LNP fiber hydrogel composites and methods

Abstract

In one aspect, a combined 1) lipid nanoparticle (LNP) and 2) fiber-hydrogel composite therapy is provided. The 1) lipid nanoparticle (LNP) and 2) fiber-hydrogel composite may be administered together or separately (e.g. sequentially) preferably together with one or more therapeutic agents. In one particular aspect, the administered LNP / fiber-hydrogel composite comprises at least one mRNA encoding one or more antigens capable of eliciting an (adaptive) immune response in a mammal, including for treatment of cancer.

Description

LNP FIBER HYDROGEL COMPOSITES AND METHODSCROSS REFERENCE TO RELATED APPLICATIONSThe present application claims priority from 1) U.S. provisional application no. 63 / 575,831 filed April 7, 2024 and 2) U.S. provisional application no. 63 / 631,402 filed April 8, 2024. Each of these applications is incorporated by reference herein in their entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCHThis invention was made with government support under grants EB028239 and AU55313 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0001] In one aspect, a combined 1) lipid nanoparticle (LNP) and 2) fiber-hydrogel composite therapy is provided. The 1) lipid nanoparticle (LNP) and 2) fiber- hydrogel composite may be administered together or separately (e.g. sequentially) preferably together with one or more therapeutic agents. In one particular aspect, the administered LNP / fiber-hydrogel composite comprises at least one mRNA encoding one or more antigens capable of eliciting an (adaptive) immune response in a mammal, including for treatment of cancer.BACKGROUND

[0002] Immunotherapy has witnessed a paradigm shift recently with the advent of mRNA lipid nanoparticles (LNPs) as an accelerated approach to combat cancer. The success of certain mRNA vaccines has demonstrated the effectiveness of mRNA constructs to deliver the encoded antigen and the safety and potency of LNPs in delivering the gene therapeutics and vaccines to relevant cells in vivo. Nonetheless, the realization of the full potential of mRNA LNP cancer vaccines requires further optimization of LNP immune activation profiles and delivery strategies to permit more efficient programming of immune responses for potent anticancer efficacy.

[0003] It would be desirable to have new compositions for mRNA LNPs.SUMMARY

[0004] We now provide new lipid nanoparticle (LNP) formulations. In one aspect, we now provide LNP fiber-hydrogel composites, including compositions that comprise a fiber-hydrogel composite together with one or more LNPs that contain or are administered in conjunction with a therapeutic agent, including an mRNA agent.

[0005] The present compositions suitably comprise 1) an LNP component and 2) a fiber- hydrogel composite components. Those components suitably can be formulated together (single formulation) and administered as a single formulation (and thus effectively simultaneously) to a subject.

[0006] Alternatively, and in certain aspects preferably, the 1) an LNP component and 2) a fiber- hydrogel composite components can be formulated separately (i.e. a first formulation comprising the 1) fiber-hydrogel composite and a second formulation comprising the 2) LNP component). Those first and second formulations can be administered together or separately. For instance, the first formulation comprising the 1) fiber-hydrogel composite can be administered (e.g. by injection) to a subject first and thereafter the second formulation comprising the 1) LNP component can be administered to the subject that has already been dosed with the first formulation comprising the fiber-hydrogel composite. An additional therapeutic agent e.g. a nucleic acid agent such as an mRNA agent suitably can be administered to the subject either separately from such first and second formulations or together with or both of such first and second formulations, e.g. admixed or as a portion of the LNP component.

[0007] The term “LNP fiber-hydrogel composite” or similar term as used herein embraces and refers to 1) an LNP component and 2) a fiber-hydrogel composite components and 3) therapeutic agent (if not a portion of the LNP component of fiber-hydrogel composite component) that are formulated together or separately and / or administered separately (but typically in a combined administration) to a subject. Use of separate formulations ) an LNP component and 2) a fiber- hydrogel composite components and 3) therapeutic agent (if not a portion of the LNP component of fiber-hydrogel composite component) is also referred to herein as a composition package.

[0008] In preferred aspects, the LNPs encapsulate or coordinate with one or more therapeutic agents including for in vivo intracellular delivery of the therapeutic agent.

[0009] In preferred aspects, an LNP / fiber-hydrogel composite system comprises at least one mRNA encoding one or more antigens capable of eliciting an (adaptive) immune response in a subject, including for treatment of cancer in the subject.

[0010] In one aspect, a lipid nanoparticle (LNP) of a present composition or composition package comprises an ionizable lipid, at least one helper lipid, a PEGylated lipid and a sterol.

[0011] We have demonstrated in vivo efficacy of the LNP composite systems , including for use of treatment or reduction of cancer. In particular aspects, the fiber-hydrogel composites are loaded with or administered in combination with LNPs containing one or more desired therapeutic agents, including an mRNA material.

[0012] In a particular aspect, we provide an mRNA LNP-incorporated microgel matrix as a programmable immunostimulating matrix that is capable of potentiating antigen expression, presentation, and / or immune cell cross-talks, and / or delivering potent antigen-specific immune responses and efficacy including in cancer therapies. We have demonstrated such systems inter alia in murine cancer models.

[0013] In one aspect, a composition or composition system or package is provided comprising: 1) one or more LNPs that are loaded or administered with one or more therapeutic agents, including a nucleic acid-based therapeutic such as an mRNA agent and 2) a polymer component suitably in a form of a fiber hydrogel composite, suitably with a porous hydrogel combined with dispersed fibers.

[0014] Preferred materials and systems and specific compositions to use as a fiber-hydrogel composite for the present composition, composition packages, kits and therapeutic methods are disclosed in U.S. Patent Publication 2021-0402061; U.S. Patent 10,471,181; and U.S. Patent 11,707,553, all incorporated herein by reference. Preferred fiber-hydrogel composite are also described in the examples which follow, including Examples 1, 2 and 3 below.

[0015] In an aspect, the fiber-hydrogel composite suitably includes fibers (e.g., nanofibers or microfibers) comprising one or more extracellular matrix proteins (ECM); a hydrogel material such as hyaluronic acid (HA); and a crosslinking agent. The hydrogel material (e.g. HA) is suitably covalently bonded to the fibers by the crosslinking agent to form a composite network.

[0016] In a particular aspect, the fiber-hydrogel composite includes fibers (e.g., nanofibers or microfibers) or microfibers comprising one or more extracellular matrix proteins (ECM); a hydrogel material such as hyaluronic acid (HA); and preferably a crosslinking agent. The hydrogel material (e.g. HA) is preferably bonded to the fibers by e.g. the crosslinking agent to form a composite network. In certain aspects, the fiber component and hydrogel material may be covalently bonded with use of a separate crosslinking agent.

[0017] Other suitable materials that can be utilized as a hydrogel material in the present composite and compositions include materials that can be formed into a gel, with one or preferably multiple functional groups (e.g. hydroxyl groups, amine groups, and thiol groups) that can react with a crosslinker. Hydrogel materials that have hydroxyl groups available for reaction with a crosslinker may be preferred in various aspects.

[0018] In aspects, the hydrogel component and the fiber component are distinct materials, i.e. the hydrogel component and the fiber component will differ in composition, molecular weight of other difference. In certain aspect, the hydrogel material will not include a collagen material.

[0019] In a particular aspect, the composition or composite comprise one or more collagen or polycaprolactone materials. In an aspect, a fiber-hydrogel composite is provided that includes fibers (e.g., nanofibers or microfibers) or microfibers comprising 1) one or more collagen or polycaprolactone materials, 2) one or more hydrogel materials such as hyaluronic acid (HA) or other material; optionally and 3) a crosslinking agent. In preferred aspects, the hydrogel material (e.g. HA) is bonded to the fibers (e.g. by the crosslinking agent if present) to form a composite network.

[0020] In an aspect, a kit is provided that suitably comprises an LNP fiber-hydrogel composite as described herein.

[0021] In an aspect, the disclosure provides a method of delivering a pharmaceutical agent in a subject including combining a pharmaceutical agent and the fiber-hydrogel composite as described herein to form a mixture; and applying the mixture to an intended site of delivery.

[0022] In an aspect, the disclosure provides an implant including an LNP fiber-hydrogel composite as described herein.

[0023] In an aspect, the disclosure provides a kit including an LNP fiber-hydrogel composite as described herein; and an applicator. In such kits, the LNP component and fiber-hydrogel component may be formulated as a single formulation, or as multiple distinct formulations and administered substantially simultaneously or spaced in time but in coordination (combined administration or therapy).

[0024] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references, the entire disclosures of which are incorporated herein by reference, provide one of skill with a general definition of many of the terms (unless defined otherwise herein) used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, the Harper Collins Dictionary of Biology (1991). Generally, the procedures of molecular biology methods described or inherent herein and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al., (2000, Molecular Cloning— A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratories); and Ausubel et al., (1994, Current Protocols in Molecular Biology, John Wiley & Sons, New-York).

[0025] The following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the 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.

[0026] As understood, the terms fiber hydrogel composite, composition or present composition and composite are used interchangeably.

[0027] Definitions

[0028] The term “a” and “an” refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0029] As used herein, “about” can mean plus or minus less than 1 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or greater than 30 percent, depending upon the situation and known or knowable by one skilled in the art.

[0030] As used herein the specification, “subject” or “subjects” or “individuals” may include, but are not limited to, mammals such as humans or non-human mammals, e.g., domesticated, agricultural or wild, animals, as well as birds, and aquatic animals. In certain embodiments, the subject is a human patient or an animal subjected to medical treatment.

[0031] As used herein, the term “hydrogel” is a type of “gel,” and refers to a water-swellable polymeric matrix, consisting of a three-dimensional network of macromolecules (e.g., hydrophilic polymers, hydrophobic polymers, blends thereof) held together by covalent or non-covalent crosslinks that can absorb a substantial amount of water (e.g., 50%, 60% 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% per unit of non-water molecule) to form an elastic gel. The hydrogel may contain “water-swellable” polymer is one that absorbs an amount of water greater than at least 50% of its own weight, upon immersion in an aqueous medium. The polymeric matrix may be formed of any suitable synthetic or naturally occurring polymer material. As used herein, the term “gel” refers to a solid three-dimensional network that spans the volume of a liquid medium and ensnares it through surface tension effects. This internal network structure may result from physical bonds (physical gels) or chemical bonds (chemical gels), as well as crystallites or other junctions that remain intact within the extending fluid. Virtually any fluid can be used as an extender including water (hydrogels), oil, and air (aerogel). Both by weight and volume, gels are mostly fluid in composition and thus exhibit densities similar to those of their constituent liquids. A hydrogel is a type of gel that uses water as a liquid medium.

[0032] In certain embodiments, the hydrogel is a composite or composite material. The term “composite” as used herein includes any association, bonding or attachments of two or more components. In some embodiments, the “hydrogel composite” as used herein include at least a polymeric fiber and a hydrogel material. The hydrogel composite contains the polymeric fiber (e.g., collagen, gelatin, etc) and hydrogel material (e.g., hyaluronic acid (HA)).

[0033] A term “functional network” as used herein means that the interactions between components results in a chemical, biochemical, biophysical, physical, or physiological benefit. In addition, a functional network may include additional components, including cells, biologicalmaterials (e.g., polypeptides, nucleic acids, lipids, carbohydrates), therapeutic compounds, synthetic molecules, and the like. In certain embodiments, the scaffold complex promotes tissue growth and cell infiltration when implanted into a target tissue present in a human subject.

[0034] The term “nanofiber” or “microfiber” can be used interchangeably to refer to fibers that are thousands of nanometers in diameter, such as 1 micron to 10 microns.

[0035] The term “nanofiber” as used herein refers to a fibrous material having at least one dimension (e.g., length, or width) less than about 999 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, or less than about 10 nm. In some embodiments, the nanofibers may have a length less than about 999 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, or less than about 10 nm. In some embodiments, the nanofibers may have a width less than about 999 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 100 nm.

[0036] The term “microfiber” as used herein refers to a fibrous material having at least one dimension (e.g., length, or width) less than about 100 pm, less than about 90 pm, less than about 80 pm, less than about 70 pm, less than about 60 pm, less than about 50 pm, less than about 40 pm, less than about 30 pm, less than about 20 pm, or less than about 10 pm. In some embodiments, the microfibers may have a length than about 10 pm, less than about 8 pm, less than about 7 pm, less than about 6 pm, less than about 5 pm, less than about 4 pm, less than about 3 pm, less than about 2 pm, or less than about 1 pm. In certain embodiments, the microfibers may have a length between about 1 pm and 10 pm, between about 5 pm and 10 pm, or between about 8 pm and 10 pm. In certain embodiments, the microfibers may have a length about 8 pm or 10 pm.

[0037] The term “fiber-hydrogel composite” as used herein refers to a composite including at least fibers such as nanofibers, microfibers, or combination thereof (e.g., polymeric fibers, or nanofibers made of extracellular matrix proteins) and hydrogel component (e.g., HA), which form functional networks. In addition, the “nanofiber-hydrogel composite,” “fiber-hydrogel composite,”“hydrogel composite,” “composite” or “complex” as used herein are interchangeably used referring to such composite including at least fibers (e.g., poly(caprolactone), collagen or gelatin nanofibers) and hydrogel component (e.g., HA).

[0038] The term “crosslinked” herein refers to a composition containing intramolecular and / or intermolecular crosslinks, whether arising through covalent or noncovalent bonding, and may be direct or include a cross-linker. “Noncovalent” bonding includes both hydrogen bonding and electrostatic (ionic) bonding.

[0039] The term “polymer” includes linear and branched polymer structures, and also encompasses crosslinked polymers as well as copolymers (which may or may not be crosslinked), thus including block copolymers, alternating copolymers, random copolymers, and the like. Those compounds referred to herein as “oligomers” are polymers having a molecular weight below about 1000 Da, preferably below about 800 Da. Polymers and oligomers may be naturally occurring or obtained from synthetic sources.

[0040] The term “extracellular matrix protein” (“ECM”) as used herein refers to a protein or its macromolecular matrix including a protein, which mimics a three-dimensional network protein existing outside of a cell (e.g., animal or mammalian cell or plant cell) and providing structural and biochemical support or adhesion to surrounding the cell. Exemplary ECM may include collagen, gelatin, elastin, decellularized matrix, or derivatives thereof.

[0041] The term “encapsulation efficiency” as used herein refers to the percentage of nucleic acid in the lipid nanoparticles that is not degraded after exposure to serum or a nuclease assay that would significantly degrade free nucleic acids.

[0042] The term “fully encapsulated” as used herein indicates that the nucleic acid in the particles is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free nucleic acids. In a fully encapsulated system, preferably less than 25% of particle nucleic acid is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10% and most preferably less than 5% of the particle nucleic acid is degraded. Fully encapsulated also indicates that the particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.

[0043] The term “lipid nanoparticle” or LNP refers to a nanoparticle that includes lipids and that is stable and dispersible in aqueous media. In exemplary embodiments, lipid nanoparticles may be from 10 nm to 500 nm in diameter, e.g., from 70 nm to 120 nm.

[0044] As used herein, the term “microparticle” refers to a small particle or particulate system, generally larger than about one micrometer (1 pm) in diameter and can be used to describe both microcapsules and microspheres.

[0045] As used herein, the term “nanoparticle” refers to a particle having one or a plurality of components, the particle having any one structural feature on a scale of less than about 1000 nm that exhibits novel properties as compared to a bulk sample of the same material or component materials. Routinely, nanoparticles have any one structural feature on a scale of less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm or less than about 100 nm. In exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10-500 nm. In other exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10-1000 nm. A spherical nanoparticle would have a diameter, for example, of between 10-100 nm or 10- 1000 nm.

[0046] A nanoparticle most often behaves as a unit in terms of its physical or biophysical properties, e.g., transport. It is noted that novel properties that differentiate nanoparticles from the corresponding bulk material typically develop at a size scale of under 1000 nm, or at a size of under 500 nm, but nanoparticles can be of a larger size, for example, for particles that are oblong, tubular, and the like. The size at which materials display different properties as compared to the bulk material is material-dependent and can be seen for many materials much larger in size than 100 nm and even for some materials larger in size than 1000 nm. Nanoparticles can be employed in a variety of drug delivery technologies (e.g., nucleic acid drug delivery technologies) and can be employed for various purposes including, but not limited to, controlled drug delivery, protection of the drugs from degradation, and protection of the body from the toxic effects of the drugs.

[0047] The term “nucleic acid” refers to a molecule of two or more nucleotides or alternative nucleotides. The term, “nucleotide” refers to a nucleoside including a phosphate group. The term “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a “nucleobase”). Examples of nucleic acids include but are notlimited to DNA, RNA, tRNA (transfer RNA), mRNA (messenger RNA), siRNA (small interfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA), ncRNA (non-coding RNA), aptamers, ribozymes, and shorter oligonucleotide sequences of any of the foregoing. Alterations of the base, sugar, and phosphate moiety of a nucleotide are encompassed by this definition.

[0048] The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes: a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and / or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.

[0049] The nucleic acid sequences may be “chimeric,” that is, composed of different regions. In the context of this disclosure “chimeric” compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide. These sequences typically comprise at least one region wherein the sequence is modified in order to exhibit one or more desired properties.

[0050] As used herein, the term “functionalized” refers to a material that is uniformly or non- uniformly modified so as to have a functional chemical moiety associated therewith (e.g., chemically modified). In some cases, functional chemical moiety is capable of reacting to permit the formation of a covalent or non-covalent bond. In some cases, functional chemical moiety can provide the material improved properties.

[0051] The terms "administered in combination with" or "co-administration", "coadministering", "combination therapy" or "combination treatment" refer to the administration of 1) a fiber- hydrogel composite as described herein and 2) an LNP and 3) a therapeutic agent (if the therapeutic is not formulated with the LNP or fiber-hydrogel composite) as described herein e.g. as separateformulations / applications (or as one single formulation / application). The co-administration can be simultaneous or sequential in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Said active agents are co- administered either simultaneously or sequentially (e.g. intravenous (i.v.) through a continuous infusion, or by injection). When multiple components (LNP / fiber-hydrogel components) are co- administered sequentially the dose is administered either on the same day in two separate administrations, or one of the agents is administered on day 1 and the second is co-administered on day 2 to day 7, preferably on day 2 to 4. Thus in one embodiment the term "sequentially" means within 7 days after the dose of the first component, preferably within 4 days after the dose of the first component; and the term "simultaneously" means at the same time.

[0052] The term "therapeutically effective amount" (or simply "effective amount") which is the amount of the respective compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. The amount of co-administration and the timing of co- administration will depend on the type (species, gender, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated.

[0053] In addition to the 1) a fiber-hydrogel composite as described herein and 2) an LNP and 3) a therapeutic agent (if the therapeutic is not formulated with the LNP or fiber-hydrogel composite) one or more further additional, distinct chemotherapeutic agents can be administered. In one embodiment such additional chemotherapeutic agents, which may be administered include, but are not limited to, anti-neoplastic agents including alkylating agents including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); Temodal(TM) (temozolamide), ethylenimines / methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguamne, azathioprine, T- deoxy coformycin (pento statin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2- chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugssuch as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; pipodophylotoxins such as etoposide and teniposide; antibiotics such as actinomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycin C, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon- alpha, IL- 2, G-CSF and GM-CSF; miscellaneous agents including platinum coordination complexes such as oxaliplatin, cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N- methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o, p-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; Gemzar(TM) (gemcitabine), progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin- releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide. Therapies targeting epigenetic mechanism including, but not limited to, histone deacetylase inhibitors, demethylating agents (e.g., Vidaza) and release of transcriptional repression (ATRA) therapies can also be combined with the antigen binding proteins. In one embodiment the chemo therapeutic agent is selected from the group consisting of taxanes (like e.g. paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxel (e.g., Abraxane and Opaxio), doxorubicin, sunitinib (Sutent), sorafenib (Nexavar), and other multikinase inhibitors, oxaliplatin, cisplatin and carboplatin, etoposide, gemcitabine, and vinblastine. In one embodiment the chemotherapeutic agent is selected from the group consisting of taxanes (like e.g. taxol (paclitaxel), docetaxel (Taxotere), modified paclitaxel (e.g. Abraxane and Opaxio). In one embodiment, the additional chemotherapeutic agent is selected from 5-fluorouracil (5-FU), leucovorin, irinotecan, or oxaliplatin. In one embodiment the chemotherapeutic agent is 5- fluorouracil, leucovorin and irinotecan (FOLFIRI). In one embodiment the chemotherapeutic agent is 5- fluorouracil, and oxaliplatin (FOLFOX).

[0054] Specific examples of combination therapies with additional chemotherapeutic agents include, for instance, therapies taxanes (e.g., docetaxel or paclitaxel) or a modified paclitaxel (e.g., Abraxane or Opaxio), doxorubicin), capecitabine and / or bevacizumab (Avastin) for the treatmentof breast cancer; therapies with carboplatin, oxaliplatin, cisplatin, paclitaxel, doxorubicin (or modified doxorubicin (Caelyx or Doxil)), or topotecan (Hycamtin) for ovarian cancer, the therapies with a multi-kinase inhibitor, MKI, (Sutent, Nexavar, or 706) and / or doxorubicin for treatment of kidney cancer; therapies with oxaliplatin, cisplatin and / or radiation for the treatment of squamous cell carcinoma; therapies with taxol and / or carboplatin for the treatment of lung cancer.

[0055] Therefore, in one embodiment the additional chemotherapeutic agent is selected from the group of taxanes (docetaxel or paclitaxel or a modified paclitaxel (Abraxane or Opaxio), doxorubicin, capecitabine and / or bevacizumab for the treatment of breast cancer.

[0056] In further aspects, methods and uses are provided for the treatment of a patient suffering from such disease as described herein, including a cancer.

[0057] Other aspects of the invention are disclosed infra.BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0059] Fig. 1 (includes Figs, la-lh) shows a preferred preparative sequence and in vivo analysis of an LNP fiber-hydrogel composite, a, Schematic of the LiNx vaccination platform, b-c, The kinetics of host cell recruitment following injection of LiNx. d, Transfection of immune cells in the composite was analyzed using Ai9 mice, e-h, Schematic and results of therapeutic or prophylactic cancer vaccine models.

[0060] Fig. 2. In vivo host cell recruitment and transfection of LiNx. a, Schematic of the LiNx vaccination platform, providing an immunostimulatory niche by effectively recruiting host immune cells to the microgel matrix (1). LiNx-delivered LNPs can preferentially transfect non- APCs (2) and recruited macrophages, followed by antigen expression, processing, and epitope presentation on MHC-I (3a) or MHC-II (4a) in different pathways. Some cross-presentation may also occur. Activated / matured APCs (3b, 4b) can migrate to LNs (5), spleen (6), and tumor; and antigen presentation to CD8+ and CD4+ T cells (7) can occur directly in LiNx (where multiple relevant cell types are recruited), LNs, spleen, or tumor, where effector T cells, activated NK cells,macrophages, etc. act in a coordinated manner, modulating the tumor microenvironment (TME) (8). DC, dendritic cell; MHC, major histocompatibility complex; TAP, transporter associated with antigen processing; TCR, T cell receptor, b-c, The kinetics of host cell recruitment following injection of three different LNP / mRNA LiNx on C57BL / 6 mice (30 pg mOVA per mouse, s.c.). The Cellaca MX High-throughput Automated Cell Counter was used to count the number of living cells within composites on both day 3 and day 7 (b). Flow cytometry was employed to determine the count of macrophages (CDl lb+CD 11c") and dendritic cells (CDl lb+CDl lc+) on day 3 (c). d-f, Ai9 mice were administered the three LiNx loaded with mCre via s.c. injections (30 pg mCre per mouse). Transfection of immune cells in the composite was analyzed by flow cytometry. The number of cells positive for tdTomato (d), as well as DCs (CDl lb+CDl lc+) cells (e) and macrophages (CD1 lb+CD11 c") (f) positive for tdTomato on day 5 and day 10 post-injection were shown. Data represent the mean± s.e.m. (n= 8 for (b-c), n=4 for (d-f) biologically independent samples). Data were analyzed using one-way ANOVA and Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ****p < 0.0001. NS, not significant.

[0061] Fig. 3. In vivo assessments of local microenvironment generated by LiNx. a, Recruitment of host cells was assessed two weeks after the administration of three different LNP / mRNA LiNx formulations on C57BL / 6 mice (30 pg mOVA per mouse, s.c.). The Cellaca MX High-throughput Automated Cell Counter was employed to count the living cells within the composites, b-d, On day 14, flow cytometry was utilized to determine the counts of CD4+ T cells (CD3+CD4+) (b), CD8+T cells (CD3+CD8+) (c), and B cells (CDllb" CD19+) (d) within the composite, e, The composition of host immune cells recruited within the composite was evaluated two weeks after administering three different LNP / mRNA LiNx formulations to C57BL / 6 mice (30 pg mOVA per mouse, s.c.). This analysis included CD4 T cells (CD3+CD4+), CD8 T cells (CD3+CD8+), B cells (CD1 lb’ CD19+), DCs (CD1 lb+CD1 lc+), macrophages (CD1 lb+CD11 c ), neutrophils (CDl lb+Ly6G+), and NK cells (CDl lb' NKp46+). f-g, An RT-PCR array was conducted using RNA isolated from the local microenvironment generated by three distinct LNP / mRNA LiNx formulations. C57BL / 6 mice received three different LNP / mRNA LiNx formulations loaded with mOVA through subcutaneous injection (30 pg mOVA per injection). The heatmap presented a panel of genes relevant to immune responses (f). Subsequent RT-PCR analysis of selected genes from the panel confirmed that D6 mRNA LiNx promoted critical proinflammatory cytokine transcripts involved in the establishment of a local immunostimulatoryniche. These included genes related to inflammatory cytokines and chemokines (IL-6, IL- la, CSF- 3, and CXCL10), Thl immune responses (TBX21, TNF, IFN-y, GZMB, NOS-2, IL-12a, IL-15, and IL-2), and genes associated with Th2 immune responses (IL-4, CCR-4, and IL- 13). Data represent the mean± s.e.m. (n= 7 biologically independent samples for a-e and n=3 biologically independent samples for f-g). Data were analyzed using one-way ANOVA and Tukey's multiple comparisons test for a-d. *P < 0.05.

[0062] Fig. 4 | In vivo assessments of antigen-specific immune activation by three different mRNA LiNx formulations, a-b, C57BL / 6 mice were administered three different LNP / mRNA LiNx formulations loaded with mOVA via s.c. injection (30 pg per mouse). The number of OVA- specific CD8 T cells within composite (a) and dLNs (b) were analyzed through flow cytometry after two weeks following a single dosage of mRNA LiNx. c-h, C57BL / 6 mice were administered with three different LNP / mRNA LiNx formulations loaded with mOVA via s.c. injection (30 pg per mouse). Mice were sacrificed two weeks after the final injection, and their splenocytes were isolated for analysis. Splenocytes were restimulated in vitro with OVA and SIINFEKL peptide (100 pg ml’1OVA and 2 pg mf1SIINFEKL) for 12 h and assessed via flow cytometry and intracellular cytokine staining to determine the percentages of CD8+IFN-y+cells (c), CD8+TNF- a+cells (d), CD8+Granzyme B+cells (e), CD4+IFN-y cells (f), CD4+TNF-a+cells (g), and CD4+IL-4+cells (h). i, Frequency of IFN-y-producing cells among restimulated splenocytes isolated from vaccinated mice on day 30 post-vaccination, assessed via ELISPOT. j, Titer of OVA-specific IgG antibodies in blood serum on day 30, determined by ELISA, k-o, C57BL / 6 mice were administered with three different LNP / mRNA LiNx formulations loaded with mOVA via s.c. injection (30 pg per mouse). Mice were sacrificed three months after the final injection, and their splenocytes were isolated for analysis. The percentages of OVA-specific CD8 T cells (CD3+CD8+OVA+cells) (k), OVA-specific effector CD8 T cells (CD3+CD8+OVA+CD44+CD62L’ cells) (1), OVA-specific central memory CD8 T cells (CD3+CD8+OVA+CD44+CD62L+cells) (m) were determined. Splenocytes were restimulated in vitro with OVA and SIINFEKL peptide (100 pg ml’1OVA and 2 pg mf1SIINFEKL) for 12 h and assessed via flow cytometry and intracellular cytokine staining to determine the percentages of CD3+CD8+TNF-a+cells (n), CD3+CD8+IFN- y+cells (o). Data represent the mean± s.e.m. (n = 7 (a-b) biologically independent samples, n = 4 (c-j) biologically independent samples, n = 8 (k-o) biologically independent samples). Data wereanalyzed using one-way ANOVA and Tukey’s multiple comparisons test for a-o. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0063] Fig. 5 | Anti-tumor efficacy of the top mRNA LNP formulations as therapeutic and prophylactic vaccines, a-d, Schematic and results of a therapeutic vaccination model for MC38- OVA in C57BL / 6 mice. Mice were inoculated s.c. with MC38-OVA and then given three s.c. injections, one week apart, of mOVA-loaded D6 (10 pg mOVA per injection) or PBS. For LiNx treatment group, mice were administered three different LNP / mRNA LiNx formulations loaded with mOVA (30 pg per mouse) or OVA protein (10 pg protein per mouse) via s.c. injection. OVA protein mixed with Alhydrogel® (1: 1) (10 pg protein per mouse) was served as a control group. Average tumor volume (b), survival curves (c), and individual tumor volume (d) are shown, e-i, Schematic and results of a therapeutic vaccine against melanoma in C57BL / 6 mice using OVA model antigen (f-g) and melanoma-associated antigens (h-i). Mice were inoculated s.c. with B16F10-OVA (f-g) or B16F10 cells (h-i) and then administered D6 LNP / mRNA LiNx formulations loaded with mOVA or Trp2 (mTrp) or GplOO (mGplOO) (30 pg per mouse) s.c. injections. Three groups received the anti-CTLA-4 mAh (100 pg per i.p. injection) treatment in combination with LiNx treatment. Average tumor volume (f, h), and survival curves (g, i) are shown. Data represent mean± s.e.m. with n = 8 (c-d), n = 6 (f-i, k— 1) biologically independent samples. Survival curves were compared using log-rank Mantel-Cox test, and the stack of P values were corrected by Holm-Sidak method for multiple comparisons with a set to 0.05. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant; i.p. intraperitoneal; α CTLA-4, anti-CTLA-4 mAb.

[0064] Fig. 6 Characterization of immune activation profile generated by D6 mRNA LiNx. C57BL / 6 mice were given three s.c. injections, one week apart, of mOVA-loaded D6 (10 pg mOVA per injection) or PBS. For LiNx treatment group, mice were administered D6 LNP / mRNA LiNx formulations loaded with mOVA (30 pg per mouse) on day 0. On day 21 post- vaccination, the mice were sacrificed, and their splenocytes were isolated and restimulated in vitro with OVA and SIINFEKL peptide (100 pg ml’1OVA and 2 pg ml-1SIINFEKL) for 12 h and the RNA was extracted and assessed using nCounter Analysis System (NanoString Technology), a- b, Volcano plot illustrates the differentially expressed genes between the D6 mRNA LiNx and PBS groups (a), as well as between the D6 mRNA LiNx and D6 mRNA LNP groups (b). P- value < 0.05; two-sided unpaired limma- moderated t test; abs fold change > 1.5. c, The heatmappresented a panel of genes relevant to immune responses for D6 mRNA LNP group and D6 mRNA LiNx group, d-e, Selected genes related to B cell signaling, Thl, Th2, Thl7, and T reg differentiation were shown, f, Bar plots showing differential pathways enriched after vaccination of D6 mRNA LiNx compared with PBS or three dosage D6 mRNA LNP group using Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Data represent mean± s.e.m. (d-e). Experiments were done with n = 6 biologically independent samples for D6 mRNA LiNx group, n = 3 biologically independent samples for PBS and D6 mRNA LNP group.

[0065] FIG. 7: In vivo host cell recruitment and transfection efficiency of LiNx. a, Schematic of the LiNx vaccination platform, providing an immunostimulatory niche by effectively recruiting host immune cells to the microgel matrix (1). LiNx-delivered LNPs can preferentially transfect non-APCs (2) and recruited macrophage-like cells, followed by antigen expression, processing, and epitope presentation on MHC-I (3) or MHC-II (4) in different pathways, b-c, Kinetics of host cell recruitment following injection of three different LNP / mRNA LiNx in C57BL / 6 mice (30 pg mOVA per mouse, s.c. ). The Cellaca MX High-throughput Automated Cell Counter was used to count the number of living cells within composites on both day 3 and day 7 post- injection (b). Flow cytometry was employed to determine the count of macrophage-like cells (CD3-CD1 Ib+Ly6g-CD11c-) and DC-like cells (CD3-CD1 lb+ Ly6g-CD1 lc+) on day 3 (c). d- f, Ai9 mice were administered the three 198 LiNx loaded with mCrel99 via s.c. injections (30 pg mCre per mouse). Transfection of cells in the composite was analysed by 200 flow cytometry. The number of cells positive for tdTomato (d), as well as DC-like cells (CD1 Ib+CDl lc+) 201 cells (e) and macrophage-like cells (CD1 Ib+CDl 1 c— ) (f) positive for tdTomato on day 5 and day 10 post- injection were shown, g-i, C57BL / 6 mice received s.c. injections of D6 LiNx loaded with either mLuc or Cy5-labeled mRNA (30 pg per mouse), (g) The transfection efficiency of D6 LiNx at the injection site and in major organs was assessed using IVIS imaging. The relative percentage of transfection and biodistribution for D6 LiNx is shown in (h). IVIS images of luciferase transfection for D6 LiNx are presented in (i). Data represent the mean s.e.m. (n = 8 for (b-c), n = 4 for (d-f) and n = 5 for (g-i) biologically independent samples). Data were analysed using one-way ANOVA and Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ****p < 0.0001. NS, not significant.

[0066] FIG. 8 In vivo assessments of local microenvironment generated by LiNx. a, Recruitment of host cells was assessed two weeks after s.c. administration of three differentLNP / mRNA LiNx formulations in C57BL / 6 mice (30 pg mOVA per mouse). The Cellaca MX High-throughput Automated Cell Counter was employed to count the living cells within the composites, b-d, On day 14, flow cytometry was utilized to determine the counts of CD3+CD4+ T cells (b), CD3+CD8+ T cells (c), and CDl lb-CD19+ B cells (d) within the composite, e, Composition of host immune cells recruited within the composite was evaluated at two weeks after s.c. administering three different LiNx formulations in C57BL / 6 mice (30 pg mOVA per mouse). This analysis included CD4 T cells, CD8 T cells, B cells, DC-like cells (CDl lb+ CDl lc+), macrophage-like cells (CDl lb+CDl lc-), neutrophils (CDl lb+Ly6G+), and NK cells (CDl lb- NKp46+). f-g, RT-PCR array analysis was conducted using RNA isolated from the local microenvironment generated by CIO, D6, and F5 LiNx formulations. C57BL / 6 mice received the three different LiNx formulations loaded with mOVA through s.c. injection (30 pg mOVA per injection). The presented heatmap shows a panel of genes relevant to immune responses (f). Subsequent RT-PCR analysis of selected genes from the panel confirmed that D6 LiNx 261 promoted critical proinflammatory cytokine transcripts involved in the establishment of a local immunostimulatory niche. These included genes related to inflammatory cytokines and chemokines (IL-6, IL-la, CSF-3, and CXCL10), Thl response (TBX21, TNF, IFN-y, GZMB, NOS-2, IL-12a, IL-15, and IL-2), and genes associated with Th2 response (IL-4, CCR-4, and IL- 13). Data represent mean s.e.m. (n = 7 biologically independent samples for a-e and n = 3 biologically independent samples for f-g). Data were analysed using one way ANOVA and Tukey's multiple comparisons test for a-d. *P < 0.05.

[0067] FIG. 9: In vivo assessments of antigen-specific immune activation 327 by three different mRNA LiNx formulations, a-b, C57BL / 6 mice were administered three different LNP / mRNA LiNx formulations loaded with mOVA via s.c. injection (30 pg per mouse). The number of OVA-specific CD8 T cells within the LiNx (a) and dLNs (b) was analysed by flow cytometry at two weeks following a single dosage of LiNx immunization, c-h, C57BL / 6 mice were administered three different LNP / mRNA LiNx formulations loaded with mOVA via s.c. injection (30 pg per mouse). Mice were sacrificed two weeks after the injection, and their splenocytes were isolated and restimulated in vitro with OVA and SIINFEKL peptides (100 pg mL-1 OVA and 2 pg mL-1 SIINFEKL) for 12 h and assessed via flow cytometry and intracellular cytokine staining to determine the percentages of CD8+IFN-y+ cells (c), CD8+TNFa+ cells (d), CD8+ Granzyme B+ cells (e), CD4+IFN-y+ cells (f), CD4+TNFa+ cells (g), and CD4+IL-4+ cells(h). i, Frequency of IFN-y-producing cells among restimulated splenocytes isolated from vaccinated mice on day 30 post-vaccination, assessed via ELISPOT. j, Titer of OVA-specific IgG antibodies in blood serum on day 30, determined by ELISA, k-o, C57BL / 6 mice were administered with three different LNP / mRNA LiNx formulations loaded with mOVA via s.c. injection (30 pg per mouse). Mice were sacrificed three months after the injection, and their splenocytes were isolated for analysis. The percentages of OVA-specific CD8 T cells (CD3+ CD8+ OVA+ cells) (k), OVA-specific effector CD8 T cells (CD3+CD8+OVA+CD44+CD62L- cells) (1), and OVA- specific central memory CD8 T cells (CD3+CD8+OVA+CD44+CD62L+ cells) (m) were determined. Splenocytes were restimulated in vitro with OVA and SIINFEKL peptide (100 pg mL-1 OVA and 2 pg mL-1 SIINFEKL) for 12 h and assessed via flow cytometry and intracellular cytokine staining to determine the percentages of CD3+CD8+TNFa+ cells (n) and CD3+CD8+IFN-y+ cells (o). (p) Titers of OVA-specific IgG antibodies in blood serum on day 90, determined by ELISA. Data represent the mean } s.e.m. (n = 7 (a-b) biologically independent samples, n = 4 (c-j) biologically independent samples, n = 8 (k-p) biologically independent samples). Data were analysed using one-way ANOVA and Tukey’s multiple comparisons test for a-o. *p < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0068] FIG. 10: Anti-tumour efficacy of the top LiNx formulations as therapeutic 412 and prophylactic vaccines, a-d, Schematic and results of a syngeneic therapeutic vaccination model for MC38-OVA in C57BL / 6 mice. Mice were inoculated s.c. with MC38-OVA and then given three s.c. injections, one week apart, of mOVA-loaded D6 LNPs (10 pg mOVA per injection) or PBS. For LiNx treatment group, mice were administered three different LiNx formulations loaded with mOVA LNPs (30 pg per mouse) or OVA protein (10 pg protein per mouse) via a single s.c. injection. OVA protein mixed with AlhydrogelR (1 : 1) (10 pg protein per mouse) served as a control group. Average tumour volume (b), survival curves (c), and individual tumour volume (d) are shown, e-i, Schematic and results of a therapeutic vaccine against another syngeneic model, B16F10 melanoma in C57BL / 6 mice, using OVA model antigen (f— g) and melanoma-associated antigens (h-i). Mice were inoculated s.c. with B16-OVA (f— g) or Bl 6F 10 cells (h-i) and then s.c. administered with D6 LiNx loaded with mOVA, mTrp2 or mGplOO (30 pg per mouse). Three groups received the anti-CTLA-4 mAb (100 pg per i.p. injection) treatment in combination with LiNx treatment. Average tumour volume (f, h), and survival curves (g, i) are shown, j-1, Schematic and results of a prophylactic vaccine against melanoma in C57BL / 6 mice using OVA modelantigen. Mice were s.c. administered with a single dose of D6 LiNx loaded with mOVA (30 pg per mouse) and then inoculated s.c. with B16-OVA cells on day 21. Average 428 tumour volume (k), and survival curves (1) are shown, (c-d), n = 6 (f— i, k-1) biologically independent samples. Survival curves were compared using log-rank Mantel -Cox test, and the stack of P values were corrected by Holm-Sidak method for multiple comparisons with a set to 0.05. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant; i.p. intraperitoneal; crCTLA-4, anti-CTLA-4 mAb.

[0069] FIG. 11 Modulation of tumour microenvironment of the D6 LiNx formulation, a-g, Schematic and analysis of the tumour microenvironment in a therapeutic vaccination model for B16-OVA in C57BL / 6 mice. Mice were inoculated subcutaneously (s.c.) with B16-OVA and subsequently received three weekly s.c. injections of mOVA-loaded D6 LNPs (10 pg mOVA per injection) or PBS. For the LiNx treatment group, mice were administered with the D6 LiNx loaded with mOVA (30 pg per mouse) via a single s.c. injection on day 4. On day 21, tumours were collected and analysed by flow cytometry to determine the percentages of (b) CD3+CD8+B220- T cells, (c) OVA-specific 480 CD3+CD8+B220- T cells, (d) CD45+CD3+CD4+ T cells, (e) NKp46+CDl lb-CD3- NK cells, (f) F4 / 80+CDl lb+CD3- macrophages, and (g) MHCII+CD1 Ic+CDl lb-Ly6G-CD3- DCs. h-k, CODEX multiplex imaging analysis of tumour tissue collected on day 14 post B16-OVA inoculation. For the LiNx treatment group, mice were administered with the D6 LiNx formulation (30 pg mOVA per mouse) via a single s.c. injection on day 4. (h) Dotplot of cell types by marker expression derived from CODEX multiplexed imaging for the pooled D6 LiNx (n = 2) and PBS-treated tumours (n = 3) using unsupervised clustering with the percentage of cells with a marker Z-score above 0.7. (i) Percentage distribution of immune and tumour cell types within the D6 LiNx (n = 2) and PBS-treated tumours (n = 3) as quantified by CODEX multiplexed imaging, (j) Comparison of the normalized percentage of immune cell subsets within the D6 LiNx-treated tumours (n = 2) and PBS-treated tumours (n = 3). (k) Cell type map of the representative D6 LiNx tumour derived from CODEX multiplexed imaging with colored data points representing selected immune and tumour cell types with other cell types lumped together in the same gray color (scale bar = 500 pm).

[0070] FIG. 12: Characterization of immune activation profile generated by the D6 LiNx. C57BL / 6 mice were given three s.c. injections, one week apart, of mOVA-loaded D6 (10pg mOVA per injection) or PBS. For LiNx treatment group, mice were administered D6 LiNx loaded with mOVA (30 pg per mouse) on day 0. On day 21 post-vaccination, the splenocytes were restimulated in vitro with OVA and SIINFEKL peptide (100 pg mL-1 OVA and 2 pg mL-1 SIINFEKL) for 12 h and the RNA was extracted and assessed using a NanoString nCounter Analysis System, a-b, Volcano plot illustrates the differentially expressed genes between the D6 LiNx and PBS groups (a), as well as between the D6 LiNx and D6 LNP groups (b). P < 0.05; two- sided unpaired limma-moderated 543 t-test; absolute fold change > 1.5. c-d, Selected genes related to B cell signaling, Thl, Th2, Thl7, and Treg differentiation are shown, e, Heatmap presenting a panel of genes relevant to immune responses for D6 LNP group and D6 LiNx group, f, Bar plots showing differential pathways enriched after vaccination of a single dose D6 LiNx compared with PBS or three doses of D6 LNPs using Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotations for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, (d-e). Experiments were conducted with n = 6 biologically independent samples for the D6 LiNx group and n = 3 biologically independent samples for PBS LiNx and D6 LNP groups, g, Schematic of the immunostimulatory niche generated by LiNx through effective recruitment of host immune cells to the microgel matrix, leading to antigen presentation to CD8+ and CD4+ T cells (including Thl, Th2, and Thl 7 cells). Thl 7 cells facilitate the migration and persistence of effector T cells and NK cells within the tumour microenvironment by promoting the secretion of chemokines CXCL9 and CXCL10 by primary tumour cells. Moreover, Thl 7 cells stimulate the release of CCL20 from tumour cells, thereby attracting CCR6+ DCs. h, Schematic and results of IL- 17 depletion experiments in the prophylactic vaccination model for OVA- expressing melanoma in C57BL / 6 mice. Mice were given one s.c. injection of D6 LiNx (30 pg mOVA per injection) or three s.c. injections, 1 week apart, of mOVA-loaded D6 LNPs (10 pg mOVA per injection) before s.c. inoculation of B16-OVA cells (3*105 cells), and antibody for IL- 17 depletion were injected every 3 d (i.p., 200 pg per mouse). Survival curves over time are shown (n = 6 biologically independent mice for the PBS group and 8 biologically independent mice for other groups).

[0071] FIG. 13: Rheological assessment and injectability of the LiNx formulation (D6 LiNx). (a) Rheological profile of D6 LiNx, showing storage modulus (G’) and loss modulus (G") across an oscillatory strain (8) range of 0-1000% in a strain sweep test, (b) Image showing the injectability of the composite through a 30-gauge needle.

[0072] FIG. 14: Scanning electron microscopy (SEM) images of D6 LiNx. The SEM imaging was taken following D6 mRNA LNPs incorporation into the NHC composite. Scanningelectron microscopy images showed that, after encapsulating LNPs within the NHC, the fibrillar microarchitecture of the NHC is preserved, with nanofibers connected to the HA hydrogel network. Scale bars: 100 pm (left) and 25 pm (right).

[0073] FIG. 15: Accelerated degradation profile at 50 °C for the NHC composite used in this study. Rheological analysis of NHC degradation was conducted at 50 °C. Samples were incubated at 50 °C in syringes and periodically tested using a rheometer to measure the storage modulus (G1). A decrease in G' over time indicates reduced stiffness, reflecting the degradation profile of the NHC scaffold. The equivalent degradation time at 37 °C was estimated based on a scaling factor. Data represent the mean ± s.d. (n = 3).

[0074] FIG. 16: Gating strategy for flow cytometry assessment of locally recruitedcells by the top LiNx formulations on days 3 and 7. Initially, viable cells were identified and gated out based on the L / D aqua-A-SSC-A plot. Lymphocytes were gated out using SSC-A and FSC-A parameters. Subsequently, singlet cells were gated out using the FSC-H-FSC- A plot. Next, CD3- CDl lb+ cells were gated, and Ly6g+ cells were further characterized as neutrophils. Additionally, Ly6g-CDl lc+ cells were identified as dendritic cells (DCs), while Ly6g-CDl lc- cells were designated as macrophages.

[0075] FIG. 17: Gating strategy for flow cytometry assessment of local cell transfection by the top LiNx formulations. Initially, lymphocytes were gated out using SSC-A and FSC-A parameters. Subsequently, singlet cells were gated out using the FSC-H-FSC-A plot and SSC-A-SSC-H plot. Viable cells were identified and gated out based on the L / D aqua-A- SSC-A plot. The analysis focused on tdTomato+ CDl lb+CDl lc- macrophages and CDl lb+CDl lc+ DCs. The fluorescence minus one (FMO) control for tdTomato+ cells was provided.

[0076] FIG. 18: Representative flow cytometry plots for tdTomato+ cells in LiNx on days 5 and 10 post-administration. Ai9 mice were administered the top three LiNx formulations loaded with mCre via s.c. injections (n = 4, 30 pg mCre per mouse). Transfection of cells in LiNx was analysed by flow cytometry. Percentages of cells positive for tdTomato are shown.

[0077] FIG. 19: In vivo transfection of CDllc-CDllb- cells by CIO, D6, and F5 mCre LiNx formulations. Ai9 mice were administered with LiNx loaded with CIO, D6, or F5 mCre LNPs via s.c. injections (30 pg mCre per mouse). Transfection of CD11 c-CDl lb- cells in the LiNx was analysed by flow cytometry. The number of CDl lc-CDl lb- cells positive for tdTomato on days 5 and 10 post-injection were shown. Data represent mean ± s.e.m. (n = 4 biologically independent samples). Data were analysed using one-way ANOVA and Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ****p < 0.0001. NS, not significant.

[0078] FIG. 20: Local retention and biodistribution profile of the labelled mRNA LNPs formulated in the D6 LiNx post-vaccination. C57BL / 6 mice received s.c. injections of D6 LiNx loaded with Cy5-labeled mRNA (30 pg per mouse). The Cy5 radiant efficiency from the D6 LiNx injection (a) was monitored over 2 weeks using IVIS imaging, (b) Cy5 radiant efficiency in major organs and at the injection site was assessed at 24 hours post- injection. IVIS biodistribution images of D6 LiNx at 24 hours post- injection were shown in (c). Data represent the mean ± s.e.m. (n = 5 biologically independent samples).

[0079] FIG. 21: Gating strategy for flow cytometry assessment of locally recruited CD4+ T cells by the LiNx formulations on day 14. Initially, viable cells were identified and gated out based on the L / D aqua-A-SSC-A plot. Lymphocytes were gated out using SSC-A and FSC-A parameters. Subsequently, singlet cells were gated out using the SSC-H-SSC-A plot. TheCD3+CD4+ cells were characterized as CD4+ T cells.

[0080] FIG. 22: Gating strategy for flow cytometry assessment of locally recruited CD8+ T cells by the top LiNx formulations on day 14. Initially, viable cells were identified and gated out based on the L / D aqua-A-SSC-A plot. Lymphocytes were gated out using SSC-A and FSC-A parameters. Subsequently, singlet cells were gated out using the FSC-H-FSC-A plot. Next, CD3+ cells were gated, and CD3+ CD8+ cells were characterized as CD8+ T cells.

[0081] FIG. 23: Gating strategy for flow cytometry assessment of locally recruited B cells by the LiNx formulations on day 14. Initially, viable cells were identified and gated out based on the L / D aqua-A-SSC-A plot. Lymphocytes were gated out using SSC-A and FSC-A parameters. Subsequently, singlet cells were gated out using the FSC-H-FSC-A plot. Next, CD3- CD1 lb- cells were gated, and Npk46- CD 19+ cells were characterized as B cells.

[0082] FIG. 24: Gating strategy for flow cytometry assessment of locally recruited NK cells, macrophages, DC cells, and neutrophils by the LiNx formulations on day 14. Initially, viable cells were identified and gated out based on the L / D aqua-A-SSC-A plot. Lymphocytes were gated out using SSC-A and FSC-A parameters. Subsequently, singlet cells were gated out using the FSC-H-FSC-A plot. First, CD3-CDl lb+ cells were gated, and within this population, Ly6g+ cells were identified and characterized as neutrophils. Subsequently, Ly6g- cells were gated. Among these, CDl lc+ cells were identified as dendritic cells (DCs), while CDl lc- cells were identified as macrophages. Finally, within the CD3-CDl lb- cell subset, Npk46+ cells were identified and characterized as NK cells.

[0083] FIG. 25: RT-PCR analysis of selected genes related to inflammatory cytokines and chemokines expression in the local microenvironment generated within the LiNx. An RTPCR array was conducted using RNA isolated from the local microenvironment generated by three distinct LiNx formulations. C57BL / 6 mice received the three different LiNx formulations loaded with mOVA through s.c. injection (30 pg mOVA per injection). The selected genes related to inflammatory cytokines and chemokines expression in the local microenvironment generated by the LiNx, including IL-6 (a), IL-la (b), CSF-3 (c), and CXCL10 (d), are shown. Data represent the mean ± s.e.m. (n = 3 biologically independent samples). Data were analysed using one-way ANOVA and Tukey's multiple comparisons. *P < 0.05, **P < 0.01. NS, not significant.

[0084] FIG. 26: RT-PCR analysis of selected genes related to Thl immune responses in local microenvironment generated by the LiNx. An RT-PCR array was conducted using RNA isolated from the local microenvironment generated by three distinct LiNx formulations. C57BL / 6 mice received three different LiNx formulations loaded with mOVA through s.c. injection (30 pg mOVA per injection). The selected genes related to Thl immune responses in local microenvironment generated by the LiNx, including TBX21 (a), TNF (b), IFN-y (c), GZMB (d), NOS-2 (e), IL-12a (f), IL-15 (g), and IL-2 (h), are shown. Data represent the mean ± s.e.m. (n = 3 biologically independent samples). Data were analysed using one-way ANOVA and Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.

[0085] FIG. 27: RT-PCR analysis of selected genes related to Th2 immune responses in the local microenvironment generated by the LiNx. An RT-PCR array was conducted usingRNA isolated from the local microenvironment generated by three distinct LNP / mRNA LiNx formulations. C57BL / 6 mice received three different LiNx formulations loaded with CIO, D6, and F5 mOVA LNPs through s.c. injection (30 pg mOVA per injection). The selected genes related to Th2 immune responses in the local microenvironment generated by LiNx, including IL-4 (a), CCR-4 (b), and IL- 13 (c), are shown. Data represent the mean ± s.e.m. (n = 3 biologically independent samples). Data were analysed using one-way ANOVA and Tukey's multiple comparisons test. *P < 0.05, **P < 0.01. NS, not significant.

[0086] FIG. 28: Gating strategy for flow cytometry assessment of OVA-specific T cells in the LiNx or draining lymph nodes on day 14 post-administration. Initially, viable cells were identified and gated out based on the L / D aqua-A-SSC-A plot. Lymphocytes were gated out using SSC-A and FSC-A parameters. Subsequently, singlet cells were gated out using the FSC-H-FSC- A plot. Next, CD3+ cells CD8+ cells were gated; and CD3+ CD8+ OVA+ cells were characterized as OVA specific T cells.

[0087] FIG. 29: Representative flow cytometry plots for OVA-specific T cells in the LiNx or draining lymph nodes on day 14 post-administration. C57BL / 6 mice were administered with the three LiNx formulations loaded with CIO, D6, and F5 mOVA LNPs via s.c. injections (n = 7, 30 pg mOVA per mouse). OVA-specific T cells in the LiNx and dLNs were analysed by flow cytometry. Percentages of cells positive for OVA tetramer are shown.

[0088] FIG. 30: In vivo assessments of antigen-specific immune activation by the LiNx loaded with mOVA D6 LNPs or empty D6 LNPs. a-c, C57BL / 6 mice were administered with the D6 LiNx formulation loaded with D6 mOVA LNPs (30 pg mOVA per mouse) or empty D6 LNPs via s.c. injection (equivalent dose of LNPs). Mice were sacrificed two weeks after the injection, and their splenocytes were isolated and restimulated in vitro with OVA and SIINFEKL peptides (100 pg mL-1 OVA and 2 pg mL-1 SIINFEKL) for 12 hours and assessed via flow cytometry and intracellular cytokine staining to determine the percentages of OVA-specific CD8 T cells (a), CD8+TNFa+ cells (b). The number of OVA-specific CD8 T cells within the LiNx (c) was analysed through flow cytometry at two weeks following a single dosage of D6 LiNx. Data represent the mean ± s.e.m. (n = 5 biologically independent samples). Data were analysed using one-way ANOVA and Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

[0089] FIG. 31: Gating strategy for flow cytometry assessments of antigen-specific immune responses generated by the LiNx formulations. Initially, lymphocytes were gated out using SSC-A and FSC-A parameters. After gating out singlet cells using the FSC-H-FSC-A and SSCA-SSC-H plots, viable cells were identified and gated out based on the L / D aqua-A-SSC-A plot. CD8+ cells or CD4+ cells were subsequently gated. Within the CD8+ or CD4+ T cell populations, those positive for IFN-y, TNFa, Granzyme-B and IL-4 were identified.

[0090] FIG. 32: Representative images of IFN- y secreting cells from the enzyme linked immunospot assay. Frequency of IFN-y-producing cells among restimulated splenocytes, assessed via ELISPOT. Splenocytes were restimulated in vitro with SIINFEKL peptide (2 pg mL-1 SIINFEKL) for 24 h.

[0091] FIG. 33: Titers of OVA-specific IgG subclass antibodies in serum samples collected on day 30 following immunization with the LiNx formulations. IgGl (a) and IgG2c (b) antibodies in serum on day 30 were determined by ELISA. Data represent the mean ± s.e.m. from a representative experiment (n = 4 biologically independent samples) of two independent experiments. Data were analysed using one-way ANOVA and Dunnett’s multiple comparisons test. *P < 0.05; ELISA, enzyme-linked immunoassay.

[0092] FIG. 34: Gating strategy for flow cytometry analysis for in vivo assessment of OVA-specific CD8+ T cells by the LiNx formulations in the spleen on day 90. Initially, lymphocytes were gated out using SSC-A and FSC-A parameters. After gating out singlet cells using the SSC-H-SSC-A plot, viable cells were further identified and gated out based on the L / D aqua-A-SSC-A plot. CD3+ cells were subsequently gated, and CD3+CD8+ cells were identified as CD8+ T cells. Within this CD8+ T cell population, cells positive for tetramer-OVA were defined as OVA-specific CD8+ T cells. Central memory T cells were defined as cells positive for both CD44 and CD62L, while effector T cells were defined as cells positive for CD44 and negative for CD62L.

[0093] FIG. 35: Representative flow cytometry plots for in vivo assessment of OVAspecific T cells in the spleen on day 90 post-administration. C57BL / 6 mice were administered with the three LiNx formulations loaded with CIO, D6, and F5 mOVA LNPs via s.c. injections (n = 8, 30 pg mOVA per mouse). OVA-specific T cells in the spleen were analysed by flow cytometry. Percentages of cells positive for OVA tetramer are shown.

[0094] FIG. 36: Gating strategy for flow cytometry assessment of cytotoxic T cell response on day 90 post-vaccination. Initially, lymphocytes from the spleen were selected using SSC-A and FSC-A parameters and then singlet cells by the SSC-H-SSC-A plot. Viable cells were identified and selected based on the live / dead Fixable Aqua-A-SSC-A plot. Next, the CD3+ and CD8+ cell populations were selected with downstream analysis focused on IFN-y and TNFa as illustrated in the representative figures.

[0095] FIG. 37: Representative flow cytometry plots for assessment of CD3+CD8+IFN-y+ cells on day 90 post-vaccination. Splenocytes from mice vaccinated with the displayed formulations were restimulated in vitro with OVA and SIINFEKL peptide (100 pg mL-1 OVA and 2 pg mL-1 SIINFEKL) for 6 h and assessed via FACS and intracellular cytokine staining to determine the percentages of CD3+CD8+IFN-y+ cells.

[0096] FIG. 38: Representative flow cytometry plots for assessment of CD3+CD8+TNFa+ cells on day 90 post-vaccination. Splenocytes from mice vaccinated with the displayed formulations were restimulated in vitro with OVA and SIINFEKL peptide (100 pg mL-1 OVA and 2 pg mL-1 SIINFEKL) for 6 h and assessed via FACS and intracellular cytokine staining to determine the percentages of CD3+CD8+TNFa+ cells.

[0097] FIG. 39: Titers of OVA-specific IgG subclass antibodies detected in serum samples collected on day 90 post-vaccination. IgGl (a) and IgG2c (b) antibodies in serum on day 90 were determined by ELISA. Data represent the mean ± s.e.m. with n = 8 biologically independent samples. Data were analysed using one-way ANOVA and Dunnett’s multiple comparisons test. **P < 0.01, ****p < 0.0001; ELISA, enzyme-linked immunoassay.

[0098] FIG. 40: Body weights of mice vaccinated with the mOVA LiNx formulations. The body weights of mice were monitored during vaccination schedule. Mice are given a single dosage of LiNx via s.c. injections on day 0 (30 pg mOVA per injection). Data represent the mean ± s.e.m. with n = 10 biologically independent samples.

[0099] FIG. 41: Serum cytokine levels of mice vaccinated with the D6 mOVA LiNx formulation at 24 h post- vaccination. Serum cytokine levels, including IFN-y (a), IL-10 (b), IL- 4 (c), IL-2 (d), and TNF-a (e), were measured in mice 24 hours post-vaccination with the D6 LiNx formulation. Mice received a single dose of LiNx via s.c. injection containing 30 pg mOVA per injection. Data represent the mean ± s.e.m. with n = 4 biologically independent samples.

[0100] FIG. 42: Anti-tumour efficacy of the LiNx formulations as therapeutic vaccines for MC38-OVA tumour model. Mice were inoculated s.c. with MC38-OVA cells and then given three s.c. injections, one week apart, of mOVA-loaded D6 LNPs or LiNx (10 pg mOVA per injection) or PBS mixed with NHC. For the LiNx treatment groups, mice were administered with a single dose of LiNx loaded with mOVA (30 pg per mouse) or OVA protein (10 pg protein per mouse) via s.c. injection. OVAprotein mixed with Alhydrogel® (1: 1) (10 pg protein per mouse) served as a control group. Individual tumour volumes are shown over time.

[0101] FIG. 43: Anti-tumour efficacy of the D6 LiNx as a therapeutic vaccine for B16- OVA tumour model. Mice were inoculated s.c. with B16-OVA cells and then given a single dose of D6 LiNx loaded with mOVA (30 pg per mouse) via s.c. injection. Two groups also received a repeated anti-CTLA-4 monoclonal antibody (mAh; 100 pg per i.p. injection) treatment alone or in combination with the LiNx. Individual tumour volumes are shown over time.

[0102] FIG. 44: Anti-tumour efficacy of the D6 LiNx as a therapeutic vaccine for B16F10 tumour model. Mice were inoculated .s. c. with B16F10 cells and then given a single dose of D6 LiNx loaded with mTrp2 or mGplOO (30 pg per mouse) via s.c. injection. Two groups also received a repeated anti-CTLA-4 monoclonal antibody (mAb; 100 pg per i.p. injection) treatment in combination with the LiNx treatment. Individual tumour volumes are shown over time.

[0103] FIG. 45: Anti-tumour efficacy of the D6 LiNx as a prophylactic vaccine for B16-OVA tumour model. Mice were administered with a single dose of D6 LiNx loaded with mOVA (30 pg per mouse) via s.c. injection and then inoculated s.c. with B16F10-OVA cells on day 21. Individual tumour volumes are shown over time.

[0104] FIG. 46: Anti-tumour efficacy of the LiNx loaded with either mOVA D6 LNPs or empty D6 LNPs in a prophylactic vaccine model for B16-OVA tumour. Mice were administered with a single dose of D6 LiNx loaded with LNPs (30 pg mOVA per mouse) or empty D6 LNPs via s.c. injection and then inoculated s.c. with B16F10-OVA cells on day 21. Survival times are shown over time (n = 8 mice).

[0105] FIG. 47: Prophylactic efficacy of the D6 LiNx in B16-OVA tumour model and resistance to rechallenge. Schematic and results of a prophylactic vaccination and rechallengemodel for B16-0VA in C57BL / 6 mice. Mice were vaccinated with the D6 mOVA LiNx (30 pg per mouse) before s.c. inoculation of OVA-expressing melanoma (B16-OVA) cells. After 100 days, the tumour free mice were rechallenged with B16-OVA cells. Mice at a similar age were included as a control group injected with PBS. Survival curves (b, c) are shown. In b, n = 6 for PBS group and n = 18 biologically independent samples for the LiNx group. In c, n = 6 for PBS group, and n = 17 biologically independent samples for the LiNx group.

[0106] FIG. 48 Representative images from the CODEX fluorescence imaging analysis on the collected tumour samples. Statining details can be found in the Experimental section. Images shown here are from tumour samples stained with selected markers includingB220 (green), CD4 (yellow), CD45 (white), CD8 (red), DAPI (blue), Ly6G (teal), and NKp46 (purple).

[0107] FIG. 49: Cell type map of a representative PBS-treated tumour sample from CODEX multiplexed imaging experiment. The coloured data points represent selected immune and tumour cell types with all other cell types lumped together in the same grey colour (scale bar = 500 pm).

[0108] FIG. 50: Immunocytes profile of the blood samples extracted from mice treated with or withouαt- IL-17 antibody. Mice were administered with theα- IL-17 antibody (i.p., 200 pg per injection every three days) over a 2-week period. Following this, blood samples were collected; and immune cell populations were analysed, (a) CD45+CD3+ T cells, CD45+CD3+CD8+ T cells, CD45+CD3+CD4+ T cells, and CD45+CD19+ B cells were examined, (b) CD3-CDl lb-NKp46+ NK cells, CD3-CDl lb+Ly6G-CDl lc+MHCII+ DCs, and CD3- CDl lb+Ly6G-CDl lc-F4 / 80+macrophages were assessed. Data represent the mean ± s.e.m. (n = 4 biologically independent samples). Data were analysed using one-way ANOVA and Tukey's multiple comparisons. NS, not significant.DETAILED DESCRIPTION

[0109] As discussed above, we now provide fiber-hydrogel composite compositions that are suitably loaded or mixed with or used in conjunction with one or more LNPas.

[0110] In one preferred aspect, we now provide mRNA LNP-loaded nanofiber-hydrogel matrix (LiNx), which promotes endogenous immune cell infiltration and can creates a programmable immunostimulating microenvironment, therefore maximizing antigen expression and presentation.We analyzed 1,080 LNPs to boost transfection efficiency in antigen-presenting cells (APC). Zhu, Y., et al. Nat. Biomed. Eng (2023). We have now found that tuning of LNP composition enables the modification of transgene expression in both APCs and non-APCs, thereby influencing the elicited immune activation profile. In one preferred aspect, we have now loaded the LNPS including D6 LNP into a nanofiber-hydrogel composite (NHC).2Z.-C. et al. Small 2022, 18, 2202309. This configuration yields a more robust immune response with a single-dose vaccination in in vivo colon carcinoma and melanoma models including mouse colon carcinoma and melanoma models (Fig. la).

[0111] LIPID NANOPARTICLES

[0112] In the context of the present disclosure, a lipid nanoparticle (LNP) delivery vehicle typically serves to transport a desired therapeutic agent to a target cell or tissue. In certain embodiments, the lipid formulation encapsulates the therapeutic agent.

[0113] Lipid nanoparticles (LNPs) have been engineered as carriers for mRNA-encoding antigens with validated biosafety profile. However, their application in protein delivery is less explored. In this study, the formulations for spleen-targeted delivery of gene-editing proteins including Cre recombinase and Cas9 were identified using this LNP screening platform. In particular, with in vitro screening with HEK293 Ai9 cell line, and further cluster-mode in vivo screening in Ai9 mice, formulations targeting the spleen for gene editing in T cells, B cells and DCs are disclosed herein.

[0114] In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNP) have a structure that includes a single monolayer or bilayer of lipids that encapsulates a compound in a solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous phase or other liquid phase in its interior, but rather the lipids from the bilayer or monolayer shell are directly complexed to the internal compound thereby encapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles having a relatively uniform dispersion of shape and size. While sources vary on what size qualifies a lipid particle as being a nanoparticle, there is some overlap in agreement that a lipid nanoparticle can have a diameter in the range of from 10 nm to 1000 nm. However, more commonly they are considered to be smaller than 120 nm or even 100 nm.

[0115] For lipid nanoparticle nucleic acid delivery systems, the lipid shell is formulated to include an ionizable cationic lipid which can complex to and associate with the negatively chargedbackbone of the nucleic acid core. Ionizable cationic lipids with apparent pKa values below about 7 have the benefit of providing a cationic lipid for complexing with the nucleic acid's negatively charged backbone and loading into the lipid nanoparticle at pH values below the pKa of the ionizable lipid where it is positively charged. Then, at physiological pH values, the lipid nanoparticle can adopt a relatively neutral exterior allowing for a significant increase in the circulation half-lives of the particles following i.v. administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutics, and low levels of cytotoxicity and immunogenicity.

[0116] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids were widely studied as synthetic materials for delivery of nucleic acid medicines. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed by cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and characterized by broad size distributions ranging from the submicron scale to a few microns. Lipoplexes, such as the Lipofectamine® reagent, have found considerable utility for in vitro transfection. However, these first-generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (Imparted by the cationic lipid) result in rapid plasma clearance, hemolytic and other toxicities, as well as immune system activation. In some aspects, proteins, nucleic acid molecules etc., provided herein and lipids or lipid formulations provided herein form a lipid nanoparticle (LNP).

[0117] In the context of the present disclosure, a lipid nanoparticle (LNP) delivery vehicle typically serves to transport a desired therapeutic agent to a target cell or tissue. In certain embodiments, the lipid formulation encapsulates the therapeutic agent.

[0118] The manufacture of lipid nanoparticles is described in detail in the examples section. Lipid nanoparticles can also be produced (e.g., allowed to self-assemble, e.g., spontaneously) by injecting a lower alkanol solution containing lipids into an aqueous solution. Various lipids can be used to achieve desired properties, such as size, surface charge, and capacity for encapsulants. Such properties can also be influenced by the composition of the aqueous solution.

[0119] Lipid nanoparticles of the invention can encapsulate a wide range of hydrophilic molecules, e.g., nucleic acids such as DNA and RNA, or alternative versions of DNA or RNA.Variations in lipid nanoparticle size can be affected by controlling process parameters. In particular, the rate at which the lower alkanol solution is injected into the aqueous solution is inversely related to the resulting lipid nanoparticle size. Similarly, minimizing variance in the rate of injection will minimize variance in lipid nanoparticle size, yielding homogeneous suspensions of lipid nanoparticles, e.g., within a single batch or among multiple batches. A precise rate of injection can be attained, e.g., through a servo pump.

[0120] Typically, the lipid nanoparticles are liposomes with a lipid bilayer surrounding an aqueous interior. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 nm and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

[0121] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed (e.g., osmolality or pH), the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and / or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

[0122] Conventional downstream processing can be employed. For example, lipid nanoparticles can be purified or concentrated, e.g., by tangential flow filtration, dialysis, or desalting column (e.g., a PD-10 desalting column). In methods involving dialysis, the filter membrane geometry, including the area of the filter and the fiber diameter can be varied to achieve an optimal rate of filtration according to known parameters, such as lipid nanoparticle size, encapsulants size, and liquid viscosity (e.g., buffer viscosity).

[0123] In certain embodiments, the nanoparticle suspension can be exchanged with a solution containing a cryoprotectant (e.g., for long term storage in, e.g., frozen or lyophilized form).Physiologically suitable cyroprotectants for lipid nanoparticles are known in the art and include, e.g., sucrose, glucose, mannitol, glycerol, and other carbohydrates and polyalcohols. In one non- limiting example, a lipid nanoparticle solution is dialyzed against a sucrose solution (e.g., a TRIS / sucrose buffer).

[0124] A sterile filtration step may also be employed, and the membrane area, pore size and filtration force can be varied, as described above. The lipid nanoparticles described herein may be made in a sterile environment.

[0125] The manufacture of lipid nanoparticles is described in examples section. Preferred lipid nanoparticles and preparation thereof are also disclosed in PCT / US2023 / 016938 (WO2023 / 192503). Lipid nanoparticles can also be produced (e.g., allowed to self-assemble, e.g., spontaneously) by injecting a lower alkanol solution containing lipids into an aqueous solution. Various lipids can be used to achieve desired properties, such as size, surface charge, and capacity for encapsulants. Such properties can also be influenced by the composition of the aqueous solution. Lipid nanoparticles of the invention can encapsulate a wide range of hydrophilic molecules, e.g., nucleic acids such as DNA and RNA, or alternative versions of DNA or RNA. Variations in lipid nanoparticle size can be affected by controlling process parameters. In particular, the rate at which the lower alkanol solution is injected into the aqueous solution is inversely related to the resulting lipid nanoparticle size. Similarly, minimizing variance in the rate of injection will minimize variance in lipid nanoparticle size, yielding homogeneous suspensions of lipid nanoparticles, e.g., within a single batch or among multiple batches. A precise rate of injection can be attained, e.g., through a servo pump.

[0126] Typically, the lipid nanoparticles are liposomes with a lipid bilayer surrounding an aqueous interior. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 nm and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

[0127] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed (e.g., osmolality or pH), the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and / or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

[0128] Conventional downstream processing can be employed. For example, lipid nanoparticles can be purified or concentrated, e.g., by tangential flow filtration, dialysis, or desalting column (e.g., a PD-10 desalting column). In methods involving dialysis, the filter membrane geometry, including the area of the filter and the fiber diameter can be varied to achieve an optimal rate of filtration according to known parameters, such as lipid nanoparticle size, encapsulants size, and liquid viscosity (e.g., buffer viscosity).

[0129] In certain embodiments, the nanoparticle suspension can be exchanged with a solution containing a cryoprotectant (e.g., for long term storage in, e.g., frozen or lyophilized form). Physiologically suitable cyroprotectants for lipid nanoparticles are known in the art and include, e.g., sucrose, glucose, mannitol, glycerol, and other carbohydrates and polyalcohols. In one non- limiting example, a lipid nanoparticle solution is dialyzed against a sucrose solution (e.g., a TRIS / sucrose buffer).

[0130] A sterile filtration step may also be employed, and the membrane area, pore size and filtration force can be varied, as described above. The lipid nanoparticles described herein may be made in a sterile environment.

[0131] LNP Compositions

[0132] In one aspect, a lipid nanoparticle (LNP) formulation comprises an ionizable lipid, at least one helper lipid, a PEGylated lipid, a sterol or combinations thereof. In certain embodiments, the at least one helper lipid comprises a cationic lipid, a zwitterionic lipid, an anionic lipid, or combinations thereof. In certain embodiments, the cationic lipid comprises l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB) or the combination thereof. In certain embodiments, the zwitterionic lipid comprises 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or thecombination thereof. In certain embodiments, the anionic lipid comprises 1,2-dimyristoyl-sn- glycero-3 -phosphate (14PA), l-stearoyl-2-oleoyl-sn-glycero-3 -phospho (l ’-rac-glycerol) (18PG) or the combination thereof. In certain embodiments, the ionizable lipid comprises DLin-MC3- DMA. In certain embodiments, the sterol comprises cholesterol.

[0133] Ionizable Lipids: Other examples of ionizable lipids include 3-(didodecylamino)- N1 ,N1 ,4-tridodecyl-l -piperazineethanamine (KL10), N 1 -[2-(didodecylamino)ethyl]-N 1 ,N4,N4- tri dodecyl- 1 ,4-piperazinedi ethanamine (KL22), 14,25 -ditri decyl- 15, 18,21 ,24-tetraaza- octatriacontane (KL25), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), l,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl- 3-[(9Z,12Z)-octadeca-9,12-dien-l-yl oxy] propan- 1 -amine (Octyl-CLinDMA), (2R)-2-({8-[(30)- cholest-5-en-3-yloxy]octyl})oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy] propan- 1 -amine (Octyl-CLinDMA (2R)), and (2S)-2-({8-[(3P)-cholest-5-en-3- yloxy] octyl } oxy)-N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-l -yloxy]propan-l -amine (Octyl-CLinDMA (2S)).

[0134] PEG Modified Lipids: In certain embodiments, the PEGylated lipid comprises DMG- PEG 2000. As used herein, the terms “PEG lipid” and “PEGylated lipid refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified l,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0135] In certain embodiments, the PEG lipid includes, but are not limited to, 1,2-dimyristoyl- sn-glycerol methoxypoly ethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG- 1,2- dimyristyloxlpropyl-3 -amine (PEG-c-DMA).

[0136] In certain embodiments, the PEG lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG- modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

[0137] In certain embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.

[0138] In certain embodiments, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG- DSG and PEG-DSPE.

[0139] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some embodiments, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0140] In certain embodiments, PEG lipids can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In some embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl ( — OH) groups on the lipid. In some embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In some embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an — OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0141] In some embodiments, an LNP fiber-hydrogel composite that includes one or more lipids described herein may further include one or more adjuvants, e.g., Glucopyranosyl Lipid Adjuvant(GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0142] FIBER-HYDROGEL COMPOSITE

[0143] In preferred systems, the administered fiber-hydrogel composite can provide a porous scaffold which allows cells and other molecules to be transported bidirectionally between the region of administration of the composite and surrounding tissues.

[0144] In one aspect, a preferred nanofiber-hydrogel composite (NHC) designed to mimic the microarchitecture and mechanical properties of the soft tissue extracellular matrix. In preferred aspects, the composite integrates a hyaluronic acid (HA) hydrogel network with e.g. polymer nanofiber (e.g. electrospun poly(s-caprolactone) (PCL) or collogen nanofibers and fragments) through covalent linking, resulting in a suitably structured composite with controlled mechanical properties, including stiffness, pore size, and degradation time. The mechanical fragmentation method enhances scalability and reduces dependency on gelation time and precursor viscosity, generating microgels from NHC.

[0145] In particular aspects, the fibers (e.g., nanofibers or microfibers) of a nanofiber-hydrogel composite (NHC) suitably include one or more extracellular matrix proteins (ECM). In certain aspects, the nanofiber-hydrogel composite (NHC) may include poly(caprolactone) and / or collagen fibers.

[0146] In one aspect, a fiber- hydrogel composite includes (i) fibers (e.g., nanofibers or microfibers) comprising one or more extracellular matrix proteins (ECM), and particularly one or more poly(caprolactone) and / or collagen materials; (ii) a hyaluronic acid (HA); and optionally (iii) a crosslinking agent. The HA is bonded (e.g., covalently or non-covalently) to the fibers (suitably by the crosslinking agent if present)to form a composite network.

[0147] In certain embodiments, the fibers (e.g., nanofibers or microfibers) have a mean diameter of less than about 10 mm, less than about 5 mm, less than about 4 mm, less than about 3 mm, less than about 2 mm, less than about 1 mm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, less than about 10 nm, less than about 5 nm, or less than about 1 nm. In certain embodiments, the fibershave a mean diameter in a range of about 10 nm to 5 mm, about 100 nm to 5 mm. In certain embodiments, the fibers have a mean diameter in a range of about 1 nm to 1,000 nm, about 1 nm to 500 nm, or about 1 nm to 100 nm.

[0148] In certain embodiments, the fibers (e.g., microfibers) have a length greater than about 1 mm, greater than about 5 mm, greater than about 10 mm, greater than about 20 mm, greater than about 30 mm, greater than about 40 mm, greater than about 50 mm, greater than about 60 mm, greater than about 70 mm, greater than about 80 mm, greater than about 90 mm, greater than about 100 mm, greater than about 200 mm, greater than about 300 mm, greater than about 400 mm, greater than about 500 mm, greater than about 600 mm, greater than about 700 mm, greater than about 800 mm, greater than about 900 mm, or of about 1 mm. In certain embodiments, the fibers (e.g., microfibers) have a length in the range of about 1 to 1,000 mm, of about 10 to 500 mm, or of about 100 to 500 mm. The diameter and length of the fibers (e.g., nanofibers or microfibers) may be determined using optical (including fluorescence) microscopy or electron microscopy.

[0149] In certain embodiments, the fibers can be cylindrical (with a similar cross-section width and height) or ribboned in shape (with the cross-section width greater than the height).

[0150] Preferably, the fibers (e.g., nanofibers or microfibers) may have an aspect ratio in a range of at least about 10 to about at least 10,000. It will be appreciated that, because of the very small diameter of the fibers, the fibers have a high surface area per unit of mass. This high surface area to mass ratio permits fiber-forming solutions or liquids to be transformed from liquid or solvated fiber-forming materials to solid fibers in fractions of a second.

[0151] In certain embodiments, the fibers (e.g., nanofibers or microfibers) are functionalized. In certain embodiments, the fibers (e.g., nanofibers or microfibers) are functionalized with groups comprising hydroxyl, amino, carboxyl, thio, acrylate, sulfonate, phosphate, maleimide, amide, as well as modified forms thereof, such as activated or protected forms. In certain embodiments, any functional groups that can be reacted with a crosslinker, especially the epoxy and vinyl sulfonyl groups of a crosslinker, may be used. Examples of functional groups that are reactive to the epoxy or vinyl sulfonyl groups of a crosslinker include hydroxyl, carboxyl, thiol, or amino groups.

[0152] The nanofibers may include, but not limited to, nanofibers, nanotubes, nanofilaments, mesh sections, branched filaments or networks. The nanofibers may also comprise any suitablechemical functional groups to facilitate the covalent or noncovalent crosslinking between the nanofibers and the polymers of the hydrogels of the invention. Method, techniques, and materials are well known in the art for making and functionalizing nanofibers. Any microfabrication methods are used to make the nanofibers. In various embodiments, the disclosed devices can be assembled and / or manufactured using any suitable microfabrication technique. Such methods and techniques are widely known in the art.

[0153] Particularly, the fibers (e.g., nanofibers or microfibers) in the fiber-hydrogel composite include one or more extracellular matrix proteins (ECMs). In certain embodiments, the fibers (e.g., nanofibers or microfibers) suitably include one or more selected from collagen, gelatin, elastin, elastin-like polypeptides, tropoelastin, decellularized matrix, and hyaluronic acid. In certain embodiments, the fibers (e.g., nanofibers or microfibers) include one or more from bovine type I collagen, gelatin, or derivatives. In certain embodiments, the fibers (e.g., nanofibers or microfibers) include one or more collogen including bovine type I collagen and a recombinant collagen which may be a human recombinant collagen material.

[0154] In certain embodiments, the fiber-hydrogel composite may include a natural extracellular matrix for fibers (e.g., nanofibers or microfibers). In certain embodiments, the fiber-hydrogel composite may include a synthetic extracellular matrix for fibers (e.g., nanofibers or microfibers).

[0155] In certain embodiments, the one or more ECMs include a collagen nanofiber, which may be naturally obtained or synthesized. In certain embodiments, the collagen nanofiber includes a type I bovine collagen nanofiber or fragments thereof. In certain embodiments, the collagen nanofibers may be obtained from natural sources, or may be fabricated or prepared from a composition (resin composition) including collagen. For example, the collagen nanofiber may be formed by electrospinning, centrifugal spinning, blow spinning, or combinations thereof. Particularly, collagen nanofibers are preferably prepared by electrospinning.

[0156] “Human recombinant collagen may be provided by any suitable method known in the art, including as disclosed in U.S. Patents 5,962,648 and 5,593,859 and W02004 / 078120. Suitably collagen will be recombinantly manufactured by culturing a cell which has been transfected with at least one gene encoding the polypeptide comprising collagen and genes encoding the oc and subunits of the post-translational enzyme prolyl 4-hydroxylase and purifying the resultant collagenmonomer therefrom. The recombinant collagen solution may be subsequently subjected to polymerization or cross-linking conditions.

[0157] Bovine collagen suitably can be a mixture of collagen type I (85%) and collagen type III (15%). An advantage of recombinant collagen is that collagen type I and collagen type III are made independen tly of one another, and so any combination of type I and type III collagen can be made. The present compositions and composites may suitably comprise human collagen type I and human collagen type III in any ratio. For example, the compositions and composites may comprise human collagen type I and human collagen type III in a ratio by weight of 100:0, 80:20, 60:40, 50:50, 40:60, 20:80 or 0: 100, or anywhere in-between. Preferably, the ratio by weight of human collagen type I: human collagen type III is greater than about 50:50, and preferably it is greater than about 70:30, for example about 80:20. Suitably, the type I human recombinant collagen makes up at least about 75% by weight of the total human recombinant collagens in the material.

[0158] In one aspect, the present composites and compositions comprise collagen, wherein the composite has a surface area greater than about 2.3 m2 / g collagen. In other aspects, the composite has a surface area greater than about 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 3.8, 4.0, and 4.4 m2 / g collagen. In a particular aspect, a composite is provided comprising collagen, wherein the composite has a surface area of or greater than about 4.0 m2 / g collagen.

[0159] The fibers (e.g., nanofibers or microfibers) suitably may preferably be fabricated by electrostatic spinning (also referred to as electrospinning). The process of electrospinning generally involves the introduction of a liquid into an electric field, so that the liquid is caused to produce fibers. These fibers are generally drawn to a conductor at an attractive electrical potential for collection. During the conversion of the liquid into fibers, the fibers harden and / or dry. This hardening and / or drying may be caused by cooling of the liquid, i.e., where the liquid is normally a solid at room temperature; by evaporation of a solvent, e.g., by dehydration (physically induced hardening); or by a curing mechanism (chemically induced hardening). Electrostatically spun fibers can be produced having very thin diameters. Parameters that influence the diameter, consistency, and uniformity of the electrospun fibers include the polymeric material and cross- linker concentration (loading) in the fiber-forming combination, the applied voltage, and needle collector distance.

[0160] The electrospun fibers (e.g., poly(caprolactone) or collagen nanofiber) may provide superior properties, e.g., high porosity in the hydrogel phase and mechanical reinforcement from the solid fiber component, which may be beneficial for optimal cell infiltration properties and structural integrity.

[0161] In certain embodiments, the hydrogel material such as HA may be covalently bonded to the fibers (e.g., nanofibers or microfibers). For example, the hydrogel material such as HA may be covalently bonded to the recombinant or type I bovine collagen nanofiber of fragments thereof. In certain embodiments, the crosslinking agent generates interfacial bonding between the collagen nanofiber and the HA. Due to bonding and interaction (e.g., covalent, non-covalent or ionic bonding), the collagen nanofiber may be retained inside or inner space of the fiber-hydrogel composite (e.g., inside of the composite network). In certain embodiments, the crosslinking agent may react with the hydroxyl groups of hydrogel material (e.g. HA) and amino groups of the collagen nanofiber to form the composite network. For example, interfacial bonding between the collagen nanofiber and the hydrogel material (e.g. HA) may increase the composite stiffness even at relatively low fiber loading density.

[0162] In certain embodiments, a storage modulus of the fiber-hydrogel composite is at least about 10 Pa, at least about 20 Pa, at least about 30 Pa, at least about 40 Pa, at least about 50 Pa, at least about 60 Pa, at least about 70 Pa, at least about 80 Pa, at least about 90 Pa, at least about 100 Pa, at least about 150 Pa, at least about 200 Pa, at least about 250 Pa, at least about 300 Pa, at least about 400 Pa, or at least about 500 Pa. In certain embodiments, a storage modulus of the fiber- hydrogel composite ranges from about 1 to about 1,000 Pa, from about 20 to about 800 Pa, from about 100 to about 500 Pa, or from about 150 to about 500 Pa. In certain embodiments, a storage modulus of the fiber-hydrogel composite ranges from about 0.5 to about 30 kPa. Storage modulus values as referred to herein may be determined by procedures set forth in Example 3 which follows, which include: use a analytical tool such as AR2 (TA Instruments) using a parallel plate geometry of 8 mm with 0.5 mm gap at 25 °C, with linear viscoelastic region being measured for the composition sample by a strain sweep with an increasing shear strain amplitude at a set frequency (1 Hz).

[0163] In certain embodiments, as discussed, an alternate hydrogel phase such as collagen, chitosan, alginate, PVA, gelatin, PEG or other glycol ethers, cellulose, or cellulose materials may be used in place of or in combination with hyaluronic acid (HA).

[0164] In certain embodiments, the fiber-hydrogel composite may be stable at the temperature and pressure suitable for terminal sterilization (e.g., autoclaving). For example, the fiber-hydrogel composite may be stable at a temperature of about 100 °C to 131 °C for at least 30 minutes (less needed at 131 °C). In certain embodiments, the composite may have increased thermal stability and / or results in shelf stability at ambient temperatures. Thus, with autoclave sterilization for example, the gels can be sterilized at the terminal manufacturing step to reduce the cost, risk, or regulatory burden of manufacturing without significant change to the mechanical behaviors of the composite.

[0165] The preferred form of interaction between the fibers (e.g., nanofibers or microfibers) and the hydrogel component includes a crosslinking moiety, generally present in an amount effective to introduce bonding between the fibers (e.g., nanofibers or microfibers) and the hydrogel material, e.g., to induce crosslinking between collagen nanofibers and hyaluronic acid.

[0166] For example, in case that high cohesive strength is desired, the fibers (e.g., nanofibers or microfibers) and the hydrogel component may be covalently crosslinked. For example, the HA hydrogel polymer component may be covalently crosslinked to the fibers, either intramolecularly or intermolecularly or through covalent bonds. In the former case, there are no covalent bonds linking the polymers to one another or to the nanostructures, while in the latter case, there are covalent crosslinks binding the polymers to one another or to the nanostructures. The crosslinks may be formed using any suitable means, including using heat, radiation, or a chemical curing (crosslinking) agent. The degree of crosslinking should be sufficient to eliminate or at least minimize cold flow under compression. Crosslinking also includes the use of a third molecule, a “cross-linker” utilized in the cross-linking process.

[0167] “ Cross-linkers” or “Cross-linking agents” may suitably include one or more selected from difunctional epoxide-based crosslinkers, 1 ,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), PEG molecules with terminal epoxide functional groups, and HA-reactive agents. Preferably, the crosslinking agent includes DVS or BDDE. In certain embodiments, a PEG crosslinking agent, which may contain terminal functional groups, such as epoxide or vinyl sulfonegroups, may be used to introduce crosslinking between the fibers (e.g., nanofibers or microfibers) and also between the fibers (e.g., nanofibers or microfibers) and the hydrogel to extend durability of the composite network and to modulate crosslinking density. In certain embodiments, the crosslinking agent does not include any spacer within its structure.

[0168] Crosslinking may also be accomplished with radiation, typically in the presence of a photoinitiator. The radiation may be ultraviolet, alpha, beta, gamma, electron beam, and x-ray radiation, although ultraviolet radiation is preferred. Useful photosensitizers are triplet sensitizers of the “hydrogen abstraction” type, and include benzophenone and substituted benzophenone and acetophenones such as benzyl dimethyl ketal, 4-acryloxybenzophenone (ABP), 1 -hydroxy - cyclohexyl phenyl ketone, 2,2-diethoxyacetophenone and 2,2-dimethoxy-2-phenylaceto-phenone, substituted alpha-ketols such as 2-methyl-2-hydroxypropiophenone, benzoin ethers such as benzoin methyl ether and benzoin isopropyl ether, substituted benzoin ethers such as anisoin methyl ether, aromatic sulfonyl chlorides such as 2-naphthalene sulfonyl chloride, photoactive oximes such as 1 -phenyl- l,2-propanedione-2-(O-ethoxy-carbonyl)-oxime, thioxanthones including alkyl- and halogen-substituted thioxanthonse such as 2-isopropylthioxanthone, 2- chlorothioxanthone, 2,4 dimethyl thioxanone, 2,4 dichlorothioxanone, and 2,4-diethyl thioxanone, and acyl phosphine oxides. Radiation having a wavelength of 200 to 800 nm, preferably, 200 to 500 nm, is preferred for use herein, and low intensity ultraviolet light is sufficient to induce crosslinking in most cases. However, with photosensitizers of the hydrogen abstraction type, higher intensity UV exposure may be necessary to achieve sufficient crosslinking. Such exposure can be provided by a mercury lamp processor such as those available from PPG, Fusion, Xenon, and others. Crosslinking may also be induced by irradiating with gamma radiation or an electron beam. Appropriate irradiation parameters, i.e., the type and dose of radiation used to effect crosslinking, will be apparent to those skilled in the art.

[0169] Suitable chemical curing agents, also referred to as chemical cross-linking “promoters,” include, without limitation, polymercaptans such as 2,2-dimercapto diethylether, dipentaerythritol hexa(3 -mercaptopropionate), ethylene bis(3 -mercaptoacetate), pentaerythritol tetra(3- mercaptopropionate), pentaerythritol tetrathioglycolate, polyethylene glycol dimercaptoacetate, polyethylene glycol di(3 -mercaptopropionate), trimethylolethane tri(3 -mercaptopropionate), trimethylolethane trithioglycolate, trimethylolpropane tri(3 -mercaptopropionate), trimethylolpropane trithioglycolate, dithioethane, di- or trithiopropane and 1,6-hexane dithiol. Thecrosslinking promoter is added to the uncrosslinked hydrophilic polymer to promote covalent crosslinking thereof, or to a blend of the uncrosslinked hydrophilic polymer and the complementary oligomer, to provide crosslinking between the two components.

[0170] In certain embodiments, a concentration of the crosslinking agent (e.g., DVS, or BDDE) ranges from about 0.01 v / v%. to about 10 v / v%, 0.05 v / v%. to about 10 v / v%, 0.05 v / v%. to about 5 v / v%, from about 0.2 v / v%. to about 10 v / v%, from about 0.5 v / v%. to about 5.0 v / v%, or from about 0.5 v / v%. to about 2.5 v / v%, based on the total volume of the fiber-hydrogel composite. In certain embodiments, a concentration of the DVS ranges from about 0.01 v / v%. to about 10 v / v%, 0.05 v / v%. to about 10 v / v%, 0.05 v / v%. to about 5 v / v%, from about 0.2 v / v%. to about 10 v / v%, from about 0.5 v / v%. to about 5.0 v / v%, or from about 0.5 v / v%. to about 2.5 v / v%, based on the total volume of the fiber-hydrogel composite.

[0171] In certain embodiments, a concentration of the HA ranges from about 0.1 w / v% to about 10 w / v%, from about 0.2 w / v% to about 10 w / v%, from about 0.2 w / v% to about 5.0 w / v%, from about 0.5 w / v% to about 10.0 w / v%, from about 0.5 w / v% to about 5.0 w / v%, from about 0.5 w / v% to about 2.0 w / v%, or from about 0.8 w / v% to about 2.0 w / v%, based on the total volume of the fiber-hydrogel composite.

[0172] In certain embodiments, a concentration of the one or more ECMs ranges from about 0.1 to about 50 w / v%, from about 0.1 to about 40 w / v%, from about 0.1 to about 30 w / v%, from about 0.1 to about 20 w / v%, from about 0.1 to about 10 w / v%, from about 1 to about 10 w / v%, from about 1 to about 5 w / v%, from about 1 to about 3 w / v%, or from about 1.5 to about 3 w / v%, based on the total volume of the fiber-hydrogel composite. In certain embodiments, a fiber loading density of the collagen nanofiber ranges from about 0.1 w / v% to about 10 w / v%, from about 1 w / v% to about 10 w / v%, from about 1 w / v% to about 5 w / v%, from about 1 w / v% to about 3 w / v% based on the total volume of the fiber-hydrogel composite.

[0173] For example, in an embodiment, a concentration of the one or more ECMs ranges from about 0.1 to about 20 w / v%, a concentration of the HA ranges from about 0.5 to about 10 w / v%, and a concentration of the crosslinking agent (e.g., DVS, or BDDE) ranges from about 0.05 to about 5.0 v / v%, based on the total volume of the fiber-hydrogel composite. Further, in an embodiment, the concentration of the one or more ECMs ranges from about 1.5 to about 3.0 w / v%, the concentration of the HA ranges from about 0.8 to about 2 w / v%, and a concentration of thecrosslinking agent (e.g., DVS) ranges from about 0.5 to about 2.5 v / v%, based on the total volume of the fiber-hydrogel composite.

[0174] The fiber-hydrogel composite may be formed to have a final device pH ranging from about 5.0 to about 9.0, from about 6.0 to about 8.0 from, or about 7.0 to about 7.4, in an isotonic solution.

[0175] The fiber-hydrogel composite may be formulated in a form of a sheet or a flowable or injectable fluid. For example, the fiber-hydrogel may be formulated in a solution that can be applied and shaped (or optionally dried) to form a sheet type gel. Further, the fiber-hydrogel composite may be formed in a fluid (e.g., aqueous suspension or dispersion) that can pass through of a 27-gauge or smaller needle.

[0176] In certain embodiments, the fiber-hydrogel composite may exhibit monocyte recruitment, monocyte polarization, or both. In certain embodiments, the fiber-hydrogel may accommodate or include macrophages.

[0177] In certain embodiments, the fiber-hydrogel composite (either alone or in combination with an LNP component (the LNP component also suitably including a therapeutic agent)) is suitably administered to a subject by injection and maintains cell viability greater than about 50%, about 60%, about 70%, about 80%, about 90%, or about 95 % after 7 days of injection.

[0178] Preferably, a fiber-hydrogel composite may be formulated such that the density, ratio of gel to fibers, and other properties are variable, while maintaining sufficient porosity and strength. A ratio of the fibers (e.g., nanofibers or microfibers) to hydrogel material can be determined by any means known in the art. For example, the ratio of ECM fibers to hydrogel material (e.g. hyaluronic acid) is from about 1: 100 to about 100: 1 on a component-mass basis, such as about 1:50 to about 50: 1, or 1: 10 to about 10: 1, such as 1:5 to about 5:1, such as about 1:3 to about 3:1. The ratio of ECM fibers to hydrogel material (e.g. hyaluronic acid) is also provided as a concentration basis, e.g., a given weight of polymeric fiber per volume of hydrogel material. For example, the concentration is from about 1 to 50 mg / mL. The hydrogel material is suitably generally connected, attached or disposed within the ECM fibers as forming the composite network.

[0179] The fiber-hydrogel composite may contain a plurality of pores present on or within a surface of the composite. The presence, size, distribution, frequency and other parameters of the pores can be modulated during the creation of the composite, hydrogel, or fibers (e.g., nanofibers or microfibers). Pore size can be from below about 1 nm to up to 100 pm, including 1, 2, 3, 4 5, 10, 15, 20, 30, 40, 50, 6070, 80, 90 or 100 pm, and the size thereof may be narrowly tailored, e.g., such that at least 40%, such as 50%, 60%, 70%, 80%, 90%, 95% or greater than 95% of the pores are in a desired size or within a desired size range.

[0180] In one aspect, preferred fibe-hydrogel composites comprise a plurality of pores present on a surface of a layer of the composite or composition, wherein the pores are present at a concentration of at least about 50 pores per cm2of the surface, and wherein at least 80% of the pores have an average pore diameter on the surface that is at least about 5 microns.

[0181] The fiber-hydrogel composite (either alone or in combination with an LNP component (the LNP component also suitably including a therapeutic agent)) may be suitable for incorporation into a tissue of a human subject, and thus they are generally “biocompatible”, meaning capable of interacting with a biological system (such as found in a human subject) without inducing a pathophysiological response therein and / or thereby. In certain embodiments, the fiber-hydrogel composite (either alone or in combination with an LNP component (the LNP component also suitably including a therapeutic agent)) is provided in order to be durably retained in the tissue, e.g., organs, nerve tissues. Alternatively, the composite may be transiently retained in the human subject and are provided as substantially biodegradable. Preferably, the ECM fibers may further include biocompatible biodegradable polymers, e.g., biocompatible biodegradable polyester.

[0182] The fiber-hydrogel composite may further include active agents in addition to an LNP.

[0183] In certain embodiments, the fiber-hydrogel composite may also include additional optional additive components. Such components are known in the art and can include, for example, fillers, preservatives, pH regulators, softeners, thickeners, pigments, dyes, refractive particles, stabilizers, toughening agents, detackifiers, pharmaceutical agents (e.g., antibiotics, angiogenesis promoters, antifungal agents, immunosuppressing agents, antibodies, and the like), and permeation enhancers. These additives, and amounts thereof, are selected in such a way that they do not significantly interfere with the desired chemical and physical properties of the hydrogel composition.

[0184] In certain embodiments, the fiber-hydrogel composite (either alone or in combination with an LNP component (the LNP component also suitably including a therapeutic agent)) may include pH regulating compounds. Compounds useful as pH regulators include, but are not limited to, glycerol buffers, citrate buffers, borate buffers, phosphate buffers, or citric acid-phosphate buffers may also be included so as to ensure that the pH of the hydrogel composition is compatible with that of an individual's body surface.

[0185] In certain embodiments, the fiber-hydrogel composite (either alone or in combination with an LNP component (the LNP component also suitably including a therapeutic agent)) may be delivered by any suitable method, such as via a syringe or bellows pack (single dose delivery systems) or a multidose system, such as a pressurized delivery system or delivery via a “bag in the can” type system. The administration may extend to a single dose delivery system including the fiber-hydrogel composite, for the treatment of wounds, to a pressurized delivery system including the fiber-hydrogel composite (e.g., an aerosol spray).

[0186] In certain embodiment, it may be advantageous to render the fiber-hydrogel composite electrically conductive for use in biomedical electrodes and other electrotherapy contexts, i.e., to attach an electrode or other electrically conductive member to the body surface. For example, the fiber-hydrogel composite may be used to attach a transcutaneous nerve stimulation electrode, an electrosurgical return electrode, or an EKG electrode to a patient's skin or mucosal tissue. These applications involve modification of the fiber-hydrogel composite so as to contain a conductive species. Suitable conductive species are ionically conductive electrolytes, particularly those that are normally used in the manufacture of conductive adhesives used for application to the skin or other body surface, and include ionizable inorganic salts, organic compounds, or combinations of both. Examples of ionically conductive electrolytes include, but are not limited to, ammonium sulfate, ammonium acetate, monoethanolamine acetate, diethanolamine acetate, sodium lactate, sodium citrate, magnesium acetate, magnesium sulfate, sodium acetate, calcium chloride, magnesium chloride, calcium sulfate, lithium chloride, lithium perchlorate, sodium citrate and potassium chloride, and redox couples such as a mixture of ferric and ferrous salts such as sulfates and gluconates. Preferred salts are potassium chloride, sodium chloride, magnesium sulfate, and magnesium acetate, and potassium chloride is most preferred for EKG applications. Although virtually any amount of electrolyte may be present in the adhesive compositions of the invention, it is preferable that any electrolyte present be at a concentration in the range of about 0.1 to about15 wt. % of the hydrogel composition. The procedure described in U.S. Pat. No. 5,846,558 to Nielsen et al. for fabricating biomedical electrodes may be adapted for use with the hydrogel compositions of the invention, and the disclosure of that patent is incorporated by reference with respect to manufacturing details. Other suitable fabrication procedures may be used as well, as will be appreciated by those skilled in the art.

[0187] In certain embodiments, an fiber-hydrogel composite may suitably include microbeads (e.g., spherical or non-spherical) in an amount of about 10% to 90% of the total weight of the composite. In certain embodiments, an fiber-hydrogel composite may suitably include microbeads in an amount of about 20% to 80% of the total weight of the composite, or in an amount of about 30% to 70% of the total weight of the composite, or in an amount of about 40% to 60% of the total weight of the composite.

[0188] In certain embodiments, the microbeads are gelated or cured before the LNP is added to the microbeads. In certain embodiments, the microbeads are gelated or cured after the biological active material is added to the microbeads. The microbeads may be gelated by chemical reaction (e.g., cross-linking) or by UV irradiation to cure or polymerize the polymers to form nanofibers.

[0189] KIT

[0190] Provided herein is a kit including the fiber-hydrogel composite ((either alone or in combination with an LNP component (the LNP component also suitably including a therapeutic agent)) as described herein and a suitable applicator, including an injection device, such as a syringe. In certain embodiments, the applicator may include an injector or injection needle.

[0191] METHODS OF USE

[0192] In an aspect, provided also are methods of producing the fiber-hydrogel composite as described herein. The method may include a step of contacting a crosslinking agent with fibers (e.g., nanofibers or microfibers) comprising one or more extracellular matrix proteins (ECMs) and hydrogel material e.g. hyaluronic acid (HA) to obtain a fiber-hydrogel composite. The hydrogel material (e.g. HA) is suitably bonded to the fibers (e.g., nanofibers or microfibers) by the crosslinking agent to form a composite network. The formed composite network may be admixed with one or more separately prepared LNPs

[0193] In certain embodiments, the method may include steps of modulating crosslinking condition. In certain embodiments, the crosslinking condition is modulated to be under a basic condition and a pH of the crosslinking condition ranges from about 9 to about 14, from about 10 to about 14, from about 10 to about 13sor from about 10 to about 12. In certain embodiments, the pH of the crosslinking condition ranges from about 10 to about 14, from about 12 to about 13.3. For example, the pH of the crosslinking condition is about 12.4, about 12.7, about 13.0 or about 13.3.

[0194] In certain embodiments, the step of contacting is performed for about 30 minutes to about 4 hours. In certain embodiments, the step of contacting is performed for more than about 30 minutes, more than about 1 hour, more than about 1.5 hour, more than about 2 hours, more than about 2.5 hours, more than about 3 hours, more than about 3.5 hours, or about 4 hours or less. In certain embodiments, the step of contacting is performed for about 30 minutes, about 1 hour, about 1.5 hour, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, or about 4 hours or less. In certain embodiments, the step of contacting is performed at a temperature of about 37 degrees C.

[0195] In certain embodiments, the contacting is performed for about less than about 2 hours. In such case, the contacting may be performed at elevated temperature, e.g., at a temperature of about 37 to 100 degrees C, about 40 to 100 degrees C, about 50 to 100 degrees C, or about 60 to 100 degrees C.

[0196] In certain embodiments, the contacting is performed for more than one day. In such case, the contacting may be performed at lower temperature, e.g., at a temperature of about 0 to 37 degrees C, about 0 to 30 degrees C, about 0 to 25 degrees C, about 0 to 20 degrees C, or about 0 to 15 degrees C.

[0197] The method of producing the fiber-hydrogel composite may further include a step of swelling the fiber-hydrogel composite. In certain embodiments, the step of swelling is performed by incubating the fiber-hydrogel composite in an aqueous buffer solution (e.g., PBS). In certain embodiments, the buffer solution may be used in greater than 10 times, greater than 50 times, greater than 100 times, greater than 200 times, greater than 300 times, greater than 400 times, greater than 500 times, or greater than 1000 times, of volume of the fiber-hydrogel composite. In certain embodiments, the swelling is performed (continuously or discontinuously) for more thanan hour, more than 2 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 15 hours, more than 20 hours, more than 24 hours, more than 30 hours, more than 40 hours, more than 50 hours, more than 60 hours, or more than 72 hours. Preferably, the swelling is performed (continuously or discontinuously) for 24 to 72 hours at room temperature.

[0198] In certain embodiments, the fiber-hydrogel composite produced herein may be in a biphasic gel or monophasic gel. For example, the fiber-hydrogel composite is a biphasic gel with similar bead sizes made by forcing the gel through screens. Alternatively, the fiber-hydrogel composite is a monophasic gel with a continuous distribution of gel bead sizes made by homogenizing the gel or mechanically disrupting the gel during the crosslinking reaction.

[0199] Therapeutic Agents

[0200] Lipid nanoparticles may include one or more therapeutics and / or prophylactics, such as a nucleic acid. The disclosure features methods of delivering a therapeutic and / or prophylactic, such as a nucleic acid to a mammalian cell or organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof comprising administering to a mammal and / or contacting a mammalian cell with a LNP including a therapeutic and / or prophylactic, such as a nucleic acid.

[0201] Therapeutics and / or prophylactics include biologically active substances and are alternately referred to as “active agents”. A therapeutic and / or prophylactic may be a substance that, once delivered to a cell or organ, brings about a desirable change in the cell, organ, or other bodily tissue or system. Such species may be useful in the treatment of one or more diseases, disorders, or conditions.

[0202] Therapeutic Agents

[0203] Polynucleotides and Nucleic Acids

[0204] In some embodiments, a therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term “polynucleotide”, in its broadest sense, includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger RNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs,shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc. In some embodiments, a therapeutic and / or prophylactic is an RNA. RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to, shortmers, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer- substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is an mRNA.

[0205] In some embodiments, a therapeutic and / or prophylactic is an mRNA. An mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and my have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.

[0206] In other embodiments, a therapeutic and / or prophylactic is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a LNP including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0207] In some embodiments, a therapeutic and / or prophylactic is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts.

[0208] Gene Editing Agents

[0209] In certain embodiments, the therapeutic agent (suitably coupled / co-administered with an LNP and fiber-hydrogel composite) is a gene editing agent, e.g, Cre recombinase, CRISPR-Cas, etc.

[0210] Programmable nucleases enable precise genome editing by, in some instances, introducing DNA double-strand breaks (DSBs) at specific genomic loci, thereby initiating geneediting. Generally, as embodied herein, a variety of gene editing systems can be employed to target genes, elements, or regions. In some embodiments, the gene editing agents comprise: Cre recombinases, CRISPR / Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators, homologues, orthologs or combinations thereof. In one embodiment, the gene editing agent is a Clustered Regularly Interspaced Short Palindromic Repeated (CRISPR)- associated endonuclease, or homologues or orthologs thereof. In another embodiment, the CRISPR-associated endonuclease is Cas9 or homologues or orthologs, thereof. In some embodiments, the gene editing system comprises a CRISPR-Cas system. In some embodiments, the gene editing system comprises meganucleases. In some embodiments, the gene editing system comprises zinc finger nucleases (ZFNs). In some embodiments, the gene editing system comprises transcription activator-like effector nucleases (TALENs). These gene editing systems can be broadly classified into two categories based on their mode of DNA recognition: ZFNs, TALENs and meganucleases achieve specific DNA binding via protein-DNA interactions, whereas CRISPR-Cas systems are targeted to specific DNA sequences by a short RNA guide molecule that base-pairs directly with the target DNA and by protein-DNA interactions. Accordingly, protein targeting or nucleic acid targeting can be employed to target the DNA loc, e.g. a tumor gene.

[0211] CRISPR-Cas Systems: The CRISPR-Cas system includes a gene editing complex comprising a CRISPR-associated nuclease, e.g., Cas9, and a guide RNA complementary to a target sequence situated on a DNA strand within the target sequence, e.g., in regulatory sequences or structural gene sequences. The mutation can comprise a deletion. The size of the deletion can vary from a single nucleotide base pair to about 10,000 base pairs. The mutation can comprise an insertion, that is, the addition of one or more nucleotide base pairs to the target sequence. The size of the inserted sequence also may vary, for example from about one base pair to about 300 nucleotide base pairs. The mutation can comprise a point mutation, that is, the replacement of a single nucleotide with another nucleotide. Useful point mutations are those that have functional consequences, for example, mutations that result in the conversion of an amino acid codon into a termination codon or that result in the production of a nonfunctional protein.

[0212] In embodiments, the CRISPR / Cas system can be a type I, a type II, or a type III system. Non-limiting examples of suitable CRISPR / Cas proteins include Cas9, CasX, CasY.l, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, SpCas9-HFl, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, ARMAN 1, ARMAN 4, Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f,Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966.

[0213] The Cas9 can be an orthologous. Six smaller Cas9 orthologues have been used and reports have shown that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter.

[0214] In addition to the wild type and variant Cas9 endonucleases described, embodiments of the disclosure also encompass CRISPR systems including “enhanced-specificity” S. pyogenes Cas9 variants (eSpCas9), which dramatically reduce off target cleavage. These variants are engineered with alanine substitutions to neutralize positively charged sites in a groove that interacts with the non-target strand of DNA. This aim of this modification is to reduce interaction of Cas9 with the non-target strand, thereby encouraging re-hybridization between target and non- target strands. The effect of this modification is a requirement for more stringent Watson-Crick pairing between the gRNA and the target DNA strand, which limits off-target cleavage (Slaymaker, I.M. et al. (2015) DOI: 10.1126 / science.aad5227).

[0215] In certain embodiments, three variants found to have the best cleavage efficiency and fewest off-target effects: SpCas9 (K855A), SpCas9 (K810A / K1003A / R1060A) (a.k.a. eSpCas9 1.0), and SpCas9(K848A / K1003A / R1060A) (a.k.a. eSPCas9 1.1) are employed in the compositions. The disclosure is by no means limited to these variants, and also encompasses all Cas9 variants (Slaymaker, I.M. et al. Science. 2016 Jan 1;351(6268): 84-8. doi: 10.1126 / science.aad5227. Epub 2015 Dec 1). The present disclosure also includes another type of enhanced specificity Cas9 variant, “high fidelity” spCas9 variants (HF-Cas9). Examples of high fidelity variants include SpCas9-HFl (N497A / R661A / Q695A / Q926A), SpCas9-HF2 (N497A / R661A / Q695A / Q926A / D1135E), SpCas9-HF3 (N497A / R661A / Q695A / Q926A / L169A), SpCas9-HF4 (N497A / R661A / Q695A / Q926A / Y450A). Also included are all SpCas9 variants bearing all possible single, double, triple and quadruple combinations of N497A, R661 A, Q695A, Q926A or any other substitutions (Kleinstiver, B. P. et al., 2016, Nature. DOI: 10.1038 / naturel 6526).

[0216] As used herein, the term “Cas” is meant to include all Cas molecules comprising variants, mutants, orthologues, high-fidelity variants and the like.

[0217] In general, CRISPR / Cas proteins comprise at least one RNA recognition and / or RNA binding domain. RNA recognition and / or RNA binding domains interact with guide RNAs. CRISPR / Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains. Active DNA-targeting CRISPR-Cas systems use 2 to 4 nucleotide protospacer-adjacent motifs (PAMs) located next to target sequences for self versus non-self discrimination. ARMAN- 1 has a strong ‘NGG’ PAM preference. Cas9 also employs two separate transcripts, CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), for RNA-guided DNA cleavage. Putative tracrRNA was identified in the vicinity of both ARMAN- 1 and ARMAN-4 CRISPR-Cas9 systems (Burstein, D. et al. New CRISPR-Cas systems from uncultivated microbes. Nature. 2017 Feb 9;542(7640):237-241. doi: 10.1038 / nature21059. Epub 2016 Dec 22).

[0218] Proteins / small molecules

[0219] In other embodiments, a therapeutic and / or prophylactic coupled / co-administered with an LNP and fiber-hydrogel composite) is a protein. Therapeutic proteins useful in the nanoparticles in the disclosure include, but are not limited to, gentamycin, amikacin, insulin, erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), Factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferons, heparin, Hepatitis B surface antigen, typhoid vaccine, and cholera vaccine. Other therapeutics and / or prophylactics include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5 -fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracy clines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids).

[0220] In certain embodiments, a therapeutic and / or prophylactic coupled / co-administered with an LNP and fiber-hydrogel composite) is a small molecule drug useful in the treatment of a particular disease, disorder, or condition. Examples of drugs useful in the lipid nanoparticles include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensive agents (e.g., clonidine and hydralazine), anti-depressants (e.g., imipramine, amitriptyline, and doxepin), anti-conversants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotic / antibacterial agents (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, narcotics, and imaging agents.

[0221] In some embodiments, a therapeutic and / or prophylactic coupled / co-administered with an LNP and fiber-hydrogel composite) is a cytotoxin, a radioactive ion, a chemotherapeutic, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic. A cytotoxin or cytotoxic agent includes any agent that may be detrimental to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, teracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol, rachelmycin (CC-1065), and analogs or homologs thereof. Radioactive ions include, but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Vaccines include compounds and preparations that are capable of providing immunity against one or more conditions related to infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, and tuberculosis and can include mRNAs encoding infectious disease derived antigens and / or epitopes.Vaccines also include compounds and preparations that direct an immune response against cancer cells and can include mRNAs encoding tumor cell derived antigens, epitopes, and / or neoepitopes. Compounds eliciting immune responses may include, but are not limited to, vaccines, corticosteroids (e.g., dexamethasone), and other species.

[0222] Methods of Treatment

[0223] Provided also are methods of using the LNP fiber- hydrogel composite to treat a subject suffering or susceptible from a disease or disorder. In particular aspects, an LNP fiber-hydrogel composite is administered to treat a subject suffering from a cancer. In general, the methods for treating a disease, disorder, or condition in a subject comprise administering a therapeutically effective dose of a presently disclosed LNP fiber-hydrogel composite to a subject in need of treatment thereof.

[0224] In certain aspects, the disease to be treated is selected from a cancer or an infection, and the subject is suffering from or susceptible to the caner or infection. In particular embodiments, the cancer is selected from basal cell carcinoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal carcinoma, gastric cancer, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, malignant pleural mesothelioma, Merkel cell carcinoma, metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, small cell lung cancer, squamous cell carcinoma, and urothelial carcinoma.

[0225] In certain embodiments, the infection comprises a viral infection. In particular embodiments, the viral infection is selected from a coronavirus infection, a Zika virus infection, influenza, a flavivirus infection, and a human immunodeficiency virus (HIV) infection.

[0226] In other embodiments, the method further comprises administering an fiber-hydrogel composite and LNP with one or more immune checkpoint inhibitors. In certain embodiments, the immune checkpoint inhibitor is selected from a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor. In particular embodiments, the one or more immune checkpoint inhibitors is selected from Ipilimumab, Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, and Cemiplimab.

[0227] As discussed above, a fiber-hydrogel composite suitably can be formulated in a luid composition and administered such as by injection. The LNP component suitably formulated witha therapeutic agent (e.g. a nucleic acid agent such as an mRNA agent) also may be administered e.g. by injection.

[0228] In one aspect, the subject may first receive administration of a fiber-hydrogel composite. Following the fiber-hydrogel composite (e.g. within 6, 12, 24, 48, 98 hours or more), one or more dosages of the LNP component (suitably together with a therapeutic agent) may be administered to the subject, suitably proximate the site where the fiber-hydrogel component has been or is or will be administered, e.g. within 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 30 cms of the site where the fiber-hydrogel component has been or is or will be administered.

[0229] In various aspects, it may be preferred to co-administer the fiber-hydrogel composite and LNP component to such proximate administration patient sites, i.e. where the fiber-hydrogel composite and LNP component are each administered (either together or as separate compositions) within 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 30 cms of the same patient site, for example where the fiber-hydrogel composite and LNP component are each administered by injection.

[0230] Pharmaceutical Compositions

[0231] Pharmaceutical compositions of the present disclosure comprise the lipid nanoparticles disclosed herein, a therapeutically effective amount of at least one therapeutic agent and / or one or more pharmaceutically acceptable excipient, carrier or diluent. As used herein, the term “pharmaceutically acceptable excipient, carrier or diluent” includes compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In certain embodiments, the pharmaceutical compositions may further comprise at least one tissue-targeting agent, for example, peptide conjugates such as antibodies or fragments thereof, aptamers and the like.

[0232] The pharmaceutical compositions may be in a form suitable for parenteral administration. Depending upon the therapeutic application, for in vivo administration, the compositions comprising anti-inflammatory lipid nanoparticles disclosed herein may be administered to a subject in need thereof intravenously, intradermally, intramuscularly, subcutaneously, sublingual, intratumorally, intracardiac, by intratracheal instillation, bronchial instillation, and / or inhalation.

[0233] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or suspension, which may be formulated according to known procedures. A sterile injectable preparation may also be a sterile injectable suspension in a non-toxic parenterally-acceptable buffer. In other embodiments, the pharmaceutical composition may be lyophilized resulting in the form of a dry powder, wherein the dry powder can be later reconstituted for administration as needed. Dry powder compositions may further comprise bulking agents, for example, sucrose or trehalose.

[0234] Pharmaceutical liquid compositions can be nebulized by use of inert gases. Nebulized suspensions may be breathed directly from the nebulizing device or the nebulizing device can be attached to face masks tent, or intermittent positive pressure breathing machine. Furthermore, solid dosage forms may also be administered via inhalation using dry-powder inhalers. Suspension or dry powder pharmaceutical compositions can be administered orally or nasally from devices which deliver the pharmaceutical composition in an appropriate manner.

[0235] The amount of a therapeutic agent that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the subject treated and the particular route of administration. For further information on routes of administration and dosage regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0236] Further provided herein is a pharmaceutical kit comprising a pharmaceutical composition comprising a fiber-hydrogel composite and LNP component and suitably one or more further therapeutic agents.

[0237] In one aspect, the fiber-hydrogel composite may be packaged separately (e.g. in a container such as sealed vial) from the LNP component( which may be packaged in a separate container such as a sealed vial). One or more therapeutic agents suitably may be formulated and packaged with the LNP component or may packaged separately (e.g. as a third component and in a third container).

[0238] In another aspect, one or more of such components may be packaged together, for example as a single formulation that each of the each of the fiber-hydrogel composite LNP component and optionally with one or more additional therapeutic agents.

[0239] Such kits may further comprise various conventional pharmaceutical kit components such as containers comprising pharmaceutically-acceptable adjuvants, diluents or carriers, and additional containers readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and / or guidelines for mixing the components, can also be included in the kit. In particular, such kits include instructions (e.g. product label) for use of the kit components, e.g. to treat a particular disease or disorder such as cancer.

[0240] In an aspect, the disclosure also provides a method of delivering a pharmaceutical agent in a subject. The method includes combining a pharmaceutical agent and the LNP fiber-hydrogel composite as described herein to form a mixture; and applying the mixture to an intended site of delivery.

[0241] EXAMPLES

[0242] Example 1 : Preparation of fiber-hydrogel composite

[0243] Nanofibers were produced by electrospinning PCL (polycaprolactone, 80 k from Sigma Aldrich). The nanofibers were spun into a random mesh. The spinning parameters were a 10% wt solution of PCL in 90% / l% w / w DCM-DMF, at a flow rate of 0.6 ml / h through a 27 gauge blunt needle 15 cm from the target metal plate. The needle voltage was +10 kV, with the target plate was negatively biased with a voltage of ~3 kV. One mL of solution was spun per round for each of the nanofiber sheet.

[0244] The fibers were then functionalized with a multistep process. Briefly, the fibers were plasma-treated to have reactive groups on the fiber surface, to which acrylic acid was conjugated by UV photoinitiation. The acrylate groups were then reacted with EDC and diazimine to form primary amines. These amines could then be reacted with SMCC to attach maleimide groups, which could readily react with the thiol groups in the hydrogel.

[0245] A composite surgical mesh was prepared using a composite gel formulation with 5.4 mg / mL of thiolated hyaluronic acid (220 KDa, thiolation degree of 25%) and 5.4 mg / mL of PEG- diacrylate (PEG-DA) with 10 mg / mL of functionalized dispersed nanofibers. The polypropylene surgical mesh used was Ethicon Prolene Soft (Product code SPMH). The meshes were cleanedwith serial ethanol soaks and allowed to dry in the biosafety cabinet before use. The lx2 cm rectangles of mesh were placed into the bottom of the 2.5x4.5 cm Teflon molds, two per mold.

[0246] An aliquot of 500 ii L of composite was pipetted on into each mold (for both meshes), then a piece of plastic was placed over the meshes, and pressed down to spread out the composite. The meshes were allowed to gel overnight in 37° C. incubator. The gelled meshes were removed and lyophilized as a final product (see FIG. 21). The mesh could then be rehydrated prior to use.

[0247] In certain embodiments, the functionalized-fiber mesh was cut into sections of 60 mg or less. A 60 mg sample is soaked in ethanol, and then added to the ceramic mortar that has been partially filled with liquid nitrogen. The fiber sample will become very rigid. Keeping the sample cool enough to maintain rigidity, the fiber sheet is cut into "5 mmx5 mm sections with scissors. When the full sheet has been cut, the fibers are ground with the mortar and pestle for ~20 min, keeping the mortar partially full with liquid nitrogen. The fiber shiny is then poured into ethanol. About 1 mg of surfactant is added to the slurry to help prevent fiber entanglement. The suspension is centrifuged for min at 300 G, and the supernatant is discarded. The fibers are allowed to dry overnight. The fibers are then weighed into a secondary centrifuge tube, so that a precise concentration of fibers can be suspended. The fibers are then soaked in ethanol to sterilize, centrifuged, had the supernatant discarded, and allowed to dry overnight in a biosafety cabinet. The fibers are then resuspended to the desired concentration in deionized water, usually 15 mg / mL.

[0248] I o form the hydrogel composite, 1 mL of the fiber-suspension is used to rehydrate 1 vial of HA-SH, resulting in a solution of 15 mg / mL fibers and 10 mg / mL of hyaluronic acid. To 900 pL of this solution, 100 uL of 10% PEG-D A stock solution is added, to give a final concentration of 13.6 mg / mL fiber, 9 mg / mL HA-SH, and 10 mg / mL PEG-DA. This is the formulation for the initial in vivo examples, but other formulations have been made by varying the constituent concentrations.

[0249] The resulting composites were milky white in color (FIG. 1C), as opposed to the transparent hydrogels without the fibers. The composite gels maintained their shape and had good handleability, while the hydrogel-alone group was more prone to tearing. The fibers in the hydrogel were disperse and ranged in length from tens to hundreds of microns. A SEM image of cross-section of a fractured, lyophilized sample composite shows the close association between the fibers and hydrogel component, as well as the high density of dispersed fibers.

[0250] Materials and Methods

[0251] Thiolated hyaluronic acid (HA) was purchased from ESI BIO (Alameda, Calif.). Poly (ethylene glycol) diacrylates was purchased from Laysan Bio, Inc (Arab, Ala.). The followings were obtained from Sigma; polyfs-caprolactone), ethylamino-maleimide, acrylic acid, Toluidine blue O, N-hydroxysuccinimide (NHS), cysteine, bovine serum albumin (BSA), acetic acid and Triton™ X-100. Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin / streptomycin, Alexa Fluor® 568 Phalloidin and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen Life Technologies. Ethyl(dimethylaminopropyl) carbodiimide (EDC) was obtained from AnaSpec, Inc. (Fremont, Calif.). All other chemicals and reagents were of analytical grades.

[0252] Electrospinning of PCL Nanofibers for Rheology Experiments:

[0253] To fabricate two different diameters of PCL fibers, 11.0 and 8.5% (w / v) PCL solution were prepared in a mixture of di chloromethane and dimethylformamide (9: 1, v / v) and a mixture of chloroform and methanol (3: 1, v / v), respectively. Each homogenous PCL solution was loaded a syringe with a metallic needle of 27 G. Then, electrospinning was performed with following parameters; 1.0 ml / h of a feeding rate, 15 kV of an applied positive voltage for a metallic needle, and 12 cm of a distance between the end of a needle to a ground. Morphology of fibers was observed using a field-emission scanning electron microscope (FESEM, JEOL 6700F) and a diameter of fibers was measured with FESEM images using Image! software (US National Institutes of Health, Bethesda, Md.).

[0254] Electrospinning for In Vivo Composites:

[0255] Spinning conditions: 16% w / v PCL (95% 45.000 Mn PCL, 5% 80,000 Mn PCL, both from Sigma) in a solvent mixture of dichloromethane and dimethylformamide (9: 1, w / w). The fibers were spun at a rate of 5.25 ml / h through a blunt 27 gauge needle separated 10 cm from the face of the grounded wheel, spinning at 1000 rpm. The applied voltage was 15 kV and the electrospinning pump was rastered back and forth across the 85 mm travel distance for 140 passes at 2 nim / sec (about 4 h). The fiber sheet was then cut into 14 cm-diameter individual sheets for functionalization.

[0256] Preparation of Surface-Functionalized Fibers with MAL:

[0257] To surface-functionalize on fibers with MAL, a surface of fibers was induced carboxyl groups by grafting poly(acrylic acid) (PAA) according to the literature with a minor modification [Interface Focus 2011, 1, 725-733], Briefly, fibers were plasma-treated under 280 mmHg with oxygen atmosphere at room temperature for 10 min to induce free radicals on a surface of fibers. Then 70 mg of fibers in 10 ml of 3 or 10% (v / v) acrylic acid solution in 0.5 mM NalCh was exposed to UV (36 mW / cm2, DYMAX Light Curing Systems 5000 Flood, Torrington, Conn.) for 90 s for photo-polymerization of PAA on fibers surface (PAA-fibers). After incubating PAA-fibers at room temperature for 20 min, PAA-fibers were washed with 20 mi of deionized water three times to remove unreacted acrylic acid. After completely air-drying PAA-fibers, a density of carboxyl groups on PAA-fibers were determined by toluidine blue O (TBO) assay with the assumption that TBO interacts with a carboxyl group on fibers at 1 : 1 of molar ratio [J Biomed Mater Res 2003, 67, 1093-1104], Briefly, PAA-fibers (1 *1 cm2) were completely immersed in 1 ml of 0.5 mM TBO solution in 0.1 mM of NaOH (pH 10) after soaking 20 pl of 50% (v / v) ethanol and reacted with gentle shaking at room temperature for 5 h. After washing them with 0.1 mM NaOH (pH 10), adsorbed TBO on a surface of PAA-fibers was desorbed using 1 nil of 50% (v / v) acetic acid with vigorous shaking at room temperature for 1 h. Then an optical density'- of supernatant was measured at 633 nm using a microplate reader (BioTeck Synergy?, Winooski, Vt). TBO in 50% (v / v) acetic acid was used as a standard.

[0258] PAA-fibers were ground to prepare fiber fragments using a cryogenic mill (Freezer / Mill 6770, SPEX SamplePrep, Metuchen, N.J.) with following parameters; 10 cycles of 1 min for milling and 3 min for cooling in liquid nitrogen. After collecting PAA-fiber fragments into a 50- ml conical tube, PAA-fiber fragments were completely dispersed in 10 ml of a mixture of isopropylacohol and distilled water (1 :1, v / v) to modify with aminoethyl-MAL on a surface of fibers. Briefly, PAA-fibers were added NHS and EDC to activate carboxyl groups of PAA on fibers. A molar ratio of carboxyl group to NHS and EDC was 1 to 4 and 4, respectively. The activation was performed with gently shaking at room temperature. After 1 h, aminoethyl-MAL was added into the carboxyl groups-activated fibers with 1 to 2 of molar ratio of carboxyl groups to aminoethyl-MAL. Then the reaction was performed with gently shaking at room temperature for 12 h. Surface-functionalized fibers with MAL were lyophilized after washing with distilled w'ater three times. Here, a density of MAL on fibers was on the assumption that all of carboxyl groups on a surface of fibers were completely substituted by MAL. oz

[0259] Preparation of Fiber-HA Hydrogel Composites:

[0260] For preparing a fiber-HA hydrogel composite, thiolated HA and PEGDA were completely dissolved in PBS (pH 7.4) to the desired concentration of 12.5 mg / mL and 100 mg / mL, respectively. MAL-fibers with the desired concentration of 25 mg / mL were completely dispersed in PBS (pH 7.4). The suspension of nanofibers, HA, PEG-DA, and PBS are then serially added to reach the formulation's desired final concentration. After homogenous mixing the composite precursor solution, for rheological studies, 100 pL of the composite precursor solution was poured into a mold (diameter=8 mm) and incubated at 37° C. for 2 h for gelation. For compression studies, 200 pL of precursor solution is added to a cylindrical Teflon mold (diameter=;:6.35 mm, h=6.35 mm) and incubated as above. To observe morphology of cross-section of a fiber-HA hydrogel composite and HA hydrogel using FESEM, a composite and HA hydrogel were dehydrated by serial ethanol washing (10 min each at 50%, 70%, 80%, 90%, 100%, and 100% Ethanol) before either critical point drying (Samdri-795, Tousimis, Rockvillle, Md.) or chemical drying (HDMS). The samples were freeze-fractured in liquid nitrogen to reveal the internal pore structure. The structure was sputter coated with a 10-nm layer of platinum (Hummer 6.2 Sputter System, Anatech UDA, Hayward, Calif.), then imaged with a field-emission SEM (JEOL 6700F, Tokyo Japan).

[0261] F or preparation of the composites for in vivo animal studies, the thiolated HA was reconstituted to 12.5 mg / mL, in PBS. The PEG-DA was dissolved to 100 mg / mL in PBS. The MAL-fibers were resuspended to 25 mg / mL in sterile PBS. The fibers were first combined with the HA solution and allowed to react for 10 min before being combined with the PEG-DA to obtain the desired final concentrations. The suspension was then immediately pipetted into the cylindrical Teflon molds (McMaster-Carr, Robbinsville, N.J.), with 300 pL into cylindrical molds 11.125 mm in diameter and 3 mm in height for the in vivo samples. The gels were then placed into the 37° C. incubator to gel overnight.

[0262] Example 2 - Collagen fiber production

[0263] Bovine source type I collagen solution was purchased from Advanced Biomatrix. Bovine collagen solution was firstly lyophilized overnight to obtain collagen powders, and then type I collagen solution (8 w / v%) was prepared in l,l,l,3,3,3-hexafluoro-2-propanol (HFIP) at room temperature for around 6 hours to make a viscous cloudy electrospinning solution. The electrospinning was performed with the following parameters: 5mL / h of the flow rate; 20-25 kVof the voltage applied to the 22-G metallic needle; 12.5 cm of the collecting distance; 900 rpm of the rotation rate of the metallic collector. This set of parameters results in a mean fiber diameter of around 600 nm. By using carbodiimide chemistry, fibers were immersed in ethanol solution (95% v / v%) containing 50 mM l-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide (EDC) and 20 mM N-hydroxysuccinimide (NHS) for 24 hours. After the crosslinking, fibers were washed in 0.75% glycine solution three times with 5 minutes each time to remove the excessive reagents and to quench the activated fiber surface. The collagen fibers were then broken down to fragments using cryomilling (Freezer / Mill 6770, SPEX SamplePrep). The fragments were filtered through different cell strainers (40 and 100 pm) to reach a relative uniform fiber length.

[0264] Example 3 : Preparation of the collagen fiber-HA hydrogel composite

[0265] A preferred NHC construct comprises composed of three components: hyaluronic acid (HA) network, bovine type I collagen nanofibers, and divinyl sulfone (DVS) crosslinker). Before incorporating the collagen nanofibers (produced in Example 2 above) into the HA network during crosslinking, we optimized the crosslinking conditions for the HA gel phase alone

[0266] Sodium hyaluronate (MW 1.5 MDa) was purchased from LifeCore. HUVECs and vascular endothelial cell culture medium were purchased from Lonza. All other chemical reagents were purchased from Sigma- Aldrich. All other cell culture reagents and supplements were obtained from Invitrogen. HA was dissolved in distilled water at a stock concentration of 25 mg / mL. DVS concentration was calculated as the ratio to the hydroxyl groups in HA (such as 1.17 w / v%, 2.34 w / v%, and 4.68 w / v%). The stock HA solution was diluted to 2 w / v% using distilled water and sodium hydroxide to get four different pHs (12.4, 12.7, 13.0 and 13.3), with other parameters set to be the same (2w / v% HA, 37°C, 3 hour reaction time). The reaction time or gelation kinetics were performed by preparing multiple samples and measuring the mechanical properties at various timepoints (30 min, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours and 16 hours) to get a timepoint where the stiffness reaches a plateau. The crosslinking of the NHCs followed the same conditions to the HA hydrogels, different fiber density (0, 1 and 3 w / v%) were added to the mixed precursors to test the gelation kinetics of the NHCs. After the gelation of hydrogels and NHCs, dialysis was performed using dialysis membranes (6000-8000 MWCO, Spectrum) against pH 7.4 phosphate buffer for 48 hours to remove the unreacted DVS, to balance the pH and to swell the samples for further studies. The mechanical properties were measured again after the swelling.Microgels were then generated with stainless steel wire cloth discs to reach a gel particle size at around 100 pm as we previously reported.

[0267] . We found that using DVS chemistry to crosslink the HA network at a pH of 12.7 was effective in forming a robust crosslinked hydrogel while minimizing the degradation of HA molecules and collagen fibers during gelation. The reaction pH had a large effect upon the resulting gel in the range of pH 12-13.3. With other parameters set to be the same (37 °C, HA concentration 2 w / v%, DVS concentration 2.93 w / v%,), while the reaction pH was set to be 12.4, 12.7, 13.0 and 13.3 prepared by different NaOH concentrations (0.001 M, 0.01 M, 0.1 M, 1 M). The reaction at pH 13.3 and 13.0 showed a dramatic degradation after 2 hours of reaction, resulting in two unstable hydrogels with low reproducibility.

[0268] The crosslinking time was optimized to around 2 hours to reach the maximal storage modulus and limit degradation. After tuning the DVS chemistry, we introduced the nanofibers to the HA network while crosslinking to generate interfacial bonding between the HA network and the nanofibers, and a reinforcement effect was observed comparing HA and NHC at a similar crosslinking density. Additionally, the composite could pass through 27-gauge needle easily after crosslinking. To further investigate this reinforcement effect quantitatively, we measured the storage modulus GO’ (normalized to a storage modulus control) of the HA hydrogel phase, the G’ of the overall NHC and the G’ of a hydrogel-nanofiber mixture without interfacial bonding. In a rheological test with 1 to 3 w / v% of fiber loading density, the G’ of the composite was ranging from 1.5 to 4 folds higher than that without interfacial bonding, and the G’ difference increased with the increase of the fiber loading and the crosslinker concentration. Furthermore, we also investigated the effect of fiber lengths on the stiffness enhancement by using different cell strainers (40 pm, 100 pm, unscreened) to screen out the large fiber fragments. In an unintuitive result, the gels with the fiber fragments with length between 40 pm to 100 pm helped generate the largest stiffness enhancement, though this relative enhancement was minimized at the highest crosslinking concentrations. It is not clear why the 40 - 100 pm fiber size range would be optimal for mechanical reinforcement, as it would generally be expected that longer continuous fibers (> 100 pm group) would increase the gel stiffness further by supporting more of the applied load (since the fibrous component is stiffer than the hydrogel phase), or by becoming more entangled (since longer fibers tend to be more prone to entanglement) with other fibers for efficient load transfer (and thus increased stiffness). It was also possible that shorter fibers (< 40 pm) would result in a stiffer gelat a given concentration by being less entangled and take on more orientations. It was not expected for the intermediate fibers to result in the most efficient mechanical reinforcement.

[0269] These tuning steps allowed us to generate NHC with G’ in the range of 450 Pa to 1500 Pa after crosslinking, and 150-Pa to 1000-Pa after swelling. To mimic the soft tissue microenvironment, HA controls (G’ = 100-Pa and G’ = 250-Pa) and composite (G’ = 250-Pa and G0’=100-Pa) were generated and particularized to microgels with diameters around 100 pm. The storage modulus for all three groups were measured not significantly different from the initial crosslinked bulk gels. Lastly, before utilizing the prepared materials for later in vitro and in vivo studies, we autoclaved the hydrogels and composites and observed that the G’ measurements were not significantly lowered after the terminal sterilization, indicating the translational potential of this material. Autoclaving was performed to sterilize the hydrogels and NHCs after gelation. Briefly, the gels were placed at the autoclave cycle at 118°C with a 5-minute sterilization step. The total sterilization cycle would take 30 minutes to complete. After the sterilization, the mechanical properties of each gel were measured again using rheological tests.

[0270] Example 4: (LiNx) preparation and in vivo use

[0271] In one preferred protocol, to prepare lipid nanoparticles, (LiNx), the synthesized fiber- hydrogel composition (sometimes referred to as NHC microgel suitably having e.g. G' of -250 Pa) was combined with LNPs using the syringe extrusion method. (Z.-C. et al. Small 2022, 18, 2202309). The kinetics of host cell recruitment was examined following injection of LiNx on C57BL / 6 mice (30 pg mOVA per mouse, s.c.). To assess cell transfection in vivo, Ai9 mice were s.c. administered with LiNx containing 30 pg mCre per mouse. Lor tumor inhibition studies, mice aged 6-8 weeks were injected with MC38-OVA or B16-OVA cells (1 x 106in prophylactic and 3 x 105in therapeutic studies, s.c.) into the right flank. Tumor growth was monitored every two days.

[0272] To investigate host cell recruitment in LiNx, we injected mOVA LiNx into mice and harvested composites at 3- and 7-days. Incorporating D6 LNPs significantly increased host immune cell recruitment compared to LiNx itself (Fig. lb). A substantial number of APCs, particularly macrophages, were detected on day 3 (Fig. 1c). We further assessed the delivery efficacy of LiNx using Cre mRNA with Ai9 mice and found that D6 LNPs loaded in the LiNx resulted in substantial tdTom+cell including macrophages and DCs in composites (Fig. Id).

[0273] The anti -tumor effect of D6 LiNx was investigated in MC38-OVA and B6-OVA therapeutic tumor models (Fig. le). D6 LiNx demonstrated potent tumor suppression, resulting in a median survival time of 75 days with 50% of mice remaining tumor-free beyond 100 days (Fig. If). Notably, D6 LiNx outperformed free LNPs given in a standard immunization schedule with three doses (median survival time of 37.5 days), emphasizing the superior efficacy of this single- dose hydrogel vaccine. Additionally, D6 LiNx was assessed in a prophylactic melanoma model (Fig. 1g), showing stronger tumor growth inhibition and a prolonged median survival time of 30 days (Fig. Ih).

[0274] Example 5: Host cell recruitment and transfection profile of mRNA LiNx

[0275] We employed DLin-MC3-DMA as the ionizable lipid, DMG-PEG2000 as the PEGylated lipid, and incorporated six helper phospholipids previously utilized in FDA-approved or experimental LNP formulations, resulting in a comprehensive library of 1,080 LNPs. After thorough screenings, three LNP formulations (CIO, D6, and F5 LNPs) were identified, exhibiting a significantly elevated expression of the OVA-derived SIINFEKL peptide on major histocompatibility complex class I (MHC-I) on BMDCs, along with an enhanced maturation level, marking them as promising candidates for further exploration in the context of mRNA LiNx development. For the preparation of LiNx, the synthesized NHC microgel (with a targeted shear storage modulus G' of -250 Pa) was mixed with mRNA LNP suspension using the syringe extrusion method.

[0276] To investigate the recruitment of host cells within various LiNx composites (CIO, D6, F5, or PBS LiNx), we subcutaneously injected the ovalbumin (OVA) encoding mRNA LiNx into the right flank of mice. The composites were harvested at 3- and 7-days post-injection and the number of viable cells within the composites was quantified using Acridine Orange / Propidium Iodide (AO / PI) staining. As depicted in Fig. 2b, a substantial number of host cells were recruited inside the scaffold for all groups at both time points. Notably, upon incorporating D6 and F5 mRNA LNP into the composites, a significant increase in the number of recruited living cells was observed. In comparison to PBS LiNx, there was a 4.2-fold increase for D6 mRNA LiNx and a 2.9-fold increase for F5 mRNA LiNx in the number of cells recruited inside the composites on day 3. By day 7, there was a 2.9-fold increase for D6 mRNA LiNx and a 2.7-fold increase for F5 mRNA LiNx in the number of cells recruited inside the composites, relative to the PBS LiNx group.Furthermore, a significant presence of APCs, including macrophages (CDl lb+CDl lc cells) and DCs (CD1 lb+CDl lc+cells), was identified within the scaffold on day 3 through flow cytometry analysis. Notably, the D6 mRNA LiNx group exhibited a markedly heightened recruitment of macrophages, with levels 4.4-fold, 8.13-fold, and 2.0-fold higher than those observed in the PBS, CIO, and F5 mRNA LiNx groups, respectively.

[0277] For an efficient hydrogel-based mRNA vaccine, the expression profile of the antigen, along with adequate recruitment of host cells, plays a crucial role in eliciting a robust immune response. To assess the in vivo delivery efficacy of CIO, D6, and F5 mRNA LiNx, we conducted subcutaneous injections using Cre-recombinase mRNA (mCre) LiNx in genetically engineered tdTomato (tdTom) reporter mice (Ai9 mice). These mice contain a LoxP-flanked stop cassette, preventing the expression of the tdTom protein until the Cre recombinase induces its removal, allowing for the expression of the fluorescent tdTom. Our findings revealed that all three LiNx formulations resulted in significant levels of tdTom+cells, including dendritic cells (DCs) and macrophages, in the composites on days 5 and 10 post-injection (Figs. 2d-f). In comparison to CIO and F5 mRNA LiNx, D6 mRNA LiNx exhibited notably higher levels of transfected cells in the composites. Specifically, on day 10, the transfected cells for D6 mRNA LiNx were approximately 129-fold and 117-fold higher than those for the CIO mRNA LiNx and F5 mRNA LiNx groups, respectively. The D6 mRNA LiNx group also showed approximately a 1000-fold higher level of transfected DCs compared to the other two groups. Additionally, the transfected macrophage level was 2125-fold and 1691-fold higher for D6 mRNA LiNx compared to CIO mRNA LiNx and F5 mRNA LiNx, respectively.

[0278] Taken together, these results above underscore the promising performance of the LiNx platform, especially D6 mRNA LiNx in facilitating in vivo host cell recruitment and the delivery efficacy of antigen- encoding mRNA within a short timeframe. The difference observed among different mRNA LiNx candidates may be attributed to differences in mRNA LNPs' transfection efficiency, stability within composites, and the distinct immunoadjuvant effects associated with various lipid compositions. mRNA LiNx effectively forms local immunostimulatory niche

[0279] The cellular profiles of infiltrated cells within the composites were assessed at a later time point, specifically on day 14 post s.c. injection using mOVA LiNx. As depicted in Fig. 3a,even after 14 days, a substantially higher number of infiltrated host cells persisted in both D6 and F5 LiNx groups. In comparison to PBS LiNx and CIO LiNx, D6 LiNx and F5 LiNx have approximately a 9-fold increase in terms of the number of infiltrated host cells within the composites. Moreover, our results indicated a significant increase in the presence of CD4+T cells (Fig. 3b), CD8+T cells (Fig. 3c), and B cells (Fig. 3d) within the D6 LiNx composites compared to other groups (with a 73 -fold higher CD4+T cell count, 200-fold higher CD8+T cell count, and 305-fold higher B cell count for the D6 LiNx group compared to the PBS LiNx group). The proportion of specific immune cell types was assessed for various treatment groups, as illustrated in Fig. 3e. At this time point, the predominant immunocytes within the composites for PBS LiNx, CIO LiNx, and F5 LiNx were neutrophils. Approximately 60% of immunocytes within the composites were neutrophils for both PBS and CIO LiNx groups, while it increased to around 80% for the F5 LiNx group. Nevertheless, in the D6 LiNx group, a notable presence of T cells and B cells was observed within the composites. Approximately 50% of the immunocytes within the composite consisted of T cells (with around 20% being CD8+T cells and 30% being CD4+T cells), while B cells constituted about 20% of the immunocytes. These findings suggest that in the case of D6 LiNx, an immunostimulatory niche formed, marked by a substantial influx of adaptive immune cells such as T cells and B cells into the composite. This scenario is highly advantageous for facilitating crosstalk among immune cells and inducing a robust immune response.

[0280] The local microenvironments influenced by various LiNx formulations were further characterized using real-time polymerase chain reaction (PCR) arrays, analyzing RNA extracted from the local injection sites on day 14 post-injection. The mRNA levels related to immune responses were detected, quantified, and analyzed (Figs. 3f-g). The findings suggest that, in contrast to other groups, the site of local injection for D6 mOVA LiNx displayed increased expression of genes associated with inflammatory cytokines and chemokines, including IL-6, IL- la, CSF-3, and CXCL10. Moreover, there were elevated levels of genes linked to Thl immune responses, such as TBX21, TNF, IFN-y, GZMB, NOS-2, IL-12a, IL-15, and IL-2, as well as genes associated with Th2 immune responses, namely IL-4, CCR-4, and IL-13.

[0281] The above results indicate that 14 days post- injection, substantial cell presence was observed in D6 LiNx composites. The majority of these cells were associated with adaptive immune responses, specifically T cells and B cells, rather than neutrophils. This indicates an ongoing crosstalk among immune cells, suggesting the potential for induction of robust immuneresponses. Moreover, the D6 LiNx group exhibited the formation of an immunostimulatory microenvironment characterized by elevated levels of immunostimulatory cytokines and the presence of Thl and Th2-related genes.

[0282] LiNx induces potent antigen-specific immune responses

[0283] The vaccination potential of the three LiNx formulations was further tested in mice following s.c. injections with a single dosage at day 0, consisting of 30 pg OVA mRNA, with PBS LiNx serving as the control. The LiNx-induced antigen-specific CD8+T cell response was initially examined by collecting and analyzing cells from both composites and draining lymph nodes (dLNs) of treated mice on day 14. As depicted in Figs. 4a-b, the D6 LiNx group demonstrated a significantly higher level of antigen-specific CD8+T cells compared to the CIO LiNx and F5 LiNx groups, with a ~64-fold and ~l l-fold increase detected inside the composites, respectively. Moreover, within the dLNs, the D6 LiNx group exhibited a ~3.7-fold and ~2.9-fold higher number of antigen-specific CD8+T cells compared to the CIO LiNx and F5 LiNx groups, respectively.

[0284] Spleens from vaccinated mice were harvested on day 14, homogenized into a cell suspension, and subjected to ex vivo antigen restimulation. The D6 LiNx group displayed increased frequencies of CD8+IFN-y+, CD8+TNF-a+, and CD8+Granzyme B+cell populations (Figs. 4c-e). Specifically, compared to the CIO and F5 LiNx treated groups, the D6 LiNx group exhibited approximately 9.0- and 6.2-fold increases in CD8+IFN-y+cell frequencies, respectively. Alongside the robust CD8+T cell response, the D6 LiNx group also showed significantly higher numbers of Thl cells (CD4+IFN-y+and CD4+TNF-a+), as depicted in Figs. 4f-g. On day 14 post- vaccination, there was a ~16.4-fold higher level of antigen-specific CD4+IFN-y+Thl cells and a 3.0-fold higher level of antigen-specific CD4+TNF-a+Thl cells for the D6 LiNx group. In contrast, no significant increases were observed in the CIO and F5 LiNx-treated groups. Furthermore, the frequency of antigen-specific CD4+IL-4+Th2 cells was examined for all groups. In comparison to the PBS LiNx-treated group, the D6 LiNx-treated group exhibited a significantly higher number of antigen-specific Th2 cells (2.8-fold increase, P = 0.0156), contrasting with the CIO andF5 LiNx groups (1.8- and 1.7-fold higher, respectively; P > 0.5).

[0285] The antigen-specific response after vaccination was further evaluated on day 30 post- vaccination. As depicted in Fig. 4i, spleens from vaccinated mice were collected on day 30, homogenized into a cell suspension, and subjected to ex vivo antigen restimulation. The resultconfirmed that D6 LiNx induced a substantially higher antigen-specific T-cell response. Comparative analysis with CIO LiNx and F5 LiNx unveiled approximately 3.7-fold and 3.8-fold higher levels of IFN-secreting cells within the D6 LiNx group. In addition, as shown in Fig. 4j, D6 LiNx induced a significantly higher OVA-specific IgG titer including both IgGl and IgG2c subclass titers, indicating a potent humoral response. However, the antibody responses generated by CIO and F5 LiNx groups were limited.

[0286] The immune memory responses of LiNx were evaluated three months post- vaccination. Substantial levels of antigen-specific CD8+T cells were still present in the spleen of mice from the D6 LiNx group (Fig. 4k). Significantly, within these T cells, there were both CD44+CD62L" effector T cells and notably higher levels of CD44+CD62L+central memory T cells. Specifically, the D6 LiNx group exhibited approximately a 23.8-fold higher level of antigen-specific effector T cells and a 10.1 -fold higher level of central memory T cells, underscoring the establishment of long-term memory immune responses (Figs. 41-m). Spleenocytes from vaccinated mice were harvested on day 90 and subjected to ex vivo antigen restimulation. As depicted in Figs. 4n-o, the D6 LiNx group exhibited increased frequencies of CD8+TNF-a+(5.0-fold higher than PBS LiNx group, P=0.0006) and CD8+IFN-y+(5.3-fold higher than PBS LiNx group, P=0.0001) cell populations. Furthermore, an examination of the biosafety profile for CIO, F5, and D6 LiNx revealed no significant differences in terms of body weight across all LiNx groups during the vaccination schedule.

[0287] The above results indicate that following a single-dose vaccination, D6 LiNx effectively elicited a more potent antigen-specific Thl-type and Th2-type immune response compared to CIO and F5 LiNx. Moreover, D6 LiNx vaccination resulted in a strong and persistent memory immune response. In contrast, no memory immune responses were observed three months after vaccination with CIO and F5 LiNx.

[0288] Anti-tumor effects induced by a single-dose vaccination of D6 mRNA LiNx

[0289] Given the potent antigen-specific immune responses induced by the D6 mRNA LiNx, we investigated its efficacy as a cancer vaccine in various therapeutic and prophylactic tumor models. C57BL / 6 mice were subcutaneously inoculated with 3 x 105MC38-OVA cells on the right posterior side on day 0. On day 4, the mice received vaccinations with CIO, D6, or F5 mRNA LiNx, each containing 30 pg of mOVA (Fig. 5a). Control groups included PBS LiNx, OVA protein(30 pg of OVA per mouse) mixed with Alhydrogel®, or LiNx. Additionally, C57BL / 6 mice were immunized on days 4, 11, and 18 with D6 LNPs containing 10 pg of mOVA as a control. As illustrated in Figure 4b-d, the D6 mRNA LiNx exhibited a significant tumor suppression effect in this treatment model, resulting in a median survival time of 75 days compared to 31 days for the negative control group. For D6 mRNA LiNx group, there were 50% of the mice remained tumor- free beyond 100 days. Nevertheless, when contrasted with D6 mRNA LiNx, the mice treated with PBS LiNx, OVA LiNx, Alhydrogel® with OVA, CIO mRNA LiNx, and F5 mRNA LiNx exhibited restricted therapeutic efficacy, leading to limited extension of median survival time. In comparison to the D6 mRNA LiNx-treated group receiving a single dose, the group treated with three doses of free D6 mRNA LNPs exhibited limited efficacy, resulting in a median survival time of 37.5 days. These findings suggest that D6 mRNA LiNx not only produced a markedly more robust antitumor effect when compared to other LiNx formulations (C10 andF5 mRNA LiNx) and negative controls (PBS LiNx, OVA LiNx, and Alhydrogel® with OVA), but also demonstrated superior efficacy compared to traditional free LNPs that requires three doses. This single-dose hydrogel vaccine has the capability of further enhancing the anti-tumor effect of mRNA LNPs.

[0290] We further evaluated the therapeutic efficacy of D6 mRNA LiNx in the B16F10 tumor model, utilizing the model OVA antigen, as well as two other clinically relevant antigens — tyrosinase- related protein 2 (Trp2) and glycoprotein 100 (GplOO). Initially, C57BL / 6 mice were subcutaneously inoculated on the right posterior side with 3 x 105B16F10-OVA cells on day 0. On day 4, mice were vaccinated with D6 mRNA LiNx containing 30 pg mOVA (Fig. 5e). As depicted in Figs. 5f-g, D6 mRNA LiNx demonstrated a notable tumor suppression effect in this treatment model, resulting in a median survival time of 30.5 days compared to 17 days for the negative control group. When D6 mRNA LiNx was administered in conjunction with an immune checkpoint inhibitor (100 pg anti-CTLA-4 monoclonal antibody, given i.p. on days 6, 13, 20, and 27), a synergistic effect was observed, extending the median survival time to 42 days. In contrast, no significant tumor suppression effect was observed in the group treated solely with α- CTLA-4 antibody compared to the PBS control.

[0291] Subsequently, D6 mRNA LiNx was evaluated in the same mouse model using clinically relevant tumor antigens Trp2 and GplOO instead of the model antigen OVA (Fig. 5e). C57BL / 6 mice were subcutaneously inoculated on the right posterior side with 3 x 105B16F10 cells on day 0. On day 4, mice were vaccinated with D6 mRNA LiNx containing 30 pg of mRNA encodingeither Trp2 or GplOO. The potent anti-tumor effect was also observed with these two antigens, resulting in substantially prolonged median survival times of 22.5 and 21.5 days for D6 mTrp2 LiNx and D6 mGplOO LiNx, respectively (Figs. 5h-i). However, no significant improvement was observed when combining these D6 mRNA LiNx with CTLA-4 anαt-ibody treatment.

[0292] Beyond the therapeutic cancer model, D6 mRNA LiNx was further assessed in a polyphyletic melanoma model. C57BL / 6 mice were immunized on day 0 with D6 mRNA LiNx containing 30 pg of mOVA. On day 21, animals were subcutaneously inoculated on the right posterior side with 1 x 106B16F10-OVA cells (Fig. 5j). The D6 mRNA LiNx treated group exhibited a significantly stronger tumor growth inhibition rate with prolonged overall survival times compared to the PBS, PBS LiNx, and OVA LiNx groups. The median survival time was 30 days for the D6 mRNA LiNx group, compared to 16 days for the PBS group and 17 days for the PBS LiNx and OVA LiNx groups (Figs. 5k-l).

[0293] LiNx amplifies the immune response of mRNA LNPsTo comprehensively evaluate the immune responses triggered by D6 mRNA LNP and mRNA LNPs, we analyzed the expression of 547 genes in splenocytes from vaccinated mice using the nCounter Analysis System (NanoString Technology). In detail, the vaccination efficacy of the D6 mRNA LiNx formulations was tested in mice through subcutaneous injections with a single dose on day 0, comprising 30 pg OVA mRNA, while three doses of D6 mRNA LNP (10 pg OVA mRNA) on days 0, 7, and 14 served as the control. Spleens from vaccinated mice were collected on day 21, homogenized into a cell suspension, and subjected to ex vivo antigen restimulation. As depicted in Fig. 6a-c, the comparison between untreated mice and the D6 mRNA LiNx treated group revealed 376 genes with significant differences, including 25 downregulated and 351 upregulated genes. Similarly, when comparing the D6 mRNA LiNx treated group with the three- dosage mRNA LNPs treated group, 344 genes exhibited significant differences, consisting of 35 downregulated and 309 upregulated genes. Upon closer examination of various subsets of related genes, including Thl, Th2, Thl7, Treg differentiation-related genes, and B cell receptor signaling genes, we observed notable differences. In comparison to the three dosages of D6 mRNA LNPs treated group, the D6 mRNA LiNx treated group exhibited upregulation in 6 out of 15 Thl differentiation- related genes, 9 out of 16 Th2 differentiation- related genes, 13 out of 19 Thl 7 differentiation-related genes, and 12 out of 30 B cell signaling-related genes (Fig. 6d-e). Regardingregulatory T cell-related genes, there were no significant differences, except for the downregulation of 1 out of 7 genes in the D6 mRNA LiNx treated group. By employing Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to establish connections between the information and higher-order functional aspects, we discovered that the LiNx platform, when utilized, augmented immune responses through multiple pathways (Fig. 6f). In addition to its positive impact on conventional Thl and Th2 cell differentiation, antigen presentation, and the B cell T cell receptor signaling pathway, a noteworthy revelation emerged: Thl 7 differentiation and the IL- 17 signaling pathway exhibited significant enhancement following D6 mRNA LiNx treatment. This unexpected finding suggests that the heightened effectiveness of D6 mRNA LiNx treatment in anticancer outcomes, as compared to D6 mRNA LNP, may be attributed to the substantial enhancement of Thl7-cell-mediated anti-tumor immunity, which is recognized for its considerable therapeutic potential.Example 6:

[0294] RESULTS

[0295] Host cell recruitment and transfection profile of mRNA LiNxThe three top-performing mRNA LNP formulations (CIO, D6, and F5 LNPs, Supplementary Table 1) identified from our previous study were selected to prepare the LiNx.16 All three mRNA 130 LNPs led to high levels of expression of the OVA-derived SIINFEKL peptide on major 131 histocompatibility complex class I (MHC-I) on BMDCs and enhanced maturation of BMDCs, 132 marking them as promising candidates for exploration in the context of the LiNx. The NHC 133 microgel with a shear storage modulus G' of 250 Pa was chosen for its previously reported 134 effectiveness in cell recruitment, migration, and retention (FIG. 13a). We mixed 135 the NHC microgel particles with mRNA LNPs using the syringe extrusion method immediately before injection (FIG. 13b). Scanning electron microscopy images revealed that, after encapsulating LNPs within the NHC, the same fibrillar microarchitecture 137 of the NHC is 138 observed, with nanofibers entrapped within the HA hydrogel network (FIG. 14). The accelerated stability test of NHC at 50°C showed that, at this temperature, NHC stiffness was 140 reduced by approximately 40% after 7 days and around 70% after 14 days (FIG. 15). Based on a scaling factor calculated empirically, the NHC scaffold used in this study can degrade over a period of 3 to 4 months at37°C. To investigate host cell recruitment within various LiNx formulations loaded with CIO, D6, or F5 containing ovalbumin (OVA)-encoding mRNA, or PBS, respectively, we subcutaneously injected the LiNx into the right flank of C57BL / 6 mice. The composites were harvested at 3 and 7 days post- injection, and the number of viable cells within the composites was quantified using Acridine Orange / Propidium Iodide (AO / PI) staining. As depicted in FIG. 7b, a substantial number of host cells were recruited inside the scaffold for all groups at both time points. Notably, the incorporation of D6 and F5 mRNA LNPs into the composites markedly increased the number of recruited cells. In comparison to PBS LiNx, there was a 4.2-fold increase for D6-mRNA LiNx and a 2.9-fold increase for F5-mRNA LiNx in the number of cells recruited inside the composites on day 3. By day 7, there was a 2.9-fold increase for D6-mRNA LiNx and a 2.7-fold increase for F5- mRNA LiNx in the number of cells recruited inside the composites relative to the PBS LiNx group. Furthermore, a notable presence of APCs, including macrophage-like cells (CD3-CDl lb+Ly6g- CD11c- cells) and DC-like cells (DCs; CD3-CD1 Ib+Ly6g-CD1 lc+ cells), was identified within the composites on day 3 by flow cytometry analysis. Notably, the D6-mRNA LiNx group exhibited the highest level of recruitment of macrophage-like cells, which were 4.4-fold, 8.13-fold, and 2.0- fold higher than those observed in the PBS, CIO, and F5-mRNA LiNx groups, respectively (FIG. 7c, FIG. 16). For an efficient vaccine, the expression profile of the antigen, along with adequate recruitment of host cells, plays a crucial role in eliciting a robust immune response.24 To assess the in vivo delivery efficacy of CIO-, D6-, and F5-mRNA LiNx, we conducted s.c. injections of LiNx loaded with Cre recombinase mRNA (mCre) in genetically engineered tdTomato (tdTom) reporter mice (Ai9 mice). These mice contain a LoxP-flanked stop cassette, preventing the expression of the tdTom protein until it is removed by Cre recombinase, allowing for the expression of tdTom. Our findings revealed that all three LiNx formulations resulted in notable levels of tdTom+ cells, including DC like cells, macrophage-like cells, and CDl lc-CDl lb- cells in LiNx 167 on days 5 and 10 post-injection (FIG. 7d-f, FIGS. 17-19). In comparison to C10- and F5-mRNA LiNx, D6-mRNA LiNx exhibited a substantially higher level of transfected cells in the composite. On day 10, the number of transfected cells in D6 LiNx was approximately 129- fold and 117-fold higher than those for the CIO LiNx and F5 LiNx groups, respectively. The D6- mRNA LiNx group also showed approximately a 1,000-fold higher number of transfected DC-like cells compared to the other two groups. Additionally, the number of transfected macrophage-like cells was 2,125-fold and 1,691-fold higher in D6 LiNx compared to CIO LiNx and F5 LiNx,respectively. The biodistribution of LiNx formulations was further examined using Cy5-labeled mRNA, as shown in FIG20a. All three mRNA LNPs were gradually released from the LiNx injection site over two weeks post-injection. Among the formulations, D6 exhibited the most delayed release profile, with approximately 25% of mRNA LNPs still retained within the LiNx platform. Additionally, at 24 hours post-vaccination, D6 LiNx showed that most LNPs remained at the injection site, with no detectable signal in other major organs (FIG. 20b-c). The decrease in signal at the injection site observed initially was likely due to the uptake and degradation of Cy5- mRNA by local or recruited cells. In terms of transfection efficiency of D6 LiNx was further validated using mLuc mRNA. The data revealed that D6 LiNx transfection was largely confined to the injection site at 24 hours post- injection, with approximately 97% of the transfection signal detected there (FIG. 7g-i) and minimal transfection observed in other organs. Taken together, these results underscore the promise of the LiNx platform, especially D6 LiNx, in facilitating in vivo host immune cell recruitment and delivering antigen-encoding mRNA.

[0296] mRNA LiNx effectively forms a local immunostimulatory niche

[0297] The cellular profiles of infiltrated cells within the composites were assessed at a later time point, specifically on day 14 post s.c. injection using mOVA LiNx. As depicted in Fig. 8a, even after 14 days, a substantially higher number of infiltrating host cells persisted in both D6 and F5 LiNx groups. In comparison to PBS LiNx (PBS+NHC) control and CIO LiNx, D6 LiNx and F5 LiNx showed approximately a 9-fold increase in terms of the number of infiltrated host cells within the composites. Moreover, a substantial increase in the presence of CD3+CD4+ T cells (Fig. 8b, Fig. 21), CD3+CD8+ T cells (Fig. 8c, Fig. 22), and CD3-CD1 Ib-Npk46-CD19+ B cells (Fig. 8d, Fig. 23) were detected in the D6 LiNx compared to other groups, showing a 73 -fold higher CD4+ T cell count, a 200-fold higher CD8+ T cell count, and a 305-fold higher B cell count for the D6 LiNx, compared to the PBS LiNx control. The proportion of specific immune cell types on day 14 was assessed for various treatment groups, as illustrated in Fig. 8e (Fig. 24). The predominant immunocytes within the composites for PBS LiNx control, CIO LiNx, and F5 LiNx were neutrophils, with approximately 60% of immunocytes within the composites were neutrophils for both PBS LiNx and CIO LiNx groups, and around 80% for the F5 LiNx. In contrast, the D6 LiNx contained substantial amounts of T cells and B cells: approximately 50% of the immunocytes within the LiNx were T cells, ~20% CD8+ T cells, and ~30% CD4+ T cells, while B cells constituted about 20% of the immunocytes. The immunostimulatory niche generated in D6LiNx, characterized by a substantial influx of T cells and B cells, is highly advantageous for facilitating crosstalk among various immune cell types. The local microenvironments influenced by various LiNx formulations were further characterized using real-time polymerase chain reaction (PCR) arrays, analyzing RNA extracted from the local injection sites on day 14 post-injection. The mRNA levels related to immune responses were detected, quantified, and analysed (Fig. 8f-g). The findings suggest 233 that, in contrast to other groups, the local injection site for D6 mOVA LiNx displayed increased expression of genes associated with inflammatory cytokines and chemokines, including IL-6, IL- la, CSF-3, and CXCL10. Moreover, there were elevated levels of genes linked to Thl immune responses, such as TBX21, TNF, IFN-y, GZMB, NOS-2, IL-12a, IL-15, and IL-2, as well as genes associated with Th2 immune responses, namely IL-4, CCR-4, and IL-13 (Figs. 25-27). The above results reveal that, at 14 days post- injection, substantial host immune cell recruitment events were observed in the LiNx groups, particularly the D6 LiNx. The majority of these cells were associated with adaptive immune responses, specifically T cells and B cells, rather than neutrophils. The close proximity of these immune cells may enable crosstalk among them, potentially advantageous for the induction of a robust immune response. Moreover, the D6 LiNx group exhibited the formation of an immunostimulatory microenvironment characterized by elevated levels of immunostimulatory cytokines, transcripts related to both Thl and Th2 responses, as well as an engaged innate inflammatory response.

[0298] LiNx induces a potent antigen- specific immune response

[0299] The vaccination outcomes of the three LiNx formulations were assessed in C57BL / 6 mice271 following s.c. injections with a single dosage consisting of 30 pg OVA mRNA, with PBS LiNx serving as the control. The LiNx-induced antigen-specific CD8+ T cell response was initially examined by collecting and analyzing cells from both LiNx and draining lymph nodes (dLNs) of the treated mice on day 14. As depicted in Fig. 9a-b and Figs. 28-29, the D6 LiNx demonstrated a substantially higher level of antigen-specific CD8+ T cells compared to the CIO and F5 LiNx groups, with ~64-fold and ~11-fold increases detected inside the niches, respectively. Moreover, within the dLNs, the D6 LiNx exhibited a 3.7-fold and 2.9-fold higher number of antigen-specific CD8+ T cells, compared to the CIO and F5 LiNx groups, respectively. In contrast, the LiNx loaded with empty D6 LNPs did not generate a detectable immune response (Fig. 30). The spleens from vaccinated mice were harvested on day 14, dissociated into a cell suspension, and subjected to ex vivo antigen restimulation. The D6 LiNx yielded increased frequencies ofCD8+IFN-y+, CD8+TNFa+, and CD8+Granzyme B+ cell populations (Fig. 9c-e, Fig. 31). Specifically, compared to the CIO and F5 LiNx-treated groups, the D6 LiNx exhibited approximately 9.0- and 6.2-fold increases in CD8+IFN-y+ cell frequencies, respectively. Alongside the robust CD8+ T cell response, the D6 LiNx also showed higher numbers of Thl cells (CD4+IFN-y+ and CD4+TNFa+), as depicted in Fig. 9f-g. On day 14 post-vaccination, there was a 16.4-fold higher level of antigen-specific CD4+IFN-y+ Thl cells and a 3.0-fold higher level of antigen specific CD4+TNFa+ Thl cells for the D6 LiNx group. In contrast, no significant increases were observed in the CIO and F5 LiNx-treated groups. Furthermore, the frequency of antigen- specific CD4+IL-4+ Th2 cells was examined for all groups (Fig. 9h). In comparison to the PBS LiNx control, the D6 LiNx group exhibited a higher number of antigen-specific Th2 cells (2.8- fold increase, P = 0.0156), in contrast to the CIO and F5293 LiNx groups (1.8- and 1.7-fold higher, respectively; P > 0.5). The antigen-specific response after vaccination was further evaluated on day 30 post- vaccination. As depicted in Fig. 9i, the splenocytes from vaccinated mice collected on day 30 were subjected to in vitro antigen restimulation. The D6 LiNx induced a substantially higher level of antigen specific T-cell response (Fig. 32). Approximately 3.7-fold and 3.8-fold higher levels of IFN-y-secreting cells were detected within the D6 LiNx group, compared with CIO LiNx and F5 LiNx, respectively. In addition, as shown in Fig. 9j, D6 LiNx induced a higher OVA301 specific IgG titer including both IgGl and IgG2c subclass titers, indicating a potent humoral response (Fig. 33) as well. The antibody responses generated by CIO and F5 LiNx groups were limited. The memory immune responses of LiNx were evaluated at three months post- vaccination.5 Substantial levels of antigen-specific CD8+ T cells were still present in the spleen of mice in the D6 LiNx group (Fig. 9k, Figs. 34-35). More importantly, within these T cells, there were both CD44+CD62L- effector T cells and higher levels of CD44+CD62L+ central memory T cells. Specifically, the D6 LiNx group exhibited approximately a 23.8-fold higher level of antigen-specific effector T cells and a 10.1 -fold higher level of central memory T cells, underscoring the establishment of long-term memory responses (Fig. 91-m). Splenocytes harvested from vaccinated mice on day 90 were also subjected to ex vivo antigen restimulation. As depicted in Fig. 9n-o, the D6 LiNx group exhibited 5.0-fold higher CD8+TNFa+ cells than the PBS LiNx control (P = 0.0006) and 5.3-fold higher CD8+IFN-y+ cells than the PBS LiNx control (P = 0.0001) (Figs. 36-38). In addition to T cell responses, a substantially higher OVA-specific IgG titer, including both IgGl and IgG2c subclass titers, was observed in the D6 LiNx-treatedgroup at 90 days post-vaccination, indicating a potent long-lasting humoral response (Fig. 9p, Fig. 39). Furthermore, an examination of the biosafety profiles for CIO, F5, and D6 LiNx showed no significant differences in body weight across all LiNx-treated groups throughout the vaccination schedule, and no significant changes in serum cytokine levels (IFN-y,0 TNF-a, IL-4, IL-10, and IL-2) post-vaccination in the D6 LiNx treated mice (Figs. 40-41). The above results indicate that, following a single-dose vaccination, D6 LiNx 322 effectively elicited more potent antigen-specific Thl and Th2 responses than CIO and F5 LiNx. Moreover, D6 LiNx vaccination resulted in a strong and persistent memory immune response. In contrast, no memory immune responses were observed three months after vaccination in the CIO and F5 LiNx groups.

[0300] Anti-tumour effects induced by a single-dose vaccination of D6 mRNA LiNx

[0301] Given the potent antigen-specific immune responses induced by the D6 LiNx, we investigated its efficacy as a cancer vaccine in therapeutic and prophylactic tumour models. In the first study, C57BL / 6 mice were s.c. inoculated with 3 x 105 OVA-expressing MC38 colorectal cancer cells on the right posterior side on day 0. On day 4, the mice received vaccinations with CIO, D6, or F5 mRNA LiNx, each containing 30 pg of mOVA (Fig. 10a). Control groups included the NHC and OVA protein (30 pg of OVA per mouse) mixed with Alhydrogel® or OVA in NHC. Additionally, C57BL / 6 mice were immunized with 3 doses of D6 LNPs containing 10 pg of mOVA on days 4, 11, and 18, as a control. As illustrated in Fig. 4b-d, the single dose D6 LiNx exhibited effective tumour suppression in this treatment model, resulting in a median survival time of 75 days compared to 31 days for the negative control group (Fig. 42). In the D6 LiNx group, 50% of the mice remained tumour-free beyond 100 days. Mice treated with the NHC, OVA in NHC, OVA in Alhydrogel®, CIO LiNx, and F5 LiNx exhibited limited levels of therapeutic efficacy, leading to low extension of median survival time. In comparison to the group treated with the standard three doses D6 mRNA LNP immunization, which resulted 367 in a median survival time of 37.5 days, a single dose of D6 LiNx produced a markedly improved antitumour effect, demonstrating the capability of LiNx in enhancing the anti-tumour effect of mRNA LNPs. We further evaluated the therapeutic efficacy of D6 LiNx in the B16-OVA melanoma tumour model, utilizing the model OVA antigen. C57BL / 6 mice were s.c. inoculated on the right posterior side with 3 x 105 B16-OVA cells on day 0. On day 4, with an average tumour size of ~25 mm3, mice were vaccinated with D6 LiNx containing 30 pg mOVA (Fig. lOe). As depicted in Fig. lOf-g, D6 LiNx demonstrated a stronger tumour suppression effect in this treatment model, resulting in amedian survival time of 30.5 days compared to 17 days for the negative control (Fig. 43). As shown in Fig. 2f, the local microenvironment at the injection site in D6 LiNx-treated group exhibited a highly elevated CTLA-4 marker, which prompted us to combine anti-CTLA-4 therapy with the LiNx vaccination to evaluate their combined effect. When D6 LiNx was administered in conjunction with an immune checkpoint inhibitor (100 pg anti-CTLA-4 monoclonal antibody, given i.p. on days 6, 13, 20, and 27), a synergistic effect was observed, extending the median survival time to 42 days. In contrast, no significant tumour suppression effect was observed in the group treated solely with anti-CTLA-4 antibody, as compared to the PBS LiNx control. Subsequently, the D6 LiNx was evaluated in the syngeneic melanoma mouse model using two clinically relevant tumour antigens, tyrosinase-related protein 2 (Trp2) and glycoprotein 100 (GplOO) (Fig. lOe). C57BL / 6 mice were s.c. inoculated on the right posterior side with 3 x 105 B16F10 cells on day 0. On day 4, with an average tumour size of ~25 mm3, mice were vaccinated with D6 LiNx containing 30 pg of mRNA encoding either Trp2 or GplOO. The potent anti-tumour effect was observed with these two antigens, resulting in prolonged median survival times of 22.5 and 21.5 days for D6 mTrp2 LiNx and D6 mGplOO LiNx, respectively (Fig. lOh-i, Fig. 44). However, no significant improvement was observed when combining these two D6 LiNx formulations with anti-CTLA-4 antibody treatment. Beyond the therapeutic cancer model, D6 LiNx was also assessed in a prophylactic B16-OVA melanoma model. C57BL / 6 mice were immunized on day 0 with D6 LiNx containing 30 pg of mOVA. On day 21, animals were s.c. inoculated on the right posterior side with 1 x 106 B16-OVA ells (Fig. lOj). A single injection of D6 LiNx yielded a stronger tumour 396 inhibition efficacy with prolonged overall survival times compared to the PBS, PBS-NHC, and OVA-NHC groups. The median survival time was 30 days for the D6 LiNx group, compared to 16-17 days for the three control groups (Fig. 10k-l, Fig. 45). In addition, LiNx loaded with empty D6 LNPs did not generate a significant anti-tumour effect (Fig. 46). To further confirm the efficacy of the D6 mOVA LiNx and assess its long-term protective effect, we conducted a tumour rechallenge study with an increased sample size (Fig. 47). C57BL / 6 mice were immunized as previously described and, on day 21, were subcutaneously inoculated with 3 x 105B16-OVA cells on the right posterior side. Survival was monitored over 100 days, and 17 of 18 mice (94.4%) remained tumour-free following the single D6 LiNx vaccination (Fig. 47b). These mice were subsequently rechallenged with 3 x 105 B16-OVA cells on the right posterior side. As shown in Fig. 47c, 11 of 17 (64%) mice remained tumour- free post-rechallenge, demonstrating the long-term protective effect of D6 LiNx. These findings further confirm the antitumour efficacy of the D6 LiNx platform and highlight its potential for sustained protection.

[0302] Modulation of the tumour microenvironment by LiNx

[0303] To assess the effect of LiNx treatment on the tumour microenvironment, we analysed the infiltrating immune cells in the tumour mass. As shown in Fig. Ila, C57BL / 6 mice were inoculated 438 with 3 x 105 B16-OVA cells on day 0. Starting on day 4, mice received three subcutaneous (s.c.) injections of mOVA-loaded D6 LNPs (10 pg mOVA per injection) or PBS at one-week intervals (days 4, 11, and 18). For the LiNx treatment group, mice were administered the D6 LiNx formulations (30 pg per mouse) via a single s.c. injection on day 4. On day 21, the mice were euthanized, and tumour tissues were collected, homogenized, and analysed by flow cytometry. As shown in Fig. 11b, treatment with the D6 LiNx resulted in a substantial increase in CD8+ T cells within the tumour microenvironment. Approximately 4.5% of the total cell population consisted of CD8+ T cells, representing a three-fold increase compared to treatment with 3 doses of the D6 LNPs, which only induced about 1.5% CD8+ T cells in the tumour. In addition, a 3.9-fold higher (1.3%) OVA-specific CD8+ T cells among the total cell population in the tumour was observed in the D6 LiNx group, compared to the 3 doses of D6 LNPs (Fig. 5c). Similarly, higher levels of CD4+ T cells, NK cells, macrophages, and DCs were also observed in the D6 LiNx group (Fig. 1 Id-g). This indicates that the D6 LiNx induced a robust antigen-specific immune response and modulated the tumour microenvironment.

[0304] To further validate the effect of the LiNx platform on the tumour 452 microenvironment, we employed CODEX multiplexed fluorescence microscopy for spatial proteomics that uses iterative imaging and DNA-barcoded antibodies to enable the simultaneous imaging of multiple markers. 25-28 We designed a panel to identify major adaptive and innate immune cell types (Fig. llh, Tables 2-4). C57BL / 6 mice were inoculated with 3 x 105 B16-OVA cells on day 0 and were treated with the D6 LiNx (30 pg mOVA per mouse) via a single s.c. injection on day 4. On day 14post-tumour inoculation, tumour tissues were collected, sectioned, and analysed. As shown in Figs, l li-k and Figs. 48-49, the LiNx group recruited substantially higher levels of adaptive immune cells, including CD8+ and CD4+ T cells, as well as B cells, infiltrating the tumour region compared to the PBS group. Additionally, tumours collected frommice treated with the D6 LiNx showed a substantial increase in NK cells and CD 169+ macrophages. In contrast, neutrophils and non-CD169+ macrophages & monocytes were among the majority of immune cells in the tumour region isolated from the PBS group. The observed elevation of CD 169+ macrophages indicates a shift in the tumour microenvironment induced by the D6 LiNx immunization. Notably, recent studies have shown that a high density of CD 169+ macrophages is associated with prolonged survival and favourable clinical outcomes in patients with tumours.29-35 Taken together, our results reveal that treatment with the D6 LiNx altered the tumour microenvironment by increasing the infiltration of T and B cells, which are involved in the adaptive immune response, and a notable elevation in NK cells and CD 169+ macrophages, which play a critical role in the antitumour process. These results highlight the potency of the D6 LiNx platform as a newly designed immunostimulatory vaccine platform.

[0305] LiNx amplifies the immune response of mRNA LNPs

[0306] To further differentiate the immune responses triggered by D6 mRNA LNPs and LiNx, we analysed the expression of 547 genes in splenocytes from vaccinated mice using the nCounter Analysis System (NanoString Technology). In detail, the D6 LiNx comprising 30 pg mOVA was s.c. injected on day 0, compared with three doses of D6 LNPs containing 10 pg mOVA each on days 0, 7, and 14. On day 21, spleens from vaccinated mice were collected, dissociated into single cell suspension, and then subjected to in vitro antigen restimulation. After 24 h, splenocytes were collected, and the RNA was extracted and examined using NanoString analysis. As depicted in Fig. 12a-b, the comparison between untreated mice and the D6 LiNx group revealed 376 distinctive genes, including 25 downregulated and 351 upregulated genes. When comparing the single-dose D6 LiNx with the three-dosage D6 LNPs treatment, 344 genes exhibited significant differences, consisting of 35 downregulated and 309 upregulated. Upon closer examination of various subsets of related genes, including Thl, Th2, Thl7, and Treg-related genes, 36 as well as B cell receptor signaling genes, the D6 LiNx-treated group exhibited upregulation in 6 out of 15 Thl -related genes, 9 out of 16 Th2-related genes, 13 out of 19 Thl 7- related genes, and 12 out of 30 B cell signaling-related genes, in comparison with a 3-dose free- D6 LNP control group (Fig. 12c-e).

[0307] Among Treg cell-related genes, there were no significant differences, except for the downregulation of 1 out of 7 genes in the D6 LiNx group. By employing Database for Annotation,Visualization, and Integrated Discovery (DAVID) functional annotations for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways to establish connections between the information and higher-order functional aspects, we discovered that the D6 LiNx platform augmented immune responses through multiple pathways (Fig. 12f). In addition to its enhancement effect on Thl and Th2 differentiation, antigen presentation, and B cell / T cell receptor 516 signaling pathways, a noteworthy revelation emerged: Thl 7 differentiation and the IL- 17 signaling pathway exhibited marked enhancement following D6 LiNx treatment. This unexpected finding suggests that the increased effectiveness of D6 LiNx in anti cancer efficacy, as compared to free D6 LNPs, may be attributed to the substantial enhancement of Thl 7 cell-mediated anti-tumour immunity, which has been recognized for its considerable therapeutic potential (Fig. 12g).37-44 To validate the functionality of the Thl 7 immune response induced by the D6 LiNx, we performed IL- 17 depletion experiments using a prophylactic vaccination model for OVA-expressing melanoma in C57BL / 6 mice. The mice received either a single s.c. injection of the D6 LiNx (30 pg mOVA per injection) or three s.c. injections of mOVA-loaded D6 LNPs (10 pg mOVA per injection) administered one week apart, followed by s.c. inoculation with B16-OVA cells. IL-17-depleting antibodies were administered i.p. every three days (200 pg per mouse). The results demonstrated that IL- 17 depletion led to approximately a 40% reduction in survival rate in the D6 LiNx treatment group (Fig. 12h). In contrast, IL- 17 depletion had no significant effect on the survival rate of the traditional 3-dose D6 mRNA LNP treatment group, underscoring the critical role of the Thl 7 immune response in the observed anti-tumour effect of the D6 LiNx formulation. Moreover, no significant changes in lymphocyte composition were detected in the blood of IL-17-treated α- C57BL / 6control mice, indicating that antibody treatment does not substantially alter the immune system (Fig. 50).

[0308] DISCUSSION

[0309] Previous reports on LNP-mediated gene delivery and mRNA vaccination revealed that both the choice of lipids45 and their molar ratios significantly impact various aspects, 22 including the encapsulation efficiency46 of nucleic acid payloads47, transfection efficiency, 22 targeting profiles within cells and tissues, 48 as well as the immune response profile.16 Despite enormous potential, the spatial and temporal control over the immune response still persists in LNP-based engineering.49 In this study, we tested a new concept of LiNx, an mRNA LNP-loaded microgel matrix, which promotes endogenous immune cell recruitment and retention, and createsa programmable immunostimulating microenvironment, therefore maximizing antigen expression and presentation locally. This configuration yields a more robust immune response with a single dose vaccination in mouse colon carcinoma and melanoma models.

[0310] Here, we employed a previously developed NHC due to its ability to persistently recruit and retain host immune cells following s.c. or i.m. injection. This is crucial for facilitating effective crosstalk between relevant immune cell types. We tested three different LNPs with potent transfection capabilities of BMDCs in the LiNx context. Notably, the D6 LiNx, among the three, establishes the most effective local niche at week 2, characterized by a high abundance of both T cells and B cells, in contrast to the predominant neutrophil or macrophage 580 composition observed in the CIO and F5 LiNx groups. The formation of a lymphoid tissue-like niche provides an immunostimulatory environment favourable for the induction of adaptive immune response. Interestingly, substantial differences were observed regarding host cell recruitment, transfection efficiency, local microenvironment, and anti-cancer immune responses among the three LiNx formulations. These variations may be attributed to differences in LNP stability, transfection profiles, and / or immunostimulatory cues provided by the LNPs themselves. In future applications of the LiNx platform, it is can be imperative to screen combinations of different LNPs and NHC. Additionally, physical properties such as the stiffness and biodegradability of NHC may also influence the transfection and immune response outcomes. Therefore, optimizing NHC for enhanced immune responses should involve a comprehensive examination of these physical attributes.

[0311] When we analysed the tumour microenvironment following treatment with the D6 LiNx platform using flow cytometry and CODEX multiplex imaging, we observed a marked increase in adaptive immune cells, particularly antigen-specific T cells, infiltrating the tumour. Notably, the D6 LiNx treatment group also exhibited a substantial presence of NK cells and CD 169+ macrophages. The elevated NK cell levels are particularly striking, given their critical role in the innate immune response and the ability to directly target and eliminate tumour cells. Similarly, CD 169+ macrophages, known for their role in antigen presentation, may facilitate T cell activation within the tumour microenvironment.29-35 These findings highlight the enhanced potency of the D6 LiNx platform, which strongly correlates with robust activation of both adaptive and innate immune responses. This dual activation is likely to contribute to a more sustained andeffective immune response against tumours. Collectively, our results underscore the potential of the D6 LiNx platform as a highly effective immunostimulatory vaccine platform.

[0312] When comparing a single dose of D6 LiNx to three doses of conventional D6 LNPs, we observed enhanced immune responses and therapeutic efficacy with D6 LiNx. This phenomenon may be attributed to the initial successful transfection of host cells by D6 LNPs in the LiNx, leading to continuous host cell recruitment and effective crosstalk among recruited cells in LiNx, creating a local environment conducive to the potentiation of the immune response. More interestingly, our analysis revealed that the LiNx elicited a more robust Th 17 response, in addition to Thl and Th2responses, suggesting the critical role of Thl7 cells in generating potent antitumour 610 efficacy in this context (Fig. 11g).

[0313] In summary, we present an effective approach to potentiate mRNA LNP cancer vaccine by creating an immune cell recruiting and retention microgel matrix as a local immunostimulating niche. The LiNx matrix does not rely on lymph node trafficking of LNPs, but rather leverages its capability to locally enhance antigen expression and presentation, as well as immune cell crosstalk, resulting in potent antigen-specific responses. Among the three tested LiNx formulations, D6 LiNx exhibited continuous host cell recruitment and differentiation, as well as Thl, Th2, and Thl 7 responses that correlate with the most potent antitumour efficacy among the three LiNx formulations and when compared to the traditional three-dose regimen for D6 LNP vaccination. The results showcase a potent strategy to augment immune responses elicited by mRNA LNPs through a biomaterials-enabled lymphoid niche. This presents a versatile vaccine platform applicable to a variety of disease treatment and prevention strategies, thereby expanding the utility of mRNA LNP-based immunotherapies.

[0314] MATERIALS AND METHODS

[0315] Materials. DLin-MC3-DMA was purchased from MedKoo Biosciences. DOPE, DSPC, 18PG, and DMG-PEG-2000 were obtained from Avanti Polar Lipids. Cholesterol was from Sigma-Aldrich. B16F10 (CRL-6475) were purchased from ATCC (American Type Culture Collection, USA). B16-OVA cells were kindly provided by the lab of Prof. Jonathan Schneck. MC38 OVA (KC-2370) was purchased from KYINNO Biotechnology. All mRNA (Cre mRNA, OVA mRNA,Trp2 mRNA, or GplOO mRNA) constructs were purchased from TriLink BioTechnologies. The anti-CTLA-4 (a-CTLA-4) monoclonal antibody (mctla4-mabl0-10) waspurchased from InvivoGen. CODEX antibody information was compiled in Supplementary Table 4. Sodium hyaluronate with a molecular weight of 1.5 x 106 Da (HA, research grade) was purchased from LifeCore Biomedical Inc. (Chaska, MN, USA). Glycidyl acrylate was obtained from TCI America Inc. (Portland, OR, USA). Poly(ethylene glycol) dithiol with an average molecular weightof 5 kDa (PEG-SH, MW 5 kDa) was from JenKem Technology (Plano, TX, USA). All other chemical reagents were purchased from Sigma Aldrich (St. Louis, 638 MO, USA) unless otherwise noted.

[0316] LNP synthesis and characterization. LNPs were synthesized by directly adding an organic phase containing the lipids to an aqueous phase containing mRNA in 1.5-mL microcentrifuge tubes. To prepare the organic phase, a mixture of DLin-MC3 DMA, cholesterol, DMG-PEG2000, and a helper lipid selected from a group consisting of DOPE, DSPC, and 18PG were dissolved in ethanol. The mRNA (Cre mRNA, OVA mRNA, Trp2 mRNA, or GplOO mRNA) was dissolved in 25 mM magnesium acetate buffer (pH 4.0). For larger scale LNP production, the aqueous and ethanol phases prepared were mixed at a 3:1 ratio in a flash complexation (FNC) device using syringe pumps and purified by dialysis against DI water using a 100-kDa MWCO cassette at 4°C for 24 h and were stored at 4°C before injection. The size, poly dispersity index, and zeta potentials of LNPs were measured using dynamic light scattering (ZetaPALS, Brookhaven Instruments). Diameters are reported as the intensity average.

[0317] Animals and primary cells. All animal procedures were performed under an animal protocol approved by the Johns Hopkins Institutional Animal Care and Use Committee (protocol #MO22E117). Male and female C57BL / 6 mice, 6-8 weeks of age, were purchased from the Jackson Laboratory. Male Ai9 mice, 6-8 weeks of age, were bred in Johns Hopkins Animal Facilities and randomly grouped. The mice were supplied with free access to pelleted feed and water. The pelleted feed generally contained 5% fiber, 20% protein, and 5-10% fat. The mice

[0318] 657 usually ate 4-5 g of pelleted feed (120 g per kg body weight) and drank 3-5 mL of water (150 mL per kg body weight) per day. The temperature of the mouse rooms was maintained at 18-26°C (64-79°F) at 30-70% relative humidity with a minimum of 10 room air changes per hour. Standard shoebox cages with corncob as bedding were used to house the mice. The LNPs or LiNx wer egiven through s.c. (right flank) injection at a predetermined dose per mouse.

[0319] Antibodies, cell isolation, and staining for flow cytometry. Antibodies used in this study areFITC, APC, Brilliant Violet 750 anti-mouse CDl lc (BioLegend #117306, 117310, 117357); Brilliant Violet 421 anti-mouse CD86 (BioLegend #105032); PE anti-mouse SIINFEKL H-2KB (ThermoFisher # 12574382); FITC, Brilliant Violet 605, Brilliant Violet 421 anti-mouse CD45 (BioLegend # 103108, 103140, 103134); APC anti-mouse CD3 (BioLegend # 100236); FITC, APC, Brilliant Violet 750 anti-mouse CD8 (Bi 667 oLegend # 100706, 100712, BD Biosciences #747502); PerCP-Cyanine 5.5 anti-mouse CD4 (BioLegend # 100540); PE anti- mouse ZFN-y (BioLegend # 505808); Brilliant Violet 421 anti-mouse IL-4 (BioLegend # 504120); PE-Cyanine 7 anti-mouse TNFa (BioLegend # 506324); and APC anti-mouse Granzyme B (BioLegend # 396408). All antibodies were diluted at a ratio of 1 : 100 before use.For isolation, re-stimulation, and staining of splenocytes, the spleen was removed and minced using a sterile blade and dissociated in 250 pL of digestion medium (45 units pL-1 collagenase I 25 units pL-1 DNase I and 30 units pL- 1 hyaluronidase). The suspension was transferred into a 15- mL tube containing 5-10 mb of digestion medium and then filtered through a 70-pm filter and washed once with PBS. Cells were pelleted at 300 'g for 5 min at 4 °C resuspended in 5 mL of red blood cell lysis buffer (BioLegend), and then incubated on ice for 5 min. Cells were then pelleted at 300 'g for 5 min at 4 °C and washed twice with PBS. Isolated splenocytes were counted using the Cellometer cell counter and ViaStain AOPI staining solution (CS2-0106-25 mL, Nexcelom) and diluted in PBS to be used for restimulation. Splenocytes were re-stimulated in vitro with OVA (InvivoGen Cat. vac-pova) and SIINFEKL peptide (InvivoGen Cat. vac-sin) (10 pg mL-1 OVA and 2 pg mL-1 SIINFEKL) for 12 h. After re-stimulation, cells were collected and centrifuged at 300'g for 5 min. The cell pellet was washed with staining buffer 3 times and stained with antibodies against surface markers (total volume 100 pL) for 30 min in the dark at 4°C. The stained cells were washed twice with 1 mL of PBS and then fixed and permeabilized using the fixation / permeabilization solution kit (BD Cat# 555028). Then, cells were stained with anti-IFN- y or other antibodies against intracellular cytokines. Flow data were acquired on Attune (ThermoFisher) and analysed using FlowJo software. For isolation and staining of cells from lymph nodes or LiNx, isolated lymph nodes or LiNx were mechanically digested through 70 pm nylon cell strainers to prepare single-cell suspensions. The cell suspension was washed once with PBS via centrifugation (300'g) for 5 min. Then, the cellswere resuspended in 100 pL of staining buffer and stained with antibodies (total volume 100 pL) for 20 min in the dark at 4°C. The stainedcells were washed twice with 1 mL of PBS and resuspended in 300 pL of staining buffer for flow cytometry analysis. Flow data were acquired on Attune (ThermoFisher) and analysed using FlowJo software.

[0320] ELISpot assay. Multiscreen filter plates (Millipore-Sigma 696 #S2EM004M99) were coated with antibodies specific for IFN-y (BD Biosciences #551881) and blocked following the manufacturer’s protocols. Then, 1 x 105 isolated splenocytes were plated per well and stimulated with SIINFEKL peptide (2 pg mL-1 SIINFEKL) for 24 h. All tests were performed in duplicate or triplicate and included assay-positive controls as well as cells from a reference donor with known reactivity. Spots were visualized with mouse IFN-y detection antibody (BD Biosciences #551881) followed by incubation with Streptavidin-HRP (BD Biosciences #557630) and AEC Substrate (BD Biosciences #551951). Plates were then sent to SKCCC Immune Monitoring Core for analysis.

[0321] Enzyme-linked immunosorbent assay (ELISA). For antibody detection, groups of C57BL / 6 mice were immunized with different vaccines on days 0, 7, and 14. On day 21, 100 pL of blood sample was drawn from the tail vein, and levels of antigen-specific IgG in the serum were measured by ELISA. Flat-bottomed 96-well plates (Nunc) were precoated with OVA protein at a concentration of 2 pg protein per well in 100 mM carbonate buffer (pH 9.6) at 4°C overnight, which were then blocked with 10% fetal bovine serum (FBS) in PBS-Tween (PBS-T). Sera obtained from immunized animals were diluted 100 times in PBS-T (PBS-0.05% Tween), pH 7.4, and then in 4-fold serial dilution. The undiluted and diluted serum was added to the wells and incubated at 37°C for 2 h. Horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotech Associates, #1013-05) was used at a dilution of 1:5,000 in PBS-T-10% FBS for labeling.

[0322] After adding the horseradish peroxidase substrates, optical densities were determined at awavelength of 450 nm in an ELISA plate reader (Bio-Rad). A sample is considered positive if its absorbance is twice as much as or higher than the absorbance of the negative control.Immunization and tumour therapy experiments. Mice aged 6-8 weeks were injected subcutaneously with B16-OVA, MC38-OVA cells (1 x 106 in the prophylactic model and 3 x 105 in the therapeutic model) or 3 x 105 Bl 6F 10 melanoma cells into the right flank. In therapeutic studies, vaccinations began when tumour sizes were less than 50 mm3 (on day 4 after tumour inoculation). Animals were immunized by subcutaneous injection of differentLNP or LiNx formulations containing OVA mRNA, mTrp2, or m GplOO as described in the main text. One dosage of LiNx was given, and a total of three doses were given for the LNP group. For combinatorial immunotherapy, at days 6, 13, and 20 and an additional at day 27 for OVA725 expressing melanoma after inoculation, some groups were intraperitoneally injected with 100 pg checkpoint inhibitor (a-CTLA-4 mAb). Tumour growth was measured three 726 times a week using a digital caliper and calculated as 0.5 x length x width x width. Mice were euthanized when the tumour volumes reached 2,000 mm3.

[0323] IL-17 depletion study. Depletions of IL-17 were done using IL-17A (cloneα- 17F3,BioXCell) at 200 pg i.p. every 3 d. The dosing was initiated at 3 d after the first vaccination and continued every 3 d until day 60. On day 21 post-vaccination, 3 x 105 B16-OVA melanoma cells were injected into the right flank. Tumour growth was measured three times a week using a digital caliper and calculated as 0.5 x length x width x width. Mice were euthanized when the tumour volumes reached 2,000 mm3.

[0324] Subsequent RT-PCR analysis: Real-time polymerase chain reaction (RT-PCR) was carried out using TaqMan™ Array Mouse Immune Response (Applied Biosystems, Cat#4414079). The results were analysed according to the 2-AACT method and normalized to the housekeeping gene GAPDH.

[0325] nCounter Analysis System (NanoString Technology): Total RNA was extracted from stimulated spleen cells using an RNA extraction kit (Zymo Research, Cat# R2062). Quality and concentration were evaluated using NanoDrop. Predesigned NanoString nCounter CodeSets targeting mouse immune responses-related genes were employed (NanoString, Cat#l 15000052).

[0326] Hybridization of RNA samples to these CodeSets was performed following the manufacturer's protocol. Post-hybridization, samples were processed on the nCounter Analysis System, and data normalization was conducted using nSolver Analysis Software with reference to housekeeping genes.

[0327] CODEX multiplexed imaging: Tumour tissues were collected, embedded in optimal cutting temperature (OCT) compound for CODEX, and immediately frozen. After freezing, explants were sectioned to a thickness of 7 pm using an EprediaTM HM525 NX cryostat and arranged on slides. The tissue arrays were stained with the validated panels of CODEX antibodies and imaged in accordance with a previously established protocol.50 Briefly, thisentailed cyclic stripping, annealing, and imaging of fluorescently labeled oligonucleotides complementary to the oligonucleotide conjugated to the antibody. Each array underwent CODEX multiplexed imaging; metadata from each CODEX run can be found in Supplementary Table 2. Raw imaging data were processed using the Akoya PhenoCylcer Fusion 2.2.0 software 755 for image stitching, drift compensation, deconvolution, and cycle concatenation. After the raw imaging data were processed, they were evaluated for specific signals. Any markers that produced an untenable pattern or a low signal-to-noise ratio were excluded from the ensuing analysis. Uploaded images were visualizedin ImageJ (https: / / imagej.net / software / fiji / ).

[0328] CODEX single-cell segmentation: To obtain quantitative single-cell information, individual cells and extracted single-cell protein expression were segmented. Processed data were segmented using the SPACEc package, which can also be downloaded here (https: / / github.com / yuqiyuqitan / SPACEc / tree / master).51 SPACEc incorporates Mesmer and Cellpose, which are both deep learning-based segmentation methods.52, 53 Mesmer was used for our segmentation, with DAPI (nuclei) along with CD45 and CD90 (surface membrane) as reference channels.

[0329] Cell-type analysis: Across the LiNx and PBS-treated tumours, 208,486 cells were identified and classified into 19 cell types and states (197652 cells) with noise excluded based on marker expression. Cell type identification was done following the strategies that were developed.54, 55

[0330] Briefly, segmented cells of appropriate sizes were selected by gating DAPI-positive cells, followed by Z-normalization of protein markers used for clustering (some phenotypic markers were notused in the unsupervised clustering). The data were overclustered with Leiden- based clustering with the scanpy Python package. Clusters were assigned a cell type based on average cluster protein expression and location within the image. Impure clusters were split or reclustered with K775 means clustering with the ski earn Python package following mapping back to the original fluorescent images.

[0331] Statistical analysis. A two-tailed Student’s t-test or a one-way analysis of variance (ANOVA) was performed when comparing two groups or more than two groups, respectively. Survival curves were compared using the log-rank Mantel-Cox test, and the stack of P values was corrected by the Holm-Sidak method for multiple comparisons with alpha set to 0.05. Statisticalanalysis was performed using Microsoft Excel and Prism 8.0 (GraphPad). A difference is considered significant ifP< 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001).

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[0387] 55. Hickey, J. W., Tan, Y., Nolan, G. P. & Goltsev, Y. Strategies 920 for accurate cell type identification in CODEX multiplexed imaging data. Front. Immunol. 12, (2021).Supplementary Table 1, Formulation details and particle sizes for the three selected LNPsSupplementary Table 2, CODEX staining cendstions and cycle mfnrmatlonNate; 0 Goad; W detete; 8d?o: Wahg OooOs af te^fote? Weate s^;W§w* et^^dr NO: Not ml 0: ctoteoa due os bod qaaW artetete^Sup^emeutajy Tahfe 3. £D£O marker staining equation*: teto; to&g: Nfe BtostoM fete twfegmund terfe; Weak: Ww tofeito fem wpmfed; W:: HotSupplementary Table 4, CODfcX antibody informationEQUIVALENTS

[0388] It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting tothe invention. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and / or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. A method for treating a disease or disorder in a subject in need thereof comprising administering to the subject:(a) a fiber-hydrogel complex comprising fibers and one or more hydrogel materials;(b) one or more lipid nanoparticles (LNPs).

2. A method for treating a cancer in a subject in need thereof comprising administering to the subject:(a) a fiber-hydrogel complex comprising fibers and one or more hydrogel materials;(b) one or more lipid nanoparticles (LNPs).

3. A metho dof claim 1 or 2 wherein the (a) fiber-hydrogel complex and (b) one or more lipid nanoparticles (LNPs) are administered in combination.

4. A method of any one of claims 1 to 3 wherein the (a) fiber-hydrogel complex and (b) one or more lipid nanoparticles (LNPs) are administered sequentially to the subject.

5. A method of any one of claims 1 to 3 wherein the (a) fiber-hydrogel complex and (b) one or more lipid nanoparticles (LNPs) are administered at substantially the same time to the subject.

6. A method of any one of claims 1 to 5 wherein administration of the (b) one or more lipid nanoparticles (LNPs) is initiated after initiation of administration of the (a) fiber-hydrogel complex.

7. A method of any one of claims 1 to 5 wherein administration of the (a) fiber-hydrogel complex is initiated after initiation of administration of the (b) one or more lipid nanoparticles (LNPs).

8. The method of any one of claims 1 to 7 wherein the (b) one or more lipid nanoparticles (LNPs) at a site of the subject within about 10, 8, 7, 6, 5, 4, 3, 2 or 1 cm where the (a) fiber- hydrogel complex is, has been or will be administered to the subject.

9. The method of any one of claims 1 , 2, 3 or 5 wherein the (a) fiber-hydrogel complex and (b) one or more lipid nanoparticles (LNPs) are administered as a single formulation.

10. The method of any one of claims 1 to 8 wherein the (a) fiber-hydrogel complex and (b) one or more lipid nanoparticles (LNPs) are administered as distinct formulations.

11. The method of any one of claims 1 to 10 wherein a therapeutic agent in administered in conjunction with the LNP.

12. The method of any one of claims 1 to 11 wherein the LNP encapsulates one or more therapeutic agents.

13. The method of claim 11 or 12 wherein the therapeutic agent comprises a carbohydrates, protein, lipids, nucleic acids or combinations thereof.

14. The method of claim 11 or 12 wherein the therapeutic agent comprises a nucleic acid agent.

15. The method of claim 11 or 12 wherein the therapeutic agent comprises an mRNA agent.

16. The method of any one of claims 11 to 15 wherein the therapeutic agent comprises a peptide therapeutic agent.

17. The method of any one of claims 11 to 16 wherein the therapeutic agent comprises a small molecule therapeutic agent.

18. The method of any one of claim 1 to 17 wherein the fiber-hydrogel complex comprises a hyaluronic acid network covalently linked to a plurality of fibers.

19. The method of claim 18 wherein the fibers have a mean length of less than about 200 micrometers.

20. The method of claim 18 or 19 wherein the fibers are poly(caprolactone) and / or collagen.

21. The method of any one of claims 1 to 20 wherein the LNP formulation comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 5 to about 50.

22. The method of any one of claims 1 to 20 wherein the LNP formulation comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 10 to about 30.

23. The method of any one of claims 1 to 22 wherein the LNP comprises an ionizable lipid, at least one helper lipid, a PEGylated lipid and a steriod.

24. The method of claim 23 wherein the at least one helper lipid comprises a cationic lipid, a zwitterionic lipid, an anionic lipid, or combinations thereof.

25. The method of any one of claims 23 or 24 wherein the cationic lipid comprises 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB) or the combination thereof.

26. The method of any one of claims 23 to 25 wherein the zwitterionic lipid comprises 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or the combination thereof.

27. The method of any one of claims 23 to 26 wherein the anionic lipid comprises 1,2- dimyristoyl-sn-glycero-3-phosphate (14PA), l-stearoyl-2-oleoyl-sn-glycero-3-phospho (l’-rac- glycerol) (18PG) or the combination thereof.

28. The method of any one of claims 23 to 27 wherein the ionizable lipid comprises DLin- MC3-DMA.

29. The method of any one of claims 23 to 28 wherein the polyethylene glycol (PEG)-modified lipid comprises DMG-PEG 2000.

30. The method of any one of claims 23 to 29 wherein the sterol comprises cholesterol.

31. The method of any one of claims 23 to 30 wherein the LNP comprises a combined molar percentage of DLin-MC3-DMA and the at least one helper lipid in a range from about 10% to about 90%.

32. The method of any one of claims 23 to 31 wherein the LNP comprises a combined molar percentage of DLin-MC3-DMA and the at least one helper lipid in a range from about 20% to about 80%.

33. The method of any one of claims 23 to 32 wherein the LNP comprises a weight ratio of cholesterol to DMG-PEG2000, ranging from about 5 to about 300.

34. The method of any one of claims 23 to 33 wherein the LNP comprises a weight ratio of DLin-MC3-DMA to the helper lipid, from 0.1 to 200.

35. The method of any one of claims 23 to 33 wherein the LNP formulation comprises a weight ratio of DLin-MC3-DMA to the helper lipid, from 1 to 100.

36. The method of any one of claims 1 to 35 wherein fibers and one or more hydrogel materials of the fiber-hydrogel composite are covalently bonded.

37. The method of any one of claims 1 to 36 wherein the fibers of the fiber-hydrogel composite comprise one or more selected from poly(caprolactone), collagen, gelatin, cellulose, modified cellulose, cellulose acetate, HPMC, ethyl cellulose, silk, chitosan, keratin, elastin, elastin-like polypeptides, tropoelastin, and hyaluronic acid.

38. The method of any one of claims 1 to 37 wherein the one or more hydrogel materials of the fiber-hydrogel composite comprise one or more of hyaluronic acid (HA), collagen, chitosan, alginate, polyvinyl acetate (PVA), gelatin, polyethylene glycol (PEG) and other glycol ethers, chondroitin sulfate or a cellulose material.

39. The method of any one of claims 1 to 38 wherein the one or more hydrogel materials of the fiber-hydrogel composite comprise one or more hyaluronic acid (HA) materials.

40. The method of any one of claims 1 to 39 wherein the one or more hydrogel materials of the fiber-hydrogel composite are covalently bonded to poly(caprolactone) or collagen nanofibers sheet or fragments thereof.

41. The method of any one of claims 1 to 40 wherein the of the fiber-hydrogel composite further comprises a crosslinking agent or reacted form of a crosslinking agent.

42. The method of claim 41 wherein the crosslinking agent comprises one or more selected from difunctional epoxide-based crosslinkers, 1 ,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), PEG molecules with terminal epoxide functional groups, or HA-reactive agents.

43. The method of any one of claims 1 to 42 wherein a concentration of the HA ranges from about 0.5 w / v% to about 2 w / v% based on the total volume of the fiber-hydrogel composite.

44. The method of any one of claims 1 to 43 wherein a fiber loading density of the nanofiber ranges from about 1 w / v% to about 3 w / v% based on the total volume of the fiber-hydrogel composite.

45. The method of any one of claims 1 to 44 wherein a concentration of the fibers ranges from about 0.1 w / v% to about 20 w / v%, a concentration of the hydrogel material ranges from about 0.5 w / v% to about 10 w / v%, and a concentration of a crosslinking agent or reacted crosslinking agent ranges from about 0.05 v / v% to about 5 v / v%, based on the total volume of the fiber-hydrogel composite.

46. The method composition of any one of claims 1 to 44 wherein the concentration of the fibers ranges from about 1.5 w / v% to about 3.0 w / v%, the concentration of the hydrogel material ranges from about 0.8 w / v% to about 2 w / v%, and a concentration of a crosslinking agent or reacted crosslinking ranges from about 0.5 v / v% to about 2.5 v / v%, based on the total volume of the fiber-hydrogel composite.

47. The method of any one of claims 1 to 46 wherein the (a) fiber-hydrogel composite and / or (b) one or more lipid nanoparticles (LNPs) are formulated as an injectable fluid.

48. The method of any one of claims 1 to 47 wherein the fiber-hydrogel composite comprises one or more porous structures.

49. A treatment kit comprising:(a) a fiber-hydrogel composite comprising fibers and one or more hydrogel materials;(b) one or more lipid nanoparticles (LNPs).

50. The kit of claim 49 wherein the (a) fiber-hydrogel composite comprising fibers and one or more hydrogel materials and (b) one or more lipid nanoparticles (LNPs) are packaged together.

51. The kit of claim 49 wherein the (a) fiber-hydrogel composite comprising fibers and one or more hydrogel materials and (b) one or more lipid nanoparticles (LNPs) are packaged together a single formulation.

52. The kit of claim 49 wherein the (a) fiber-hydrogel composite comprising fibers and one or more hydrogel materials; and (b) one or more lipid nanoparticles (LNPs) are packaged separately.

53. The kit of any one of claims 49 to 52 wherein the kit comprises a therapeutic agent in addition to (a) fiber-hydrogel composite comprising fibers and one or more hydrogel materials and (b) one or more lipid nanoparticles (LNPs).

54. The kit of claim 53 wherein the one or more lipid nanoparticles (LNPs) encapsulate the therapeutic agent.

55. The kit of claim 53 or 54 wherein the therapeutic agent comprises a carbohydrates, protein, lipids, nucleic acids or combinations thereof.

56. The kit of any one of claims 53 to 55 wherein the therapeutic agent comprises a nucleic acid agent.

57. The kit of any one of claims 53 to 56 wherein the therapeutic agent comprises an mRNA agent.

58. The kit of any one of claims 53 to 57 wherein the therapeutic agent comprises a peptide therapeutic agent.

59. The kit of any one of claims 53 to 58 wherein the therapeutic agent comprises a small molecule therapeutic agent.

60. The kit of any one of claims 49 to 59 wherein the LNP formulation comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 5 to about 50.

61. The kit of any one of claims 49 to 59 wherein the LNP formulation comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 10 to about 30.

62. The kit of any one of claims 49 to 55 wherein the LNP comprises an ionizable lipid, at least one helper lipid, a PEGylated lipid and a steriod.

63. The kit of claim 62 wherein the at least one helper lipid comprises a cationic lipid, a zwitterionic lipid, an anionic lipid, or combinations thereof.

64. The kit of claim 63 wherein the cationic lipid comprises l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB) or the combination thereof.

65. The kit of claim 63 or 64 wherein the zwitterionic lipid comprises 1 ,2-dioleoyl-sn-glycero- 3 -phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or the combination thereof.

66. The kit of any one of claims 63 to 65 wherein the anionic lipid comprises 1 ,2-dimyristoyl- sn-glycero-3 -phosphate (14PA), l-stearoyl-2-oleoyl-sn-glycero-3 -phospho (l’-rac-glycerol) (18PG) or the combination thereof.

67. The kit of any one of claims 62 to 66 wherein the ionizable lipid comprises DLin-MC3- DMA.

68. The kit of any one of claims 62 to 67 wherein the polyethylene glycol (PEG)-modified lipid comprises DMG-PEG 2000.

69. The kit of any one of claims 62 to 68 wherein the sterol comprises cholesterol.

70. The kit of any one of claims 49 to 69 wherein the LNP comprises a combined molar percentage of DLin-MC3-DMA and the at least one helper lipid in a range from about 10% to about 90%.

71. The kit of any one of claims 49 to 69 wherein the LNP comprises a combined molar percentage of DLin-MC3-DMA and the at least one helper lipid in a range from about 20% to about 80%.

72. The kit of any one of claims 49 to 71 wherein the LNP comprises a weight ratio of cholesterol to DMG-PEG2000, ranging from about 5 to about 300.

73. The kit of any one of claims 49 to 72 wherein the LNP comprises a weight ratio of DLin- MC3-DMA to the helper lipid, from 0.1 to 200.

74. The kit of any one of claims 49 to 72 wherein the LNP formulation comprises a weight ratio of DLin-MC3-DMA to the helper lipid, from 1 to 100.

75. The kit of any one of claims 49 to 74 wherein fibers and one or more hydrogel materials of the fiber-hydrogel composite are covalently bonded.

76. The kit of any one of claims 49 to 75 wherein the fibers of the fiber-hydrogel composite comprise one or more selected from poly(caprolactone), collagen, gelatin, cellulose, modified cellulose, cellulose acetate, HPMC, ethyl cellulose, silk, chitosan, keratin, elastin, elastin-like polypeptides, tropoelastin, and hyaluronic acid.

77. The kit of any one of claims 49 to 76 wherein the one or more hydrogel materials of the fiber-hydrogel composite comprise one or more of hyaluronic acid (HA), collagen, chitosan,alginate, polyvinyl acetate (PVA), gelatin, polyethylene glycol (PEG) and other glycol ethers, chondroitin sulfate or a cellulose material.

78. The kit of any one of claims 49 to 77 wherein the one or more hydrogel materials of the fiber-hydrogel composite comprise one or more hyaluronic acid (HA) materials.

79. The kit of any one of claims 49 to 78 wherein the one or more hydrogel materials of the fiber-hydrogel composite are covalently bonded to poly(caprolactone) or collagen nanofibers sheet or fragments thereof.

80. The kit of any one of claims 49 to 79 wherein the fiber-hydrogel composite further comprises a crosslinking agent or reacted form of a crosslinking agent.

81. The kit of claim 80 wherein the crosslinking agent comprises one or more selected from difunctional epoxide-based crosslinkers, 1 ,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), PEG molecules with terminal epoxide functional groups, or HA-reactive agents.

82. The kit of any one of claims 49 to 81 wherein a concentration of the HA of the fiber- hydrogel composite ranges from about 0.5 w / v% to about 2 w / v% based on the total volume of the fiber-hydrogel composite.

83. The kit of any one of claims 49 to 82 wherein a fiber loading density of the fiber of the fiber-hydrogel composite ranges from about 1 w / v% to about 3 w / v% based on the total volume of the fiber-hydrogel composite.

84. The kit of any one of claims 49 to 83 wherein a concentration of the fibers of the fiber- hydrogel composite ranges from about 0.1 w / v% to about 20 w / v%, a concentration of the hydrogelI l lmaterial ranges from about 0.5 w / v% to about 10 w / v%, and a concentration of a crosslinking agent or reacted crosslinking agent ranges from about 0.05 v / v% to about 5 v / v%, based on the total volume of the fiber-hydrogel composite.

85. The kit of any one of claims 48 to 78 wherein the concentration of the fibers of the fiber- hydrogel composite ranges from about 1.5 w / v% to about 3.0 w / v%, the concentration of the hydrogel material ranges from about 0.8 w / v% to about 2 w / v%, and a concentration of a crosslinking agent or reacted crosslinking agent ranges from about 0.5 v / v% to about 2.5 v / v%, based on the total volume of the fiber-hydrogel composite.

86. The kit of any one of claims 49 to 85 wherein the (a) fiber-hydrogel composite a complex comprising fibers and one or more hydrogel materials and / or (b) one or more lipid nanoparticles (LNPs) are formulated as an injectable fluid.

87. The kit of any one of claims 49 to 85 wherein the fiber-hydrogel composite comprises one or more porous structures.

88. A composition package for coordinated or combined administration to a subject, comprising: a fiber-hydrogel composite complex comprising fibers and one or more hydrogel materials; and one or more lipid nanoparticles (LNPs) associated with the complex.

89. The composition package of claim 88 wherein the LNP further comprises a therapeutic agent.

90. The composition package of claim 89 wherein the therapeutic agent comprises a carbohydrates, protein, lipids, nucleic acids or combinations thereof.

91. The composition package of claim 89 or 90 wherein the therapeutic agent is encapsulated by the LNP.

92. The composition package of any one of claims 88 to 91 wherein the LNP comprises a nucleic acid therapeutic agent.

93. The composition package of any one of claims 88 to 92 wherein the LNP comprises an mRNA therapeutic agent.

94. The composition package of any one of claims 88 to 93 wherein the LNP comprises a peptide therapeutic agent.

95. The composition package of any one of claims 88 to 94 wherein LNP comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 5 to about 50.

96. The composition package of any one of claims 88 to 95 wherein LNP comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 10 to about 30.

97. The composition package of any one of claims 88 to 96 wherein an LNP of the composition package comprises an ionizable lipid, at least one helper lipid, a PEGylated lipid and a steriod.

98. The composition package of claim 97 wherein the at least one helper lipid comprises a cationic lipid, a zwitterionic lipid, an anionic lipid, or combinations thereof.

99. The composition package of claim 98 wherein the cationic lipid comprises l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB) or the combination thereof.

100. The composition package of any one of any one of claims 98 to 99 wherein the zwitterionic lipid comprises 1 ,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or the combination thereof.

101. The composition package of any one of claims 98 to 100 wherein the anionic lipid comprises 1 ,2-dimyristoyl-sn-glycero-3 -phosphate (14PA), l-stearoyl-2-oleoyl-sn-glycero-3- phospho (l’-rac-glycerol) (18PG) or the combination thereof.

102. The composition package of any one of claims 97 to 101 wherein the ionizable lipid comprises DLin-MC3-DMA.

103. The composition package of any one of claims 97 to 102 wherein the polyethylene glycol (PEG)-modified lipid comprises DMG-PEG 2000.

104. The composition package of any one of claims 97 to 103 wherein the sterol comprises cholesterol.

105. The composition package of any one of claims 88 to 104 wherein the LNP comprises a combined molar percentage of DLin-MC3-DMA and the at least one helper lipid in a range from about 10% to about 90%.

106. The composition package of any one of claims 88 to 105 wherein the LNP comprises a combined molar percentage of DLin-MC3-DMA and the at least one helper lipid in a range from about 20% to about 80%.

107. The composition package of any one of claims 88 to 106 wherein the LNP comprises a weight ratio of cholesterol to DMG-PEG2000, ranging from about 5 to about 300.

108. The composition package of any one of claims 88 to 107 wherein the LNP comprises a weight ratio of DLin-MC3-DMA to the helper lipid, from 0.1 to 200.

109. The composition package of any one of claims 88 to 108 wherein the LNP comprises a weight ratio of DLin-MC3-DMA to the helper lipid, from 1 to 100.

110. The composition package of any one of claims 88 to 109 wherein fibers and one or more hydrogel materials of the fiber-hydrogel composite are covalently bonded.

111. The composition package of any one of claims 88 to 110 wherein the fibers of the fiber- hydrogel composite comprise one or more selected from poly(caprolactone), collagen, gelatin, cellulose, modified cellulose, cellulose acetate, HPMC, ethyl cellulose, silk, chitosan, keratin, elastin, elastin-like polypeptides, tropoelastin, and hyaluronic acid.

112. The composition package of any one of claims 88 to 111 wherein the one or more hydrogel materials of the fiber-hydrogel composite comprise one or more of hyaluronic acid (HA), collagen, chitosan, alginate, polyvinyl acetate (PVA), gelatin, polyethylene glycol (PEG) and other glycol ethers, chondroitin sulfate or a cellulose material.

113. The composition package of any one of claims 88 to 112 wherein the one or more hydrogel materials of the fiber-hydrogel composite comprise one or more hyaluronic acid (HA) materials.

114. The composition package of any one of claims 88 to 113 wherein the one or more hydrogel materials of the fiber-hydrogel composite are covalently bonded to poly(caprolactone) or collagen nanofibers sheet or fragments thereof.

115. The composition package of any one of claims 88 to 114 wherein the composition package further comprises a crosslinking agent or reacted form of a crosslinking agent.

116. The composition package of claim 115 wherein the crosslinking agent comprises one or more selected from difunctional epoxide-based crosslinkers, 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), PEG molecules with terminal epoxide functional groups, or HA- reactive agents.

117. The composition package of any one of claims 88 to 116 wherein a concentration of the HA ranges from about 0.5 w / v% to about 2 w / v% based on the total volume of the fiber-hydrogel composite.

118. The composition package of any one of claims 88 to 117 wherein a fiber loading density of the fiber of the fiber-hydrogel composite ranges from about 1 w / v% to about 3 w / v% based on the total volume of the fiber-hydrogel composite.

119. The composition package of any one of claims 88 to 118 wherein a concentration of the fibers of the fiber-hydrogel composite ranges from about 0.1 w / v% to about 20 w / v%, a concentration of the hydrogel material of the fiber-hydrogel composite ranges from about 0.5 w / v% to about 10 w / v%, and a concentration of a crosslinking agent or reacted crosslinking agent of thefiber-hydrogel composite ranges from about 0.05 v / v% to about 5 v / v%, based on the total volume of the of the fiber-hydrogel composite.

120. The composition package of any one of claims 88 to 119 wherein the concentration of the fibers of the fiber-hydrogel composite ranges from about 1.5 w / v% to about 3.0 w / v%, the concentration of the hydrogel material of the fiber-hydrogel composite ranges from about 0.8 w / v% to about 2 w / v%, and a concentration of a crosslinking agent or reacted crosslinking of the fiber- hydrogel composite of ranges from about 0.5 v / v% to about 2.5 v / v%, based on the total volume of the fiber-hydrogel composite.

121. The composition package of any one of claims 88 to 120 wherein one or both of the a) fiber-hydrogel composite complex comprising fibers and one or more hydrogel materials and b) one or more lipid nanoparticles (LNPs) associated with the complex are formulated as a single or distinct injectable fluids.

122. The composition package of any one of claims 88 to 120 wherein one or both of the a) fiber-hydrogel composite complex comprising fibers and one or more hydrogel materials and b) one or more lipid nanoparticles (LNPs) associated with the complex are formulated as a single or unitary injectable fluid.

123. The composition package of any one of claims 88 to 120 wherein one or both of the a) fiber-hydrogel composite complex comprising fibers and one or more hydrogel materials and b) one or more lipid nanoparticles (LNPs) associated with the complex are formulated as distinct injectable fluids.

124. The composition package of any one of claims 88 to 123 wherein the fiber-hydrogel composite comprises one or more porous structures.

125. A LNP fiber-hydrogel composition for administration to a subject, the composition comprising: a complex comprising fibers and one or more hydrogel materials; one or more lipid nanoparticles (LNPs) associated with the complex.

126. The composition of claim 125 further comprising a therapeutic agent.

127. The composition of claim 126 wherein the therapeutic agent comprises a carbohydrates, protein, lipids, nucleic acids or combinations thereof.

128. The composition of claim 126 or 127 wherein the therapeutic agent is encapsulated by the LNP.

129. The composition of any one of claims 125 to 128 wherein the LNP comprises a nucleic acid agent.

130. The composition of any one of claims 125 to 129 wherein the LNP comprises an mRNA agent.

131. The composition of any one of claims 125 to 130 wherein the LNP comprises a peptide therapeutic agent.

132. The composition of any one of claims 125 to 131 wherein the LNP comprises a peptide therapeutic agent.

133. The composition of any one of claims 125 to 132 wherein LNP comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 5 to about 50.

134. The composition of any one of claims 125 to 133 wherein LNP formulation comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 10 to about 30.

135. The composition of any one of claims 125 to 134 wherein an LNP of the composition comprises an ionizable lipid, at least one helper lipid, a PEGylated lipid and a steriod.

136. The composition of claim 135 wherein the at least one helper lipid comprises a cationic lipid, a zwitterionic lipid, an anionic lipid, or combinations thereof.

137. The composition of claim 136 wherein the cationic lipid comprises l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB) or the combination thereof.

138. The composition of claim 136 wherein the zwitterionic lipid comprises 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or the combination thereof.

139. The composition of claim 136 wherein the anionic lipid comprises 1,2-dimyristoyl-sn- glycero-3 -phosphate (14PA), l-stearoyl-2-oleoyl-sn-glycero-3 -phospho (l ’-rac-glycerol) (18PG) or the combination thereof.

140. The composition of any one of claims 135 to 139 wherein the ionizable lipid comprises DLin-MC3-DMA.

141. The composition of any one of claims 135 to 140 wherein the polyethylene glycol (PEG)- modified lipid comprises DMG-PEG 2000.

142. The composition of any one of claims 135 to 141 wherein the sterol comprises cholesterol.

143. The composition of any one of claims 125 to 142 wherein the LNP comprises a combined molar percentage of DLin-MC3-DMA and the at least one helper lipid in a range from about 10% to about 90%.

144. The composition of any one of claims 125 to 143 wherein the LNP comprises a combined molar percentage of DLin-MC3-DMA and the at least one helper lipid in a range from about 20% to about 80%.

145. The composition of any one of claims 125 to 144 wherein the LNP comprises a weight ratio of cholesterol to DMG-PEG2000, ranging from about 5 to about 300.

146. The composition of any one of claims 125 to 145 wherein the LNP comprises a weight ratio of DLin-MC3-DMA to the helper lipid, from 0.1 to 200.

147. The composition of any one of claims 125 to 146 wherein the LNP comprises a weight ratio of DLin-MC3-DMA to the helper lipid, from 1 to 100.

148. The composition of any one of claims 125 to 147, wherein fibers and one or more hydrogel materials of the fiber-hydrogel composite are covalently bonded.

149. The composition of any one of claims 125 to 148 wherein the fibers of the fiber-hydrogel composite comprise one or more selected from poly(caprolactone), collagen, gelatin, cellulose,modified cellulose, cellulose acetate, HPMC, ethyl cellulose, silk, chitosan, keratin, elastin, elastin-like polypeptides, tropoelastin, and hyaluronic acid.

150. The composition of any one of claims 125 to 149 wherein the one or more hydrogel materials of the fiber-hydrogel composite comprise one or more of hyaluronic acid (HA), collagen, chitosan, alginate, polyvinyl acetate (PVA), gelatin, polyethylene glycol (PEG) and other glycol ethers, chondroitin sulfate or a cellulose material.

151. The composition of any one of claims 125 to 150 wherein the one or more hydrogel materials of the fiber-hydrogel composite comprise one or more hyaluronic acid (HA) materials.

152. The composition of any one of claims 125 to 151 wherein the one or more hydrogel materials of the fiber-hydrogel composite are covalently bonded to poly(caprolactone) or collagen nanofibers sheet or fragments thereof.

153. The composition of any one of claims 125 to 152 wherein the composition further comprises a crosslinking agent or reacted form of a crosslinking agent.

154. The composition of claim 153 wherein the crosslinking agent comprises one or more selected from difunctional epoxide-based crosslinkers, 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), PEG molecules with terminal epoxide functional groups, or HA-reactive agents.

155. The composition of any one of claims 125 to 154 wherein a concentration of the HA of the fiber-hydrogel composite ranges from about 0.5 w / v% to about 2 w / v% based on the total volume of the fiber-hydrogel composite.

156. The composition of any one of claims 125 to 155 wherein a fiber loading density of the fiber of the fiber-hydrogel composite ranges from about 1 w / v% to about 3 w / v% based on the total volume of the fiber-hydrogel composite.

157. The composition of any one of claims 125 to 156 wherein a concentration of the fibers of the fiber-hydrogel composite ranges from about 0.1 w / v% to about 20 w / v%, a concentration of the hydrogel material of the fiber-hydrogel composite ranges from about 0.5 w / v% to about 10 w / v%, and a concentration of a crosslinking agent or reacted crosslinking agent ranges from about 0.05 v / v% to about 5 v / v%, based on the total volume of the of the fiber-hydrogel composite.

158. The composition of any one of claims 125 to 156 wherein the concentration of the fibers of the fiber-hydrogel composite ranges from about 1.5 w / v% to about 3.0 w / v%, the concentration of the hydrogel material of the fiber-hydrogel composite ranges from about 0.8 w / v% to about 2 w / v%, and a concentration of a crosslinking agent or reacted crosslinking agent ranges from about 0.5 v / v% to about 2.5 v / v%, based on the total volume of the of the fiber-hydrogel composite.

159. The composition of any one of claims 125 to 158 wherein the composition is formulated as an injectable fluid.

160. The composition of any one of claims 125 to 158 wherein the of the fiber-hydrogel composite comprises one or more porous structures.

161. A vaccine comprising a composition package or composition of any of claims 88 to 160.162 . The vaccine according to claim 161 wherein the composition package or composition according to any of claims 88 to 160 elicits an adaptive immune response.163 A kit comprising a composition package, composition or vaccine of any one of claims 88 to 162.

164. The kit of claim 163 further comprises an applicator for administering the composition package, composition or vaccine to a subject.

165. The kit of claim 164 wherein the applicator is an injector with a needle or cannulas.

166. The kit of any one of claims 163 to 165 further comprising instructions for use of the kit to treat a subject.

166. A method of treating a subject, comprising administering to the subject a composition package, composition or vaccine of any one of claims 88 to 162.

167. The method of claim 166 wherein the therapeutic agent comprises a carbohydrates, protein, lipids, nucleic acids or combinations thereof.

168. The method of any one of claims 166 to 167 wherein the subject is suffering from cancer, and the administering treats the cancer.

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