WO2017024193A1 - Immunomodulatory extracellular matrix nanoparticles - Google Patents

Abstract

The present invention relates to novel compositions and methods for inducing immune responses with extracellular matrix nanoparticles. The invention also relates to methods of preparing the extracellular matrix nanoparticles. The compositions of the present invention play roles in post-injury regeneration, vaccination, immune tolerance, and cancer progression. The invention has immunotherapeutic applications for use in the clinic.

Description

IMMUNOMODULATORY EXTRACELLULAR MATRIX NANOPARTICLES

Related Applications

This application is an International Patent Application which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 62/201,950, filed on August 6, 2015 entitled, "Immunomodulatory Extracellular Matrix Nanoparticles", which is incorporated herein by reference in its entirety. Background of the Invention

The extracellular matrix (ECM) of tissues is a critical component of the tissue microenvironment in homeostasis, wound healing, and disease. There is an unmet need for therapies aimed at treating disorders of wound healing, homeostasis, and infectious disease as well as other pathologies.

Summary of the Invention

The present invention is based, at least in part, upon the discovery that extracellular matrix nanoparticles (ECM-NP) possessing immunomodulatory properties can be prepared from tissues. Furthermore, such extracellular matrix nanoparticles appear to exert immunogenic and immunomodulatory properties upon antigen presenting cells. Due to the close association of antigen presenting cells and several biological processes, ECM-NPs can impact subsequent processes such as wound repair, regeneration, vaccination, immune tolerance, and cancer progession oncogenesis.

In one aspect, the invention provides a composition that includes extracellular matrix (ECM) materials that are formulated as one or more nanoparticles.

In one embodiment, the ECM is milled to an average particle size of less than 1 micron in diameter to produce the ECM materials. Optionally, the ECM is milled to an average particle size of about 1-1000 nm in diameter to produce the ECM materials.

In certain embodiments, the ECM is cryogenically milled to produce the ECM materials. In some embodiments, the ECM materials are derived from one or more decellularized tissues. In a related embodiment, the one or more decellularized tissues include bladder and/or heart tissues.

Another aspect of the invention provides a method of preparing ECM materials that involves: obtaining cellular tissues; decellularizing the tissues into ECM material; milling the decellularized ECM to reduce the particle size; suspending the ECM in aqueous solution; and homogenizing the ECM in solution, thereby further fragmenting the ECM until a desired nanoparticle size is achieved, thereby preparing ECM materials.

In one embodiment, the step of obtaining cellular tissues involves obtaining mammalian tissue. Optionally, the mammalian tissue includes tissue obtained from one or more of the following sources: porcine, equine, caprine, canine, feline, murine, bovine, non- human primate, and/or human.

In another embodiment, the tissue includes one or more of the following tissues: cardiac, liver, bladder, small intestine, large intestine, spleen, lymph node, dermis, muscle, kidney, stomach, lung, pancreas, brain, adipose tissue and/or ocular tissue.

In certain embodiments, the decellularization is performed by a means that is mechanical, chemical, enzymatic and/or any combination thereof.

In one embodiment, the milling further involves lyophilization, cryogenic milling and/or any combination thereof.

In another embodiment, the aqueous solution further includes a buffer.

Another aspect of the invention provides a method of treating a disease, disorder or condition associated with an antigen presenting cell (APC) in a subject, the method involving administering an extracellular matrix nanoparticle (ECM-NP) to a subject suffering from or at risk of suffering from a disease, disorder or condition associated with an antigen presenting cell in an amount sufficient to treat the disease, disorder or condition in the subject.

In one embodiment, the antigen presenting cells are selected from the following: macrophages, dendritic cells, B cells, and/or activated epithelial cells.

In another embodiment, the disease, disorder or condition is selected from the following: injury, trauma, infection, sepsis, cancer, heart disease, liver disease, kidney disease, neurodegenerative disorder, neurological disorder, immune disorder and/or autoimmune disease.

Definitions

By "agent" is meant any small compound (small molecule or otherwise), antibody, cytokine, growth factor, nucleic acid molecule, or polypeptide, or fragments thereof.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "antigen presenting cell/ APC" is meant a cell that displays foreign and self antigens complexed with major histocompatibility complexes (MHCs) on their surfaces; this process is known as antigen presentation. T-cells may recognize these complexes using their T-cell receptors (TCRs). Antigen presenting cells (APC) process antigens and present them to T-cells. APCs can include but are not limited to: dendritic cells, macrophages, B cells, and activated epithelial cells.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes,"

"including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

By "cytokine" is meant a category of small proteins (-5-20 kDa) that are important in cell signaling. They are released by cells and affect the behavior of other cells. Cytokines can also be involved in autocrine signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a given cytokine may be produced by more than one type of cell. They act through receptors, and are especially important in the immune system; cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and

responsiveness of particular cell populations. Some cytokines enhance or inhibit the action of other cytokines in complex ways. They are important in health and disease, specifically in host responses to infection, immune responses, inflammation, trauma, sepsis, cancer, and reproduction.

"Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

By "effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

By "extracellular matrix" is meant a collection of extracellular molecules secreted by cells that provides structural and biochemical support to the surrounding cells. Because multicellularity evolved independently in different multicellular lineages, the composition of ECM varies between multicellular structures; however, cell adhesion, cell-to-cell communication and differentiation are common functions of the ECM. The animal extracellular matrix includes the interstitial matrix and the basement membrane. Interstitial matrix is present between various animal cells (i.e., in the intercellular spaces). Gels of polysaccharides and fibrous proteins fill the interstitial space and act as a compression buffer against the stress placed on the ECM. Basement membranes are sheet-like depositions of ECM on which various epithelial, endothelial, neural, skeletal muscle, and other specialized cell types rest. The plant ECM includes cell wall components, like cellulose, in addition to more complex signaling molecules. Some single-celled organisms adopt multicelluar biofilms in which the cells are embedded in an ECM composed primarily of extracellular polymeric substances (EPS).

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids, or more.

By "gene" is meant a locus (or region) of DNA that encodes a functional RNA or protein product, and is the molecular unit of heredity.

By "immunomodulation therapy" is meant the "treatment of disease by inducing, enhancing, or suppressing an immune response". Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while

immunotherapies that reduce or suppress are classified as suppression immunotherapies.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By "modulate" is meant alter (increase or decrease). Such alterations are detected by standard art known methods such as those described herein. By "nanoparticle" is meant particles between 1 and 999 nanometers in size (sub- micron). In nanotechnology, a particle is defined as a small object that behaves as a whole unit with respect to its transport and properties. Particles are further classified according to diameter. In certain embdodiments, a "nanoparticle" is between 1 and 800 nm in size; in certain other embodiments, a "nanoparticle" is between 1 and 600 nm in size; in certain additional embodiments, a "nanoparticle" is between 1 and 600 nm in size; optionally, a "nanoparticle" is between 1 and 450 nm in size; in certain embodiments, a "nanoparticle" is between 1 and 300 nm in size; in further embodiments, a "nanoparticle" is between 1 and 220 nm in size; optionally, a "nanoparticle" is between 1 and 100 nm in size. In certain embodiments, ultrafine particles include nanoparticles and tend to be between 1 and 100 nanometers in size, fine particles tend to be sized between 100 and 2,500 nanometers, and coarse particles cover a range between 2,500 and 10,000 nanometers.

By "nucleic acid" is meant biopolymers, or large biomolecules, essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. Each nucleotide has three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base. If the sugar is deoxyribose, the polymer is DNA. If the sugar is ribose, the polymer is RNA. Together with proteins, nucleic acids are the most important biological macromolecules; each are found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information— in other words, information is conveyed through the nucleic acid sequence, or the order of nucleotides within a DNA or RNA molecule. Strings of nucleotides strung together in a specific sequence are the mechanism for storing and transmitting hereditary, or genetic information via protein synthesis. Nucleic acids include but are not limited to: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), messenger RNA (mRNA), ribosomal RNA

(rRNA), transfer RNA (tRNA), micro RNA (miRNA), and small interfering RNA (siRNA).

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or

100%.

By "reference" is meant a standard or control condition.

A "reference sequence" is a defined sequence used as a basis for sequence comparison or a gene expression comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 40 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 or about 500 nucleotides or any integer thereabout or there between.

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

By "subject" is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;

aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

A "therapeutically effective amount" is an amount sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

Other features and advantages of the invention will be apparent to those skilled in the art from the following detailed description and claims.

Brief Description of the Drawings

Figure 1A depicts transmission electron microscopy images of extracellular matrix- nanoparticles (ECM-NP) from porcine cardiac and bladder tissues. Figure IB depicts bar graphs showing the z-average diameter (nm) and zeta potential for porcine cardiac and bladder ECM-NPs measured by dynamic light scattering.

Figure 1C depicts a graph of the number % size distribution of cardiac and bladder ECM-NPs as determined by dynamic light scattering prior to filtration.

Figure ID depicts NF-kB signaling activation in macrophages following exposure to bladder ECM-NPs. (red corresponds with nuclear localization of p65 subunit NF-kB complex over the blue stained nuclei; the actin cytoskeleton is stained with phalloidin in teal).

Figures 2A through 2G depicts in vivo imaging results of administering fluorescently labeled ECM-NPs to mice. Figure 2A shows the ventral view of an imaged mouse three days post- injection of ECM-NP in the tail. Figure 2B shows the ventral view of an imaged mouse three days after intraperitoneal injection of ECM-NP. Figure 2C shows the right aspect view of an imaged mouse three days after intraperitoneal injection of ECM-NP as well as the zoomed in view of its ear punch injury site. Figure 2D shows the ventral view of an imaged mouse three days after subcutaneous injection of ECM-NP. Figure 2E shows the right aspect view of a mouse three days after intramuscular (TA muscle) injection of ECM-NP. Figure 2F shows the right aspect view of an imaged mouse three days after foot pad injection of ECM- NP. Figure 2G shows the ventral aspect view of an imaged mouse three days after foot pad injection of ECM-NP as well as the zoomed in view of the footpad and leg. Figures 3 A and 3B depict flow cytometry analysis of systemic macrophage polarization and MHC Class II expression in the spleen of a mouse volumetric muscle loss model (n=3 animals). Figure 3A shows the FACS analysis of MHC Class II expressed in splenic macrophages (defined by the macrophage specific marker F4/80) exposed to skeletal muscle ECM-NPs or synthetic polystyrene NPs (control). Figure 3B shows the graphs of the % of F4/80+ macrophages gated as positive or negative for MHC Class II expression after exposure to ECM-NPs versus control. Statistical significance is defined as p<0.05 using a student's t-test and is denoted by (*).

Figure 4 depicts different passages of lung and cadiac tissue nanoparticles after homogenization.

Figure 5 depicts how homogenization conditions affect size.

Figure 6 depicts how filtration can control size.

Figure 7 depicts the process to generate ECM nanoparticles. Figure 8 depicts ECM co-localization with cells.

Figure 9 depicts ECM completely carpets wells at 1 mg/ml.

Figure 10 depicts NF-κΒ p65 translocating from the cytoplasm to the nucleus.

Figure 11 depicts less nuclear co-localization in 10 μg/ml vs 100 μg/ml.

Figure 12 depicts decelled sections of cardiac, bladder and liver ECM.

Figure 13 depicts Transmission Electron Microscopy (TEM) yields small globular ECM-NPs and big collagen strands.

Figure 14 depicts cardiac and bladder TEM images.

Figure 15 depicts sizing of cardiac and bladder ECMs.

Figure 16 depicts sizing of ECM post-filtration using TEM imaging.

Figure 17 depicts graphs of cardiac and bladder ECMs pre- and post-filtration.

Figure 18 depicts immunofluorescence of NF-κΒ activation by ECM-NPs.

Figure 19 depicts the strategy for studying mouse bone marrow macrophage internalization of ECM-NPs.

Figure 20 depicts ECM-NP uptake after washing.

Figure 21 depicts fluorescence analysis of a cell population with and without Ml polarization.

Figure 22 depicts FITC detection across NP treated cells.

Figure 23 depicts that PEGylation inhibits uptake.

Figures 24A and 24B depict FITC analysis for PEGylation uptake.

Figure 25 depicts additional FITC-PEG uptake data.

Figure 26 depicts FITC-PEG uptake in the left graph and intensity of uptake in the right graph.

Figures 27 A and 27B depict graphs of the fold change in expression of Ml (Fig. 27 A) and M2 (Fig. 27B) genes following exposure to cardiac, small intestine, and bladder ECM-NPs.

Figure 28 depicts effect of ECM source tissue on macrophage gene expression. Figure 29 depicts cardiac ECM-NPs vs polystyrene-NPs.

Figure 30 depicts graphs comparing polystyrene-NPs and cardiac ECM-NPs on polarization of gene expression.

Figure 31 depicts cryomilled ECM vs. ECM-NPs.

Figure 32 depicts graphs comparing cryomilled-ECM and cardiac ECM-NPs on polarization of gene expression.

Figure 33 depicts macrophage polarization with cardiac NPs and uptake inhibitors (cytochalsin D, chlorpromazine, and 5(N,N dimethyl) amiloride hydrochloride (DMA)).

Figure 34 depicts graphs comparing uptake inhibitors and cardiac ECM-NPs on polarization of gene expression.

Figure 35 depicts macrophage polarization with cardiac NPs and cytokines.

Figure 36 depicts graphs comparing cytokine alone and combined with cardiac ECM- NPs on polarization of gene expression.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, upon the discovery that extracellular matrix nanoparticles (ECM-NP) possessing immunomodulatory properties can be prepared from tissues. The invention provides for novel extracellular matrix nanoparticles, which exert immunogenic and immunomodulatory properties upon antigen presenting cells. Due to the close association of antigen presenting cells and several biological processes, the present invention has applications in processes/pathologies such as wound repair, regeneration, vaccination, immune tolerance, and cancer progession/oncogenesis.

In certain aspects of the present invention, extracellular matrix (ECM) scaffolds composed of decellularized tissues promote regenerative responses in part by polarizing the wound immune environment. Injectable nanoparticles, traditionally synthetic, can be efficiently delivered with a wide biodistribution. Therefore, the present invention provides for ECM-NPs that would rapidly present bioactive ECM components to sites of

inflammation, as well as other sites of antigen presenting cells. Targeting of the ECM-NPs of the invention (e.g., via use/attachment of antibodies, aptamers or the like) is also

contemplated. The parameters of the invention are set forth in additional detail below. Introduction

The extracellular matrix (ECM) of tissues is a critical component of the tissue microenvironment in homeostasis, wound healing, and disease. Biologic scaffolds prepared from decellularized tissues capture the complexity of the native ECM, and promote site- appropriate remodeling. The biologic effects are due in part to the activation and modulation of infiltrating immune cells, such as macrophages. Nanoparticles, which have been used in drug delivery, induce distinct cell responses from the bulk material. In certain embodiments of the present invention, preparation of a nanoparticle formulation of ECM, and the characterization of its immunomodulatory properties is demonstrated.

Extracellular Matrix (ECM)

ECM Biology

Due to its diverse nature and composition, the ECM can serve many functions, such as providing support, segregating tissues from one another, and regulating intercellular communication. The extracellular matrix regulates a cell's dynamic behavior. In addition, it sequesters a wide range of cellular growth factors and acts as a local store for them. Changes in physiological conditions can trigger protease activities that cause local release of such stores. This allows the rapid and local growth factor-mediated activation of cellular functions without de novo synthesis.

Formation of the extracellular matrix is essential for processes like growth, wound healing, and fibrosis. An understanding of ECM structure and composition also helps in comprehending the complex dynamics of tumor invasion and metastasis in cancer biology as metastasis often involves the destruction of extracellular matrix by enzymes such as serine proteases, threonine proteases, and matrix metalloproteinases.

The stiffness and elasticity of the ECM has important implications in cell migration, gene expression, and differentiation. Cells actively sense ECM rigidity and migrate preferentially towards stiffer surfaces in a phenomenon called durotaxis. They also detect elasticity and adjust their gene expression accordingly which has increasingly become a subject of research because of its impact on differentiation and cancer progression.

Components of the ECM are produced intracellularly by resident cells and secreted into the ECM via exocytosis. Once secreted, they then aggregate with the existing matrix. The ECM is composed of an interlocking mesh of fibrous proteins and glycosaminoglycans (GAGs). GAGs are carbohydrate polymers and are usually attached to extracellular matrix proteins to form proteoglycans (hyaluronic acid is a notable exception, see below).

Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+), which attracts water molecules via osmosis, keeping the ECM and resident cells hydrated. Proteoglycans may also help to trap and store growth factors within the ECM. Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan (PG) in which two or three HS chains are attached in close proximity to cell surface or ECM proteins. It is in this form that HS binds to a variety of protein ligands and regulates a wide variety of biological activities, including developmental processes, angiogenesis, blood coagulation, and tumor metastasis. In the extracellular matrix, especially basement membranes, the multi-domain proteins perlecan, agrin, and collagen XVIII are the main proteins to which heparan sulfate is attached. Chondroitin sulfates contribute to the tensile strength of cartilage, tendons, ligaments, and walls of the aorta. They have also been known to affect neuroplasticity. Keratan sulfates have a variable sulfate content and, unlike many other GAGs, do not contain uronic acid. They are present in the cornea, cartilage, bones, and the horns of animals.

Hyaluronic acid (or "hyaluronan") is a polysaccharide consisting of alternating residues of D-glucuronic acid and N-acetylglucosamine, and unlike other GAGs, is not found as a proteoglycan. Hyaluronic acid in the extracellular space confers upon tissues the ability to resist compression by providing a counteracting turgor (swelling) force by absorbing significant amounts of water. Hyaluronic acid is thus found in abundance in the ECM of load-bearing joints. It is also a chief component of the interstitial gel. Hyaluronic acid is found on the inner surface of the cell membrane and is translocated out of the cell during biosynthesis. Hyaluronic acid acts as an environmental cue that regulates cell behavior during embryonic development, healing processes, inflammation, and tumor development. It interacts with a specific transmembrane receptor, CD44.

Collagens are the most abundant protein in the ECM. In fact, collagen is the most abundant protein in the human body and accounts for 90% of bone matrix protein content. Collagens are present in the ECM as fibrillar proteins and give structural support to resident cells. Collagen is exocytosed in precursor form (procollagen), which is then cleaved by procollagen proteases to allow extracellular assembly. Disorders such as Ehlers Danlos Syndrome, osteogenesis imperfecta, and epidermolysis bullosa are linked with genetic defects in collagen-encoding genes. Elastins, in contrast to collagens, give elasticity to tissues, allowing them to stretch when needed and then return to their original state. This is useful in blood vessels, the lungs, in skin, and the ligamentum nuchae, and these tissues contain high amounts of elastins.

Elastins are synthesized by fibroblasts and smooth muscle cells. Elastins are highly insoluble, and tropoelastins are secreted inside a chaperone molecule, which releases the precursor molecule upon contact with a fiber of mature elastin. Tropoelastins are then deaminated to become incorporated into the elastin strand. Disorders such as cutis laxa and Williams syndrome are associated with deficient or absent elastin fibers in the ECM.

Fibronectins are glycoproteins that connect cells with collagen fibers in the ECM, allowing cells to move through the ECM. Fibronectins bind collagen and cell- surface integrins, causing a reorganization of the cell's cytoskeleton and facilitating cell movement. Fibronectins are secreted by cells in an unfolded, inactive form. Binding to integrins unfolds fibronectin molecules, allowing them to form dimers so that they can function properly. Fibronectins also help at the site of tissue injury by binding to platelets during blood clotting and facilitating cell movement to the affected area during wound healing.

Laminins are proteins found in the basal laminae of virtually all animals. Rather than forming collagen-like fibers, laminins form networks of web-like structures that resist tensile forces in the basal lamina. They also assist in cell adhesion. Laminins bind other ECM components such as collagens, nidogens, and entactins.

The ECM can exist in varying degrees of stiffness and elasticity, from soft brain tissues to hard bone tissues, the elasticity of the ECM can differ by several orders of magnitude. This property is primarily dependent on collagen and elastin concentration, and it has recently been shown to play an influential role in regulating numerous cell functions. Cells can sense the mechanical properties of their environment by applying forces and measuring the resulting backlash. This plays an important role because it helps regulate many important cellular processes including cellular contraction, cell migration, cell proliferation, differentiation and cell death (apoptosis). Inhibition of nonmuscle myosin II blocks most of these effects, indicating that they are indeed tied to sensing the mechanical properties of the ECM.

Differing mechanical properties in ECM exert effects on both cell behaviour and gene expression. Although the mechanism by which this is done has not been thoroughly explained, adhesion complexes and the actin-myosin cytoskeleton, whose contractile forces are transmitted through transcellular structures are thought to play key roles in the yet to be discovered molecular pathways. ECM elasticity can direct cellular differentiation, the process by which a cell changes from one cell type to another. In particular, naive mesenchymal stem cells (MSCs) have been shown to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. MSCs placed on soft matrices that mimic brain differentiate into neuron- like cells, showing similar shape, RNAi profiles, cytoskeletal markers, and transcription factor levels. Similarly stiffer matrices that mimic muscle are myogenic, and matrices with stiffnesses that mimic collagenous bone are osteogenic.

Stiffness and elasticity also guide cell migration, this process is called durotaxis. The term was coined when the tendency of single cells to migrate up rigidity gradients (towards more stiff substrates) was discovered. The molecular mechanisms behind durotaxis are thought to exist primarily in the focal adhesion, a large protein complex that acts as the primary site of contact between the cell and the ECM. This complex contains many proteins that are essential to durotaxis including structural anchoring proteins (integrins) and signaling proteins (adhesion kinase (FAK), talin, vinculin, paxillin, a-actinin, GTPases etc.) which cause changes in cell shape and actomyosin contractility. These changes are thought to cause cytoskeletal rearrangements in order to facilitate directional migration.

Many cells bind to components of the extracellular matrix. Cell adhesion can occur in two ways; by focal adhesions, connecting the ECM to actin filaments of the cell, and hemidesmosomes, connecting the ECM to intermediate filaments such as keratin. This cell- to-ECM adhesion is regulated by specific cell-surface cellular adhesion molecules (CAM) known as integrins. Integrins are cell-surface proteins that bind cells to ECM structures, such as fibronectin and laminin, and also to integrin proteins on the surface of other cells.

Fibronectins bind to ECM macromolecules and facilitate their binding to transmembrane integrins. The attachment of fibronectin to the extracellular domain initiates intracellular signalling pathways as well as association with the cellular cytoskeleton via a set of adaptor molecules such as actin.

There are many cell types that contribute to the development of the various types of extracellular matrix found in plethora of tissue types. The local components of ECM determine the properties of the connective tissue. Fibroblasts are the most common cell type in connective tissue ECM, in which they synthesize, maintain, and provide a structural framework; fibroblasts secrete the precursor components of the ECM, including the ground substance. Chondrocytes are found in cartilage and produce the cartilagenous matrix.

Osteoblasts are responsible for bone formation. Extracellular matrix has been found to cause regrowth and healing of tissue. In terms of injury repair and tissue engineering, the extracellular matrix serves two main purposes. First, it prevents the immune system from being triggered by the injury and responding with inflammation and scar tissue. Next, it facilitates the surrounding cells to repair the tissue instead of forming scar tissue.

ECM-NP Preparation

In some embodiments, the present invention describes the preparation of ECM nanoparticles. ECM-NPs are prepared by sequentially fragmenting decellularized tissues. Decellularization can include any mechanical, chemical, and/or enzymatic methods that preserve the insoluble ECM while removing the soluble cellular components of a tissue. In some embodiments, decellularization is performed with acids, detergents (e.g., Triton-XlOO), and/or DNAse. In some embodiments, enzymes used in decellularization includes: nucleases, DNAses, RNAses, endonucleases, and exonucleases. Other detergents that can be used include but are not limited to sodium dodecyl sulfate, sodium deoxycholate, Tween-20,

Triton X-200, and CHAPS. Proteolytic enzyme treatments may also be used such as trypsin or brief pepsin treatment in acid, or deglycoslyation. Other chemical treatments such as solvent lipid extraction (e.g. chloroform, alcohols), alkali treatment (sodium hydroxide), or hypertonic/hypotonic salt/sugar solutions may be used to lyse and remove cells. Mechanical processing includes but is not limited to removal (by cutting or peeling away) of specific layers in a tissue, sonication, barocyclic pressure cycling, slicing, cutting into smaller pieces (manually or using a blender type device. Immersion (inserting tissues in solution) or perfusion (pumping solution through native fluid conducting structures for example, blood vessels, bronchi of the lung, or bile duct of liver) decellularization methods may be used.

In some embodiments, any mammalian tissue can be used to prepare ECM-NPs.

Mammalian tissues for decellularization include but are not limited to: porcine, bovine, equine, caprine, canine, feline, ovine, murine, non-primate human and human tissues. ECM- NPs can be derived from tissues including but not limited to: cardiac, liver, bladder, small intestine, large intestine, spleen, lymph node, dermis, muscle, kidney, stomach, lung, pancreas, brain, adipose tissue and ocular tissues.

In some embodiments, ECM from decellularized tissues is lyophilized and cryogenically milled to reduce the ECM to particles to the 20-200 micron scale. ECM particles are then suspended in water or buffer and fragmented further via high pressure homogenization to create ECM nanoparticles (less than 1 micron in size). In some embodiments, high pressure homogenization is achieved using an Avestin Emulsiflex-C5 high pressure homogenizer (or equivalent). ECM suspension is passed through the homogenizer at increasing homogenizing pressures. Over the first 2-4 cycles, pressure is gradually increased from 5,000 psi to a maximum range of 25,000-30,000 psi. The ECM suspension is then homogenized for an additional 4-7 passes at 25,000-30,000 psi with sample cooling in ice water between passes. In some embodiments, ECM nanoparticles are produced in which greater than 90% of the ECM volume (and over 99% of discrete particles) is below 1 micron in size.

In some embodiments, adjusting the homogenizing pressure conditions and number of passes may be used to prepare larger or smaller particle sizes. In some embodiments, ECM- NPs have a diameter smaller than one micron. In some embodiments, ECM-NPs have a diameter in the range 1-1000 nm. In some embodiments, ECM-NPs have a diameter in the range 100-900 nm. In some embodiments, ECM-NPs have a diameter in the range 200-800 nm. ECM-NP size distribution can be purified by filtration using specific molecular weight cutoffs, for example 220 nm, 450 nm, and 800 nm filter pore sizes.

Nanoparticles

Nanoparticle characterization is necessary to establish understanding and control of nanoparticle synthesis and applications. Characterization is done by using a variety of different techniques, mainly drawn from materials science. Common techniques are electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), x-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, Rutherford backscattering spectrometry (RBS), dual polarisation interferometry and nuclear magnetic resonance

(NMR). The technology for nanoparticle tracking analysis (NTA) allows direct tracking of the Brownian motion; which allows the sizing of individual nanoparticles in solution. The majority of these nanoparticle characterization techniques are light-based, but a non-optical nanoparticle characterization technique called Tunable Resistive Pulse Sensing (TRPS) has been developed that enables the simultaneous measurement of size, concentration and surface charge for a wide variety of nanoparticles. This technique, which applies the Coulter Principle, allows for particle -by-particle quantification of these three nanoparticle characteristics with high resolution. The surface coating of nanoparticles is crucial to determining their properties. In particular, the surface coating can regulate stability, solubility, and targeting. A coating that is multivalent or polymeric confers high stability. Functionalized nanomaterial-based catalysts can be used for catalysis of many known organic reactions.

For biological applications, the surface coating should be polar to give high aqueous solubility and prevent nanoparticle aggregation. In serum or on the cell surface, highly charged coatings promote non-specific binding, whereas polyethylene glycol linked to terminal hydroxyl or methoxy groups repel non-specific interactions. Nanoparticles can be linked to biological molecules that can act as address tags, to direct the nanoparticles to specific sites within the body, specific organelles within the cell, or to follow specifically the movement of individual protein or RNA molecules in living cells. Common address tags are monoclonal antibodies, aptamers, streptavidin or peptides. These targeting agents should ideally be covalently linked to the nanoparticle and should be present in a controlled number per nanoparticle. Multivalent nanoparticles, bearing multiple targeting groups, can cluster receptors, which can activate cellular signaling pathways, and give stronger anchoring.

Monovalent nanoparticles, bearing a single binding site, avoid clustering and so are preferable for tracking the behavior of individual proteins. Nanoparticles linked with specific biomolecules can activate receptors and further modulate the immune response. Linking nanoparticles to fluorescent compounds can be used to monitor nanoparticle biodistribution, tissue accumulation, and clearance over time.

Methods of Treating Diseases associated with Antigen Presenting Cells (APC)

In some embodiments, the present invention provides methods of treating diseases, disorders or conditions associated with APCs. ECM-NPs can be used to stimulate and activate APCs. ECM-NPs, in association with APCs, can treat infections, increase immunity, regulate immune tolerance, treat inflammation, and prevent sepsis. In other embodiments, ECM-NPs can also down modulate APCs when their hyperactivity is causing disease.

Immunomodulation

The invention also includes methods for immunomodulating antigen presenting cells

(APCs). In some embodiments, antigen presenting cells are activated by ECM-NPs of the present invention. ECM-NPs stimulate APCs to increase expression of cytokines important for regulating the immune system. In some embodiments, the ECM-NPs can modulate immune tolerance, immune function, inflammation, vaccination, infection, auto-immunity, and oncogenesis/cancer progression.

In some embodiments, antigen presenting cells include but are not limited to dendritic cells, macrophages, B cells, activated epithelial cells, and cells activated by INF-γ. Dendritic cells have the broadest range of antigen presentation, and are probably the most important APC. Activated DCs are especially potent TH cell activators because they express co- stimulatory molecules such as B7. Certain B-cells, which express (as B cell receptor) and secrete a specific antibody, can internalize the antigen, which bind to its BCR and present it incorporated to MHC II molecule.

Macrophages are a type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the types of proteins specific to the surface of healthy body cells on its surface in a process called phagocytosis. They are found in essentially all tissues. Macrophages play a critical role in non-specific defense (innate immunity), and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. In humans, dysfunctional macrophages cause severe diseases such as chronic granulomatous disease that result in frequent infections. Macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of cytokines. Macrophages that encourage inflammation are called Ml macrophages, whereas those that decrease inflammation and encourage tissue repair are called M2 macrophages. This difference is reflected in their metabolism, where macrophages have the unique ability to metabolize one amino acid, arginine, to either a "killer" molecule (nitric oxide) or a "repair" molecule (ornithine).

Human macrophages are produced by the differentiation of monocytes in tissues. They can be identified using flow cytometry or immunohistochemical staining by their specific expression of proteins such as CD14, CD40, CDl lb, CD64, F4/80 (mice)/EMRl (human), lysozyme M, MAC-l/MAC-3 and CD68.

Wound repair

APCs are also involved in mechanisms of wound healing/regeneration following injury. In some embodiments, ECM-NPs of the present invention can be used to aid or enhance wound healing/regeneration after injury. ECM-NPs can aid antigen present cells in enhancing wound repair and regeneration. In some embodiments, ECM scaffolds composed of decellularized tissues promote regenerative responses in part by polarizing the wound environment. In some embodiments, injuries can heal faster with less scarring. Furthermore, ECM-NPs can be deployed with engineered tissues at sites of injury to aid the recovery process. Treatment

In some embodiments, ECM-NPs of the present invention can be administered to subjects suffering from or at risk of suffering from diseases involving wound repair, homeostasis, and inflammation and infection. In some embodiments, ECM-NPs can be administered to subjects through the following routes: topical, intraperitoneal, intravenous, intramuscular, intrathecal, intracranial, intravascular, intracerebroventricular, intracerebral, intra-arterial, intraarticular, intraocular, intracardiac, intravitreal, intra-osseous, intradermal, intralesional, intrauterine, intravaginal, subcutaneous, nasal, oral, sublingual, parenteral, enteral, rectal, transdermal, transmucosal, sublabial, insufflation, and inhaled.

In some embodiments, ECM-NPs are prepared as pharmaceutical formulations. In some embodiments, the pharmaceutical formulations include but are not limited to: pills, capsules, tablets, granules, powders, salts, crystals, liquids, serums, syrups, oil, suspensions, gels, creams, pastes, films, patches, and vapors.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the figures, are incorporated herein by reference.

EXAMPLES EXAMPLE 1: Materials and Methods

Porcine cardiac and bladder was decellularized by mechanical and chemical treatments with acid, Triton X-100, and DNAse, and decellularization was characterized via hematoxylin and eosin (H&E) staining. Each ECM scaffold was then homogenized under high pressure to prepare ECM nanoparticles (ECM-NPs). Dynamic light scattering was used to determine the size distribution and zeta potential of ECM-NPs, and structure examined with transmission electron microscopy. Mouse bone marrow macrophages were isolated and exposed to ECM-NPs at several concentration, and their effect on macrophage activation was determined by immunofluorescence for NF-κΒ signaling and expression of Ml and M2 genes via qRT-PCR. This was compared to Ml (LPS+IFNy) and M2 (IL-4) cytokine exposure. Further, TNF-a secretion following ECM-NP exposure was evaluated via ELISA.

EXAMPLE 2: Preparation and physical characterization of ECM-NPs

Both bladder and cardiac tissues were decellularized, removing the majority of cell components and nuclei creating an ECM scaffold, which was successfully fragmented into globular-shaped ECM-NPs (Fig. 1A) as demonstrated by electron microscopy.

ECM-NPs were prepared by sequentially mechanically fragmenting decellularized tissues. This method is applicable to any tissue type from any mammalian species. These tissue sources include but are not limited to cardiac, liver, urinary bladder, small intestine, spleen, lymph node, dermis, and adipose tissue, from porcine and/or human species. Tissue decellularization may be achieved by any chemical, enzymatic, and/or mechanical methods that selectively remove soluble cellular components of a tissue while preserving the insoluble ECM. The resulting ECM is lyophilized and cryogenically milled to reduce the ECM to particles to the 20-200 micron scale. ECM particles are then suspended in water or buffer and fragmented further via high pressure homogenization to create ECM nanoparticles (less than 1 micron in size). High pressure homogenization is achieved using an Avestin

Emulsiflex-C5 high pressure homogenizer (or equivalent). ECM suspension is passed through the homogenizer at increasing homogenizing pressures. Over the first 2-4 cycles, pressure is gradually increased from 5,000 psi to a maximum range of 25,000-30,000 psi. The ECM suspension is then homogenized for an additional 4-7 passes at 25,000-30,000 psi with sample cooling in ice water between passes. ECM -NPs are produced in which greater than 90% of the ECM volume (and over 99% of discrete particles) is below 1 micron in size. Adjusting the homogenizing pressure conditions and number of passes may be used to prepare larger or smaller particle sizes.

Bladder ECM-NPs were larger than cardiac with z-avg diameters of 817 and 628 nm, respectively, though cardiac ECM-NPs had a greater zeta potential of -29 mV (Fig. IB). ECM-NPs had a zeta potential range of -22 to -35 mV. ECM-NPs had a range of particle sizes with ECM-NPs derived from bladder tissues displaying a wider size distribution than those derived from cardiac tissue (Fig. 1C).

EXAMPLE 3: Functional characterization of ECM-NPs in vitro

ECM-NPs induced mixed classical and alternative macrophage phenotypes in vitro via elevated IL-Ιβ, TNF-a, IL-10, and Arg-1 gene expression, which were not induced by polystyrene nanoparticles. ECM nanoparticles have been shown to rapidly modulate the phenotype of bone marrow derived macrophages in vitro, increasing TNF-a, IL-Ιβ, iNOS, and IL-10 gene expression, and cytokine secretion of TNF-a and IL-10 in a dose dependent fashion. This immune activation is not seen with 200 nm polystyrene nanoparticles, or with the same amount of cryomilled ECM (prior to nanoparticle formation). ECM nanoparticles enhance IL-10 gene expression compared to polarizing cytokines (IL-4 and/or LPS+IFN-γ), and also increase expression in the presence of these cytokines.

ECM-NPs rapidly activated NF- κΒ mediated pathways in macrophages with translocation of the p65 subunit within one hour of exposure (Fig. ID) as demonstrated by immunofluorescence. ECM-NP exposure polarized macrophages towards a mixed M1/M2 phenotype. Bladder ECM-NPs increased expression of M2 genes Arg-1 and IL-10, and the Ml gene TNF-a to a greater extent than cardiac (Fig. 27A and 27B). IL-Ιβ expression was similar between cardiac and bladder ECM-NPs, and FIZZ-1 was not expressed with either treatment. TNF-a was also rapidly secreted in response to ECM-NPs, with similar amounts for cardiac and bladder ECM-NPs. As demonstrated by ELISA, TNF-a was secreted by macrophages in a dose dependent manner at 3.9+3 ng/ml, which surpassed classical stimulation alone.

EXAMPLE 4: Functional characterization in vivo

Muscle ECM-NPs were prepared, filtered, and conjugated with near infrared dye labels for imaging. The labeled ECM-NPs were injected into hairless mice for tracking distribution of the ECM-NPs. Routes of administering injections include: footpads, tails, intraperitoneal, subcutaneous, and intramuscular injection. Labeled ECM-NPs were distributed systemically within 30 minutes of mouse footpad injection, as assessed via in vivo imaging, and were observed in secondary lymphoid tissues after 6 days (Figures 2A through 2G).

To study the effects of ECM-NPs on sites of injury, ECM-NPs were injected into proximal and distal stumps of volumetric muscle loss (VML) in injured mouse muscle. Mice were sacrificed and harvested one week later, and FACS analysis was performed.

Macrophage polarization occurred in muscle tissue, and systemic polarization occurred in the spleen. ECM-NPs increased MHC Class II expression in F4/80+ cells within the mouse spleen (Figures 3A and 3B).

In summary, a method to prepare ECM nanoparticles from decellularized tissues has been identified and described. ECM-NPs derived from both cardiac and bladder sources were immunomodulatory in vitro, inducing a mixed classic/alternative macrophage phenotype, that was notably distinct from polystyrene nanoparticles. ECM-NP retention and trafficking to lymphoid tissues in vivo suggested the potential for systemic immunotherapy using ECM-NPs, in addition to local immune polarization. Exemplary diseases, disorders and/or conditions presently contemplated for ECM-NP treatment include trauma, regeneration, cancer, transplant tolerance, autoimmune diseases and disorders, among others.

Invention Outcomes and Conclusions

Creation of nano-sized tissue-derived ECM materials has been newly described herein. Conventional milling technology (e.g. cryogenic milling) is not sufficient for creating sub-micron sized particles. While previous strategies have been used to create particles from purified ECM components, such as collagen, the currently identified/produced ECM -NPs are composed of whole ECM, containing numerous types of proteins, proteoglycans, and glycosaminoglycans, rather than individual purified components or synthetic polymers. Synthetic nanoparticles have few inherent immunological properties, in the absence of additional immune- stimulating chemicals and cytokines. ECM-NPs have been newly developed and have now been demonstrated to possess immune-activating properties in vitro. In addition, the ECM-NPs of the invention have exhibited in vivo biocompatibility, as observed when injected intravenously into mice.

ECM-NPs were prepared from both cardiac and bladder tissues, and were shown to modulate macrophage polarization. ECM-NP source tissue differentially induced Ml and M2 polarization, with bladder tissue-derived ECM-NP having induced a regulatory phenotype that exhibited increased IL-10 and TNF-a expression, as compared to cardiac - derived ECM-NP.

REFERENCES

1. Brown BN, Ratner BD, Goodman SB, Amar S, Badylak SF. Macrophage

polarization: an opportunity for improved outcomes in biomaterials and regenerative medicine. Biomaterials. 2012. 33(15):3792-802.

2. Owens DE, Peppas NA. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. International Journal of Pharmaceutics. 2006. 307(1):93-102. Equivalents

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

What is claimed is:

1. A composition comprising extracellular matrix (ECM) materials that are formulated as one or more nanoparticles.

2. The composition of claim 1, wherein the ECM is milled to an average particle size of less than 1 micron in diameter to produce the ECM materials.

3. The composition of claim 1 or claim 2, wherein the ECM is milled to an average particle size of about 1-1000 nm in diameter to produce the ECM materials.

4. The composition of any of the above claims, wherein the ECM is cryogenically milled to produce the ECM materials.

5. The composition of any of the above claims, wherein the ECM materials are derived from one or more decellularized tissues.

6. The composition of claim 5, wherein the one or more decellularized tissues comprise bladder and/or heart tissues.

7. A method of preparing ECM materials comprising:

obtaining cellular tissues,

decellularizing the tissues into ECM material,

milling the decellularized ECM to reduce the particle size,

suspending the ECM in aqueous solution, and

homogenizing the ECM in solution, thereby further fragmenting the ECM until a desired nanoparticle size is achieved.

8. The method of claim 7, wherein the step of obtaining cellular tissues comprises obtaining mammalian tissue.

9. The method of claim 8, wherein the mammalian tissue comprises tissue obtained from one or more sources selected from the group consisting of porcine, equine, caprine, canine, feline, murine, bovine, non-human primate, and human.

10. The method of claim 7, wherein the tissue comprises one or more tissues selected from the group consisting of cardiac, liver, bladder, small intestine, large intestine, spleen, lymph node, dermis, muscle, kidney, stomach, lung, pancreas, brain, adipose tissue and ocular tissues.

11. The method of claim 7, wherein the decellularization is performed by a means selected from the group comprising mechanical, chemical, enzymatic and/or any combination thereof.

12. The method of claim 7, wherein the milling further comprises lyophilization, cryogenic milling and/or any combination thereof.

13. The method of claim 7, wherein the aqueous solution further comprises a buffer.

14. A method of treating a disease, disorder or condition associated with an antigen presenting cell (APC) in a subject, the method comprising administering an extracellular matrix nanoparticle (ECM-NP) to a subject suffering from or at risk of suffering from a disease, disorder or condition associated with an antigen presenting cell in an amount sufficient to treat the disease, disorder or condition in the subject.

15. The method of claim 14, wherein the antigen presenting cells are selected from the group consisting of macrophages, dendritic cells, B cells, and activated epithelial cells.

16. The method of claim 15, wherein the disease, disorder or condition is selected from the group consisting of injury, trauma, infection, sepsis, cancer, heart disease, liver disease, kidney disease, neurodegenerative disorder, neurological disorder, immune disorder and autoimmune disease.