Lipid nanoparticle compositions for microneedle-based transdermal delivery

Abstract

The present disclosure describes microneedle array compositions comprising a plurality of microneedles projecting from a substrate. Each microneedle of the plurality of microneedles comprises a penetrating tip and a base that is integrally connected with the substrate, wherein each microneedle of the plurality of microneedles is a porous microneedle composed of a degradable hyaluronic acid polymer comprising a disulfide bond coupled to a terminal amine group. Each microneedle of the plurality of microneedles comprises one or more lipid nanoparticles comprising: an amount of an ionizable lipid, an amount of a neutral lipid, an amount of cholesterol, an amount of one or more polyethylene glycol lipids (PEG-lipids), and an amount of a N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium (DOTAP) molecule.

Description

[0001] Attorney Docket No. 29618-0522WO1

[0002] LIPID NANOPARTICLE COMPOSITIONS FOR MICRONEEDLE-BASED

[0003] TRANSDERMAL DELIVERY

[0004] CLAIM OF PRIORITY

[0005] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 718,992, filed on November 11, 2024. The entire content of the foregoing is hereby incorporated by reference.

[0006] TECHNICAL FIELD

[0007] The present disclosure describes compositions comprising degradable hyaluronicbased hydrogels comprising lipid nanoparticles (LNPs) including microneedle arrays (MNs) and methods of preparing the same. The disclosure also describes methods of delivering a therapeutic agent in a subject in need thereof using these compositions. The hydrogel MNs can include an amino-modified hyaluronic acid polymer comprising a disulfide bond. The methods of delivering a therapeutic agent can include contacting a skin surface of the subj ect with the microneedle array compositions and applying pressure such that the tips of the plurality of microneedles comprising the LNPs perforate and / or penetrate the skin surface, thereby releasing the LNPs and associated-therapeutic agent in the tissue.

[0008] BACKGROUND

[0009] Microneedles (MN) have become an emerging tool for biomedical applications, attracting both scientific and industrial interests. Despite being a sought-after technology, MN-based platforms displaying both therapeutic and diagnostic capacities within the same device have rarely been explored as synchronizing both capacities has been proven challenging. Accordingly, there is an unmet need for MN-based platforms capable of therapeutic and diagnostic applications.

[0010] SUMMARY

[0011] Certain aspects of the present disclosure are directed to microneedle array comprising: a plurality of microneedles projecting from a substrate, each microneedle of the plurality of microneedles comprising a penetrating tip and a base that is integrally connected with the substrate, wherein each microneedle of the plurality7of microneedles is a porous microneedle composed of a degradable hyaluronic acid polymer comprising a disulfide bond coupled to a terminal amine group, and wherein each microneedle of the plurality of microneedles Attorney Docket No. 29618-0522WO1 comprises one or more lipid nanoparticles comprising: an amount of an ionizable lipid; an amount of a neutral lipid; an amount of cholesterol; an amount of one or more polyethylene glycol lipids (PEG-lipids) ranging from about 2.5% to about 10% molar percentage; and an amount of a N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium (DOTAP) molecule ranging from about 5% to about 50% molar percentage.

[0012] In some embodiments, the microneedle array further comprises an excipient at a concentration of about 8% (w / v). In some embodiments, the excipient is sucrose. In some embodiments, the amount of the ionizable lipid ranges from about 16% to about 50%. In some embodiments, the amount of the ionizable lipid is about 24%. In some embodiments, the ionizable lipid is selected from the group consisting of Dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA). 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino} octanoate (SM-102), and 4A3-SC8. In some embodiments, the ionizable lipid is 4A3-SC8. In some embodiments, the amount of the neutral lipid ranges from about 10% to about 24%.

[0013] In some embodiments, the amount of the neutral lipid is about 24%. In some embodiments, the neutral lipid is a phosphatidylcholine lipid or a phosphatidylethanolamine lipid. In some embodiments, the phosphatidylcholine lipid or the phosphatidylethanolamine lipid is selected from the group consisting of di oleoylphosphatidylethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoylphosphatidylcholine (DSPC), dipal-mitoylphosphatidylcholine (DPPC), 1 -palmitoyl-2-oleoyl-sn-gly cero-3-phosphocholine (POPC), and l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). In some embodiments, the phosphatidylcholine lipid or the phosphatidylethanolamine lipid is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE).

[0014] In some embodiments, the amount of cholesterol ranges from about 32% to about 48%. In some embodiments, the amount of cholesterol is about 48%. In some embodiments, the amount of the one or more PEG-lipids is about 5%. In some embodiments, the one or more PEG-lipids comprise one or both of l,2-dimyristoyl-rac-glycero-3- methoxypoly ethylene glycol (DMG-PEG) and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-|maleimide(polyethylene glycol)] (DSPE-PEG-maleimide). In some embodiments, the amount of the DOTAP is about 15%. In some embodiments, the degradable hyaluronic acid polymer comprises the following chemical structure: Attorney Docket No. 29618-0522WO1

[0015] In some embodiments, each microneedle of the plurality of microneedles has a height of about 100 pm to about 1,500 pm and a base having a radius of about 100 pm to about 1,500 pm. In some embodiments, each microneedle has a height of about 600 pm and a base having a radius of about 150 pm. In some embodiments, the substrate is a polymeric, biodegradable substrate. In some embodiments, the polymeric, biodegradable substrate comprises poly(D,L-lactide-co-glycolide) polymer. In some embodiments, the one or more lipid nanoparticles further comprise a therapeutic agent. In some embodiments, the therapeutic agent comprises a chemokine, a chemotherapeutic, a nucleic acid, a protein, a macromolecule, a nanoparticle, a chemical-based drug, or any combination thereof. In some embodiments, the nucleic acid comprises messenger RNA (mRNA), circular RNA (circRNA). small interfering RNA (siRNA), self-amplifying RNA (saRNA), or any combination thereof.

[0016] Certain aspects of the present disclosure are directed to a method of preparing a microneedle array, the method comprising: casting a hyaluronic acid polymer solution into a microneedle mold; optionally centrifuging the microneedle mold containing the hyaluronic acid polymer solution; optionally freeze-drying the microneedle mold containing the hyaluronic acid polymer solution; casting a crosslinker into the microneedle mold containing the freeze-dried hyaluronic acid polymer; optionally centrifuging the microneedle mold containing the freeze-dried hyaluronic acid polymer and the crosslinker, thereby crosslinking the hyaluronic acid polymer and forming a hyaluronic acid hydrogel; optionally freeze-drying the microneedle mold containing the hyaluronic acid hydrogel; casting a solution comprising one or more lipid nanoparticles and an excipient into the microneedle mold containing the freeze-dried hyaluronic acid hydrogel; and casting a biodegradable polymer into the microneedle mold to form a microneedle substrate, wherein the one or more lipid Attorney Docket No. 29618-0522WO1 nanoparticles comprise an amount of one or more PEG-lipids ranging from about 2.5% to about 10% molar percentage, and an amount of a DOTAP molecule ranging from about 5% to about 50% molar percentage.

[0017] In some embodiments, the microneedle array is any of the microneedle arrays disclosed herein. In some embodiments, the hyaluronic acid of the hyaluronic acid polymer solution comprises the following chemical structure:

[0018] Certain aspects of the present disclosure are directed to a method of transdermally delivering a therapeutic agent to a subject in need thereof, the method comprising: contacting any of the microneedle arrays disclosed herein with a skin surface of the subject; and applying pressure on the microneedle array such that the penetrating tip of each microneedle of the plurality of microneedles penetrates the skin surface, thereby releasing the therapeutic agent.

[0019] In some embodiments, the method further comprises maintaining the microneedle array in place for about 5 minutes to about 24 hours after the penetrating tip of each microneedle of the plurality of microneedles penetrates the skin surface. In some embodiments, the therapeutic agent comprises a cell, a chemokine, a chemotherapeutic, a nucleic acid, a protein, a macromolecule, a nanoparticle, an exosome, a chemical-based drug, or any combination thereof. In some embodiments, the nucleic acid comprises messenger RNA (mRNA), circular RNA (circRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), or any combination thereof.

[0020] Certain aspects of the present disclosure are directed to a nanoparticle comprising: an amount of an ionizable lipid; an amount of a neutral lipid; an amount of cholesterol; an amount of one or more polyethylene glycol lipids (PEG-lipids) ranging from about 2.5% to about 10% molar percentage; and an amount of a N-(2,3-dioleoyloxy)propvl-N,N,N- Attorney Docket No. 29618-0522WO1 trimethylammonium (DOTAP) molecule ranging from about 5% to about 50% molar percentage.

[0021] The terms “subject” or “patient” as used herein refer to any mammal (e.g., a human or a veterinary subject, e.g., a dog, cat, horse, cow, goat, sheep, mouse, rat, or rabbit) to which a composition or method of the present disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. The subject may seek or need treatment, require treatment, is receiving treatment, will receive treatment, or is under care by a trained professional for a particular disease or condition.

[0022] The term “composition” as used herein can refer to a microneedle array composition, a precursor composition (e.g., a composition before crosslinking polymerization), and / or a hydrogel composition (e.g., a hydrogel composition after crosslinking polymerization), as provided by the corresponding context of the disclosure.

[0023] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a micelle” includes mixtures of micelles, reference to “a micelle” includes mixtures of two or more such micelles, and the like.

[0024] As used herein, the term “therapeutic agent” is any molecule that is encapsulated, conjugated, fused, dispersed, embedded, mixed, or otherwise affixed to any of the compositions described herein and is useful for a disease therapy.

[0025] As used herein, the term “payload” refers to an agent delivered by a chemically- modified hydrogel composition described herein (e.g., chemically-modified hyaluronic acid hydrogel microneedles or other chemically-modified hyaluronic acid hydrogel compositions).

[0026] By the term “nanoparticle” is meant a particle that has a diameter between about 2 nm to about 200 nm (e.g., between 10 nm and 200 nm, between 2 nm and 100 nm, between 2 nm and 40 nm, between 2 nm and 30 nm, between 2 nm and 20 nm, between 2 nm and 15 nm, between 100 nm and 200 nm, and between 150 nm and 200 nm). Non-limiting examples of nanoparticles include the nanoparticles described herein. Additional examples of nanoparticles are known in the art.

[0027] By the term "nucleic acid” is meant any single- or double-stranded polynucleotide (e.g., DNA or RNA having a semi-synthetic or a synthetic origin). The term nucleic acid includes oligonucleotides containing at least one modified nucleotide (e.g., containing a modification in the base and / or a modification in the sugar) and / or a modification in the phosphodiester bond linking two nucleotides. Exemplary nucleic acids for use in accordance with the present disclosure include, but are not limited to, one or more of DNA, RNA, Attorney Docket No. 29618-0522WO1 hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, mRNAs, antisense RNAs, ribozymes, catalytic DNA. RNAs that induce triple helix formation, aptamers, vectors, and the like. Additional non-limiting examples of nucleic acids are described herein and are known in the art.

[0028] As used herein, the expression “pharmaceutically acceptable” applies to a composition that contains composition ingredients that are compatible with other ingredients of the composition as well as physiologically acceptable to the recipient (e.g., a mammal such as a human) without the resulting production of excessive undesirable and unacceptable physiological effects or a deleterious impact on the mammal being administered the pharmaceutical composition. A composition as described herein can comprise one or more earners, useful excipients, and / or diluents.

[0029] As used herein, the term “hydrogel” refers to a polymeric material having a three- dimensional physical or covalently cross-linked networks that have an affinity for an aqueous medium and are able to absorb a large amount of water while maintaining a semisolid morphology (e g., they do not normally dissolve in the aqueous medium unless they are triggered to do so).

[0030] As used herein, the term “aqueous medium” as used herein refers to water or a solution based primarily on water such as phosphate-buffered saline (PBS), or water containing one or more salts dissolved therein.

[0031] As used herein, the term “crosslink” refers to an interconnection between polymer chains via chemical bonding, such as, but not limited to, covalent bonding, ionic bonding, or affinity interactions that are caused by a chemical composition (e.g., a crosslinker).

[0032] As used herein, the term “biodegradable” refers to a substance which may be broken down by microorganisms, or which spontaneously breaks down over a relatively short time (within about 14 days to about 6 months) when exposed to environmental conditions commonly found in nature. For example, the compositions described herein may be degraded by a reducing agent (e.g., TCEP) that is contacted with the composition.

[0033] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment 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 embodiment. 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. Furthermore, the use of the term “about,” as used Attorney Docket No. 29618-0522WO1 herein, refers to an amount that is near the stated amount by about 10% including increments therein. For example, "about’ ’ can mean a range including the particular value and ranging from 10% below that particular value and spanning to 10% above that particular value.

[0034] As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology7that do not contain those elements or features.

[0035] Where values are described in the present disclosure in terms of ranges, endpoints are included. Furthermore, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0036] Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur according to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

[0037] 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. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0038] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. Attorney Docket No. 29618-0522WO1

[0039] DESCRIPTION OF DRAWINGS

[0040] FIG. 1A is a schematic of the screening process of lipid nanoparticle (LNP) formulations for use in HA-PEG hydrogel microneedles (MNs) for transdermal delivery. The chemical components of the LNP formulation and the chemical structure of the HA-SS-NEL crosslinked with NHS -terminated 8-arm PEG forming a digestible HA hydrogel through a disulfide bond are also shown. An image of microneedles loaded with LNPs is also shown.

[0041] FIG. IB is a schematic of the LNPs of the disclosure.

[0042] FIG. 1C illustrates the fabrication process of the HA-based, LNP -loaded hydrogel MNs. HA-based MNs were fabricated by casting an aqueous amine-modified HA (HA-SS- NH2) solution into a PDMS mold by centrifugation and crosslinking using the NHS- terminated 8-arm PEG crosslinker. LNPs were loaded and a poly(lactic-co-gly colic acid) (PLGA) back layer was added.

[0043] FIGs. 2A-2D show the comparative stability analysis of lipid nanoparticles for mRNA and saRNA formulations. FIG. 2A. Molar ratio of components in lipid nanoparticle (LNP) formulations used to encapsulate mRNA and saRNA. FIG. 2B. LNP sizes by DLS between five identical formulations encapsulating mRNA and saRNA respectively. FIG. 2C. Zeta potential by DLS of the LNP formulations . FIG. 2D. LNP size stability' by DLS over four days, measured immediately after dialysis (Day 0) and at subsequent time points (Day 1 and Day 3).

[0044] FIGs. 3A-3D show the screening LNP-MN formulations for different lipid components. FIG. 3A. Molar ratios of components for lipid nanoparticle (LNP) formulations. FIG. 3B. Encapsulation efficiency of PEG, DOTAP, and ionizable lipid libraries measured by RiboGreen® assay. FIG. 3C. Cell viability’ of B16 cells 24 h posttransfection with LNPs by tox fluorescence assay. FIG. 3D. Gene expression levels by luciferase assay 24 h post-transfection of Bl 6 cells by LNPs.

[0045] FIGs. 4A-4C show the evaluation of the activation of LNPs in microneedles with gene expression. Percentage of gene expression in Bl 6 cells transfected by LNPs with different (FIG. 4A) DOTAP molar% and (FIG. 4B) PEG molar % released from a microneedle matrix compared to free LNPs following 24 hrs treatment with 62.5ng, 125ng and 187.5 ng of mRNA concentrations. FIG. 4C is a schematic illustrating the evaluation process.

[0046] FIGs. 5A-5F shows the excipient screening for lyophilization stability. (FIG. 5A) Encapsulation efficiency of 2.5% PEG-LNPs with varying sucrose concentrations (0, 2.5, 5, Attorney Docket No. 29618-0522WO1

[0047] 10, 20% w / v), measured by Ribogreen assay, comparing lyophilized (L) and non-lyophilized samples. (FIG. 5B) Size and (FIG. 5C) zeta potential by dynamic light scattering (DLS) (Malvern Analytical). (FIG. 5D) Viability of B16 cells 24 h post-transfection with LNPs by tox fluorescence assay. (FIG. 5E) Gene Expression levels by luciferase assay 24 h posttransfection of B16 cells by LNPs. FIG. 5F is a schematic illustrating the optimization process for LNP stability after lyophilization.

[0048] FIGs. 6A-6B In vivo testing of LNP-loaded microneedles. Mice were anesthetized, shaved, and microneedle patches were applied for 24 h. After 24 h, microneedles were removed and mice were administered D-luciferin (150 mg / kg) by intraperitoneal injection. Mice were imaged by the IVIS™ in vivo imaging system for bioluminescence after reaching the plateau of the bioluminescence signal. The bioluminescence signal is compared in FIG. 6A between the standard LNP formulation (1.5% PEG), and one of the formulations that retained the highest activity and overall luminescence signal in vitro (5% PEG). FIG. 6B shows an image of Cy5-labeled lipid nanoparticles transferred from hydrogel microneedles in mice after 24 hrs using IVIS™ with the left three mice treated with loaded microneedles and the right two mice treated with empty microneedles.

[0049] DETAILED DESCRIPTION

[0050] Lipid nanoparticles (LNPs) have emerged as a versatile platform for the delivery of nucleic acids and other therapeutic agents, with broad applications in gene therapy and related fields. In some embodiments, integration of microneedle-based transdermal delivery systems with LNP technology enhances the potential of these formulations by enabling minimally invasive administration, providing direct access to skin-resident immune cells, and improving storage stability. While LNPs have demonstrated efficacy for gene delivery, microneedle-based transdermal delivery systems have not been fully optimized for clinical translation. Accordingly, there remains a need for compositions and methods that combine the advantages of LNPs with microneedle technology7in a stable and effective manner.

[0051] The compositions described herein include biocompatible, chemically crosslinkable hyaluronic acid-based hydrogels comprising LNP compositions, including microneedle arrays. In some examples, the microneedle (MN) array compositions described herein are used for targeted, transdermal drug delivery. Methods of using and / or preparing these MN array compositions are also provided herein.

[0052] Certain embodiments of the present disclosure address this need by providing hyaluronic acid (HA)-based hydrogel microneedles incorporating LNP formulations Attorney Docket No. 29618-0522WO1 optimized for stability and performance. In some embodiments, LNP-microneedle formulations are prepared using lipid component libraries that vary in composition, including PEG-lipids at molar ratios of about 1.5%, about 2.5%, about 5%, and about 10% (including variants A and B), cationic lipids such as DOTAP at about 5%, about 15%, about 25%, and about 50%, and ionizable lipids selected from MC3, SM-102, and 4A3-SC8. These formulations may be screened and optimized for encapsulation efficiency, particle stability, and compatibility with hydrogel microneedle matrices. In some embodiments, the compositions described herein are further evaluated for in vivo performance, including delivery efficiency and therapeutic outcomes. Some embodiments of the various compositions and methods described herein may provide one or more of the following advantages.

[0053] Certain embodiments of the present disclosure provide biocompatible, crosslinkable hyaluronic acid (HA)-based hydrogels incorporating lipid nanoparticle (LNP) compositions. As discussed above, there is an unmet need for delivery systems capable of supporting both therapeutic and diagnostic applications. The compositions and methods described herein address this need by enabling efficient, localized delivery of therapeutic agents. In some embodiments, the microneedle array compositions and methods disclosed herein deliver a payload encapsulated within optimized LNP formulations. In some embodiments, these LNP compositions are specifically engineered to penetrate tissue via microneedles and release their cargo effectively. Importantly, the LNP formulation can be adjusted to control the rate and profile of therapeutic agent delivery, providing flexibility for different clinical applications.

[0054] Additionally, in some embodiments, the LNP platform enables encapsulation and delivery of a broader array of therapeutic cargos, including nucleic acids, peptides, proteins, and small molecules, than would not be possible with microneedles alone.

[0055] The HA-based microneedles provide a minimally invasive platform for delivering these cargos across nano- and micro-scale sizes. Further, in some embodiments, the microneedlebased delivery methods allow precise, localized administration within the skin, reducing off- target effects and systemic side effects commonly associated with conventional drug delivery. In some embodiments, the microneedle arrays are non-invasive and pain-free, promoting high patient compliance while minimizing infection risk and eliminating needle-bome disease concerns.

[0056] In addition, certain embodiments provide hydrogel compositions with tunable properties, including swelling ratio, mechanical strength, degradation rate, and drug release Attorney Docket No. 29618-0522WO1 profile. These properties can be adjusted by vary ing the molecular weight and concentration of one or more crosslinkers. Moreover, the hydrogel compositions may exhibit on-demand degradation triggered by exposure to a reducing agent. Collectively, in some embodiments, these features provide a flexible, customizable drug delivery platform suitable for diverse therapeutic applications.

[0057] In some embodiments, the LNP formulations described herein successfully encapsulate nucleic acids such as messenger RNA (mRNA) and self-amplifying RNA (saRNA), achieving particle sizes of approximately 150 nm and maintaining stability7for at least four days post-dialysis. In certain aspects, encapsulation efficiencies exceed about 85%. Formulations containing PEG-lipids at about 1.5%, about 2.5%, and about 10% generally exhibit higher cell viability7compared to certain DOTAP-containing and ionizable lipid variants, which may exhibit viability below about 75%. In some embodiments, gene expression levels achieved by these formulations exceed about 10A5 units, and notably, DOTAP-containing and ionizable lipid formulations consistently maintain expression levels above this threshold.

[0058] Furthermore, certain embodiments provide optimized mRNA-loaded LNP formulations comprising about 15% DOTAP and about 2.5% PEG-lipid, which demonstrate enhanced gene delivery7efficiency when incorporated into HA-based hydrogel microneedles. In some embodiments, lyophilization stability of these formulations is improved by inclusion of cryoprotectants such as sucrose at concentrations between about 5% and about 10% (w / v), thereby maintaining encapsulation efficiency, particle size, zeta potential, cell viability, and gene expression following reconstitution. These findings support the use of such formulations for stable and efficient delivery' of nucleic acids in microneedle-based platforms.

[0059] Compositions

[0060] LNP Compositions

[0061] The present disclosure features LNP compositions that can be incorporated within one or more microneedles of a microneedle array. The LNP compositions loaded in MN array s can be used for active transdermal delivery of target nucleic acids. In some aspects, the instant disclosure provides for lipid nanoparticles that comprise a permanently cationic lipid, cholesterol, helper lipid(s), PEGylated lipid(s), and ionizable amine-containing lipid(s). The present disclosure demonstrates that certain LNP formulations provide for optimal LNP stability and release from MN arrays. In some instances, such LNP formulations comprise from about 5% to about 50% (molar percentage) of L2-Dioleoyl-3-trimethylammonium Attorney Docket No. 29618-0522WO1 propane (often DOTAP, also referred to as 18: 1TAP), which is a di-chain (gemini) cationic surfactant molecule represented by Formula I.

[0062] Formula I.

[0063] In some embodiments, the LNP formulation comprises about 5% to about 50% (molar percentage) of a cationic surfactant represented by Formula I, which may include 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP, also referred to as 18:1 TAP) or other dichain (gemini) cationic surfactants within the scope of Formula I. In some aspects, provided herein are lipid nanoparticles comprising defined amounts of DOTAP, an ionizable lipid, a neutral lipid, cholesterol, and one or more PEG-lipids, such that the LNPs are stable within the HA-based MN arrays described herein and are configured to be readily released into tissue upon application of the HA-based MN arrays. In many instances, the percentage of the ionizable lipid, the neutral lipid (e.g., phospholipid), the cholesterol, and one or more PEG- lipids in the LNPs is selected to accommodate the incorporation of DOTAP into the particle. Specifically, in instances where the amounts of DOTAP in a particle are selected to range from 5% to 50% (molar percentage) (e.g., about 5% to about 15%, about 5% to about 25%, about 15% to about 25%, about 15% to about 50%, or about 25% to about 50%) of the total amounts of the ionizable lipid, the neutral lipid (e.g., phospholipid), the cholesterol, and the one or more PEG-lipids in the particles are adjusted to conform with the amounts of DOTAP in the particle.

[0064] For example, the amounts of the lipids, other than DOTAP, in the particle can be adjusted as follows: amounts of ionizable lipid can be adjusted to range from about 16% to about 50%(molar percentage); amounts of a neutral lipid can be adjusted to range from about 10% to about 24% (molar percentage), amounts of cholesterol can be adjusted to range from about 32% to about 48% (molar percentage); and amounts of the one or more PEG-lipids can be adjusted from about 2.5% to about 10% of the total amounts of lipids (% of total lipids) in the LNP. Attorney Docket No. 29618-0522WO1

[0065] In some embodiments, the LNPs include about 15% (molar percentage) of DOTAP. In some embodiments, the LNPs include about 5% of DOTAP. In some embodiments, the LNPs include about 5% to about 50% DOTAP (molar percentage). In certain aspects, the amount of DOTAP may be selected from one or more subranges within this range, such as about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 30%, about 5% to about 35%, about 5% to about 40%, about 5% to about 45%, or about 5% to about 50%; about 10% to about 15%. about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 10% to about 40%, about 10% to about 45%, or about 10% to about 50%; about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 35%, about 15% to about 40%, about 15% to about 45%. or about 15% to about 50%; about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 20% to about 40%, about 20% to about 45%, or about 20% to about 50%; about 25% to about 30%, about 25% to about 35%, about 25% to about 40%, about 25% to about 45%, or about 25% to about 50%; about 30% to about 35%, about 30% to about 40%. about 30% to about 45%, or about 30% to about 50%; about 35% to about 40%, about 35% to about 45%, or about 35% to about 50%; about 40% to about 45%, or about 40% to about 50%; about 45% to about 50%. In some embodiments, the LNPs include about 5% to about 15% DOTAP.

[0066] In some embodiments, the LNPs include about 24% of an ionizable lipid. In some embodiments, the LNPs include about 16% to about 50% of an ionizable lipid (e.g., about 16% to about 20%, about 16% to about 25%, about 16% to about 30%, about 16% to about 35%, about 16% to about 40%, about 16% to about 45%, or about 16% to about 50%; about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 20% to about 40%, about 20% to about 45%, or about 20% to about 50%; about 25% to about 30%, about 25% to about 35%, about 25% to about 40%, about 25% to about 45%, or about 25% to about 50%; about 30% to about 35%, about 30% to about 40%, about 30% to about 45%, or about 30% to about 50%; about 35% to about 40%, about 35% to about 45%, or about 35% to about 50%; about 40% to about 45%, or about 40% to about 50%; and about 45% to about 50%). In some embodiments, the ionizable lipid content may also be about 16%. about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

[0067] In some embodiments, the LNPs include about 24% of a neutral lipid. In some embodiments, the LNPs include about 10% to about 24% of a neutral lipid (e.g., about 10% to about 15%, about 10% to about 20%, about 10% to about 24%; about 15% to about 20%, Attorney Docket No. 29618-0522WO1 about 15% to about 24%; and about 20% to about 24%). In some embodiments, the neutral lipid content may also be about 10%, about 15%, about 20%, or about 24% of a neutral lipid.

[0068] In some embodiments, the LNPs include about 48% of cholesterol. In some embodiments, the LNPs include about 32% to about 48% of cholesterol (e.g., about 32% to about 35%, about 32% to about 40%, about 32% to about 45%, or about 32% to about 48%; about 35% to about 40%, about 35% to about 45%, or about 35% to about 48%; about 40% to about 45%. or about 40% to about 48%; and about 45% to about 48%). In some embodiments, the cholesterol content may also be about 32%, about 35%, about 40%, about 45%, or about 48%.

[0069] In some embodiments, the LNPs include one or more PEG-lipids comprising DMG- PEG and / or DSPE-PEG-maleimide (DSPE-PEG-mal). In some embodiments, the LNPs include about 5% DMG-PEG (e.g., DMG-PEG-2000). In some embodiments, the LNPs include about 2.5% to about 10% of one or more PEG-lipids (e.g., DMG-PEG), such as about 2.5% to about 5%, about 2.5% to about 7.5%, or about 2.5% to about 10%; about 5% to about 7.5%, or about 5% to about 10%; and about 7.5% to about 10%. In some embodiments, the PEG-lipid content may also be about 2.5%, about 5%. about 7.5%, or about 10%.

[0070] In some embodiments, the amount of DOTAP in the lipid nanoparticles (LNPs) is selected from about 5 mol% to about 50 mol% of total lipid. In certain embodiments, the amounts of the remaining lipids are determined by proportional scaling of a base formulation such that, when DOTAP is present at x mol%. the remaining (100-x) mol% is distributed among the neutral lipid (e.g., DOPE), cholesterol, the ionizable lipid (e.g., 4A3-SC8), and one or more PEG-lipids (e.g., DMG-PEG-2000) in the same molar ratios as 23.8:47.6:23.9:4.75, respectively. In other embodiments, the amounts of the remaining lipids fall within the following ranges, each expressed as a percentage of the remaining (100-x) mol%: ionizable lipid (e.g., 4A3-SC8) from about 16% to about 50%; neutral lipid (e.g., DOPE) from about 10% to about 24%; cholesterol from about 32% to about 48%; and one or more PEG-lipids (e.g., DMG-PEG-2000) from about 2.5% to about 10%.

[0071] In some instances, the disclosure provides a base LNP formulation comprising: about 0.0% of DOTAP; about 23.8% of a DOPE neutral lipid; about 47.6% of cholesterol; about 23.9% of a 4A3-SC8 ionizable lipid; and about 4.75% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0072] In some embodiments, the disclosure provides an LNP formulation comprising: about 4.8% of DOTAP; about 22.6% of a DOPE neutral lipid; about 45.3% of cholesterol; about Attorney Docket No. 29618-0522WO1

[0073] 22.8% of a 4A3-SC8 ionizable lipid; and about 4.5% of one or more PEG-lipids (e.g., DMG- PEG-2000).

[0074] In some instances, the disclosure provides an LNP formulation comprising: about 13. 1% of DOTAP; about 20.7% of a DOPE neutral lipid; about 41.3% of cholesterol; about 19.1% of a 4A3-SC8 ionizable lipid; and about 4.1% of one or more PEG-lipids (e.g., DMG- PEG-2000).

[0075] In some embodiments, the disclosure provides an LNP formulation comprising: about 13.1% of DOTAP; about 20.7% of a DOPE neutral lipid; about 41.3% of cholesterol; about 20.8% of a 4A3-SC8 ionizable lipid; and about 4.1% of one or more PEG-lipids (e.g., DMG- PEG-2000).

[0076] In some instances, the disclosure provides an LNP formulation comprising: about 19.9% of DOTAP; about 19.0% of a DOPE neutral lipid; about 38.1% of cholesterol; about 19. 1% of a 4A3-SC8 ionizable lipid; and about 3.8% of one or more PEG-lipids (e.g., DMG- PEG-2000).

[0077] In some embodiments, the disclosure provides an LNP formulation comprising: about 33.3% of DOTAP; about 15.9% of a DOPE neutral lipid; about 31.7% of cholesterol; about 16.0% of a 4A3-SC8 ionizable lipid; and about 3.2% of one or more PEG-lipids (e g., DMG- PEG-2000).

[0078] In some instances, the disclosure provides an LNP formulation comprising: about 50.0% of an ionizable lipid; about 10.0% of a DOPE neutral lipid; about 38.5% of cholesterol; and about 1.5% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0079] In some embodiments, the disclosure provides an LNP formulation comprising: about 35.0% of an ionizable lipid; about 16.0% of a DOPE neutral lipid; about 46.5% of cholesterol; and about 2.5% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0080] In some instances, the disclosure provides an LNP formulation comprising: about 35.0% of an ionizable lipid; about 16.0% of a DOPE neutral lipid; about 44.0% of cholesterol; and about 5.0% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0081] In some embodiments, the disclosure provides an LNP formulation comprising: about 35.0% of an ionizable lipid; about 16.0% of a DOPE neutral lipid; about 39.0% of cholesterol; and about 10.0% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0082] In some instances, the disclosure provides an LNP formulation comprising: about 45.0% of an ionizable lipid; about 10.0% of a DOPE neutral lipid; about 35.0% of cholesterol; and about 10.0% of one or more PEG-lipids (e.g., DMG-PEG-2000). Attorney Docket No. 29618-0522WO1

[0083] In some embodiments, the disclosure provides an LNP formulation comprising: about 35.0% of an ionizable lipid (e.g., DLin-MC3); about 16.0% of a DOPE neutral lipid; about 46.5% of cholesterol; and about 2.5% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0084] In some instances, the disclosure provides an LNP formulation comprising: about 50.0% of an ionizable lipid (e.g., DLin-MC3); about 10.0% of a DOPE neutral lipid; about 38.5% of cholesterol; and about 1.5% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0085] In some embodiments, the disclosure provides an LNP formulation comprising: about 35.0% of an ionizable lipid (e.g., SM-1 2); about 16.0% of a DOPE neutral lipid; about 46.5% of cholesterol; and about 2.5% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0086] In some instances, the disclosure provides an LNP formulation comprising: about 50.0% of an ionizable lipid (e.g., SM-102); about 10.0% of a DOPE neutral lipid; about 38.5% of cholesterol; and about 1.5% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0087] In some embodiments, the disclosure provides an LNP formulation comprising: about 35.0% of a 4A3-SC8 ionizable lipid; about 16.0% of a DOPE neutral lipid; about 46.5% of cholesterol; and about 2.5% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0088] In some instances, the disclosure provides an LNP formulation comprising: about 50.0% of a 4A3-SC8 ionizable lipid; about 10.0% of a DOPE neutral lipid; about 38.5% of cholesterol; and about 1.5% of one or more PEG-lipids (e.g., DMG-PEG-2000).

[0089] In some embodiments, the LNP formulations include an excipient. In some embodiments, the LNP formulations include the excipient at a concentration of about 8% weight / volume (w / v). In some embodiments, the LNP formulations include the excipient at a concentration of about 5% w / v to about 10% w / v (e.g., about 5% w / v to about 8% w / v, about 5% w / v to about 9% w / v, about 6% w / v to about 8% w / v, about 6% w / v to about 9% w / v, about 6% w / v to about 10% w / v, about 7% w / v to about 8% w / v, about 7% w / v to about 9% w / v, or about 7% w / v to about 10% w / v). In some embodiments, the excipient acts like or is a cryoprotectant. In some embodiments, the excipient is sucrose. In some embodiments, the inclusion of sucrose in the LNP formulations provide the LNPs with stability7during freeze- drying of the LNP -loaded microneedle arrays.

[0090] In some embodiments, the LNPs provided herein can be spherical or ellipsoidal, or can have an amorphous shape. In some embodiments, the LNPs provided herein (e.g., conjugated or non-conjugated LNPs) can have a diameter (between any two points on the exterior surface of the LNP) of between about 100 nanometers (nm) to about 250 nm (e.g., between about 100 nm to about 150 nm. between about 100 nm to about 200 nm. between about 100 nm to about 250 nm, between about 125 nm to about 150 nm, between about 150 Attorney Docket No. 29618-0522WO1 nm to about 175 nm, between about 150 nm to about 200 nm, between about 150 nm to about 250 nm). In some embodiments, LNPs having a diameter of between about 100 nm to about 250 nm localize to the tissue (e.g., epidermis, dermis, and / or subcutaneous tissue) in a subject. In some embodiments, LNPs having a diameter of between about 100 nm to about 150 nm localize to the epidermis, dermis, and / or subcutaneous tissue of a subject upon release from the microneedles of a microneedle array.

[0091] The LNP compositions of the disclosure can be prepared by various techniques which are presently known in the art. Multilamellar vesicles (MLVs) may be prepared conventional techniques, for example, by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion which results in the formation of MLVs. Unilamellar vesicles (ULVs), such as the LNPs of the disclosure, can then be formed by homogenization, sonication, or extrusion of the multi-lamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0092] In many instances, the LNPs, LNP formulations, and compositions (e.g., LNP compositions, LNP -loaded hydrogel compositions, LNP -loaded microneedle compositions, and / or LNP -loaded microneedle array compositions) of the disclosure comprise at least the following five lipid components:

[0093] Permanently Cationic Lipids

[0094] Structurally, synthetic and / or natural lipids usually contain three parts: (i) cationic or ionizable head groups, (ii) linker groups, and (iii) hydrophobic tails. The chemical diversity' of each part results in a number of structurally distinct ionizable lipids that can be produced by combinatorial chemistry. Conventional permanently charged cationic lipids previously used for nucleic acid delivery (e.g., DOTAP) are believed to readily interact with negatively charged serum proteins and aggregate in the bloodstream, which was believed to lead to rapid clearance of LNP by mononuclear phagocytes. Thus, the relatively high hemolytic activity of cationic lipids was believed to increase the risk of toxic side effects, such as hemoglobin release due to red cell membrane damage.

[0095] The disclosure demonstrates that the presence of certain ratios or amounts of permanently cationic lipids (e.g., DOTAP) in a particle can preferably allow the LNP to be stably suspended or incorporated within the HA-based hydrogel and enable release of the LNPs in vitro and in vivo upon application of the LNP -loaded MN array. In some Attorney Docket No. 29618-0522WO1 embodiments, the particles of the disclosure include DOTAP. In some embodiments, the particles of the disclosure include DOTAP, l,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), dimethyldioctadecylammonium (DDAB), 1,2- dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), or any combination thereof.

[0096] Ionizable Cationic Lipids

[0097] Ionizable cationic lipids are traditional components in many existing LNP formulation(s). Their acid dissociation constants (pKa) determine the ionization and surface charge of the LNP, further affecting its stability and toxicity. To avoid these problems, ionizable cationic lipids with pKa values ty pically ranging from 6.0 to 7.0 have been developed and deployed, most notably in vaccine formulations. This ionizable lipid-based LNP (iLNP) ensures efficient encapsulation of nucleic acids under acidic conditions and reduces toxicity during recycling under physiological conditions. After entering endosomes / lysosomes (which have a pH below surface pKa), LNPs can be positively charged again to facilitate endosome escape and release mRNA into the cytoplasm. It has been reported that LNPs with pKa values of 6.2-6.5 and 6.6-6.9 favored hepatic delivery of siRNA in vivo and intramuscular administration of mRNA vaccines, respectively.

[0098] Depending on the number of amino heads, ionizable cationic lipids can be classified as either monoamino or polyamino lipids. Non-limiting examples of monoamino acid ionizable cationic lipids contemplated in particles of the disclosure include DLin-MC3-DMA (MC3), SM-102, and ALC-0315. Non-limiting examples of monoamino acid ionizable cationic lipids contemplated in particles of the disclosure include 3060no, cKK-E12, C12- 200, 5A2-SC8, TT3, and FTT5. In some embodiments, the ionizable lipid is selected from the group consisting of Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 9- Heptadecanyl 8-{(2-hydroxyethyl)|6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), and 4A3-SC8. In some embodiments, the ionizable lipid is selected from the group consisting of 4A3-SC8, SM-102, DLin-MC2-DMA, DLin-MC3-DMA, DSDMA, DODMA, DLinDMA, DLenDMA, y-DLenDMA. DLin-K-DMA, DLin-C2K-DMA, DLin-K-C3-DM A, DLin-K- C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, or DLin-MP-DMA. In some embodiments, the ionizable lipid is 4A3-SC8.

[0099] PEG-lipi

[0100] Although PEG-lipids generally constitute the smallest molar percentage of the lipid components in LNPs (typically about 2.5 mol% and up to about 10 mol%), they have several Attorney Docket No. 29618-0522WO1 effects on the properties of lipid nanoparticles, including influencing particle size and zeta potential. A variety of PEG lipids are contemplated for use with the LNPs of the disclosure, including terminally modified PEG lipids.

[0101] PEG lipids for use in the present disclosure can be, for example, maleimide terminally modified PEG lipids that can be conjugated with cell targeting peptides. PEG lipids for use with the instant LNPs can have the general structure — (CELCEEOjn — or — (CH2CH2O) nCELCEL. This general structure can further be modified with heterobifunctional maleimide linker. The disclosure contemplates that a variety of PEG molecules can be incorporated into its LNPs, including polyethylene glycol) (PEG) maleimide (e.g., PEG-2000 maleimide), polyalkylene glycols, polypropylene or polybutylene glycols, methoxy poly (ethylene glycol), or methoxy poly (ethylene glycol) propionic acid (mPEG-acid) where n can be from about 1 to about 400. An LNP comprising a thiol reactive motive conjugated to a PEG molecule (e.g., heterobifunctional maleimide PEG) can be decorated with various types of peptides that are displayed on the surface of the LNP molecule.

[0102] In some instances, a PEG molecule is linked to a thiol reactive group for further conjugation to a peptide. In some instances the PEG molecule is a maleimide conjugated PEG molecule. Reactive PEGs can be used for amine pegylation, thiol pegylation, or N- terminal pegylation. The amine in the N-terminus and / or the carboxyl group in the C- terminus can react with a targeting peptide.

[0103] In some instances, a PEG-lipid of the one or more PEG-lipids that is suitable for use in the particles of the disclosure is l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol (DMG-PEG). In some embodiments, a PEG-lipid of the one or more PEG-lipids that is suitable for use in the particles of the disclosure is DMG-PEG 2000. In some instances, the PEG molecule is methoxy poly (ethylene glycol) succinimidyl proprionate (mPEG-SPA). In some instances, a PEG molecule is a methoxy poly (ethylene glycol) propionic acid (mPEG- acid). In some cases, the polyethylene glycol molecule weighs from about 1,000 kilodaltons to about 5,000 kilodaltons. The covalent attachment of a targeting peptide to an LNP via a thiol reactive linkage can change the physicochemical characteristics of the LNP. Examples of physicochemical characteristics that can be altered by binding to a PEG include its zeta potential, its PDI, and the overall hydrodynamic size of the LNP.

[0104] Non-limiting examples of commercially available PEGs suitable for use in the particles of the disclosure include, but are not limited to those available fromNektar Therapeutics, San Carlos, CA, such as mPEG-NEL (Mw about 10 kDa, about 20 kDa), methoxy PEG Succinimidyl a-Methylbutanoate (SMB), SMB-PEG-SMB, methoxy PEG Attorney Docket No. 29618-0522WO1

[0105] Succinimidyl Propionate (mPEG-SPA), Branched PEG N-Hydroxysuccinimide (mPEG2- NHS), mPEG-CM-HBA-NHS, NHS-HBA-CM-PEG-CM-HBA-NHS, mPEG-ButyrALD, ButyrALD-PEG-ButyrALD, Branched PEG ButyrALD (mPEG2-ButyrALD), Orthopyridylthioester (mPEG-OPTE), mPEG Maleimide (MAL), MAL-PEG-MAL, Branched PEG Mai eimide (mPEG2-MAL), Forked Maleimide (mPEG-MAL2 and mPEG2-MAL2), mPEG- Ortho-pyridyldisulfide (mPEG-OPSS) , OPSS-PEG-OPSS, mPEG-SH, SH-PEG-SH, Amine- PEG-Acid. Boc-PEG-NHS, Fmoc-PEG-NHS. MAL-PEG-NHS, Vinylsulfone-PEG-NHS, Acrylate-PEG-NHS Ester.

[0106] Non-limiting examples of PEGs that can be used in amine pegylation include, for example, PEGs manufactured by Jenken Technology USA such as: Y-shape PEG NEIS Esters, Y-shape PEG Carboxyl, Glucose PEG NHS Ester. Galactose PEG NHS Ester, Methoxy PEG Succinimidyl Carboxymethyl Ester, Methoxy PEG Carboxyl, Methoxy PEG Succinimidyl Butanoate, Methoxy PEG Succinimidyl Hexanoate, Methoxy PEG Hexanoic Acid, Methoxy PEG Succinimidyl Succinamide, Methoxy PEG Succinimidyl Glutaramide, Methoxy PEG Succinimidyl Carbonate, Methoxy PEG Nitrophenyl Carbonate. Methoxy PEG Succinimidyl Succinate, Methoxy PEG Succinimidyl Glutarate. Non-limiting examples of PEGs that can be used in thiol pegylation include Y-shape PEG Maleimide, Methoxy PEG Maleimide, Methoxy PEG Vinylsulfone, Methoxy PEG Thiol. Non-limiting examples of PEGs that can be used in N-terminal pegylation include, for example. PEGs manufactured by Jenken Technology USA such as: Y-shape PEG Aldehyde, Y-shape PEG Acetaldehyde, Y- shape PEG Propionaldehyde, Methoxy PEG Propionaldehyde.

[0107] In some embodiments, the molecular weight of a PEG molecule used in an LNP of the disclosure can be, for example, no greater than 5 kilodaltons (kDa), no greater than 4.5 kilodaltons, no greater than 4 kilodaltons, no greater 3.5 than kilodaltons (kDa), no greater than 3 kilodaltons (kDa), no greater than 2.5 kilodaltons (kDa), no greater than 2 kilodaltons (kDa), no greater than 1.5 kilodaltons (kDa), or no greater than 1 kilodaltons (kDa).

[0108] In some cases, the molecular weight of a PEG molecule can be greater than 1 kilodalton (kDa). greater than 1.5 kilodaltons (kDa), greater than 2 kilodaltons (kDa), greater than 2.5 kilodaltons (kDa). greater than 3 kilodaltons (kDa), greater than 3.5 kilodaltons (kDa), greater than 4 kilodaltons (kDa), or greater than 4.5 kilodaltons (kDa).

[0109] In some cases the molecular weight of a PEG oligomer can be from about 1 kilodalton (kDa) to about 5 kilodaltons (kDa), from about 1 kilodalton (kDa) to about 2 kilodaltons (kDa), from about 1 kilodaltons (kDa) to about 3 kilodaltons (kDa). from about 1 kilodaltons (kDa) to about 4 kilodaltons (kDa), from about 1 kilodaltons (kDa) to about 5 kilodaltons Attorney Docket No. 29618-0522WO1

[0110] (kDa), from about 1.5 kilodaltons (kDa) to about 2 kilodaltons (kDa), from about 1.5 kilodaltons (kDa) to about 3 kilodaltons (kDa), from about 1.5 kilodaltons (kDa) to about 3.5 kilodaltons (kDa), from about 1.5 kilodaltons (kDa) to about 4 kilodaltons (kDa), from about 1.5 kilodaltons (kDa) to about 4.5 kilodaltons (kDa), from about 1.5 kilodaltons (kDa) to about 5 kilodaltons (kDa), from about 2 kilodaltons (kDa) to about 3 kilodaltons (kDa), from about 2 kilodaltons (kDa) to about 3.5 kilodaltons (kDa), from about 2 kilodaltons (kDa) to about 4 kilodaltons (kDa), from about 2 kilodaltons (kDa) to about 4.5 kilodaltons (kDa), from about 2 kilodaltons (kDa) to about 5 kilodaltons (kDa).

[0111] In some embodiments, the molecular weight of a maleimide-terminally modified PEG lipid is about 2 kilodaltons (kDa). In some embodiments, the molecular weight of a PEG molecule is from about 1 kilodaltons (kDa) to about 5 kilodaltons (kDa).

[0112] Neutral-lipids - Helper Lipids - Phospholipids

[0113] Phospholipids are neutral “helper” lipids that contribute to the formation of lipid nanoparticles and the escape of endosomes. In many instances, a particle of the disclosure comprises a neutral lipid that is a phosphatidylcholine lipid or a phosphatidylethanolamme lipid. The phosphatidylcholine lipid or the phosphatidylethanol amine lipid can be selected from the group comprising l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 -palmitoyl-2-oleoyl- glycero-3-phosphocholine (POPC), and l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). In some embodiments, the LNP formulations include a neutral lipid that is DOPE.

[0114] Cholesterol

[0115] The inclusion of cholesterol in nucleic acid-containing LNP formulations is based primarily on two major findings obtained with liposomal formulations of small molecule therapeutics: 1) cholesterol is an exchangeable molecule that can accumulate within liposomes during circulation, 2) cholesterol dramatically reduces the amount of surfacebound proteins and improves the circulating half-life.

[0116] Functionalized HA Polymer of the LNP -Loaded Hydrogel MNs

[0117] Hyaluronic acid (HA) is a viscoelastic, biocompatible, biodegradable, non-toxic, and non-immunogenic natural linear polysaccharide with high water affinity. HA is known to play a role in the regeneration and reconstruction of soft tissues. In some embodiments, a Attorney Docket No. 29618-0522WO1 chemically-modified HA can be included in the LNP-loaded microneedle array compositions of the present disclosure. In some embodiments, the structure of each microneedle (e.g., from the base to the tip) is composed of the chemically-modified HA described herein. In some embodiments, the chemically modified HA can be an amino-modified hyaluronic acid comprising a disulfide bond (HA-SS-NH2). In some embodiments, the chemically-modified HA in the hyaluronic acid polymer solution comprises the following chemical structure:

[0118] In some embodiments, the HA polymer that is chemically modified has a molecular weight ranging from about 0.5 kilodalton (kDa) to about 20,000 kDa (e.g., about 0.5 kDa to about 60 kDa, about 1 kDa to about 60 kDa, about 5 kDa to about 60 kDa, about 10 kDa to about 60 kDa, about 20 kDa to about 60 kDa, about 30 kDa to about 60 kDa, about 40 kDa to about 60 kDa, about 50 kDa to about 60 kDa, about 60 kDa to about 75 kDa, about 60 kDa to about 100 kDa. about 60 kDa to about 200 kDa, about 60 kDa to about 300 kDa, about 60 kDa to about 400 kDa, about 60 kDa to about 500 kDa, about 60 kDa to about 600 kDa, about 60 kDa to about 700 kDa, about 60 kDa to about 800 kDa, about 60 kDa to about 900 kDa, about 60 kDa to about 1000 kDa, about 60 kDa to about 2000 kDa, about 60 kDa to about 3000 kDa, about 60 kDa to about 4000 kDa, about 60 kDa to about 5000 kDa, about 60 kDa to about 6000 kDa, about 60 kDa to about 7000 kDa, about 60 kDa to about 8000 kDa, about 60 kDa to about 9000 kDa, about 60 kDa to about 10,000 kDa, about 60 kDa to about 11,000 kDa, about 60 kDa to about 12,000 kDa, about 60 kDa to about 13,000 kDa, about 60 kDa to about 14,000 kDa, about 60 kDa to about 15,000 kDa. about 60 kDa to about 16,000 kDa, about 60 kDa to about 17,000 kDa, about 60 kDa to about 18,000 kDa, about 60 kDa to about 19,000 kDa, about 60 kDa to about 20,000 kDa. about 1000 kDa to about 5000 kDa, Attorney Docket No. 29618-0522WO1 about 1000 kDa to about 10,000 kDa, about 1000 kDa to about 15,000 kDa, about 1000 kDa to about 20,000 kDa.

[0119] In some embodiments, the chemically -modified HA polymer includes one or more side chains including one or more functional groups (e.g., a disulfide group and an amine group), each side chain having a length ranging from about 3 carbon atoms to about 100 carbon atoms (e.g.. about 3 carbon atoms to about 4 carbon atoms, about 3 carbon atoms to about 5 carbon atoms, about 3 carbon atoms to about 6 carbon atoms, about 3 carbon atoms to about 7 carbon atoms, about 3 carbon atoms to about 8 carbon atoms, about 3 carbon atoms to about 9 carbon atoms, about 3 carbon atoms to about 10 carbon atoms, about 3 carbon atoms to about 15 carbon atoms, about 3 carbon atoms to about 20 carbon atoms, about 3 carbon atoms to about 25 carbon atoms, about 3 carbon atoms to about 30 carbon atoms, about 3 carbon atoms to about 35 carbon atoms, about 3 carbon atoms to about 40 carbon atoms, about 3 carbon atoms to about 45 carbon atoms, about 3 carbon atoms to about 50 carbon atoms, about 3 carbon atoms to about 55 carbon atoms, about 3 carbon atoms to about 60 carbon atoms, about 3 carbon atoms to about 65 carbon atoms, about 3 carbon atoms to about 70 carbon atoms, about 3 carbon atoms to about 75 carbon atoms, about 3 carbon atoms to about 80 carbon atoms, about 3 carbon atoms to about 85 carbon atoms, about 3 carbon atoms to about 90 carbon atoms, about 3 carbon atoms to about 95 carbon atoms, about 3 carbon atoms to about 99 carbon atoms, about 10 carbon atoms to about 50 carbon atoms, about 50 carbon atoms to about 100 carbon atoms, or more).

[0120] In some embodiments, the chemically-modified HA polymer includes one or more side chains having a same chain length. In some embodiments, the chemically-modified HA polymer includes one or more side chains having a different chain length. In some embodiments, the one or more side chains include an amine group and a disulfide group. In some embodiments, the one or more side chains include an amine group. In some embodiments, the one or more side chains include a disulfide group. In some embodiments, the chemically modified hyaluronic acid comprises one or more side functional groups. In some embodiments, the chemically modified hyaluronic acid comprises one or more side chains including one or more side functional groups. In some embodiments, the side functional groups comprise one or more disulfide groups, thiol groups, urea groups, carboxylic ester groups, carboxylic acid groups, carboxylic acid salts, latent carboxylic acid Attorney Docket No. 29618-0522WO1 groups, quaternary amine groups, tertiary' amine groups, secondary amine groups, primary amine groups, azides, alkynes. poly(alkylene ether) groups, and any combinations thereof.

[0121] In some embodiments, the plurality of microneedles comprises a degradable hyaluronic acid polymer comprising a disulfide bond. In some embodiments, the degradable hyaluronic acid polymer is cross-linkable. In some embodiments, the degradable hyaluronic acid polymer is an amino-modified hyaluronic acid prior to being crosslinked (e.g., in a precursor state). In some embodiments, the hyaluronic acid polymer can be synthesized by activating sodium hyaluronate with N-(3-(dimethylamino)propyl)carbodiimide (EDC) and N- hydroxysuccinimide (NHS).

[0122] In some embodiments, the activated hyaluronic acid can be mixed with cysteamine dihydrochloride and reacted for about 12 hours. In some embodiments, the activated hyaluronic acid can be mixed with cysteamine dihydrochloride at a ratio ranging from about 1 : 1 to about 1:20 (e.g., about 1 : 1 to about 1: 10, about 1:2 to about 1 : 10, about 1:3 to about 1 : 10, about 1:4 to about 1: 10, about 1 :5 to about 1: 10, about 1:6 to about 1 :10, about 1 :7 to about 1: 10. about 1:8 to about 1 : 10, about 1 :9 to about 1: 10, about 1: 10 to about 1 : 15, or about 1 : 10 to about 1 :20). In some embodiments, the activated hyaluronic acid can be mixed with excess cysteamine dihydrochloride. The addition of cysteamine dihydrochloride to the activated hyaluronic acid functionalizes the hyaluronic acid wi th a terminal amino group and the disulfide bond.

[0123] In some embodiments, the plurality of LNP-loaded microneedles comprises a degradable hyaluronic acid polymer having an on-demand degradation dependent upon the cleavage of the disulfide bond. For example, in some embodiments, the degradable hyaluronic acid polymer can have a degradation profile that is controlled by the addition of a reducing agent that cleaves the disulfide bond. In some embodiments, each microneedle of the plurality of microneedles is a degradable microneedle configured to be degraded upon exposure to the reducing agent. In some embodiments, the disulfide bond is configured to be cleaved upon exposure to the reducing agent. In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, the reducing agent is glutathione, dithiothreitol, beta-mercaptoethanol, or any combination thereof.

[0124] In some embodiments, the disulfide bond is configured to be cleaved by exposure to about 1 millimolar (mM) to about 100 mM of TCEP (e.g., about 1 mM to about 10 mM, about 2 mM to about 10 mM, about 3 mM to about 10 mM, about 4 mM to about 10 mM, about 5 mM to about 10 mM, about 6 mM to about 10 mM. about 7 mM to about 10 mM, about 8 mM to about 10 mM, about 9 mM to about 10 mM, about 10 mM to about 20 mM, Attorney Docket No. 29618-0522WO1 about 10 tnM to about 30 mM. about 10 mM to about 40 mM, about 10 mM to about 50 mM, about 10 mM to about 60 mM. about 10 mM to about 70 mM, about 10 mM to about 80 mM, about 10 mM to about 90 mM, about 10 mM to about 100 mM). In some embodiments, the disulfide bond is configured to be cleaved by exposure to about 10 mM of TCEP.

[0125] Generally, a hydrogel may be formed by using at least one, or one or more ty pes of hydrogel precursors, and setting or solidifying the one or more types of hydrogel precursors in an aqueous solution to form a three-dimensional network, wherein formation of the three- dimensional network may cause the one or more types of hydrogel precursors to gel. As used herein, the term “hydrogel precursor” refers to any uncrosslinked hyaluronic acid polymer that may be used to form a hydrogel. Examples of hydrogel precursors include, but are not limited to, the amino-modified hyaluronic acid comprising a disulfide bond (HA-SS-NH2). In some embodiments, the hydrogel precursor includes a chemically-modified polymer. The chemically -modified HA polymer may form a three-dimensional network in an aqueous medium to form a hydrogel. In some embodiments, the chemically -modified HA polymer comprises a disulfide bond and terminal amine group.

[0126] In some embodiments, the primary amine of the functionalized hyaluronic acid can be reacted with a chemical crosslinker. In some embodiments, the hyaluronic acid polymer precursor (e.g., the uncrosslinked hyaluronic acid polymer) comprises a terminal amino group. In some embodiments, the primary amine group enables the functionalized hyaluronic acid to be chemically crosslinked. In some embodiments, the chemical crosslinker is polyethylene glycol (PEG) comprising a succinimidyl functional group (e.g., -NHS). In some embodiments, the crosslinker comprises a buffer (e.g., phosphate buffer). In some embodiments, the chemical crosslinker is a multi-arm PEG. In some embodiments, the chemical crosslinker is a 3-arm PEG, a 4-arm PEG, a 6-arm PEG, an 8-arm PEG, or any combination thereof. In some embodiments, the chemical crosslinker is an 8-arm PEG. As used herein, the term “multi-arm PEG” refers to a molecule having multiple linear PEG chains attached to a central core. For example, a 3-arm PEG refers to a molecule having 3 PEG chains coupled to its central core, a 4-arm PEG refers to a molecule having 4 PEG chains coupled to its central core, a 6-arm PEG refers to a molecule having 6 PEG chains coupled to its central core, a 8-arm PEG refers to a molecule having 8 PEG chains coupled to its central core.

[0127] In some embodiments, the crosslinker (e.g., PEG-NHS) is present at a concentration of about 1% w / v to about 70% w / v (e.g., about 1% to about 10% w / v, about 1% to about 15% w / v, about 1% to about 20% w / v, about 1% to about 25% w / v, about 1% to about 29% w / v, Attorney Docket No. 29618-0522WO1 about 5% to about 10% w / v, about 5% to about 15% w / v, about 5% to about 20% w / v, about 5% to about 25% w / v, about 5% to about 30% w / v, about 10% to about 15% w / v, about 10% to about 20% w / v, about 10% to about 25% w / v, about 10% to about 30% w / v, about 1% to about 50% w / v, about 1% to about 60% w / v, about 1% to about 70% w / v, about 50% to about 60% w / v, about 50% to about 70% w / v, about 5% to about 50% w / v, about 10% to about 50% w / v, about 20% to about 50% w / v, about 30% to about 50% w / v, or about 40% to about 50% w / v) in a buffer (e.g.. phosphate buffer). In some embodiments, the crosslinker (e.g., PEG-NHS) is present at a concentration of about 10% w / v to in a buffer (e.g., phosphate buffer). In some embodiments, the crosslinker (e.g., PEG-NHS) is present at a concentration of about 50% w / v.

[0128] In some embodiments, the chemical crosslinker is PEG having a molecular weight ranging from about 5 kilodalton (kDa) to about 200 kDa (e.g., about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 5 kDa to about 25 kDa, about 5 kDa to about 30 kDa, about 5 kDa to about 35 kDa, about 5 kDa to about 40 kDa, about 5 kDa to about 50 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa. about 10 kDa to about 30 kDa, about 10 kDa to about 35 kDa, about 10 kDa to about 40 kDa, about 10 kDa to about 50 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, about 15 kDa to about 30 kDa, about 15k Da to about 35 kDa, about 15 kDa to about 40 kDa. about 15 kDa to about 50 kDa, about 20 kDa to about 25 kDa, about 20 kDa to about 30 kDa. about 20 kDa to about 35 kDa, about 20 kDa to about 40 kDa, about 20 kDa to about 50 kDa, about 25 kDa to about 30 kDa, about 25 kDa to about 35 kDa, about 25 kDa to about 40 kDa, about 25 kDa to about 50 kDa, about 30 kDa to about 35 kDa, about 30 kDa to about 40 kDa, about 30 kDa to about 50 kDa, about 35 kDa to about 40 kDa, about 35 kDa to about 50 kDa. or about 40 kDa to about 50 kDa, about 5 kDa to about 100 kDa, about 5 kDa to about 150 kDa, about 5 kDa to about 200 kDa, about 50 kDa to about 100 kDa, about 50 kDa to about 150 kDa, about 50 kDa to about 200 kDa, about 100 kDa to about 150 kDa, about 100 kDa to about 200 kDa, or about 150 kDa to about 200 kDa). In some embodiments, the chemical crosslinker is PEG having a molecular weight of about 10 kDa. In some embodiments, the chemical crosslinker is PEG having a molecular weight of about 40 kDa.

[0129] In some embodiments, the chemical crosslinker comprises one or more PEG polymers having different molecular weights (e.g., any of the above-described molecular weights of PEG). For example, in some embodiments, the chemical crosslinker is a combination of a first PEG having a first molecular weight and a second PEG having a second molecular Attorney Docket No. 29618-0522WO1 weight that is different than the first molecular weight. In some embodiments, the first PEG has a molecular weight of about 10 kDa, and the second PEG has a molecular weight of about 40 kDa. In some embodiments, the chemical crosslinker comprises a first PEG polymer and a second PEG polymer at a ratio of about 0: 100 wt% to about 100:0 wt% (e.g., about 0: 100 wt% to about 10:90 wt%, about 0: 100 wt% to about 20:80 wt%, about 0: 100 wt% to about 30:70 wt%, about 0: 100 wt% to about 40:60 wt%, about 0: 100 wt% to about 50:50 wt%, about 0: 100 wt% to about 60:40 wt%, about 0: 100 wt% to about 70:30 wt%, about 0: 100 wt% to about 80:20 wt%, about 0: 100 wt% to about 90: 10 wt%, about 1 :90 wt% to about 100:0 wt%, about 20:80 wt% to about 100:0 wt%, about 30:70 wt% to about 100:0 wt%, about 40:60 wt% to about 100:0 wt%, about 50:50 wt% to about 100:0 wt%, about 60:40 wt% to about 100:0 wt%, about 70:30 wt% to about 100:0 wt%, about 80:20 wt% to about 100:0 wt%, or about 90: 10 wt% to about 100:0 wt%). In some embodiments, the chemical crosslinker comprises a first PEG polymer and a second PEG polymer at a ratio of about 70:30 wt%. In some embodiments, the chemical crosslinker comprises a first PEG polymer having a molecular weight of about 40 kDa and a second PEG polymer having a molecular weight of about 10 kDa at a ratio of about 70:30 wt%, respectively.

[0130] While the above-discussed hyaluronic acid polymer has been described and illustrated with respect to certain material formulations and methods of preparation, in some embodiments, a hyaluronic acid polymer that is otherwise substantially similar in formulation and function to the above-discussed hyaluronic acid polymer may include one or more materials formulations that are different from the ones discussed above or may be prepared using methods that are modified as compared to the methods described above. For example, while the hyaluronic acid polymer has been described and illustrated as including a terminal amino group, in some embodiments, a hyaluronic acid polymer that is otherwise substantially similar in formulation and function to the above-described hyaluronic acid polymer may alternatively include a thiol group instead of the amino group.

[0131] While above-discussed hyaluronic acid polymer has been described and illustrated as being crosslinked with a PEG crosslinker comprising a succinimidyl functional group, in some embodiments, a PEG crosslinker that is otherwise substantially similar in formulation and function to the above-discussed PEG crosslinker may include an ortho-pyridyl disulfide (OPSS) functional group instead of a succinimidyl functional group. For example, in some embodiments, the PEG crosslinker including the OPSS functional group crosslinks a chemically-modified HA polymer that is functionalized with a thiol group. In some embodiments, a PEG crosslinker that is otherwise substantially similar in formulation and Attorney Docket No. 29618-0522WO1 function to the above-discussed PEG crosslinker may include an maleimide functional group instead of a succinimidyl functional group.

[0132] For example, in some embodiments, the PEG crosslinker including the maleimide functional group crosslinks a chemically -modified HA polymer that is functionalized with a thiol group. In some embodiments, a PEG crosslinker that is otherwise substantially similar in formulation and function to the above-discussed PEG crosslinker may include an acrylate functional group instead of a succinimidyl functional group. For example, in some embodiments, the PEG crosslinker including the acrylate functional group crosslinks a chemically -modified HA polymer that is functionalized with a thiol group and / or an amine group. In some embodiments, the chemical crosslinker comprises one or more amine groups and a disulfide bond (e.g., NHS-SS-NHS).

[0133] LNP-Locided Hydrogel MN Array Compositions

[0134] The present disclosure features LNP-loaded hydrogel microneedle array compositions that can include a plurality of LNP-loaded hydrogel microneedles projecting from a substrate. In some embodiments, the plurality of LNP-loaded hydrogel microneedles of the disclosure are degradable, porous, drug-eluting hydrogels that may facilitate the delivery of therapeutic agent-loaded LNPs through the structural barriers of tissues (e.g., skin) upon application, as illustrated in FIG. 1A. For example, the microneedle arrays of the disclosure can be applied to a skin surface to deliver LNPs encapsulating a therapeutic cargo directly to an injured site, a disease site, and / or a transplant site in the skin of a patient. To this end, the LNP-loaded hydrogel microneedle array compositions described herein can further include one or more LNPs encapsulating or otherwise associated with one or more therapeutic agents.

[0135] In some embodiments, the LNP-loaded hydrogel microneedle array comprises a substrate from which the plurality of LNP-loaded hydrogel microneedles project therefrom. In some embodiments, the substrate is configured to be an anchor for LNP-loaded hydrogel microneedle array administration and retrieval. For example, when in use, the user (e.g., a clinician) can grasp the microneedle array by a substrate surface or an edge of the surface prior to applying the microneedle array on a skin surface of the patient.

[0136] In some embodiments, the substrate is a substantially planar surface. In some embodiments, the substrate is a backing layer. In some embodiments, the substrate is a polymeric substrate. In some embodiments, the substrate is a biodegradable substrate. In some embodiments, the substrate is a non-biodegradable substrate. In some embodiments, the substrate comprises a non-soluble polymer. In some embodiments, the substrate Attorney Docket No. 29618-0522WO1 comprises poly(D,L-lactide-co-glycolide) (PLGA) polymer. In some embodiments, the substrate comprises a water-soluble polymer. In some embodiments, the substrate comprises poly(vinyl alcohol) (PVA). In some embodiments, the substrate comprises polyethylene glycol diacrylate. Non-limiting examples of polymers that can be used to prepare a substrate include poly(caprolactone), poly(ethylene) glycol, poly(vinyl) pyrrolidone, poly(2-hydroxy ethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), polyethylene glycol), poly(methacrylic acid), polylactides (PLA), polyglycolides (PGA), polyanhydrides, polyorthoesters, polycyanoacrylate polycaprolactone, cellulose, lignin, alginate, chitosan, starch, or any combination thereof.

[0137] In some embodiments, each LNP -loaded hydrogel microneedle of the plurality of LNP-loaded hydrogel microneedles comprises a penetrating tip and a base that is integrally connected with the substrate. In some embodiments, each LNP-loaded hydrogel microneedle of the plurality of LNP-loaded hydrogel microneedles comprises a penetrating tip and a base that is removably connected with the substrate (e.g.. each LNP-loaded hydrogel microneedle can be configured to be detached from the substrate via a trigger mechanism such as the dissolution of the substrate). In some embodiments, each LNP-loaded hydrogel microneedle has an elongate body having a proximal end and a distal end. In some embodiments, the elongate body generally tapers from the proximal end, near the base, to the distal end, near the penetrating tip. In some embodiments, each LNP-loaded hydrogel microneedle has a pyramidal or conical shape such that the microneedles taper to a point or a tip that is configured to perforate and / or penetrate a skin surface. The dimensions and geometry of the LNP-loaded hydrogel microneedles can vary' as desired.

[0138] In some embodiments, each LNP-loaded hydrogel microneedle has a height ranging from about 100 pm to about 1,500 pm (e.g., about 100 pm to about 600 pm, about 200 pm to about 600 pm, about 300 pm to about 600 pm, about 400 pm to about 600 pm, about 500 pm to about 600 pm, about 600 pm to about 700 pm, about 600 pm to about 800 pm, about 600 pm to about 900 pm, about 600 pm to about 1000 pm, about 600 pm to about 1100 pm, about 600 pm to about 1200 pm, about 600 pm to about 1300 pm, about 600 pm to about 1400 pm, or about 600 pm to about 1500 pm). In some embodiments, each LNP-loaded hydrogel microneedle has a height of about 600 pm. The height of each LNP-loaded hydrogel microneedle can be measured from the base at the proximal end of the microneedle to the tip at the distal end of the LNP-loaded hydrogel microneedle. In some embodiments, Attorney Docket No. 29618-0522WO1 each LNP -loaded hydrogel microneedle has a height that is sufficient to penetrate the stratum comeum and pass into the epidermis and / or the dermis.

[0139] In some embodiments, each LNP -loaded hydrogel microneedle has a base (e.g., a circular base or a rectangular base) having a width (e.g., a radius or rectangular width) ranging from about 100 pm to about 10,000 pm (e.g., about 100 pm to about 150 pm, about 125 pm to about 150 pm, about 150 pm to about 200 pm. about 150 pm to about 300 pm, about 150 pm to about 400 pm, about 150 pm to about 500 pm, about 150 pm to about 600 pm, about 150 pm to about 700 pm, about 150 pm to about 800 pm, about 150 pm to about 900 pm, about 150 pm to about 1000 pm, about 150 pm to about 1100 pm, about 150 pm to about 1200 pm, about 150 pm to about 1300 pm, about 150 pm to about 1400 pm, about 150 pm to about 1500 pm, about 150 pm to about 5000 pm. about 150 pm to about 7,500 pm, about 150 pm to about 10,000 pm, about 1500 pm to about 5000 pm, about 1500 pm to about 7,500 pm, or about 1500 pm to about 10,000 pm). In some embodiments, each LNP- loaded hydrogel microneedle has a circular base having a radius of about 150 pm. In some embodiments, each LNP-loaded hydrogel microneedle has a rectangular base having a width of about 300 pm. The radius of a circular base of each LNP-loaded hydrogel microneedle can be defined as the distance from the center of the circular base to an edge of the circular base. The width of a rectangular base of each LNP-loaded hydrogel microneedle can be defined as the distance from a first edge of the rectangular base to a second, directly opposing edge of the rectangular base.

[0140] In some embodiments, each LNP-loaded hydrogel microneedle has a tip width ranging from about 1 pm to about 500 pm (e.g., about 1 pm to about 5 pm, about 1 pm to about 10 pm, about 1 pm to about 15 pm, about 1 pm to about 20 pm, about 1 pm to about 25 pm, about 1 pm to about 30 pm, about 5 pm to about 10 pm, about 5 pm to about 15 pm, about 5 pm to about 20 pm, about 5 pm to about 25 pm, about 5 pm to about 30 pm, about 10 pm to about 15 pm, about 10 pm to about 20 pm, about 10 pm to about 25 pm, about 10 pm to about 30 pm, 15 pm to about 20 pm, about 15 pm to about 25 pm, about 15 pm to about 30 pm. about 20 pm to about 25 pm, about 20 pm to about 30 pm, about 25 pm to about 30 pm. about 1 pm to about 300 pm, about 1 pm to about 400 pm. about 1 pm to about 500 pm, about 300 pm to about 400 pm, about 300 pm to about 500 pm, about 10 pm to about 300 pm, about 50 pm to about 300 pm, about 100 pm to about 300 pm, about 150 pm to about 300 pm, or about 250 pm to about 300 pm). In some embodiments, each LNP- loaded hydrogel microneedle has a tip width of about 300pm. Attorney Docket No. 29618-0522WO1

[0141] In some embodiments, the LNP-loaded hydrogel microneedle array can have any suitable shape or size. In some embodiments, the LNP-loaded hydrogel microneedle array is an array of LNP-loaded hydrogel microneedles having dimensions ranging from about 5 x 5 to about 20 x 20 (e.g., about 5 x 5 to about 11 x 11, about 6 x 6 to about 11 x 11, about 7 x 7 to about 11 x 11, about 8 x 8 to about 11 x 11, about 9 x 9 to about 11 x 11, about 10 x 10 to about 11 x 11, about 11 x 11 to about 15 x 15, about 11 x 11 to about 20 x 20. In some embodiments, the LNP-loaded hydrogel microneedle array is an 11 x 11 array of LNP-loaded hydrogel microneedles. In some embodiments, a density of the plurality of LNP-loaded hydrogel microneedles of the LNP-loaded hydrogel microneedle array can range between about 100 microneedles / cm2to about 1000 microneedles / cm2or more (e.g., about 100 microneedles / cm2to about 500 microneedles / cm2. about 100 microneedles / cm2to about 600 microneedles / cm2, about 100 microneedles / cm2to about 700 microneedles / cm2, about 100 microneedles / cm2to about 800 microneedles / cm2, about 900 microneedles / cm2to about 500 microneedles / cm2, about 100 microneedles / cm2to about 950 microneedles / cm2, about 500 microneedles / cm2to about 600 microneedles / cm2. about 500 microneedles / cm2to about 700 microneedles / cm2, about 500 microneedles / cm2to about 800 microneedles / cm2, about 500 microneedles / cm2to about 900 microneedles / cm2, about 500 microneedles / cm2to about 1000 microneedles / cm2, or more)._In some embodiments, a density of the plurality of microneedles of the microneedle array is about 500 microneedles / cm2.

[0142] In some embodiments, the LNP-loaded hydrogel microneedle array can be arranged in a variety of ways. In some embodiments, the LNP-loaded hydrogel microneedle array can be arranged with a tip-to-tip spacing between LNP-loaded hydrogel microneedles ranging from about 50 pm to about 1000 pm (e.g., about 50 pm to about 600 pm, about 100 pm to about 600 pm, about 200 pm to about 600 pm, about 300 pm to about 600 pm, about 400 pm to about 600 pm, about 500 pm to about 600 pm, about 600 pm to about 700 pm, about 600 pm to about 800 pm, about 600 pm to about 900 pm, or about 600 pm to about 1000 pm). In some embodiments, the LNP-loaded hydrogel microneedle array can be arranged wi th a tip- to-tip spacing between LNP-loaded hydrogel microneedles of about 600 pm.

[0143] Physical Properties of LNP-loaded Hydrogel MN Array Compositions

[0144] The physical properties of the LNP-loaded hydrogel microneedle array compositions of the disclosure include, but are not limited to, swelling ratio, mechanical strength, degradation rate, and drug release profile can be finely tuned by modulating the concentration, type, and / or molecular weight of the chemical crosslinker, the reducing agent, Attorney Docket No. 29618-0522WO1 and / or the LNPs. For example, in some embodiments, the molecular weight of the crosslinker can have a significant impact on the swelling ratio and mechanical strength of the amino-functionalized HA polymer. In some embodiments, the degradation rate is dependent on the concentration of the reducing agent that the amino-functionalized HA polymer is exposed to.

[0145] In some embodiments, the molecular weight of the crosslinker (e.g., PEG) is directly proportional to the swelling ratio of the hydrogel microneedles when measured after an extended period of time (e g., 24 hours). In some embodiments, the molecular weight of the crosslinker does not have a significant impact on the sw elling ratio of the hydrogel microneedles when measured after a short period of time (e g., at about 2 hours at most). In some embodiments, crosslinking the HA polymer with a crosslinker (e.g., PEG) having a high molecular weight (e.g., 40 kDa) can lead to greater swelling ratio (e.g., about 1800%) after about 24 hours.

[0146] In some embodiments, the LNP-loaded hydrogel microneedles have a swelling ratio ranging from about 600% to about 1300% (e.g., about 600% to about 700%, about 600% to about 800%, about 600% to about 900%, about 600% to about 1000%, about 600% to about 1100%, about 600% to about 1200%, about 600% to about 1250%, about 900% to about 1000%, about 900% to about 1100%, about 900% to about 1200%, or about 900% to about 1300%) after contacting the functionalized HA hydrogels with an aqueous environment for at least about 15 minutes to about 2 hours.

[0147] In some embodiments, the LNP-loaded hydrogel microneedles have a swelling ratio ranging from about 1300% to about 1800% (e.g., about 1300% to about 1400%, about 1300% to about 1500%, about 1300% to about 1600%, about 1300% to about 1700%, about 1300% to about 1750%, about 1400% to about 1500%, about 1400% to about 1600%, about 1400% to about 1700%, about 1400% to about 1800%, about 1500% to about 1600%, about 1500% to about 1700%, about 1500% to about 1800%, about 1600% to about 1700%, about 1 00% to about 1800%, or about 1700% to about 1800%) after contacting the functionalized HA hydrogels with an aqueous environment for at least about 2 hours to about 48 hours (e.g., about 2 hours to about 12 hours, about 2 hours to 24 hours, about 2 hours to 36 hours, about 2 hours to 48 hours, about 12 hours to 24 hours, about 12 hours to 36 hours, about 12 hours to 48 hours, about 24 hours to 36 hours, about 24 hours to 48 hours, or about 36 hours to 48 hours).

[0148] In some embodiments, the molecular weight of the crosslinker (e.g., PEG) is inversely proportional to the mechanical strength of the LNP-loaded hydrogel microneedles. For Attorney Docket No. 29618-0522WO1 example, crosslinking the HA polymer with a crosslinker (e.g., PEG) having a low molecular weight (e.g., 10 kDa) can lead to greater mechanical strength that results in more efficient skin perforation and / or penetration as compared to a crosslinker (e.g., PEG) having a higher molecular weight (e.g., 40 kDa). In some embodiments, crosslinking the HA polymer with a combination of crosslinkers (e.g., PEG) having a low molecular weight (e.g., 10 kDa) and a higher molecular weight (e.g., 40 kDa) at specific ratios (e.g., 70% (wt%) 40 kDa and 30% (wt%) 10 kDa) can lead to greater mechanical strength that results in more efficient skin perforation and / or penetration as compared to a single crosslinker (e.g., PEG) having a higher molecular weight (e.g., 40 kDa).

[0149] In some embodiments, the degradation of the crosslinked HA polymer is controlled by the time and concentration of the reducing agent that the hydrogel is exposed to. In some embodiments, the crosslinked HA polymer hydrogels have on-demand degradation (e.g., the degradation can be rapidly dissolved or degraded wdthin 30 seconds or less). In some embodiments, the concentration of the reducing agent is directly proportional to the degradation time. In some instances, the degradation of the hydrogels is almost instantaneous (e.g., within about 5 minutes or less) when exposed to about 100 mM of a reducing agent (e.g., TCEP).

[0150] In some embodiments, the crosslinked HA polymer hydrogel is degraded within about 15 seconds (s) to about 10 minutes (min.) (e.g., about 15 s to about 1 min., about 15 s to about 2 min., about 15 s to about 3 min., about 15 s to about 4 min., about 15 s to about 5 min., about 15 s to about 6 min., about 15 s to about 7 min., about 15 s to about 8 min., about 15 s to about 9 min., about 15 s to about 10 min., about 30 s to about 1 min., about 30 s to about 2 min., about 30 s to about 3 min., about 30 s to about 4 min., about 30 s to about 5 min., about 30 s to about 6 min., about 30 s to about 7 min., about 30 s to about 8 min., about 30 s to about 9 min., about 30 s to about 10 min., about 1 min. to about 2 min., about 1 min. to about 3 min., about 1 min. to about 4 min., about 1 min. to about 5 min., or about 1 min. to about 10 min.) when exposed to about 75 mM to about 150 mM (e.g., about 75 mM to about 100 mM, about 80 mM to about 100 mM, about 90 mM to about 100 mM, about 100 mM to about 110 mM, about 100 mM to about 115 mM, about 100 mM to about 120 mM. about 100 mM to about 130 mM, about 100 mM to about 140 mM, or about 100 mM to about 150 mM) of a reducing agent (e.g., TCEP).

[0151] In some embodiments, the crosslinked HA polymer hydrogel is degraded within about 30 seconds (s) to about 20 minutes (min.) (e.g., about 30 s to about 1 min., about 30 s to about 2 min., about 30 s to about 3 min., about 30 s to about 4 min., about 30 s to about 5 Attorney Docket No. 29618-0522WO1 min., about 30 s to about 6 min., about 30 s to about 7 min., about 30 s to about 8 min., about 30 s to about 9 min., about 30 s to about 10 min., about 1 min. to about 2 min., about 1 min. to about 3 min., about 1 min. to about 4 min., about 1 min. to about 5 min., about 1 min. to about 10 min., about 30 s to about 11 min., about 30 s to about 12 min., about 30 s to about 13 min., about 30 s to about 14 min., about 30 s to about 15 min., about 30 s to about 16 min., about 30 s to about 17 min., about 30 s to about 18 min., about 30 s to about 19 min., about 30 s to about 19 min., about 1 min. to about 15 min., about 1 min. to about 20 min., about 5 min. to about 10 min., about 5 min. to about 15 min., about 5 min. to about 20 min., about 10 min. to about 15 min., about 10 min. to about 20 min., or about 15 min. to about 20 min.).

[0152] In some embodiments, the crosslinked HA polymer hydrogel is degraded when exposed to about 75 mM to about 150 mM (e.g., about 75 mM to about 100 mM. about 80 mM to about 100 mM, about 90 mM to about 100 mM, about 100 mM to about 110 mM, about 100 mM to about 115 mM, about 100 mM to about 120 mM, about 100 mM to about 130 mM, about 100 mM to about 140 mM, or about 100 mM to about 150 mM) of a reducing agent (e.g., TCEP).

[0153] In some embodiments, the crosslinked HA polymer hydrogel is degraded within about 10 min. to about 30 min. (e g., about 10 min. to about 12 min., about 10 min. to about 15 min., about 10 min. to about 20 min., about 10 min. to about 25 min., about 10 min. to about 28 min., about 15 min. to about 20 min., about 15 min. to about 25 min., about 15 min. to about 30 min., about 20 min. to about 25 min., about 20 min. to about 30 min., or about 25 min. to about 30 min.) when exposed to about 5 mM to about 15 mM (e.g., about 5 mM to about 7.5 mM, about 5 mM to about 10 mM, about 5 mM to about 12.5 mM, about 5 mM to about 15 mM, about 10 mM to about 12.5 mM, or about 10 mM to about 15 mM) of a reducing agent (e.g., TCEP).

[0154] In some embodiments, the crosslinked HA polymer hydrogel is degraded within about 10 min. to about 30 min. (e.g., about 10 min. to about 12 min., about 10 min. to about 15 min., about 10 min. to about 20 min., about 10 min. to about 25 min., about 10 min. to about 28 min., about 15 min. to about 20 min., about 15 min. to about 25 min., about 15 min. to about 30 min., about 20 min. to about 25 min., about 20 min. to about 30 min., or about 25 min. to about 30 min.).

[0155] In some embodiments, the crosslinked HA polymer hydrogel is degraded when exposed to about 5 mM to about 15 mM (e.g., about 5 mM to about 7.5 mM, about 5 mM to about 10 mM, about 5 mM to about 12.5 mM. about 5 mM to about 15 mM, about 10 mM to about 12.5 mM, or about 10 mM to about 15 mM) of a reducing agent (e.g., TCEP). Attorney Docket No. 29618-0522WO1

[0156] In some embodiments, the crosslinked HA polymer hydrogel is degraded within about 50 hours (h) to about 150 h (e.g., about 50 h to about 75 h, about 50 h to about 100 h, about 50 h to about 125 h, about 50 h to about 150 h, about 75 h to about 100 h, about 75 h to about 125 h, about 75 h to about 150 h, about 100 h to about 125 h, about 100 h to about 150 h, about 125 h to about 150 h) when exposed to about 0.5 mM to about 2 mM (e.g., about 0.5 mM to about 1 mM, about 0.5 mM to about 1.25 mM, about 0.5 mM to about 1.5 mM, about 0.5 mM to about 1.75 mM, about 0.5 mM to about 1.9 mM, about 1 mM to about 1.25 mM. about 1 mM to about 1 .5 mM, about 1 mM to about 1 .75 mM, about 1 mM to about 2 mM, about 1.25 mM to about 1.5 mM, about 1.25 mM to about 1.75 mM, about 1.25 mM to about 2 mM, about 1.5 mM to about 1.75 mM, about 1.5 mM to about 2 mM, or about 1.75 mM to about 2 mM) of a reducing agent (e.g.. TCEP).

[0157] In some embodiments, the crosslinked HA polymer hydrogel is degraded within about 50 hours (h) to about 150 h (e.g., about 50 h to about 75 h, about 50 h to about 100 h, about 50 h to about 125 h, about 50 h to about 150 h, about 75 h to about 100 h, about 75 h to about 125 h, about 75 h to about 150 h, about 100 h to about 125 h, about 100 h to about 150 h, about 125 h to about 150 h).

[0158] In some embodiments, the crosslinked HA polymer hydrogel is degraded when exposed to 0.5 mM to about 2 mM (e.g., about 0.5 mM to about 1 mM, about 0.5 mM to about 1.25 mM, about 0.5 mM to about 1.5 mM, about 0.5 mM to about 1.75 mM. about 0.5 mM to about 1.9 mM, about 1 mM to about 1.25 mM, about 1 mM to about 1.5 mM. about 1 mM to about 1.75 mM, about 1 mM to about 2 mM, about 1.25 mM to about 1.5 mM, about 1.25 mM to about 1.75 mM, about 1.25 mM to about 2 mM, about 1.5 mM to about 1.75 mM, about 1.5 mM to about 2 mM, or about 1.75 mM to about 2 mM) of a reducing agent (e.g., TCEP).

[0159] In some embodiments, the drug concentration and drug-release profile of therapeutic agents encapsulated within or otherwise associated with the LNPs in the LNP-loaded hydrogel microneedles can be precisely tuned. For example, the concentration of LNPs incorporated into the HA-based hydrogel microneedles can be increased to deliver a higher dose of the therapeutic agent, or decreased to provide a lower dose. In certain embodiments, the drug-release profile can be modulated by adjusting one or more parameters, such as the crosslinking density of the hydrogel, the molecular weight of the HA polymer, the ratio of LNPs to hydrogel matrix, or the physicochemical properties of the LNPs themselves (e.g., lipid composition, PEG-lipid content, and surface charge). Additionally, in some embodiments, release kinetics may be controlled through external triggers, such as exposure Attorney Docket No. 29618-0522WO1 to reducing agents, pH changes, or enzymatic degradation, enabling on-demand or sustained release. Collectively, these features allow customization of both the dose and release rate to meet specific therapeutic requirements.

[0160] Therapeutic Cargoes

[0161] In some embodiments, the LNPs of the LNP-loaded hydrogel microneedles described herein include one or more therapeutic agents (e.g., as a drug delivery’ payload). In some embodiments, the one or more therapeutic agents are encapsulated within the LNPs. In some embodiments, the therapeutic agents are encapsulated in, carried by, or otherwise loaded in or on the LNPs in the LNP-loaded hydrogel microneedles. In some embodiments, one or more therapeutic agents are conjugated to a surface of the LNPs in the LNP-loaded hydrogel microneedles. In some embodiments, a first therapeutic agent is encapsulated in, carried by, or otherwise loaded in or on the LNPs, while a second therapeutic agent is disposed outside the LNPs or is not associated with the LNPs but is within the same LNP-loaded hydrogel microneedle. For example, in some embodiments, the second therapeutic agent is dispersed, embedded, suspended, and / or mixed within the plurality’ of microneedles or other chemically- modified HA polymeric structures but not associated with the LNPs. In some embodiments, the first and second therapeutic agents are the same. In some embodiments, the first and second therapeutic agents are the different.

[0162] In some embodiments, the LNPs in the LNP-loaded hydrogel microneedles encapsulate a therapeutic agent at an encapsulation efficiency ranging from about 20% to about 95% (e.g., about 20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 55%, about 20% to about 60%, about 20% to about 65%. about 20% to about 70%, about 20% to about 75%, about 20% to about 80%, about 20% to about 85%, about 20% to about 90%, about 20% to about 95%, about 25% to about 30%, about 50% to about 55%, about 50% to about 60%, about 55% to about 60%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%. or about 65% to about 95%).

[0163] In some embodiments, the therapeutic agent is a hydrophobic therapeutic agent, a hydrophilic therapeutic agent, an amphiphilic therapeutic agent, or any combination thereof. In some embodiments, the therapeutic agent comprises a chemokine, a chemotherapeutic, a nucleic acid, a protein, a macromolecule, a nanoparticle, an exosome, a chemical-based drug, or any combination thereof. In some embodiments, the therapeutic agent is an immunosuppressor and / or the immunoregulator. Attorney Docket No. 29618-0522WO1

[0164] In some embodiments, the therapeutic agent comprises a nucleic acid. In some embodiments, the therapeutic agent can include a naturally occurring, modified, or synthetic nucleic acid such as, but not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), viral vectors, chromosomes, aptamers, nucleosomes, or any combination thereof. In some embodiments, the nucleic acid comprises messenger RNA (mRNA), circular RNA (circRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), or any combination thereof. In some embodiments, the therapeutic agent is mRNA. In some embodiments, the therapeutic agent is circRNA. In some embodiments, the therapeutic agent is siRNA. In some embodiments, the therapeutic agent is saRNA.

[0165] Non-limiting examples of nucleic acids include DNA such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, and RNA / DNA hybrids. Non-limiting examples of nucleic acids also include RNA such as various types of coding and non-coding RNA. Non-limiting examples of the different types of RNA include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), viral RNA, CRISPR RNA (crRNA), trans-activating CRISPR RNA (tracrRNA), single guide RNA (sgRNA), and crRNA / tracrRNA hybrid. The RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or single-stranded RNA. The RNA can be circular RNA. The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA). Further exemplary nucleic acids include, but are not limited to, recombinant nucleic acids, recombinant DNA, cDNA, genomic DNA. dsDNA, RNA, siRNA, mRNA, saRNA, miRNA, IncRNA, tRNA, and shRNA.

[0166] In some embodiments, the nucleic acid is homologous to a nucleic acid in a cell. In some embodiments, the nucleic acid is heterologous to a nucleic acid in a cell. In some embodiments, the nucleic acid is in the form of a plasmid. In some embodiments, the nucleic acid is a therapeutic nucleic acid. In some embodiments, the nucleic acid encodes a therapeutic polypeptide.

[0167] In some embodiments, the therapeutic agent comprises C-C motif chemokine 22 (CCL22) and / or interleukin-2 (IL-2). In some embodiments, the therapeutic agent is a chemokine receptor. In some embodiments, the chemokine receptor comprises CC chemokine receptor 1 (CCR1), CC chemokine receptor 2 (CCR2), CC chemokine receptor 3 Attorney Docket No. 29618-0522WO1

[0168] (CCR3), CC chemokine receptor 4 (CCR4), CC chemokine receptor 5 (CCR5), CC chemokine receptor 6 (CCR6). CC chemokine receptor 7 (CCR7), CC chemokine receptor 8 (CCR8), CC chemokine receptor 9 (CCR9), CC chemokine receptor 10 (CCR10), CC chemokine receptor 11 (CCR11 ), CXC chemokine receptor 1 (CXCR1), CXC chemokine receptor 2 (CXCR2), CXC chemokine receptor 3 (CXCR3), CXC chemokine receptor 4 (CXCR4), CXC chemokine receptor 5 (CXCR5), CXC chemokine receptor 6 (CXCR6), or any combination thereof.

[0169] In some embodiments, the therapeutic agent is a cytokine. In some embodiments, the immunosuppressor and / or the immunoregulator is a cytokine. In some embodiments, the cytokine comprises an interleukin (e.g., IL-2, IL-4, IL-7, IL-9, IL-13 and IL-15, IL-3, IL-6, IL-11, IL-13. IL-17A-F. IL-21, IL-22. IL-23, IL10, IL-35), transforming growth factor-beta (TGF-0), interferon-gamma (IFN-y), tumor necrosis factor-alpha (TNF-a), or any combination thereof.

[0170] In some embodiments, the therapeutic agent is an anti-neoplastic agent or a chemotherapeutic. As used herein, an “anti-neoplastic agent” is any substance used to treat cancer. This includes treatments to a primary tumor (e.g., to inhibit tumor growth), treatments to reduce invasiveness of a primary tumor, and treatments to inhibit metastasis. Anti- neoplastic agents include chemotherapeutic agents (e.g., small organic molecules, generally with a molecular weight less than 1 kDa), therapeutic proteins (e.g., antibodies, restriction enzyme, tumor suppressor protein) and therapeutic nucleic acids (e.g., DNAs (e.g.. triplexforming olignucleeotide, cDNA encoding an anti-neoplastic agent (e.g., an antibody)) and RNAs (e.g., siRNA, antisense RNA, ribozyme)). Anti-neoplastic agents often, though not exclusively, block one or more aspects of cell growth and / or proliferation (e.g., they can be cytostatic and / or cytotoxic). As used herein the term “chemotherapeutic agent” or “chemotherapeutic” (or “chemotherapy”, in the case of treatment with a chemotherapeutic agent) is meant to encompass any non-proteinaceous (i.e., non-peptidic), non-nucleic acid chemical compound useful in the treatment of cancer.

[0171] In some embodiments, the chemotherapeutic comprises methotrexate, cisplatin, doxorubicin, docetaxel, erlotinib, paclitaxel, paraclitaxel, 5 -fluorouracil and gemcitabine. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; Attorney Docket No. 29618-0522WO1 acetogenins (e.g., bullatacin and bullatacinone); camptothecin (including synthetic analogues topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophy cins (particularly cryptophy cin 1 and cryptophy cin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan. novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e g. calicheamicin, especially calicheamicin gammall and calicheamicin phill); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubincin (AdramycinTM) (including morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin. tubercidin. ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as demopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine. 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine. floxundine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2.2', 2"- trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and Attorney Docket No. 29618-0522WO1 anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytosine arabinoside (“Ara-C”); cyclophosphamide; thiopeta; taxoids, e.g. paclitaxel (TAXOL®, Bristol Meyers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GemzarTM); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin (DDP) and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitroxantrone; vancristine; vinorelbine (Navelbine™); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0172] Also included in the definition of "chemotherapeutic agent” are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston™); inhibitors of the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); and antiandrogens such as flutamide, nilutamide, bical utamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0173] In some embodiments, an anti-neoplastic agent is a tyrosine kinase inhibitor. For example, ZD1839 (Iressa™ of AstraZeneca K.K.) shows a competitive effect for ATP in ATP binding site of EGFR (epidermal growth factor receptor) ty rosine kinase, and inhibits tyrosine kinase activity by inhibiting autophosphorylation of tyrosine kinase. Another inhibitor of EGFR tyrosine kinase activity is erlotinib (N-(3-ethynylphenyl)-6,7-bis(2- methoxy ethoxy )quinazolin-4-amine). Imatinib mesylate (GLEEVEC™, formerly STI-571) can inhibit the ty rosine kinase activity7of both BCR-Abl and c-kit. Sorafenib (Nexavar™) is a small molecular inhibitor of Raf kinase, PDGF (platelet-derived growth factor) VEGF receptor 2 & 3 kinases and c-Kit.

[0174] Anti-neoplastic agents can also include therapeutic antibodies, including those raised against tumor antigens or antigens associated with myelodysplasia. Therapeutic antibodies include antibody fragments, as discussed above, monoclonal antibodies, chimeric antibodies and humanized antibodies. Exemplary therapeutic antibodies include the following. IMC- C225 or cetuximab (Erbitux™), is an EGFR-targeted monoclonal antibody that recognizes Attorney Docket No. 29618-0522WO1 the receptor portion of EGFR on the cell surface and inhibits the autophosphorylation of EGFR; thereby inhibiting its tyrosine kinase activity. Herceptin (trastuzumab) is a monoclonal antibody directed against the Her2 / Neu protein (which is homologous to EGFR and whose overexpression is associated with more aggressive disease and poorer prognosis, particularly in breast cancers). Rituximab (RITUXAN™) is an antibody raised against the CD20 protein on lymphoma cells and which selectively depletes normal and malignant CD20+ pre-B and mature B cells. Alemtuzumab (CAMPATH™) is a monoclonal antibody that specifically targets the CD52 antigen found on B and T lymphocytes; it is used for the treatment of chronic lymphocytic leukemia (CLL) and lymphoma. Gemtuzumab zogamicin (MYLOTARG™) is an antibody conjugate that combines a specific antibody directed against CD33 with a chemotherapeutic drug (zogamicin), and is indicated for the treatment of relapsed adult acute myelocytic leukemia.

[0175] In some embodiments, the therapeutic agent comprises a protein. In some embodiments, the therapeutic agent comprises a polypeptide, or peptide, including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and / or an interleukin), a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof, can be natural, synthetic or humanized, a peptide hormone, a receptor, or a signaling molecule. In some embodiments, the therapeutic agent comprises insulin. In some embodiments, the therapeutic agent comprises a hormone (e.g. a thyroid hormone such as levothyroxine or synthetic triiodothyronine). In some embodiments, the protein comprises an antibody (e.g., a single variable domain on a heavy chain (VHH) antibody, a nanobody®). In some embodiments, the antibody is anti-CD3 monoclonal antibody. In some embodiments, the antibody comprises an anti -programmed death- 1 (PD-1) monoclonal antibody, an anti-programmed death ligand- 1 (PD-1) monoclonal antibody, an anti-vascular endothelial growth factor receptor (VEGFR) monoclonal antibody, an anti- cytotoxic T-lymphocyte-associated protein 4 (CTLA4) monoclonal antibody, or any combination thereof. In some embodiments, the antibody comprises an anti-CD3 monoclonal antibody, an anti-IL-6 monoclonal antibody, an anti-CD28 monoclonal antibody, an anti- CD52 monoclonal antibody, or any combination thereof. In some embodiments, the therapeutic agent comprises a macromolecule. In some embodiments, macromolecule comprises glucose. In some embodiments, the therapeutic agent includes, but is not limited to, at least one of a protein, a polypeptide, a peptide, a nucleic acid, a virus, a virus-like particle, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid and a carbohydrate or a combination thereof Attorney Docket No. 29618-0522WO1

[0176] (e.g., chromosomal material comprising both protein and DNA components or a pair or set of effectors, wherein one or more convert another to active form, for example catalytically).

[0177] In some embodiments, the therapeutic agent comprises an anti-inflammatory therapeutic agent. In some embodiments, the therapeutic agent is a corticosteroid. Exemplar}' chemical-based drug for inclusion in the compositions include, but are not limited to, an antibacterial agent, an anti-fungal agent, an anti-viral agent, an anti-acanthamoebal agent, an immunosuppressive agent, an anti-vascular endothelial growth factor (anti-VEGF) agent, a growth factor, or any combination thereof.

[0178] In some embodiments, the therapeutic agent is a psychotropic drug (e.g., an antidepressant or an anti-epileptic drug). Non-limiting examples of anti-depressants include a tricyclic antidepressant (e.g. secondary’ amine tricyclic antidepressant or a tertiary amine tricyclic antidepressant), a selective serotonin reuptake inhibitor, a serotonin and noradrenaline reuptake inhibitor, a reversible monoamine oxidase inhibitor and a monoamine oxidase inhibitor, nortriptyline, desipramine, amitripty line, imipramine, fluoxetine, citalopram, paroxetine, fluvoxamine, escitalopram (lexapro), sertraline, venlafaxine, moclobemide, phenelzine, duloxetine, and tranylcypromine. Non-limiting examples of antiepileptic drugs includes carbamazepine, valproate, ethosuximide and phenyloin. In some embodiments, the psychotropic drug is arapiprazole, olanzapine, quetiapine, risperidone, or ziprasidone.

[0179] In some embodiments, the therapeutic agent is an anti-inflammatory’ therapeutic agent. Non-limiting examples of suitable anti-inflammatory agents include a steroidal antiinflammatory' drug (e.g., prednisolone), a non-steroidal anti-inflammatory drug (e.g., bromfenac), an mTOR inhibitor (e.g., rapamycin), a calcineurin inhibitor, a synthetic or natural anti-inflammatory protein, methylprednisolone, prednisolone, hydrocortisone, fludrocortisone, prednisone, celecoxib, ketorolac, piroxicam, diclorofenac, ibuprofen, and ketoprofen, rapamycin, cyclosporin, and tacrolimus / FK-506. In some embodiments, the therapeutic agent is an anti-hypertensive drug. Non-limiting examples of anti-hypertensive drugs include a vasodilator (e.g., minoxidil), an angiotensin-converting enzyme (ACE) inhibitors (e.g.. captopril, benazepril, enalapril, enalaprilat, fosinopril, lisinopril, quinapril, ramipril, and trandolapril or others with similar molecular mechanisms), a calcium channel blocker (e.g., nifedipine, verapamil, nicardipine, diltiazem, isradipine, amlodipine, nimodipine, felodipine, nisoldipine, bepridil or others with similar molecular mechanisms), a beta-blocker (e.g., atenolol, metoprolol, propranolol, timolol, nadolol, acebutolol, pindolol, Attorney Docket No. 29618-0522WO1 sotalol, labetalol, oxprenolol or others with similar molecular mechanisms), methyldopa, hydralazine hydrochloride, labetalol, adenosine, nifedipine, and magnesium sulfate.

[0180] In some embodiments, the therapeutic agent is a growth factor. In some embodiments, the growth factor includes, but is not limited to, epithelial growth factor, fibroblast growth factor, nene growth factor, hepatocyte growth factor, or any combination thereof. Further non-limiting examples of suitable grow th factors include transforming growth factors (TGFs) (e.g., beta transforming growth factors such as, TGF-01, TGF- 2. TGF-P3), fibroblast growth factors (FGFs), platelet derived growth factors (PDGFs), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors, bone morphogenetic proteins (e.g., BMP-1, BMP -2, BMP-3, BMP-4, BMP-5, BMP- 6, BMP-7, BMP-8. BMP-9); heparin-binding growth factors (e.g., fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor (IGF)), Inhibins (e.g., Inhibin A, Inhibin B), growth differentiating factors (for example, GDF-1), and Activins (e.g.,, Activin A, Activin B, Activin AB), and biologically active analogs, fragments, and derivatives of such growth factors.

[0181] In some embodiments, the therapeutic agent is a vaccine. In some embodiments, the therapeutic agent is an mRNA vaccine. In some embodiments, the therapeutic agent is a vaccine comprise an RNA that encodes highly immunogenic antigens capable of eliciting potent neutralizing antibodies responses against coronavirus antigens, such as Severe Acute Respiratory’ Syndrome (SARS)-CoV-2 coronavirus antigens. In some embodiments, the therapeutic agent is a "booster" vaccine. As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster” refers to an extra administration of the prophylactic (vaccine) composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition.

[0182] In some embodiments, the hydrogel composition further includes a pharmaceutically acceptable carrier. As used herein, the expression “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible w ith the other ingredients of the formulation and is compatible with administration to a subject, for example a human. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry Attorney Docket No. 29618-0522WO1 a risk of toxicity, irritation, allergic response, immunogenicity', or any other complication that excessively outweighs its therapeutic benefits. Examples of pharmaceutically acceptable carriers include, but are not limited to, a solvent or dispersing medium containing, for example, water, pH buffered solutions (e.g., phosphate buffered saline (PBS), HEPES, TES, MOPS, etc.), isotonic saline, Ringer’s solution, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), alginic acid, ethyl alcohol, and suitable mixtures thereof. In some embodiments, the pharmaceutically acceptable carrier can be a pH buffered solution (e g. PBS).

[0183] In some embodiments, the pharmaceutically acceptable carrier is a topical carrier. In some embodiments, the composition is formulated for topical use. In some embodiments, the composition is topically administered to a tissue (e.g.. a skin tissue) of a patient. In some embodiments, the composition can be applied to a tissue (e.g., a skin tissue) for topical, targeted delivery of a therapeutic agent.

[0184] Methods of LNP-Loaded Hydrogel Microneedle Fabrication

[0185] The present disclosure features methods of preparing an LNP-loaded hydrogel microneedle array using the above-described hyaluronic acid polymer comprising a disulfide bond and a terminal amine group. A variety7of methods for manufacturing microneedles are available and any suitable method for manufacturing microneedles or microneedle arrays are contemplated for use with the compositions and methods disclosed herein. In some embodiments, the LNP-loaded hydrogel microneedles are manufactured using any suitable method, including, but not limited to molding (e.g., self-molding, micro-molding, microembossing, microinjection, roll-to-roll processing, and the like).

[0186] In some embodiments, the methods include casting the hyaluronic acid polymer solution into a microneedle mold. In some embodiments, the microneedle mold comprises an array of negative microneedle projections (e.g., an array of 11 x 11 microneedles). In some embodiments, the negative microneedle projections have a height within any of the height ranges described elsewhere herein (e.g., of about 600 pm) and circular base having a radius within any of the radius or width ranges described elsewhere herein (e.g.. a radius of about 150 pm). The microneedle mold can be composed of any suitable material (e g., poly dimethylsiloxane (PDMS)).

[0187] Next, in some embodiments, the methods include centrifuging the microneedle mold containing the hyaluronic acid polymer solution. In some embodiments, the centrifugation step forces the HA polymer solution throughout the mold and helps to evenly distribute the Attorney Docket No. 29618-0522WO1

[0188] HA polymer solution. In some embodiments, the microneedle mold containing the hyaluronic acid polymer solution is centrifuged at a speed of about 3000 revolutions per minute (RPM) to about 5000 (e.g., about 3000 RPM to about 4200 RPM, about 3500 RPM to about 4200 RPM, about 4000 RPM to about 4200 RPM, about 4200 RPM to about 4500 RPM, or about 4200 RPM to about 5000 RPM). In some embodiments, the microneedle mold containing the hyaluronic acid polymer solution is centrifuged at about 4200 RPM. In some embodiments, the microneedle mold containing the hyaluronic acid polymer solution is centrifuged for about 1 min. to about 10 mins (e.g., about 1 min. to about 5 min., about 2 min. to about 5 min., about 3 min. to about 5 min., about 4 min. to about 5 min., about 5 min. to about 6 min., about 5 min. to about 7 min., about 5 min. to about 8 min., about 5 min. to about 9 min., or about 5 min. to about 10 min.). In some embodiments, the microneedle mold containing the hyaluronic acid polymer solution is centrifuged for about 5 minutes.

[0189] Next, in some embodiments, the methods include freeze-drying the microneedle mold containing the hyaluronic acid polymer solution. In some embodiments, the microneedle mold containing the hyaluronic acid polymer solution is freeze-dried for about 12 hours or more. Next, in some embodiments, the methods include casting a crosslinker (e.g., PEG- NHS) solution into the microneedle mold containing the freeze-dried hyaluronic acid polymer to crosslink the hyaluronic acid polymer and form a hydrogel. In some embodiments, the centrifugation step forces the crosslinker solution throughout the mold and helps to evenly distribute the crosslinker solution throughout the freeze-dried polymer composition. In some embodiments, the methods described herein enable a gradual gelation of the HA hydrogel and ensure a successful polymerization of the hydrogel matrix (e.g., from the tip to the base of each microneedle) that has a homogenous composition.

[0190] In some embodiments, the microneedle mold containing the crosslinker solution and freeze-dried polymer composition is centrifuged at a speed of about 3000 revolutions per minute (RPM) to about 5000 (e.g., about 3000 RPM to about 4200 RPM, about 3500 RPM to about 4200 RPM, about 4000 RPM to about 4200 RPM, about 4200 RPM to about 4500 RPM, or about 4200 RPM to about 5000 RPM). In some embodiments, the microneedle mold containing the crosslinker solution and freeze-dried polymer composition is centrifuged at about 4200 RPM. In some embodiments, the microneedle mold containing the crosslinker solution and freeze-dried polymer composition is centrifuged for about 1 min. to about 10 mins (e.g., about 1 min. to about 5 min., about 2 min. to about 5 min., about 3 min. to about 5 min., about 4 min. to about 5 min., about 5 min. to about 6 min., about 5 min. to about 7 min., about 5 min. to about 8 min., about 5 min. to about 9 min., or about 5 min. to about 10 min.). Attorney Docket No. 29618-0522WO1

[0191] In some embodiments, the microneedle mold containing the crosslinker solution and freeze- dried polymer composition is centrifuged for about 5 minutes. In some embodiments, excess polymer and / or crosslinker solution is carefully removed after the centrifugation step. In some embodiments, the methods include freeze-drying the microneedle mold containing the crosslinked hydrogel. In some embodiments, the microneedle mold containing the crosslinked hydrogel is freeze-dried for about 12 hours or more.

[0192] Next, in some embodiments, a solution containing one or more LNPs encapsulating or otherwise associated with one or more therapeutic agents is deposited in the microneedle mold containing the freeze-dried, crosslinked hydrogel. In some embodiments, the microneedle mold containing the added therapeutic agent loaded-LNPs is spun for about 1 second (s) to about 60 s (e.g., about 1 s to about 15 s, about 1 s to about 30 s, about 1 s to about 45 s, about 1 s to about 60 s, about 15 s to about 30 s, about 15 s to about 45 s, about 15 s to about 60 s). In some embodiments, the microneedle mold containing the added therapeutic agent loaded-LNPs is subjected to a vacuum pressure for about 1 min. to about 5 min. (e g., about 1 min. to about 2 min., about 1 min. to about 3 min., about 1 min. to about 4 min., or about 1 min. to about 5 min.). In some embodiments, the microneedle mold containing the added therapeutic agent loaded-LNPs is subjected to a vacuum pressure instead of being spun. In some embodiments, the microneedle mold containing the added therapeutic agent loaded-LNPs is subjected to a vacuum pressure in addition to being spun (e.g., before or after microneedle mold is spun).

[0193] Next, in some embodiments, the methods include adding a backing layer to the microneedle mold containing the freeze-dried LNP-loaded hyaluronic acid hydrogel. In some embodiments, the backing layer is added immediately after spinning the microneedle mold containing the added therapeutic agent-loaded LNPs. In some embodiments, the backing layer is the substrate of the microneedle array. In some embodiments, the backing layer is a polymeric layer. In some embodiments, the backing layer comprises a biodegradable polymer. In some embodiments, the biodegradable polymer is a poly(D,L-lactide-co- glycolide) (PLGA) polymer. In some embodiments, any suitable biodegradable polymer can be used to prepare the backing layer. Non-limiting examples of biodegradable polymers include, but are not limited to, polymers of hydroxy acids such as lactic acid and glycolic acid polylactide, polyglycolide, polylactide-co-glycolide, and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone). In some embodiments, the backing layer is formed by adding Attorney Docket No. 29618-0522WO1 a polymeric solution to the mold. In some embodiments, the polymeric solution is added dropwise until covering the entire surface of the mold.

[0194] In some embodiments, the methods further include drying the microneedle mold containing the LNP -loaded hyaluronic acid hydrogel and the backing layer. In some embodiments, the microneedle mold is dried for at least about 12 hours. In some embodiments, the microneedle mold is dried at about room temperature (e.g.. about 20 °C to about 25 °C). Lastly, In some embodiments, the methods include removing the microneedle mold from the microneedle array. In some embodiments, the microneedle mold is peeled off. In some embodiments, the microneedle mold is stored in a low-humidity environment (e.g., about 1% relative humidity' (rh) to about 20% rh) or a no-humidity' environment (e.g., 0% rh).

[0195] Methods of Therapeutic Agent Delivery

[0196] The present disclosure features methods of transdermally delivering a therapeutic agent to a subj ect in need thereof using the LNP -loaded HA-based hydrogel microneedle array compositions described herein (e.g., the LNP-loaded hydrogel microneedle arrays composed of the above-described LNPs and hyaluronic acid polymer comprising a disulfide bond and a terminal amine group).

[0197] In some embodiments, the methods include contacting any of the LNP-loaded hydrogel microneedle array compositions described herein with a skin surface of the subject. Next, in some embodiments, the methods further include applying pressure on the LNP- loaded hydrogel microneedle array such that the penetrating tip of each LNP-loaded hydrogel microneedle of the plurality of LNP-loaded hydrogel microneedles penetrates the skin surface. In some embodiments, the tip portion of the LNP-loaded hydrogel microneedles perforates and / or penetrates the stratum comeum. In some embodiments, the LNP-loaded hydrogel microneedles penetrate the epidermis and / or the dermis. In some embodiments, the LNP-loaded hydrogel microneedles create a pathway or pore within a skin tissue (e.g., within the stratum comeum, epidermis, and / or dermis) and release therapeutic agent-loaded LNPs. In some embodiments, once released, the LNPs and / or the therapeutic agent released by the LNPs can directly go into the systemic circulation without facing the barrier of the stratum comeum. In some embodiments, once released, the LNPs and / or the therapeutic agent released by the LNPs can directly go into the lymphatic circulation without facing the barrier of the stratum comeum. In some embodiments, once released, the LNPs and / or the therapeutic agent released by the LNPs can remain within a localized area of the tissue near the microneedle injection site. In some embodiments, the LNPs and / or the therapeutic agent Attorney Docket No. 29618-0522WO1 released by the LNPs is internalized by cells at, near, and / or neighboring the microneedle injection site.

[0198] In some embodiments, the methods include maintaining the LNP-loaded hydrogel microneedle array in place for about 5 minutes to about 1 week (e.g., about 5 min. to about 10 min., about 5 min. to about 15 min., about 5 min. to about 20 min., about 5 min. to about 30 min., about 5 min. to about 1 hour (h), about 5 min. to about 2 h, about 5 min. to about 3 h, about 5 min. to about 6 h, about 5 min. to about 12 h, about 5 min. to about 18 h. about 5 min. to about 24 h, about 5 min. to about 36 h, about 5 min. to about 48 h, about 5 min. to about 72 h, about 5 min. to 4 days, about 5 min. to 5 days, about 5 min. to 6 days, about 5 min. to 7 days, about 10 min. to about 15 min., about 10 min. to about 20 min., about 10 min. to about 30 min., about 10 min. to about 1 hour (h), about 10 min. to about 2 h, about 1 h to about 2 h, about 1 h to about 3 h, about 1 h to about 6 h, about 1 h to about 12 h, about 1 h to about 24 h, about 1 day to about 2 days, about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 6 days, about 1 day to about 7 days, or more) after the penetrating tip of each LNP-loaded hydrogel microneedle of the plurality of LNP-loaded hydrogel microneedles penetrates the skin surface.

[0199] Indications

[0200] The present disclosure features methods of treating a disease in a subject in need thereof using the chemically-modified hyaluronic acid compositions described herein (e.g., the LNP-loaded microneedle arrays composed of the above-described hyaluronic acid polymer comprising a disulfide bond and a terminal amine group). The LNP-loaded hydrogel MN array compositions of the disclosure may be used for the prevention and / or treatment of a wide variety of diseases; particularly cancers and skin disorders. Both benign and malignant tumors, as well as metastases of a rimary tumor, can be treated. The LNP-loaded hydrogel MN array compositions of the disclosure may also be used for the prevention and / or treatment a variety of infectious diseases.

[0201] As used herein, “prevention” includes prophylaxis, delay of onset of symptoms or blocking onset of symptoms altogether. As used herein, “treatment,” refers to inhibition of progression of a neoplastic disease, malignant disorder, or skin disorder, stasis of symptoms, prolongation of survival, partial or full amelioration of symptoms, and partial or full eradication of a neoplastic condition, disease, or disorder or full eradication of a skin condition, disease, or disorder. Attorney Docket No. 29618-0522WO1

[0202] Thus, treatment includes partial or total alleviation of symptoms, or reduction of signs or symptoms of illness, and specifically includes, without limitation, prolongation of survival. The expected progression-free survival times may be measured in months to years, depending on prognostic factors including the number of relapses, stage of disease, and other factors. Prolongation of survival includes without limitation an increase in survival time of at least 1 month, about at least 2 months, about at least 3 months, about at least 4 months, about at least 6 months, about at least 1 year, about at least 2 years, about at least 3 years, or more. Overall survival can also be measured in months to years. The patient’s symptoms may remain static or may decrease.

[0203] Non-limiting indications that can be treated using the hydrogel compositions of the disclosure include those involving undesirable or uncontrolled cell proliferation. Such indications include benign tumors, various types of cancers such as primary tumors and tumor metastasis, restenosis (e.g. coronary, carotid, and cerebral lesions), hematological disorders, abnormal stimulation of endothelial cells (atherosclerosis), insults to body tissue due to surgery, abnormal wound healing, abnormal angiogenesis, diseases that produce fibrosis of tissue, liver fibrosis, kidney fibrosis, lung fibrosis, scleroderma, atherosclerosis, repetitive motion disorders, disorders of tissues that are not highly vascularized, and proliferative responses associated with organ transplants.

[0204] Generally, cells in a benign tumor retain some or all of their differentiated features and do not divide in a completely uncontrolled manner. A benign tumor is usually localized and nonmetastatic. Specific types of benign tumors that can be treated using the present disclosure include, but are not limited to, hemangiomas, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas and pyogenic granulomas.

[0205] In a malignant tumor, cells become undifferentiated, do not respond to the body's grow th control signals, and multiply in an uncontrolled manner. Malignant tumors are invasive and capable of spreading to distant sites (metastasizing). Malignant tumors are generally divided into two categories: primary and secondary. Primary tumors arise in a particular tissue and remain in that tissue (i.e., they remain in situ). A secondary tumor, or metastasis, is a tumor which originates in one region of the body and spreads to another region. Common routes for metastasis of a malignant cell are direct growth into adjacent structures, dissemination through the vascular or lymphatic systems, and tracking along tissue planes and body spaces (peritoneal fluid, cerebrospinal fluid, etc.). Attorney Docket No. 29618-0522WO1

[0206] Primary' and metastatic tumors that can be treated by the methods disclosed herein include, but are not limited to, lung cancer (including, but not limited to, lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, non-small-cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including, but not limited to, ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma); colorectal cancer (including, but not limited to, colon cancer, rectal cancer); anal cancer; pancreatic cancer (including, but not limited to, pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostate cancer; ovarian carcinoma (including, but not limited to, ovarian epithelial carcinoma or surface epithelial-stromal tumour including serous tumour, endometrioid tumor and mucinous cystadenocarcinoma, sex-cord-stromal tumor); liver and bile duct carcinoma (including, but not limited to, hepatocelluar carcinoma, cholangiocarcinoma, hemangioma); esophageal carcinoma (including, but not limited to, esophageal adenocarcinoma and squamous cell carcinoma); non-Hodgkin's lymphoma; bladder carcinoma; carcinoma of the uterus (including, but not limited to, endometrial adenocarcinoma, uterine papillary serous carcinoma, uterine clear-cell carcinoma, uterine sarcomas and leiomyosarcomas, mixed mullerian tumors); glioma, glioblastoma, medullablastoma, and other tumors of the brain; kidney cancers (including, but not limited to, renal cell carcinoma, clear cell carcinoma, Wilm's tumor); cancer of the head and neck (including, but not limited to, squamous cell carcinomas); cancer of the stomach (including, but not limited to. stomach adenocarcinoma, gastrointestinal stromal tumor); multiple myeloma; testicular cancer; germ cell tumors; neuroendocrine tumors; cervical cancer; carcinoids of the gastrointestinal tract, breast, and other organs; and signet ring cell carcinoma.

[0207] Mesenchymal tumors include, but are not limited to, sarcomas, fibrosarcomas, haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, and leiomy sarcoma.

[0208] Specific types of cancers or malignant tumors, either primary or secondary, that can be treated using compositions disclosed herein also include, but are not limited to, skin cancer, bone cancer, brain cancer, cancer of the lary nx, gall bladder, pancreas, parathyroid, thyroid, adrenal, neural tissue, head and neck, bronchi, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteosarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor. Attorney Docket No. 29618-0522WO1 islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas, intestinal ganglioneuromas, hyperplastic comeal nerve tumor, marfanoid habitus tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoides, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcomas, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, glioblastoma multiforme, lymphomas, malignant melanomas and epidermoid carcinomas.

[0209] Proliferative responses and / or immune responses associated with organ transplantation that, for example, contribute to transplant rejection and associated complications can also be treated using the methods and compositions of the present disclosure. Specifically, these proliferative responses can occur during transplantation of organs such as the heart, lung, liver, and kidney; as well as following bone marrow or other hematopoietic cell transplantations. Furthermore, immune responses associated with skin transplantation (e.g., allografts) can also be treated using the methods and compositions of the present disclosure.

[0210] Non-limiting skin indications that can be treated using the hydrogel compositions of the disclosure include, but are not limited to, topic dermatitis, contact dermatitis, drug- induced delayed type cutaneous allergic reactions, toxic epidermal necrolysis, cutaneous T- cell lymphoma, bullous pemphigoid, alopecia areata, alopecia totalis, alopecia universalis, androgenetic alopecia, vitiligo, acne rosacea, prurigo nodularis, scleroderma, herpes simplex viral skin infections, acne, rosacea, eczema, keloids, psoriasis, pruritus, scleroderma, post- inflammatory hyperpigmentation, melasma, skin cancers, bums, lupus erythematosus, or any combination thereof.

[0211] In some embodiments, the hydrogel microneedle array compositions of the disclosure may be utilized to treat and / or prevent a variety of infectious diseases. Non-limiting skin infectious diseases that can be treated using the hydrogel compositions of the disclosure include, but are not limited to, Severe Acute Respiratory' Syndrome (SARS), Middle East Respiratory Syndrome (MERS). and COVID-19 (Coronavirus Disease 2019). In some embodiments, the hydrogel microneedle array compositions treat and / or prevent the variety of infectious diseases by delivering a vaccine (e.g., an RNA vaccine) comprising and / or encoding highly immunogenic antigens capable of eliciting potent neutralizing antibodies responses against coronavirus antigens, such as SARS-CoV-2 coronavirus antigens. Attorney Docket No. 29618-0522WO1

[0212] EXAMPLES

[0213] Certain embodiments of the present disclosure are further described in the following examples, which do not limit the scope of any embodiments described in the claims.

[0214] Example 1 - Synthesis of HA-Modified Polymer and LNP Loading

[0215] To generate the hyaluronic acid backbone polymer, the carboxyl groups of sodium hyaluronate (60kDa, LifeCore Biomedical) were activated using N-(3-dimethylaminopropyl)- N’ -ethylcarbodiimide (1 :4 molar ratio) and N-hydroxysuccinimide (1 :2 molar ratio) at room temperature for 30 minutes, prior to reaction with cystamine dihydrochloride (1: 10 molar ratio) at room temperature for 12 hours. The resultant amino-modified hyaluronic acid was dialyzed in water for one week, then freeze dried and stored at -20°C until use. Hydrogel surrogates were created by preparing a 10% w / v solution of amino-modified hyaluronic acid in phosphate buffer with 8% w / v sucrose and freeze dried. An 8-arm PEG-NHS solution (10% w / v in phosphate buffer with 8% w / v sucrose) was generated by mixing PEG-NHS polymer with concentrated lipid nanoparticles. This was added in equal volume to the original hyaluronic acid solution and freeze dried. The pellet was resuspended in cell culture media and incubated with B16-F10 cells for 24 hrs in a transwell dish and compared to a dose-matched fresh LNP solution. Luciferase expression and cell vi abi 1 i ty were assessed using One-Gio + Tox Reporter Luciferase and Cell Viability assay.

[0216] Example 2 - Microneedle (MN) Patch Preparation

[0217] Microneedle patches were fabricated using a centrifugation method to cast polymers into a poly dimethylsiloxane mold. Each microneedle patch consisted of an 11 by 11 array with each needle having a radius of 150 pm and a height of 600 pm. To generate the hyaluronic acid backbone polymer, the carboxyl groups of sodium hyaluronate (60kDa, LifeCore Biomedical) were activated using N-(3-dimethylaminopropyl)-N’- ethylcarbodiimide (1 :4 molar ratio) and N-hydroxysuccinimide (1 :2 molar ratio) at room temperature for 30 minutes, prior to reaction with cystamine dihydrochloride (1: 10 molar ratio) at room temperature for 12 hours. The resultant amino-modified hyaluronic acid was dialyzed in water for one week, then freeze dried and stored at -20°C until use.

[0218] For the preparation of the microneedle patch, amino-modified hyaluronic acid (10% w / v) was cast into the mold by centrifugation and then freeze dried. After removal from the lyophilizer, a second layer consisting of an 8-arm PEG-NHS crosslinker (10% w / v, Attorney Docket No. 29618-0522WO1

[0219] PEGworks) was deposited on the mold, centrifuged, and again freeze dried. Next, the drug layer containing either concentrated lipid nanoparticles with 8% w / v sucrose (mCherry or MY01 microneedle groups) or 8% w / v sucrose only (empty microneedles) was added by centrifugation. Finally, the backing layer was added by dropwise addition of 15% w / v poly(D,L-lactide-co-glycolide) (Resomer RG 505, Sigma). Microneedle patches were allowed to dry at 4°C for at least three days prior to being peeled off the molds and stored at 4°C until further use.

[0220] Example 3 - LNP Fabrication for HA-Based MNs for Drug Delivery mRNA-loaded lipid nanoparticles were prepared at an N:P molar ratio of 6: 1. To generate the organic phase, 4-(dimethylamino)-butanoic acid (D-Lin-MC3-DMA, MedChemExpress), cholesterol, l,2-dioleoyl-.w-glycero-3-phosphoethanolamine (DOPE, Avanti), and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000, Avanti) were mixed in pure ethanol at a predetermined molar ratio. The aqueous phase was generated by diluting mRNA in 20 mM acetate buffer (pH 4). Lipid nanoparticles were synthesized by mixing the aqueous phase and the organic phase at a volumetric ratio of 3: 1 by pipetting or pulse vortexing depending on the preparation scale. The lipid nanoparticles were then dialyzed in phosphate buffered saline for at least 2 h using a Pur-A-Lyzer™ dialysis kit (Sigma) at 4°C and concentrated using centrifugal ultrafiltration devices (Amicon™, Millipore). Encapsulation efficiency was determined using the Quant-iT Ribogreen RNA assay (Invitrogen) following the manufacturer’s protocol. Size, poly dispersity index, and surface charge measurements were obtained by dynamic light scattering and zeta potential measurements using a Nanosizer ZS (Malvern Instruments).

[0221] LNPs encapsulating mRNA and saRNA were fabricated using the pipette method, with saRNA being of particular interest due to its enhanced stability, reduced immunogenicity, and prolonged-expression kinetics [2], A diverse library of LNPs were generated for assessment, the molar ratios of key lipid components: ionizable lipids (such as SM-102, 4A3-SC8, and MC3), charged structural lipids (DOTAP, assessed at 0-50%), and PEG lipids (ranging from 1.5-10%) systematically varied. Comparative stability testing was conducted to evaluate the impact of formulation changes on LNP size and zeta potential over time through Dynamic Light Scattering (DLS).

[0222] LNPs were then incorporated into hydrogel microneedle matrix. Stabilizing excipients like sucrose were tested and included at different weight percentages to enhance lyophilization stability. The LNP formulations were then characterized by parameters Attorney Docket No. 29618-0522WO1 including particle size, zeta potential, encapsulation efficiency was characterized using ribogreen assay. LNPs loaded in surrogates were incubated with B16 cells for 24 hours, after which cell viability7and gene expression was assessed using One-Gio + Tox Reporter Luciferase and Cell Viability7assay.

[0223] Example 4 - Optimization of LNP Formulations

[0224] In each of the compositions listed under the 0% - 50% DOTAP formulation names in Table 1. below, the percentage of DOTAP refers to the number of moles of DOTAP relative to the total moles of lipid in the base formulation, which, in some examples, comprises DOPE, Cholesterol, 4A3-SC8, and DMG-PEG2k. In some embodiments, the base formulation is 23.8% DOPE, 47.6% Cholesterol, 23.9% 4A3-SC8, and 4.75% DMG-PEG- 2000. In each of the compositions listed under the 1.5% - 10.5% PEG formulation names in Table 1. below, the percentage of PEG largely depends on altering the % of DMG-PEG2000 lipid and DOPE lipids unless otherwise indicated. The remaining formulations in Table 1. (i.e., MC3 A. MC3 B, SM-102 A, SM-102 B, 4A3-SC8 A, and 4A3-SC8 B) are ionizable lipid formulations. All DOTAP and PEG formulations included sucrose (8% w / v) as an excipient to support stability during freeze drying of the microneedle matrices.

[0225] Table 1. LNP Formulations. Attorney Docket No. 29618-0522WO1

[0226] Example 5 - Impact of DOTAP and PEG Concentrations on Gene Expression Induced by LNPs Loaded in MNs

[0227] FIGs. 4A-4B demonstrate that modification to standard lipid nanoparticle formulations (0% DOTAP in the first graph, and PEG 1.5% and PEG 2.5% in the second graph) are required to achieve potent activity in hydrogel microneedles. In FIG. 4A, the molar percentage of DOTAP (relative to the total other lipid components formulation) in the lipid nanoparticle formulation is modified to identify a formulation that is stable in the microneedle hydrogel matrix at the same efficacy level as fresh LNPs that have not been encapsulated in the hydrogel matrix. In FIG. 4B, the molar percentage of the PEG lipid in the lipid nanoparticle formulation is varied, with better stability of gene expression seen at higher PEG molar percentages. In all graphs, percent active refers to the expression level of the hydrogel microneedles loaded nanoparticles relative to fresh lipid nanoparticles.

[0228] Example 6 - In Vivo Testing of LNP-Loaded, HA-based MNs

[0229] Lipid nanoparticle-loaded microneedles were synthesized as described above in Examples 3 and 4. Mice were anesthetized, shaved, and microneedle patches were applied for 24 h. After 24 h, microneedles were removed and mice were administered D-luciferin (150 mg / kg) by intraperitoneal injection. Mice were imaged by the IVIS™ in vivo imaging system for bioluminescence after reaching the plateau of the bioluminescence signal. The bioluminescence signal is compared in FIG. 6A between the standard LNP formulation (1.5% PEG), and one of the formulations that retained the highest activity and overall luminescence signal in vitro (5% PEG). Increasing the molar % of PEG in the formulation can improve colloidal stability and reduce the potential for interactions with the hydrogel Attorney Docket No. 29618-0522WO1 matrix by shielding electrostatic interactions. Particles that had been released from the microneedle matrix in vitro were observed to have higher sizes (>300 nm) for formulations with lower % PEG lipid, indicative of aggregation or loss of colloidal stability.

[0230] FIG. 6B shows an image of Cy5-labeled lipid nanoparticles transferred from hydrogel microneedles in mice after 24 hrs using IVIS™ with the left three mice treated with loaded microneedles and the right two mice treated with empty microneedles.

[0231] Example 7 - Use of LNP-Loaded, HA-based MNs for Cancer Immunotherapy

[0232] An LNP -loaded, HA-based microneedle patch is fabricated as described in Examples 1-4, with the LNPs containing mRNA sequences encoding tumor-associated antigens and immune-stimulatory cytokines. When applied to a patient's skin, the microneedles penetrate the stratum comeum and deliver the LNPs into immune-active tissue, where the mRNA is expressed by resident cells. This localized delivery promotes activation of cytotoxic T lymphocytes targeting tumor cells, while minimizing systemic exposure and reducing adverse effects compared to conventional intravenous administration.

[0233] Example 8 - Use of LNP-Loaded, HA-based MNs for Vaccine Delivery

[0234] An LNP -loaded, HA-based microneedle patch is fabricated as described in Examples 1-4, with the LNPs encapsulating mRNA encoding a viral antigen. Upon application to the patient's skin, the microneedles penetrate the stratum comeum and deliver the LNPs into the epidermal and dermal layers rich in antigen-presenting cells. The mRNA is subsequently translated within host cells, inducing expression of the antigen and eliciting a robust immune response. This approach enables needle-free vaccination with improved patient compliance and potential for room-temperature stability.

[0235] References

[0236] 1. Cullis, P. R. & Hope, M. J. Lipid nanoparticle systems for enabling gene therapies. Mol. Ther. 25, 1467-1475 (2017)

[0237] 2. Kairuz, D., Samudh, N., Ely, A., Arbuthnot, P. & Bloom, K. Production, characterization, and assessment of permanently cationic and ionizable lipid nanoparticles for use in the delivery of self-amplifying RNA vaccines. Pharmaceutics 15, 1173 (2023).

[0238] 3. Phatale, V., Vaiphei, K. K., Jha, S., Patil, D., Agrawal, M. & Alexander, A. Overcoming skin barriers through advanced transdermal drug delivery approaches. J. Control. Release 351, 361-380 (2022). Attorney Docket No. 29618-0522WO1

[0239] OTHER EMBODIMENTS

[0240] It is to be understood that while certain embodiments have been described within the detailed description, the present disclosure is intended to illustrate and not limit the scope of any embodiment defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

Attorney Docket No. 29618-0522WO1WHAT IS CLAIMED IS:

1. A microneedle array comprising: a plurality of microneedles projecting from a substrate, each microneedle of the plurality of microneedles comprising a penetrating tip and a base that is integrally connected with the substrate, wherein each microneedle of the plurality of microneedles is a porous microneedle composed of a degradable hyaluronic acid polymer comprising a disulfide bond coupled to a terminal amine group, and wherein each microneedle of the plurality7of microneedles comprises one or more lipid nanoparticles comprising: an amount of an ionizable lipid; an amount of a neutral lipid; an amount of cholesterol; an amount of one or more polyethylene glycol lipids (PEG-lipids) ranging from about 2.5% to about 10% molar percentage; and an amount of a N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium (DOTAP) molecule ranging from about 5% to about 50% molar percentage.

2. The microneedle array of claim 1, further comprising an excipient at a concentration of about 8% (w / v).

3. The microneedle array of claim 2, wherein the excipient is sucrose.

4. The microneedle array of any one of claims 1-3, wherein the amount of the ionizable lipid ranges from about 16% to about 50%.

5. The microneedle array of claim 4, wherein the amount of the ionizable lipid is about 24%.

6. The microneedle array of any one of claims 1-5. wherein the ionizable lipid is selected from the group consisting of Ditinol eyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino} octanoate (SM-102), and 4A3-SC8.

7. The microneedle array of claim 6, wherein the ionizable lipid is 4A3-SC8.Attorney Docket No. 29618-0522WO18. The microneedle array of any one of claims 1-7, wherein the amount of the neutral lipid ranges from about 10% to about 24%.

9. The microneedle array of claim 8, wherein the amount of the neutral lipid is about 24%.

10. The microneedle array of any one of claims 1-9, wherein the neutral lipid is a phosphatidylcholine lipid or a phosphatidylethanolamine lipid.

11. The microneedle array of claim 10, wherein the phosphatidylcholine lipid or the phosphatidylethanolamine lipid is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-distearoylphosphatidylcholine (DSPC), dipal- mitoylphosphatidylcholme (DPPC), 1 -palmitoyl-2-oleoyl-sn-gly cero-3- phosphocholine (POPC), and l-stearoyl-2-oleoyl-sn-glycero-3 -phosphocholine (SOPC).

12. The microneedle array of claim 10, wherein the phosphatidylcholine lipid or the phosphatidylethanolamine lipid is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE).

13. The microneedle array of any one of claims 1-12, wherein the amount of cholesterol ranges from about 32% to about 48%.

14. The microneedle array of claim 13, wherein the amount of cholesterol is about 48%.

15. The microneedle array of any one of claims 1-14, wherein the amount of the one or more PEG-lipids is about 5%.

16. The microneedle array of any one of claims 1-15, wherein the one or more PEG-lipids comprise one or both of l ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)] (DSPE-PEG-maleimide).

17. The microneedle array of any one of claims 1-16, wherein the amount of the DOTAP is about 15%.Attorney Docket No. 29618-0522WO118. The microneedle array of any one of claims 1-17, wherein the degradable hyaluronic acid polymer comprises the following chemical structure:

19. The microneedle array of any one of claims 1-18, wherein each microneedle of the plurality of microneedles has a height of about 100 pm to about 1,500 pm and a base having a radius of about 100 pm to about 1,500 pm.

20. The microneedle array of claim 19, wherein each microneedle has a height of about 600 pm and a base having a radius of about 150 pm.

21. The microneedle array of any one of claims 1-20, wherein the substrate is a polymeric, biodegradable substrate.

22. The microneedle array of claim 21, wherein the polymeric, biodegradable substrate comprises poly(D,L-lactide-co-glycolide) polymer.

23. The microneedle array of any one of claims 1-22, wherein the one or more lipid nanoparticles further comprise a therapeutic agent.

24. The microneedle array of claim 23, wherein the therapeutic agent comprises a chemokine, a chemotherapeutic, a nucleic acid, a protein, a macromolecule, a nanoparticle, a chemical-based drug, or any combination thereof.Attorney Docket No. 29618-0522WO125. The microneedle array of claim 24, wherein the nucleic acid comprises messenger RNA (mRNA). circular RNA (circRNA), small interfering RNA (siRNA), selfamplifying RNA (saRNA), or any combination thereof.

26. A method of preparing a microneedle array, the method comprising: casting a hyaluronic acid polymer solution into a microneedle mold; optionally centrifuging the microneedle mold containing the hyaluronic acid polymer solution; optionally freeze-drying the microneedle mold containing the hyaluronic acid polymer solution; casting a crosslinker into the microneedle mold containing the freeze-dried hyaluronic acid polymer; optionally centrifuging the microneedle mold containing the freeze-dried hyaluronic acid polymer and the crosslinker, thereby crosslinking the hyaluronic acid polymer and forming a hyaluronic acid hydrogel; optionally freeze-drying the microneedle mold containing the hyaluronic acid hydrogel; casting a solution comprising one or more lipid nanoparticles and an excipient into the microneedle mold containing the freeze-dried hyaluronic acid hydrogel; and casting a biodegradable polymer into the microneedle mold to form a microneedle substrate, wherein the one or more lipid nanoparticles comprise an amount of one or more PEG-lipids ranging from about 2.5% to about 10% molar percentage, and an amount of a DOTAP molecule ranging from about 5% to about 50% molar percentage.

27. The method of claim 26, wherein the microneedle array is the microneedle array of any one of claims 1-8.

28. The method of claim 26, wherein the hyaluronic acid of the hyaluronic acid polymer solution comprises the following chemical structure:Attorney Docket No. 29618-0522WO129. A method of transdermally delivering a therapeutic agent to a subject in need thereof, the method comprising: contacting the microneedle array of any one of claims 1 -8 with a skin surface of the subject; and applying pressure on the microneedle array such that the penetrating tip of each microneedle of the plurality of microneedles penetrates the skin surface, thereby- releasing the therapeutic agent.

30. The method of claim 29, further comprising maintaining the microneedle array in place for about 5 minutes to about 24 hours after the penetrating tip of each microneedle of the plurality of microneedles penetrates the skin surface.

31. The method of claims 29 or 30, wherein the therapeutic agent comprises a cell, a chemokine, a chemotherapeutic, a nucleic acid, a protein, a macromolecule, a nanoparticle, an exosome, a chemical-based drug, or any combination thereof.

32. The method of claim 31, wherein the nucleic acid comprises messenger RNA (mRNA). circular RNA (circRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), or any combination thereof.

33. A nanoparticle comprising: an amount of an ionizable lipid; an amount of a neutral lipid;Attorney Docket No. 29618-0522WO1 an amount of cholesterol: an amount of one or more polyethylene glycol lipids (PEG-lipids) ranging from about 2.5% to about 10% molar percentage; and an amount of a N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium (DOTAP) molecule ranging from about 5% to about 50% molar percentage.

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