the normalized values are reported along with the P-actin normalization factor

[0327] Next, it was determined if CDA@Mn-ZIF could be formulated with a protein and be used as a vaccine. The ability to test CDAtyMii-ZIF in a Mtb model was investigated for two reasons: cGAS-STING is important for intracellular bacterial recognition, particularly in Mtb, where the bacteria go so far to inhibit this pathway by overexpressing phosphodiesteraces [A71, A72], Additionally, adjuvants that target cGAS-STING to induce protective immunity with subunit vaccines are relatively underexplored but a CDN-based STING agonist Mtb subunit vaccine has shown promising results as an intranasal vaccine in pre-clinical animal models [A73] . To test CDAtyMn-ZIF as an adjuvant in an Mtb model, to the ability to combine it with a well-characterized subunit protein was investigated. The CysVac2 fusion protein is made of the Mtb antigens Ag85B and CysD [A74] . It has been shown to induce protective immunity in mouse models when formulated with AdvaxCpG — a commercially available adjuvant made from polysaccharide and CpG oligonucleotide--- and administered parenterally |A75] or intrapulmonary [A76], Therefore, as a well-characterized vaccine fusion protein, CysVac2 was a good candidate to test the adjuvant activity of the CDA@Mn-ZIF. An intramuscular (I.M.) vaccination route w as chosen as this is currently the standard used in humans for new- subunit TB vaccines. Mice were inoculated three times at two-week intervals with either 10, 3. or 1 pg of CysVac2 and 125 pg CDA@Mn-ZIF via the I.M. route. A full timeline of the experiments can be seen in Figure 17A. A cohort of mice chosen as the positive control w ere vaccinated with BCG — the commercial standard for vaccination against TB. Two weeks after the final vaccination, peripheral blood mononuclear cells (PBMCs) were isolated and immunophenotyped for activated T-cells, as it is well established that the immune response to TB is cell-mediated [A77] . Thl cells promote cell-mediated immune responses and are required for the host’s defenses against intracellular infections [A78], The CDA@Mn-ZIF, CysVac2 combo (CMZ / CysVac2) resulted in dose-dependent production of IL-17, IFN-y, IL-2, and TNF in the PBMCs after restimulation with antigen (Figure 17B-Figure 17F). As expected, adjuvant alone could not produce activated T-cells but interestingly, BCG vaccinated mice also did not show a CysVac2 T-cell response, given that Ag85B is produced by BCG. Seeing that CMZ / CysVac2 generated a potent antigen-specific T-cell response in the blood, the ability to test the vaccine's efficacy in a Mtb challenge experiment was investigated. After aerosol infection with a low dose of Mtb, there was a significant reduction of colony -forming units (CFUs) in the lungs compared to saline-treated mice (Figure 17G). The data suggest strong protective immunity induced from CDA@Mn-ZIF resulted in a significant reduction, approximately 0.3 Logic CFU reduction compared to the saline treated group, in mycobacterial burden regardless of the CysVac2 concentration. The significant reduction in bacterial burden in the CMZ / CysVac2 group is exciting, especially considering that this fusion protein contains only two known antigens commonly investigated for TB vaccine development. Since BCG is a whole-cell vaccine, enumerable epitopes and antigenic targets on its surface often result in better outcomes in mouse models; however, these results have not translated well into human models [A79-A84], Consequently, BCG serves as a positive control more than a benchmark, particularly in mice. Interestingly, the dose-dependent response in PBMC cytokine expression did not translate to a dose-associated relationship in mycobacterial burden. There are multiple variables that could contribute to a lack of dose dependence in the challenge including the different T-cell profiles in the blood may not be reflected in the lung throughout the infection period. Additionally, there are many immune factors including immunoglobulin levels and cells other than T-cells involved during infection of a highly virulent strain, which could mask the dose dependence. Prior work using CysVac2 combined with commercially available adjuvants has shown similar levels of CFU reduction, suggesting CDA@Mn-ZIF is a promising adjuvant to promote protection against TB infection [A75],

[0328] Conclusion. Previous research has demonstrated the effectiveness of ZIF-8 as both a thermostable coating and an intracellular delivery platform useful in vaccines. By combining the potent metallo-immunotherapeutic metal Mn into the structure of ZIF-8 to generate Mn-ZIF, it was possible to add a third dimension to its function — immune adjuvanting activity. The mixed metal approach also reduced cytotoxicity, providing a greater range of safe dosing options. The STING adjuvanting small molecule CDA was effectively combined with Mn-ZIF and showed its protection and delivery. CDA@Mn-ZIF further enhanced the immune adjuvanting potential through synergistic activation of STING. Excitingly, Mn-ZIF alone activated APCs and produced proinfl ammatory cytokines, and this effect was further augmented by incorporating CDA. The combination presents a dose-sparing effect of costly adjuvants as only very small amounts of CDA are needed when combined with Mn and a MOF carrier. With these results, CDAV Mn-ZIF was tested in a Mtb model where significant activation of T-cells in blood against Mtb antigens was observed and post challenge the combination of CDA@Mn-ZIF and CysVac2 fusion protein led to a significant reduction in Mtb in the lungs. These findings suggest CDA@Mn-ZIF is an effective adjuvant in in-vivo models and potentially a strong candidate to adjuvant other antigens.

[0329] Materials and Methods

[0330] Materials: TCEP-HC1 tris(2-carboxyethyl) phosphine hydrochloride was purchased from Goldbio. Zinc(II) acetate dihydrate, 2-methylimidazole, and manganese(II) acetate tetrahydrate, L -glutamine solution, sodium hydroxide, 5(6)-carboxyfluorescin and fluorometric intracellular ROS kit (deep red) were purchased from Millipore Sigma. Deep Blue Cell Viability kit, FITC anti -mouse CDl lc antibody. Alexa Fluor® 594 anti-mouse CD80 antibody, and Pacific Blue™ anti-mouse CD86 antibody were purchased from Biolegend. CDA was purchased from Invivogen. Nitric acid (trace metal grade) was purchased from Thermo Fisher Scientific. HyClone phosphate buffered saline solution, HyClone Dulbecco's modified eagle's medium (DMEM), HyClone RPMI 1640 medium, and penicillin-streptomycin were purchased from Cytiva. FB Essence was purchased from Avantor. TBK1, Phospho-TBKl, IRF-3, Phospho- IRF3, beta-actin, HRP-linked anti-rabbit IgG and SignalFire ECL reagent were purchased from Cell Signaling Technology

[0331] Instruments: SEM micrographs were captured on a Zeiss Supra 40. PXRD spectra were collected using Rigaku SmartLab X-ray Diffractometer. DLS measurements for size and zeta potential were carried out using Malvern Analytical Zetasizer Nano ZS. Fluorescence intensity measurements on 96-well plates were carried out using Biotek Synergy' H4 Hybrid microplate reader. Epifluorescence images were taken on EVOS FL digital inverted fluorescence microscope. Cell counting was carried out using Thermo Countess II. Zinc and manganese quantification was done using Agilent 7900 ICP-MS. CDA quantification was done using Thermo Fisher NanoDrop and Agilent 1100 series LC. X-ray photoelectron spectra was recorded using PHI VersaProbe II Scanning XPS Microprobe. Flow cytometry was performed on the BD Fortessa.

[0332] Cells and animals: RAW 264.7 cells were received as a gift from Dr. Rockford Draper (Department of Biological Sciences. UT Dallas). 4T1 cells were received as gifts from Dr. Laurentiu Pop (Department of Radiation Oncology, UT Southwestern). HEK 293 cells were received as a gift from Mateusz Durbacz (Department of Molecular Biology and Hamon Center for Regenerative Science and Medicine, UT Southwestern). Female C57BL / 6 mice (8-10 weeks) were purchased from Charles River Laboratories and were housed under the protocol #19-06, which was approved by IACUC. C57BL / 6 mice (8-10-week-old female, ABR Bioresources. Moss Vale, NSW, Australia) were used for the in pre-clinical assessment of the vaccine formulation. Mice were acclimated for one week prior to commencement of vaccination. All procedures were approved by the Animal Welfare Committee of the Local Sydney Health District under protocol # 2023 / 002.

[0333] Protein: CysVac2 was prepared by Sydney Analytical (University of Sydney, Camperdown, NSW, Australia) from transfected ClearColi BL21(DE3) bacteria (Gene Target Solutions Pty Ltd, Dural, NSW, Australia). The protein was purified using size exclusion and LPS removed to ensure limited reactivity. The LPS concentration was determined by Pierce Chromogenic Endotoxin Quant Kit (ThermoFisher Scientific) and by assessing functional activity in a RAW-Blue® Assay (gift from Dr. Nicholas Shields, University of Sydney), which were used to monitor the NF-kB and AP-1 responses upon pattern recognition receptor stimulation. The RAW-Blue® assay showed no activity in the CysVac2 preparation.

[0334] Mn-ZIF synthesis: Four stocks were prepared in MilliQ water, a 1 M solution of zinc (II) acetate dihydrate, 3 M 2-methylimidazole (HMIM), 0.5 M TCEP-HC1, and 1 M manganese (II) acetate tetrahydrate with lOmM TCEP. For a 15 ml reaction of 50% Mn-ZIF reactants were added in the following order: (1) 550 pl H2O, (2) 450 pl TCEP, (3) 800 pl Mn, (4) 400 pl Zn, and (5) 12.8 ml HMIM. After adding HMIM, the reaction w as vortexed and left static at RT for 20 mins, then centrifuged at 4,300 * G for 15 m. The supernatant was discarded, the pellets were washed twice with water and subsequently dried under a high vacuum overnight.

[0335] Sample preparation for in vitro and in vivo experiments: 10 mg of ZIF-8 or 15 / 30 / 50% Mn-ZIF was weighed on an analytical balance and suspended in a glass vial with endotoxin-free, sterile water to make a 10 mg / ml stock. The stock solution was vortexed and sonicated to ensure proper dispersion.

[0336] In vitro cytotoxicity. Biolegend’s resazurin-based “Deep Blue Cell Viability” kit was used for cytotoxicity and IC50 calculations. 25,000 cells per well were seeded in a 96-w-ell plate overnight in 50 pl of media and incubated at 37° C and 5% humidity. The following morning, 50 pl of 2x desired final concentration of the sample was added to n = 4 wells. The plate was placed back in the incubator for 24 h. 30 min before the 24 h time point, 10 pl of lyse buffer was added to appropriate wells as a negative control. At 24 h, 10 pl of the resazurin reagent was added to each well and mixed before being returned to the incubator to incubate for 4 h before being read at an excitation of 530 nm and an emission of 590 nm on a microplate reader. % viability was normalized to media only, and lysed cell control, and data are presented as average ± standard deviation (n = 4 with outlier analysis done in GraphPad Prism with Grubbs’ method). IC50 was determined using the linear regression function in Microsoft Excel.

[0337] CDA loading on Mn-ZIF. To prepare CDA@Mn-ZIF, 1 mg of Mn-ZIF was mixed with 30 pg CDA in methanol or water. This sample was placed on a rotisserie at RT for 24 h. After the 24 h period, the CDA@Mn-ZIF was centrifuged at 17,000 x G for 10 m, and the supernatant was collected for concentration determination by LC and NanoDrop UV-VIS. CDA@Mn-ZIF w as washed once with water and then dried overnight in a high vacuum chamber for storage or directly resuspended in DI water for use.

[0338] Determination of CDA concentration by LC. A standard curve of CDA was first prepared by diluting a 1 mg / mL stock to 25 pg / mL and making half serial dilutions down to 1.5625 pg / mL. These standards were run through polymeric reverse phase (PLRP) column on the LC with UV detector set to 260 nm wavelength. Water and acetonitrile with 0.1% formic acid w ere used as eluting solvents. 5 pL of each sample was injected on the following gradient: 20% acetonitrile for 2 minutes, an increasing gradient from 20% to 60% for 30 minutes, hold at 60% for 3 minutes, and a decrease down to 20% over 2 minutes. Unknown samples were run under the same conditions, and concentration was determined by peak area integration on GraphPad.

[0339] Association and uptake experiments: 50% Mn-ZIF was prepared with 5,6- carboxyfluorescein (CF) and rhodamine B (Rh) encapsulated by replacing the water in the reaction with saturated solutions of each dye. For association by epi, 30,000 RAW 264.7 cells were seeded in an 8-well chamber slide overnight. 60 pg / mL Mn-ZIF(Rh) was added and incubated for 6 h then samples w ere washed with PBS and imaged. For uptake by flow, 200,000 cells per well were plated in a 96-well u-bottom plate and incubated overnight. Samples were added and allowed to incubate for 4 h and 8 h. then the cells were washed multiple times w ith PBS and then FACS buffer to remove material that was not taken in. Samples were run on a BD FortessaLSR flow cytometer. Bone marrow dendritic cell cultures. Femurs and tibias were isolated from naive C57BL / 6 mice and placed in PBS. Surgical scissors were used to snip both ends of the bone, and a 25 G needle was used to flush the marrow out using RPMI over a 70 pm single cell suspension filter. Cells were centrifuged at 500 x G for 5 m, and the supernatant was removed. RBC lysis buffer was used to remove red blood cells, after which the cells were counted and suspended in 10 mL RPMI with 20 ng / mL of GM-CSF in a T-75 at a concentration of 2 x 106cells / ml. 10 mL of RPMI with GM-CSF was added three days later, and media was refreshed on days 6 and 9.

[0340] BMDC activation and cytokine production . Non-adherent cells were collected on day 10 from the previously described cultures and seeded at a concentration of 200,000 cells per well in a 96-well plate with the final volume being 200 pL with samples. Final concentration of each sample was 1.7 pg / ml of CDA, 36 pg / ml of MnCh • 4 H2O, and 60 pg / ml of ZIF-8, 60 pg / ml Mn-ZIF loaded with 1.7 pg / ml CDA. Samples w ere incubated for 20 h, at which point the plate was spun, and supernatants were taken and frozen for cytokine ELISA. BMDCs were then w ashed with PBS and follow ed a staining protocol. First, samples were incubated with Zombie UV (biolegend) in PBS, followed by adding a staining cocktail of CD11c FITC, CD80 AF647, and CD86 Pacific Blue (biolegend) and left on the ice. Samples w ere washed twice with cell staining buffer, resuspended, and ran on flow. TNF-a (biolegend), IL-6 (biolegend), IFN-y (biolegend), and IFN-P (R&D Systems) ELlSAs were run according to manufactures protocol and absorbance was read on a Synergy H4 plate reader (Biotek).

[0341] Western blot: Raw72.647 cells were seeded in a 6-well plate at I xlO6cells per well overnight. Media w as then removed and replaced with 2 ml of media with a final concentration of each sample as follows: 1.7 pg / ml of CDA. 36 pg / ml of MnCh • 4 FLO, and 60 pg / ml of ZIF- 8, 60 pg / ml Mn-ZIF and 60 pg / ml CDA@Mn-ZIF (1.7 pg / ml CDA). After 18 hours of incubation, supernatants with samples were removed and 200 pl of RIP A lyse buffer was added to each well, and cells were scraped and transferred to 15 ml centrifuged tubes. A Qsonica QI 25 probe sonicator then sonicated each sample at 50% intensity7for three 15s cycles on ice. Samples were centrifuged at 14,000 * g to pellet debris, and the supernatant was transferred to 500 pl tubes. Protein concentrations were normalized by Bradford assay and then run for 45 min on a 4 - 15% gradient gel at 200 V. The gel w as transferred by semi-dry7method to a nitrocellulose membrane and blocked with 3% milk buffer for 1 hour at RT. The blot was incubated overnight with 1 / 1000 dilutions of P-TBK-1 and P-IRF-3 antibodies in 5% BSA in TBST with rocking at 4°C. After 16 hours the blot was washed three times with TBST for 5 min each and incubated with 1 / 3000 dilution of secondary antibody for 1 h before adding substrate and reading on a Biorad gel imager (ChemiDoc Touch). The blot was stripped with stripping buffer and re-probed for TBK-1 and IRF-3, then P-actin as a loading control.

[0342] Vaccination and Inf ection Mice (5 per group) were vaccinated in both hind quadri cep muscles with 25 pL of the CDA@Mn-ZIF formulation mixed with either 10, 3 or 1 pg of CysVac2 protein 3-times at 2-week intervals. As a positive experimental control, mice were vaccinated subcutaneously with 5xl04CFU of BCG Danish (AJ Vaccines). Mice were then rested for 4 weeks during which (2 weeks after the final inoculation) they were bled to obtain peripheral blood mononuclear cells (PBMC) for immune assessment. At the end of 4 weeks, mice were infected with a low dose aerosol of Mtb, strain H37Rv (TMCC#107) and rested for another 4 weeks.

[0343] Intracellular cytokine assay: PBMC were obtained from blood taken from each mouse. Approximately 100 pL of blood was collected from a tail vein into EDTA tubes (source), diluted in endotoxin- free PBS and then PBMC obtained by centrifugation over a Histopaque-1083 (source) gradient. PBMCs were washed, counted, and resuspended to IxlO6cells / mL in completed-RPMI-1640 (10% FBS, pen / strep, L-glut). Cells were cultured with a final concentration of 10 pg / mL of CysVac2 protein for 5 hours, after which GolgiPlug was added according to manufacturer’s protocol and incubated for a further 11 hours. Cells were collected and the prepared for flow cytometry analysis using the following fluorochrome-conjugated mAb: IL17-Pacific Blue, IL2-PE, TNF-PerCP-Cy5.5, CD4-Alexaflour700, IFN-y-PECy7, CD62L- BV650, CD44-FITC, Live Dead Blue. Data was collected using a LSR Fortessa (BD Biosciences), running FACSDiva Software. Approximately 106cells per mouse were collected. FlowJo® software (BD Biosciences) was used to analyze the data.

[0344] Determination of Colony Forming Units (CFU): The number of CFU in the lungs of mice was determined by plating 10-fold serial dilution of organ homogenates on 7H10 + OADC agar (Bacto). Cultures were incubated at 37°C for 14-21 days, after which colonies were counted.

[0345] Equation El: The Mole % Mn was found by converting ppm of Mn and Zn, collected by ICP-MS, to mmol and then taking Mn as a percent of the combined amount of Mn and Zn.

[0346] (( - 1000

[0347] Figure Captions

[0348] Figure lA-Figure ID: Synthesis of Mn-ZIF and delivery' of Mn, Zn, and CDA for immune activation in-vitro and in-vivo. Figure 1A) Synthetic overview of Mn-ZIF and CDA@Mn-ZIF. Zn2+, Mn2+, a reducing agent TCEP, and 2-methylimidazole (HMIM) were mixed in water and left static at RT to afford Mn-ZIF. CDA was incorporated post-synthetically by incubating with Mn-ZIF suspended in water and methanol at RT on a rotisserie to make CD A( r / Mn-ZIF. Figure IB) Uptake of the multivariate MOF was measured to demonstrate the delivery and release of Mn, Zn, and CDA into cells. Figure 1C) The immune-stimulatory effect of Mn and CDA deliver}' to trigger the cGAS-STING pathway. First, Mn and Zn can bind to cGAS to trigger cGAMP production that activates STING. Additionally, Mn and CDA can directly activate STING, causing it to phosphorylate TANK-binding kinase 1 (TBK-1), which phosphorylates and dimerizes interferon regulatory factor 3 (IRF-3). Phosphorylated IRF-3 dimers translocate to the nucleus, turning on gene expression of pro-inflammatory cytokines. Figure ID) CDA@Mn-ZIF was combined with the Mtb fusion protein CysVac2 and used a vaccine in a pre-clinical mouse Mtb challenge experiment.

[0349] Figure 2A-Figure 2E: Characterization of Mn-ZIF. Figure 2A) PXRD diffractogram of different Mn ratios starting with ZIF-8 control with 0% Mn up to 50% Mn. SEM micrographs of Figure 2B) ZIF-8 control, Figure 2C) 15% Mn-ZIF, Figure 2D) 30% Mn-ZIF, and Figure 2E) 50% Mn-ZIF.

[0350] Figure 3A-Figure 3D: In vitro cytotoxicity measured by resazurin assay. Viabili ty of Figure 3 A) RAW 264.7 cells, Figure 3B) 4T1 cells, and Figure 3C) HEK 293 cells were measured after treatment with different doses of 0% Mn-ZIF, 15% Mn-ZIF, 30% Mn-ZIF, and 50% Mn-ZIF and plotted. Figure 3D) Calculated IC50 values of each sample on the three cell lines. Statistical analysis was performed using Two-way ANOVA with Tukey’s multiple comparison test, confidence interval of 95%. Data is presented as mean ± standard deviation. Figure 4A-Figure 4F: Characterization of CDA@Mn-ZIF, association, and uptake. Figure 4A) Scheme of post-synthetic loading of CDA onto Mn-ZIF. Figure 4B) CDA loading after incubating each concentration of CDA with 1 mg / mL of Mn-ZIF. Figure 4C) Surface charge of Mn-ZIF before and after loading with CDA measured by ^-potential. Figure 4D) Epifluorescent micrographs of CDA@Mn-ZIF loaded with rhodamine in RAW 264.7 cells stained with Hoechst and lysotracker green after 6 h. Figure 4E) Representative histogram of RAW 264.7 cell uptake of CF encapsulated in Mn-ZIF at 4 h and 8 h. Figure 4F) Average uptake at 4 h and 8 h of CF, Mn-ZIF(CF). CDA@Mn-ZIF(CF) calculated by geometric mean of FITC intensity where n = 5. Statistical analysis was performed using Two-way ANOVA with Tukey?s multiple comparison test, confidence interval of 95%. Data is presented as mean ± standard deviation. Figure 5A-Figure 5D: Immune activating potential of 50% Mn-ZIF and CDA@Mn-ZIF.

[0351] Figure 5 A) BMDC activation measured by flow cytometry. Activation was defined as CD1 lc+cells double positive for CD80+and CD86+. Figure 5B) Western blot of cGAS-STING downstream proteins with molecular weights in kDa annotated on the left. Figure 5C) TNF-a and Figure 5D) IL-6 produced by BMDCs after 24 h incubation with each sample measured by ELISA on the cell supernatant. Statistical analysis was performed with Welches t-test, confidence interval of 95%. Data is presented as mean ± standard deviation.

[0352] Figure 6. PXRD showing amorphous nature beyond 50% substitution.

[0353] Figure 7A. Survey XPS spectra of ZIF-8 vs 50% Mn-ZIF

[0354] Figure 7B. Ols XPS spectra of ZIF-8 vs 50% Mn-ZIF

[0355] Figure 8. Raw data from a 4 h LDH test on RAW cells. In this assay, dead cells release LDH into the media, which generates a signal, and living cells create a minimal signal. Lysed groups were treated with lyse buffer to induce cell death and serve as a positive control, whereas cells treated with Mn prevent the lysed cells from generating signals.

[0356] Figure 9. Controls experiment with the deep blue viability assay. In this assay, living cells generate a fluorescent signal, and lysed cells do not. Lysed Mn-ZIF works as a proper control here and also doesn’t inhibit living cells from generating signal.

[0357] Figure 11. Standard CDA concentration curve based on the LC curve's integrated peak area. Supernatants of CDA@Mn-ZIF were collected after loading and run on LC to find the concentration of CDA not bound to Mn-ZIF; this was used to calculate loading efficiency. The peak area was 6.57, which equates to 1.7 pg / ml CDA, meaning 28.3 pg / ml or 94% of the starting CDA was absorbed to the Mn-ZIF.

[0358] Figure 12. Average CDA released from 2 different samples of post-synthetically loaded CDA@Mn-ZIF. Samples of CDA@Mn-ZIF were suspended in water and placed on a rotisserie. At each time point, the samples were centrifuged, and 20 pl supernatant was taken. Aliquots were run on LC, and samples were resuspended by vortex and placed on a rotisserie until the next time point.

[0359] Figure 13A-Figure 13B. Epiflourescent images of Figure 13A) CF@Mn-ZIF on the GFP channel and Figure 13B) RhtyMn-ZIF on the RFP channel.

[0360] Figure 17A-Figure 17G: Adjuvant effect of CDA@Mn-ZIF in-vivo in a pre-clinical Tb mouse model. Figure 17A) Timeline of experiments including vaccination, blood draw, challenge, and sacrifice for lung CFU counting. Percentage of CD4+CD44hlCD62L10T cells secreting Figure 17B) IL-17, Figure 17C) IFN-y, Figure 17D) IL-2, and Figure 17E) TNF from PBMCs after in vitro stimulation with CysVac2. PBMC w as taken approximately 2 weeks after the final vaccination. Figure 17F) Example flow cytometry gating strategy for phenotyping of PBMCs. Figure 17G) Mean Logio colony forming units (CFU) in the lungs of C57BL / 6 mice inoculated with CDA@Mn-ZIF formulated with either 10, 3, or 1 pg of CysVac2 via the I.M. route, 3-times at 2-week intervals. Mice were rested and infected with low-dose aerosol (50-100 CFU) of Mtb Erdman KOI, and CFU was determined at day 30 post-infection. N=5 mice per group. Statistical analysis was performed using a one-way ANOVA. *p<0.05, **p<0.0I, ***p<0.005, ****p<0.0001. Data is presented as mean ± standard deviation.

[0361] Figure 18: LC traces of CDA and supernatant CDA@Mn-ZIF used to calculate absorption percent.

[0362] Figure 19: 24-hour cytotoxicity results for CDA@Mn-ZIF in RAW 264.7 cells. ICso was calculated to be 61.0 ± 6.3 pg / mL

[0363] Figure 20A-Figure 20C: Protection of CDA by Mn-ZIF. Figure 20 A) Structure of CDA before and after hydrolysis degradation by SVPD into pApA. Figure 20B) Reaction scheme followed for the experiment where CDA@Mn-ZIF was mixed with SVPD and then heated for 5 min. to stop the enzyme, centrifuged and pellet collected. The pellet was then treated with acetic acid and left on a rotisserie to break down the Mn-ZIF and release CDA. This was centrifuged again to collect any debris, and then the supernatant ran on LC. Figure 20C) LC traces of pApA, CDA, CDA@Mn-ZlF. and SVPD-treated CDA@Mn-ZIF. CDA@Mn-ZlF served as a control to ensure the enzyme treatment and CDA recovery process was not too harsh.

[0364] Figure 21. Uncropped chemiluminescent scans of each western blot used in Figure 5B, and colorimetric scan of the ladder used to confirm molecular weight.

[0365] Figure 22: Graphs of quantified western blot bands.

[0366] Figure 23A-Figure 23B: Cytokine production measured by ELISA in supernatants of BMDC’s incubated for 20 hours with each sample. Figure 23 A) IFN-y production and Figure 23B) IFN-0 production. Statistical analysis was performed with Welches t-test, confidence interval of 95%. Data is presented as mean ± standard deviation.

[0367] Figure 24: Amount of relative ROS generated by ZIF-8, Mn-ZIF, and CDA@Mn-ZIF compared to untreated control cells for RAW 264.7 cells. 1 pg / mL LPS was used as a positive control and generated a 186 ± 16 % increase in ROS compared to untreated cells. Data is presented as mean ± standard deviation.

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[0452] EXEMPLARY ASPECTS

[0453] In view of the described compositions and methods, herein below are described certain more particularly described aspects of the inventions. The particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teaching described herein or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulas literally used therein.

[0454] Example 1 : A manganese doped metal organic framework (Mn-MOF) comprising a metal organic framework (MOF) comprising a zeolitic imidazolate framework (ZIF) comprising metal ions connected by imidazolate linkers, wherein the metal ions comprise Mn and Zn.

[0455] Example 2: The Mn-MOF of any examples herein, particularly example 1, wherein the ZIF is ZIF-8.

[0456] Example 3: The Mn-MOF of any examples herein, particularly example 1 or example 2, wherein the Mn-MOF comprises from greater than 0% to 50% (mol%) Mn based on the total amount of metal ions.

[0457] Example 4: The Mn-MOF of any examples herein, particularly examples 1-3, wherein the Mn-MOF comprises from 10% to 50% Mn.

[0458] Example 5: The Mn-MOF of any examples herein, particularly examples 1-4. wherein the Mn-MOF comprises 15% Mn, 30% Mn, or 50% Mn.

[0459] Example 6: The Mn-MOF of any examples herein, particularly examples 1-5, wherein the Mn-MOF comprises 50% Mn.

[0460] Example 7: The Mn-MOF of any examples herein, particularly examples 1-6. wherein the Mn-MOF is crystalline.

[0461] Example 8: The Mn-MOF of any examples herein, particularly examples 1-7, wherein the Mn-MOF is nanostructured.

[0462] Example 9: The Mn-MOF of any examples herein, particularly examples 1-8. wherein the Mn-MOF is in the form of a plurality of particles.

[0463] Example 10: The Mn-MOF of any examples herein, particularly example 9, wherein the plurality of particles have an average particle size of from 100 nanometers to 2 micrometers.

[0464] Example 11 : The Mn-MOF of any one of any examples herein, particularly example 9 or example 10, wherein the plurality of particles have an average particle size of from 500 nanometers to 1 micrometer, or from 400 nanometers to 700 nanometers.

[0465] Example 12: The Mn-MOF of any examples herein, particularly examples 9-11, wherein the plurality^ of particles have a particle shape that is a rounded cubic structure.

[0466] Example 13: The Mn-MOF of any examples herein, particularly examples 9-12, yvherein the plurality of particles have a sodalite topology.

[0467] Example 14: The Mn-MOF of any examples herein, particularly examples 1-13, further comprising a therapeutic agent loaded on and / or in the Mn-MOF.

[0468] Example 15: The Mn-MOF of any examples herein, particularly example 14, wherein the therapeutic agent is conjugated to the Mn-MOF. for example electrostatically.

[0469] Example 16: The Mn-MOF of any examples herein, particularly example 14 or example 15, wherein the therapeutic agent comprises an anticancer agent, an anti-inflammatory agent, an antimicrobial agent, an immunotherapy agent, or a combination thereof.

[0470] Example 17: The Mn-MOF of any examples herein, particularly examples 14-16. wherein the therapeutic agent comprises a STING agonist.

[0471] Example 18: The Mn-MOF of any examples herein, particularly examples 14-17, wherein the therapeutic agent comprises a cyclic dinucleotide (CDN).

[0472] Example 19: The Mn-MOF of any examples herein, particularly examples 14-18, wherein the therapeutic agent comprises cyclic di -adenosine monophosphate (CDA).

[0473] Example 20: A method of making the Mn-MOF of any examples herein, particularly examples 1-19.

[0474] Example 21 : The method of any examples herein, particularly example 20, wherein the method comprises in situ incorporation of Mn.

[0475] Example 22: The method of any examples herein, particularly example 20 or example 21, wherein the method comprises dispersing a Zn salt, a Mn salt, a reducing agent, and an imidazolate in a green solvent.

[0476] Example 23 : The method of any examples herein, particularly example 22, wherein the method comprises adding reactants in the following order: the green solvent, the reducing agent, the Mn salt, the Zn salt, and the imidazolate, thereby forming a mixture.

[0477] Example 24: The method of any examples herein, particularly example 23, wherein the method further comprises agitating the mixture, such as by vortexing.

[0478] Example 25: The method of any examples herein, particularly example 24, wherein, after agitating, the mixture is left static for an amount of time.

[0479] Example 26: The method of any examples herein, particularly examples 20-25, wherein the method further comprises isolating the Mn-MOF.

[0480] Example 27: The method of any examples herein, particularly examples 20-26. wherein the method further comprises washing and / or drying the Mn-MOF.

[0481] Example 28: The method of any examples herein, particularly examples 22-27, wherein the Zn salt comprises zinc(II) acetate dihydrate.

[0482] Example 29: The method of any examples herein, particularly examples 22-28. wherein the Mn salt comprises manganese(II) acetate tetrahydrate.

[0483] Example 30: The method of any examples herein, particularly examples 22-29, wherein the imidazolate comprises 2-methylimidazole.

[0484] Example 31 : The method of any examples herein, particularly examples 22-30, wherein the reducing agent comprises sodium ascorbate, sodium citrate, tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof.

[0485] Example 32: The method of any examples herein, particularly examples 22-31, wherein the reducing agent comprises citrate. tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof.

[0486] Example 33: The method of any examples herein, particularly examples 22-32, wherein the reducing agent comprises tris(2-carboxyethyl)phosphine (TCEP).

[0487] Example 34: The method of any examples herein, particularly examples 22-33, wherein the green solvent comprises water.

[0488] Example 35: The method of any examples herein, particularly examples 22-34, wherein the green solvent consists essentially of water.

[0489] Example 36: The method of any examples herein, particularly examples 22-35, wherein the green solvent consists of water.

[0490] Example 37: The method of any examples herein, particularly examples 20-36, wherein the method is substantially free of organic solvents.

[0491] Example 38: The method of any examples herein, particularly examples 20-37, wherein the method is substantially free of methanol.

[0492] Example 39: The method of any examples herein, particularly examples 20-38, wherein the method is conducted at room temperature.

[0493] Example 40: The method of any examples herein, particularly examples 20-39, wherein the Mn-MOF is formed in an amount of time of from 10 minutes to 30 minutes, such as from 15 to 25 minutes.

[0494] Example 41 : The method of any examples herein, particularly examples 20-40, wherein the method is a one pot method.

[0495] Example 42: The method of any examples herein, particularly examples 20-41, wherein the method is substantially biocompatible.

[0496] Example 43: The method any examples herein, particularly examples 20-41, further comprising contacting the Mn-MOF with a therapeutic agent to thereby load the therapeutic agent in and / or on the Mn-MOF.

[0497] Example 44: The method of any examples herein, particularly example 43, wherein the therapeutic agent is conjugated to the Mn-MOF via electrostatic interactions.

[0498] Example 45: A pharmaceutical composition comprising the Mn-MOF of any examples herein, particularly examples 1-19.

[0499] Example 46: The pharmaceutical composition of any examples herein, particularly example 45, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

[0500] Example 47 : A method of use of the Mn-MOF of any examples herein, particularly examples 1-19 or the pharmaceutical composition of any examples herein, particularly examples 45-46.

[0501] Example 48: A method of treating, preventing, or ameliorating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the Mn-MOF of any examples herein, particularly examples 1-19 or the pharmaceutical composition of any examples herein, particularly examples 45-46.

[0502] Example 49: The method of any examples herein, particularly example 48, wherein the disease comprises cancer.

[0503] Example 50: The method of any examples herein, particularly example 48, wherein the disease is an infectious disease, e.g. an infection, such as a microbial infection.

[0504] Example 51 : The method of any examples herein, particularly example 48, wherein the disease is tuberculosis.

[0505] Example 52: The method of any examples herein, particularly examples 48-51. wherein the method comprises immunotherapy.

[0506] Example 53: The method of any examples herein, particularly examples 48-52, wherein the Mn-MOF or pharmaceutical composition activates the STING pathway to thereby treat, prevent, and / or ameliorate the disease.

[0507] Example 54: The method of any examples herein, particularly examples 48-53, wherein the Mn-MOF or pharmaceutical composition activates antigen presenting cells to thereby treat, prevent, and / or ameliorate the disease.

[0508] Example 55: The method of any examples herein, particularly examples 48-54, wherein the method comprises cell uptake followed by degradation of the Mn-MOF, thereby providing delivery and release of Mn and the therapeutic agent (when present) to the cell cytosol to thereby treat, prevent, and / or ameliorate the disease.

[0509] Example 56: The method of any examples herein, particularly example 55, wherein the method triggers cGAS-STING activation, resulting in proinflammatory cytokine production and / or bone marrow dendritic cell activation.

[0510] Example 57: A vaccine comprising the Mn-MOF of any examples herein, particularly examples 1-19 or the pharmaceutical composition of any examples herein, particularly examples 45-46.

[0511] Example 58: An immunomodulator comprising the Mn-MOF of any examples herein, particularly examples 1-19 or the pharmaceutical composition of any examples herein, particularly examples 45-46.

[0512] Other advantages which are obvious and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

[0513] The methods of the appended claims are not limited in scope by the specific methods described herein, which are intended as illustrations of a few aspects of the claims and any methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the methods in addition to those shown and described herein are intended to fall w ithin the scope of the appended claims. Further, while only certain representative method steps disclosed herein are specifically described, other combinations of the method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

CLAIMSWhat is claimed is:

1. A manganese doped metal organic framework (Mn-MOF) comprising a metal organic framework (MOF) comprising a zeolitic imidazolate framework (ZIF) comprising metal ions connected by imidazolate linkers, wherein the metal ions comprise Mn and Zn.

2. The Mn-MOF of claim 1, wherein the ZIF is ZIF-8.

3. The Mn-MOF of claim 1 or claim 2, wherein the Mn-MOF comprises from greater than0% to 50% (mol%) Mn based on the total amount of metal ions.

4. The Mn-MOF of any one of claims 1-3, wherein the Mn-MOF comprises from 10% to50% Mn.

5. The Mn-MOF of any one of claims 1-4, wherein the Mn-MOF comprises 15% Mn. 30% Mn, or 50% Mn.

6. The Mn-MOF of any one of claims 1-5, wherein the Mn-MOF comprises 50% Mn.

7. The Mn-MOF of any one of claims 1-6, wherein the Mn-MOF is crystalline.

8. The Mn-MOF of any one of claims 1-7, wherein the Mn-MOF is nanostructured.

9. The Mn-MOF of any one of claims 1-8, wherein the Mn-MOF is in the form of a plurality of particles.

10. The Mn-MOF of claim 9, wherein the plurality’ of particles have an average particle size of from 100 nanometers to 2 micrometers.

11. The Mn-MOF of any one of claim 9 or claim 10, wherein the plurality of particles have an average particle size of from 500 nanometers to 1 micrometer, or from 400 nanometers to 700 nanometers.

12. The Mn-MOF of any one of claims 9-11, wherein the plurality of particles have a particle shape that is a rounded cubic structure.

13. The Mn-MOF of any one of claims 9-12, wherein the plurality of particles have a sodalite topology.

14. The Mn-MOF of any one of claims 1-13, further comprising a therapeutic agent loaded on and / or in the Mn-MOF.

15. The Mn-MOF of claim 14, wherein the therapeutic agent is conjugated to the Mn-MOF, for example electrostatically.

16. The Mn-MOF of claim 14 or claim 15. wherein the therapeutic agent comprises an anticancer agent, an anti-inflammatory agent, an antimicrobial agent, an immunotherapy agent, or a combination thereof.

17. The Mn-MOF of any one of claims 14-16, wherein the therapeutic agent comprises a STING agonist.

18. The Mn-MOF of any one of claims 14-17, wherein the therapeutic agent comprises a cyclic dinucleotide (CDN).

19. The Mn-MOF of any one of claims 14-18, wherein the therapeutic agent comprises cyclic di-adenosine monophosphate (CDA).

20. A method of making the Mn-MOF of any one of claims 1-19.

21. The method of claim 20, wherein the method comprises in situ incorporation of Mn.

22. The method of claim 20 or claim 21. wherein the method comprises dispersing a Zn salt, a Mn salt, a reducing agent, and an imidazolate in a green solvent.

23. The method of claim 22, wherein the method comprises adding reactants in the following order: the green solvent, the reducing agent, the Mn salt, the Zn salt, and the imidazolate, thereby forming a mixture.

24. The method of claim 23, wherein the method further comprises agitating the mixture, such as by vortexing.

25. The method of claim 24, wherein, after agitating, the mixture is left static for an amount of time.

26. The method of any one of claims 20-25, wherein the method further comprises isolating the Mn-MOF.

27. The method of any one of claims 20-26, wherein the method further comprises washing and / or drying the Mn-MOF.

28. The method of any one of claims 22-27, wherein the Zn salt comprises zinc(II) acetate dihydrate.

29. The method of any one of claims 22-28, wherein the Mn salt comprises manganese(II) acetate tetrahydrate.

30. The method of any one of claims 22-29, wherein the imidazolate comprises 2- methylimidazole.

31. The method of any one of claims 22-30, wherein the reducing agent comprises sodium ascorbate, sodium citrate, tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof.

32. The method of any one of claims 22-31, wherein the reducing agent comprises citrate, tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof.

33. The method of any one of claims 22-32, wherein the reducing agent comprises tris(2- carboxyethyl)phosphine (TCEP).

34. The method of any one of claims 22-33, wherein the green solvent comprises water.

35. The method of any one of claims 22-34, wherein the green solvent consists essentially of water.

36. The method of any one of claims 22-35, wherein the green solvent consists of water.

37. The method of any one of claims 20-36, wherein the method is substantially free of organic solvents.

38. The method of any one of claims 20-37, wherein the method is substantially free of methanol.

39. The method of any one of claims 20-38, wherein the method is conducted at room temperature.

40. The method of any one of claims 20-39, wherein the Mn-MOF is formed in an amount of time of from 10 minutes to 30 minutes, such as from 15 to 25 minutes.

41. The method of any one of claims 20-40, wherein the method is a one pot method.

42. The method of any one of claims 20-41, wherein the method is substantially biocompatible.

43. The method any one of claims 20-41, further comprising contacting the Mn-MOF with a therapeutic agent to thereby load the therapeutic agent in and / or on the Mn-MOF.

44. The method of claim 43, wherein the therapeutic agent is conjugated to the Mn-MOF via electrostatic interactions.

45. A pharmaceutical composition comprising the Mn-MOF of any one of claims 1-19.

46. The pharmaceutical composition of claim 45, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

47. A method of use of the Mn-MOF of any one of claims 1-19 or the pharmaceutical composition of any one of claims 45-46.

48. A method of treating, preventing, or ameliorating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the Mn-MOF of any one of claims 1-19 or the pharmaceutical composition of any one of claims 45- 46.

49. The method of claim 48, wherein the disease comprises cancer.

50. The method of claim 48, wherein the disease is an infectious disease, e.g. an infection, such as a microbial infection.

51. The method of claim 48, wherein the disease is tuberculosis.

52. The method of any one of claims 48-51, wherein the method comprises immunotherapy.

53. The method of any one of claims 48-52, wherein the Mn-MOF or pharmaceutical composition activates the STING pathway to thereby treat, prevent, and / or ameliorate the disease.

54. The method of any one of claims 48-53, wherein the Mn-MOF or pharmaceutical composition activates antigen presenting cells to thereby treat, prevent, and / or ameliorate the disease.

55. The method of any one of claims 48-54, wherein the method comprises cell uptake followed by degradation of the Mn-MOF, thereby providing delivery and release of Mn and the therapeutic agent (when present) to the cell cytosol to thereby treat, prevent, and / or ameliorate the disease.

56. The method of claim 55, wherein the method triggers cGAS-STING activation, resulting in proinflammatory cytokine production and / or bone marrow dendritic cell activation.

57. A vaccine comprising the Mn-MOF of any one of claims 1-19 or the pharmaceutical composition of any one of claims 45-46.

58. An immunomodulator comprising the Mn-MOF of any one of claims 1-19 or the pharmaceutical composition of any one of claims 45-46.

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