Compositions and methods comprising microparticle-transferring macrophages

Abstract

The present disclosure generally relates to compositions and methods comprising conditioned macrophages. The present disclosure further generally relates to methods of treating diseases and conditions comprising the use of conditioned macrophages, such as for the treatment of fibrosis. In some aspects, the methods described herein comprise the use of conditioned macrophages for treating pulmonary fibrosis.

Description

[0001] COMPOSITIONS AND METHODS COMPRISING MICROPARTICLE-TRANSFERRING MACROPHAGES

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 552,465, filed February 12, 2024; and U.S. Provisional Patent Application No. 63 / 637,112, filed April 22, 2024, the disclosures of each of which are hereby incorporated herein by reference in their entireties.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The Sequence Listing concurrently submitted herewith as an XML-formatted text file named “046528-7137WOl_sequence_listing.xml” created on February 3, 2025, and having a size of 24,263 bytes is herein incorporated by reference in its entirety pursuant to 37 C.F.R. 1 52(e)(5).

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under NHLBI R01 HL130037 awarded by the National Heart, Lung, and Blood Institute. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] Macrophages are innate immune cells that participate as key effector cells in inflammatory and fibrotic diseases, such as pulmonary fibrosis. Fibro-inflammatory diseases generally result from chronic inflammation caused by a variety of factors including persistent infections, autoimmune reactions, allergic responses and tissue injury. Examples are systemic sclerosis, inflammatory bowel disease, lung fibrosis and others. They are a leading cause of morbidity and mortality. Living with fibro-inflammatory diseases greatly impact patients’ lives, both physically and emotionally and they account for more than one third of deaths worldwide. In particular, pulmonary fibrosis is a lung disease in which the lung becomes damaged and scarred in response to recurring harmful events such as entering pathogens, allergens, and / or foreign substances, as well as lung damage due to unknown causes as in idiopathic pulmonary fibrosis (IPF). Due to the recurrent nature of these events, the initiated pro-inflammatory response is unresolved and exaggerated, resulting in tissue damage and triggering an overactive pro-reparative phase with extensive deposition of extracellular matrix (ECM), leading to fibrosis and loss of lung function. So far, no cure exists for IPF, and, in particular, no treatment exists that enables reversal of established fibrosis.

[0010] Macrophage-based cell therapy for treatment of disease is generally limited by phenotype control of the macrophages, which are known to rapidly shift phenotype in response to microenvironmental cues in the body, as well as by the high numbers of host macrophages, which host macrophages can override the effects of any adoptively transferred macrophages.

[0011] As such, a need exists for compositions and methods for treatment of diseases, such as inflammatory and fibrotic diseases, using improved macrophage-based approaches. The present disclosure addresses this need.

[0012] BRIEF SUMMARY

[0013] In one aspect, the present disclosure generally relates to a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of macrophages comprising one or more microparticles designed to promote microparticle transfer; and / or b) a plurality of macrophages comprising one or more microparticles designed to inhibit microparticle transfer. In some aspects, a) the one or more microparticles designed to promote microparticle transfer comprises one or more microparticles comprising biophysical properties designed to promote microparticle transfer; and / or b) the one or more microparticles designed to inhibit microparticle transfer comprises one or more microparticles comprising biophysical properties designed to inhibit microparticle transfer.

[0014] Furthermore, in one aspect, the present disclosure generally relates to a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising microparticles sized to promote transfer; and / or b) a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer. In some aspects, the composition comprises both a) and b). In some aspects, the microparticles sized to promote transfer are smaller in diameter relative to the diameter of the microparticles sized to inhibit transfer. In some aspects, the microparticles sized to promote transfer are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is an anti-fibrotic agent, an anti-inflammatory agent, a pro-reparative agent, a pro- regenerative agent, a chemotherapeutic agent, or an anti-cancer agent. In some aspects, the anti- fibrotic agent is a TGFB pathway inhibitor. In some aspects, the anti -fibrotic agent is pirfenidone, nintedanib, or a combination thereof. In some aspects, the microparticles sized to inhibit transfer are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is dexamethasone. In some aspects, the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL). In some aspects, the conditioned macrophages of (a) and / or the conditioned macrophages of (b) are derived from a subject’s monocytes. In some aspects, the conditioned macrophages of (a) and / or the conditioned macrophages of (b) comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

[0015] Moreover, in one aspect, the present disclosure generally relates to a method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of any of the compositions described herein to the subject in need thereof. In some aspects, the microparticles sized to promote transfer are transferred to a cell of the subject. In some aspects, the cell is a macrophage, a fibroblast, a neuron, a tumor cell, a non-macrophage lymphocyte, a neutrophil, an epithelial cell, an endothelial cell, a progenitor cell, and / or a non-myeloid cell.

[0016] Furthermore, in one aspect, the present disclosure generally relates to a method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles sized to promote transfer. In some aspects, the method further comprises administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer. In some aspects, the disease or condition comprises pulmonary fibrosis. In some aspects, the microparticles sized to promote transfer are transferred to a cell of the subject. In some aspects, the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast. In some aspects, the microparticles sized to promote transfer are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is an anti-fibrotic agent. In some aspects, the anti-fibrotic agent is a TGFB pathway inhibitor. In some aspects, the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof. In some aspects, the microparticles sized to inhibit transfer are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is dexamethasone. In some aspects, the microparticles sized to promote transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL). In some aspects, the microparticles sized to promote transfer are smaller in diameter relative to the diameter of the microparticles sized to inhibit transfer. In some aspects, the conditioned macrophages comprising microparticles sized to promote transfer are derived from a subject’s monocytes. In some aspects, the conditioned macrophages comprising microparticles sized to promote transfer comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. In some aspects, the microparticles sized to inhibit transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL). In some aspects, the conditioned macrophages comprising microparticles sized to inhibit transfer are derived from a subject’s monocytes. In some aspects, the conditioned macrophages comprising microparticles sized to inhibit transfer comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

[0017] Moreover, in one aspect, the present disclosure generally relates to a method of treating a pulmonary fibrosis in a subject in need thereof, comprising administering to the subject: a) an effective amount of a plurality of conditioned macrophages comprising microparticles sized to promote transfer; and b) an effective amount of a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer. In some aspects, the microparticles sized to promote transfer are transferred to a cell of the subject. In some aspects, the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a neuron, a non- myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast. In some aspects, the microparticles sized to promote transfer are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is an anti-fibrotic agent. In some aspects, the anti-fibrotic agent is a TGFB pathway inhibitor. In some aspects, the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof. In some aspects, the microparticles sized to inhibit transfer are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is dexamethasone. In some aspects, the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer comprise poly(lactic-co- glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL). In some aspects, the conditioned macrophages of (a) and / or the conditioned macrophages of (b) are derived from a subject’s monocytes. In some aspects, the conditioned macrophages of (a) and / or the conditioned macrophages of (b) comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

[0018] Furthermore, in one aspect, the present disclosure generally relates to a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising an amount of microparticles, wherein the amount of microparticles is an amount that promotes microparticle transfer; and / or a plurality of conditioned macrophages comprising an amount of microparticles, wherein the amount of microparticles is an amount that inhibits microparticle transfer. In some aspects, the amount of microparticles that inhibits microparticle transfer is between about 10 pg / 106cells to about 100 pg / 106cells. In some aspects, the amount of microparticles that promotes microparticle transfer is from about 250 pg / 106cells to about 1000 pg / 106cells. In some aspects, the microparticles are from about 0.01 pm to about 20.0 pm in diameter. In some aspects, the microparticles are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is an anti-fibrotic agent, an anti-inflammatory agent, a pro-reparative agent, a pro- regenerative agent, a chemotherapeutic agent, or an anti-cancer agent. In some aspects, the anti- fibrotic agent is a TGFB pathway inhibitor. In some aspects, the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof. In some aspects, the therapeutic agent is dexamethasone. In some aspects, the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL). In some aspects, the macrophages are derived from a subject’s monocytes. In some aspects, the macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

[0019] Moreover, in one aspect, the present disclosure generally relates to a method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of a composition as described herein to the subject in need thereof. In some aspects, the microparticles are transferred to a cell of the subject. In some aspects, the cell is a macrophage, a fibroblast, a neuron a tumor cell, a non-macrophage lymphocyte, a neutrophil, an epithelial cell, an endothelial cell, a progenitor cell, and / or a non-myeloid cell.

[0020] Furthermore, in one aspect, the present disclosure generally relates to a method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a composition as described herein to the subject in need thereof. In some aspects, the disease or condition comprises pulmonary fibrosis. In some aspects, the microparticles are transferred to a cell of the subject. In some aspects, the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a neuron, a non- myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast.

[0021] Furthermore, in one aspect, the present disclosure generally relates to a method of treating a pulmonary fibrosis in a subject in need thereof, comprising administering to the subject an effective amount of a composition as described herein to the subject in need thereof. In some aspects, the microparticles are transferred to a cell of the subject. In some aspects, the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a neuron, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast.

[0022] Moreover, in one aspect, the present disclosure generally relates to a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising a microparticle functionalized with an agent designed to promote microparticle transfer; and / or b) a plurality of conditioned macrophages comprising a microparticle functionalized with an agent designed to inhibit microparticle transfer.

[0023] Furthermore, in one aspect, the present disclosure generally relates to a method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of a composition as described herein to the subject in need thereof. Moreover, in one aspect, the present disclosure generally relates to a method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles functionalized with at least one agent, wherein the agent comprises dexamethasone. In some aspects, the microparticles comprise microparticles designed to promote transfer. In some aspects, the microparticles are sized to promote transfer. In some aspects, the microparticles comprise an amount of microparticles that promotes microparticle transfer. In some aspects, the microparticles are further functionalized with an agent that promotes microparticle transfer. In some aspects, the fibrotic disease or condition is pulmonary fibrosis or is muscle fibrosis. In some aspects, the fibrotic disease or condition is pulmonary fibrosis. In some aspects, the conditioned macrophages accumulate in the lungs of the subject. In some aspects, the microparticles are transferred to a cell of the subject. In some aspects, the cell of the subject is a macrophage, a non-macrophage lymphocyte, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, a neutrophil, and / or a fibroblast In some aspects, the treatment decreases expression of CD206, MERTK and CD301 in host macrophages of the subject. In some aspects, the treatment decreases the total collagen content of the lungs of the subj ect. In some aspects, the treatment decreases the collagen content of the lungs of the subject relative to the lungs of an untreated subject having pulmonary fibrosis. In some aspects, the treatment decreases the total amount of fibrotic lung tissue of the subject. In some aspects, the treatment decreases the amount of fibrotic lung tissue of the subject relative to an untreated subject having pulmonary fibrosis. In some aspects, the treatment increases the number of endothelial cells in the subject as compared to an untreated subject. In some aspects, the conditioned macrophages are derived from the subject’s monocytes. In some aspects, the conditioned macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. In some aspects, the microparticles comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

[0024] Furthermore, in one aspect, the present disclosure generally relates to a method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of: (a) a plurality of conditioned macrophages comprising microparticles functionalized with a first agent, wherein the first agent comprises dexamethasone; and (b) a plurality of conditioned macrophages comprising microparticles functionalized with a second agent. In some aspects, the second agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is an anti-fibrotic agent. In some aspects, the anti-fibrotic agent is a TGFB pathway inhibitor. In some aspects, the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof. In some aspects, the fibrotic disease or condition is pulmonary fibrosis or is muscle fibrosis. In some aspects, the fibrotic disease or condition is pulmonary fibrosis. In some aspects, the conditioned macrophages accumulate in the lungs of the subject. In some aspects, the microparticles are transferred to a cell of the subject. In some aspects, the cell of the subject is a macrophage, a non-macrophage lymphocyte, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, a neutrophil, and / or a fibroblast In some aspects, the treatment decreases expression of CD206, MERTK and CD301 in host macrophages of the subject. In some aspects, the treatment decreases the total collagen content of the lungs of the subj ect. In some aspects, the treatment decreases the collagen content of the lungs of the subject relative to the lungs of an untreated subject having pulmonary fibrosis. In some aspects, the treatment decreases the total amount of fibrotic lung tissue of the subject. In some aspects, the treatment decreases the amount of fibrotic lung tissue of the subject relative to an untreated subject having pulmonary fibrosis. In some aspects, the treatment increases the number of endothelial cells in the subject as compared to an untreated subject. In some aspects, the conditioned macrophages are derived from a subject’s monocytes. In some aspects, the conditioned macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. In some aspects, the microparticles comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL). In some aspects, the microparticles of (a) and / or the microparticles of (b) comprise microparticles designed to promote transfer. In some aspects, the microparticles of (a) and / or the microparticles of (b) are sized to promote transfer. In some aspects, the microparticles of (a) and / or the microparticles of (b) comprise an amount of microparticles that promotes microparticle transfer. In some aspects, the microparticles of (a) and / or the microparticles of (b) are further functionalized with an agent that promotes microparticle transfer.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0026] FIG. 1 presents a schematic representation of macrophage microparticle transfer assay in which transfer of microparticles to bystander macrophages was analyzed.

[0027] FIG. 2A-FIG. 2B present graphical representations of data related to microparticle transfer to bystander macrophages. FIG. 2A presents a graphical representation of the ratio of the percent GFP negative / polystyrene (PS) beads positive cells for various ratios of % GFP to % bystander cells for either a coculture with murine bone marrow derived macrophages (BMDM) or a coculture with bone marrow mononuclear cells (BMC). FIG. 2B presents a graphical representation of the ratio of the percent GFP negative / PS beads positive cells for various ratios of % GFP to % bystander cells for either a coculture with BMDM or a coculture with BMC. n=3 for each of FIG. 2A and FIG. 2B .

[0028] FIG. 3 A-FIG. 3B present graphical representations of data related to polystyrene (PS) microparticle transfer from conditioned macrophages (expressing green fluorescent protein, GFP) to bystander macrophages by analyzing the mean fluorescence intensity (MFI) of PS microparticles after coculture. FIG. 3 A presents a graphical representation of data related to the MFI of PS microparticles of bystander macrophages for various different ratios of % GFP to % bystanders for either a coculture with BMDM or a coculture with BMC. FIG. 3B presents a graphical representation of data related to the MFI for PS beads of GFP positive cells for various different ratios of % GFP to % bystanders for either a coculture with BMDM or a coculture with BMC.

[0029] FIG. 4 presents a schematic representation of a poly(lactic-co-glycolic acid) (PLGA) microparticle transfer assay in which transfer of microparticles from conditioned macrophages to bystander macrophages was analyzed.

[0030] FIG. 5 presents a graphical representation of data related to microparticle transfer to bystander macrophages after 48 hours of co-culture with conditioned macrophages.

[0031] FIG. 6 presents a schematic representation of a macrophage microparticle transfer assay in which transfer of microparticles from conditioned macrophages to bystander macrophages in different environments was analyzed. FIG. 7A-FIG. 7B present graphical representations of data related to microparticle transfer from conditioned BMCs to bystander macrophages in different conditions FIG. 7A presents a graphical representation of the transfer of microparticles (MPs) to bystander macrophages for MO, inflammatory conditions (lipopolysaccharide and interferon-gamma, LPS / IFNg), and T helper 2 (Th2) cytokines interleukin-4 and interleukin- 13 (IL4 / IL13) groups at 48 h and at 7 days. FIG. 7B presents a graphical representation of the viability of the total cells for each of the basal (MO), LPS / IFNg, and IL4 / IL13 groups at 48 h and at 7 days. The * denotes that 48h LPS / IFNg was removed from analysis in FIG. 7A and FIG. 7B as almost all cells were dead.

[0032] FIG. 8 presents images of phagocytosis / efferocytosis at the indicated timepoints.

[0033] FIG. 9 presents a schematic representation of a live cell imaging assay analyzing transfer of microparticles from conditioned macrophages to bystander macrophages.

[0034] FIG. 10 present images of data related to PLGA microparticle (red) transfer from GFP+ MP loaded macrophages (green) to bystander macrophages (unstained). The red arrow indicates MP transfer.

[0035] FIG. 11 presents images of data related to PLGA microparticle (red) transfer from GFP+ MP loaded macrophages (green) to bystander macrophages (unstained). The red arrow indicates MP transfer.

[0036] FIG. 12 presents images of data related to PLGA microparticle (red) transfer from GFP+ MP loaded macrophages (green) to bystander macrophages (unstained). The red arrow indicates MP transfer.

[0037] FIG. 13 presents images of data related to PLGA microparticle (red) transfer from GFP+ MP loaded macrophages (green) to bystander macrophages (unstained). The red arrow indicates MP transfer.

[0038] FIG. 14 presents an image of data related to cellular extension between GFP+(green) Dex MP loaded macrophages to bystander macrophages (blue) containing red fluorescent Dex MP.

[0039] FIG. 15 presents graphical representations of data related to phenotype analysis of bystander macrophages after cocultivation with MP-loaded macrophages, comparing blank MP to Dex MP as well as comparing different sized MPs. FIG. 16 presents a graphical representation of data related to MP-positive host cells seven days after implantation of scaffolds loaded with blank MP or Dex MP-loaded macrophages into the subcutaneous space of mice, a model of fibro-inflammatory disease.

[0040] FIG. 17 presents a graphical representation of data related to the percentage of MP receiving host macrophages.

[0041] FIG. 18 presents graphical representations of data related to the phenotype of host macrophages seven days after scaffold implantation. Comparison between phenotype after implantation of a scaffold without loaded macrophages (Sc), scaffold with unloaded macrophages (Sc + BMDM), scaffold with blank MP loaded macrophages (Sc + BMDM + blank MP) and scaffolds with Dex MP loaded macrophages (Sc + BMDM + Dex MPs). Equal colored points represent scaffolds that were implanted in the same mouse.

[0042] FIG. 19 presents graphical representations of data related to the phenotype of the total, MP+(+), and MP-(-) host macrophages seven days after scaffold implantation of scaffolds loaded with blank MP and Dex MP loaded macrophages. Equal colored points represent scaffolds that were implanted in the same mouse.

[0043] FIG. 20 presents a graphical representation of data related to the percentage of MP positive non myeloid cells (CD45 negative) seven days after implantation of scaffolds with blank MP or Dex MP loaded macrophages.

[0044] FIG. 21 presents a schematic representation of an assay for analyzing microparticle uptake by macrophages.

[0045] FIG. 22 presents images of control, blank MP, and Dex MP macrophages 48h after treatment with PLGA microparticles and following incubation with PS beads.

[0046] FIG. 23 presents a graphical representation of data related to the % of PS positive macrophages for each group (control, blank MP, Dex MP) after a 30 minute incubation time with PS beads following a 48h treatment with PLGA microparticles.

[0047] FIG. 24A-FIG. 24B present a table and a graphical representation of data related to the expression of both anti- and pro-fibrotic genes for untreated, blank MPs, Dex MPs, and soluble Dex groups. FIG. 24A presents a table profiling gene expression data. FIG. 24B presents a graphical representation of MMP8 gene expression for untreated, blank MPs, Dex MPs, and soluble Dex groups. FIG. 25 presents a schematic representation of an assay for dexamethasone-loaded macrophage cell therapy for pulmonary fibrosis.

[0048] FIG. 26 presents graphical representations of data related to the effects of transplantation of conditioned macrophages to fibrotic lungs.

[0049] FIG. 27 presents a schematic representation of a mouse study as described herein. 5 days after isolation of GFP+ cells, mature macrophages were treated with 1.5 pm or 2.5 pm size blank or Dexamethasone loaded microparticles in a dose of 100 pg or 250 pg per IxlO6macrophages for 4 hours. A control group of macrophages was left untreated. After 4 hours cells were detached, and free-floating MPs were removed. Cells were frozen and stored at -80°C for 10 days. One day before the surgery, cells were thawed and seeded into a well plate. 2 hours before the surgery, 500k cells were seeded onto a collagen scaffold (5 mm x 2 mm). 7 days after implantation, scaffolds were digested and host and administered cells were analyzed via flow cytometry.

[0050] FIG. 28 presents a graphical representation of data related to the cell number of host cell populations (non-myeloid cells (CD45-), non-macrophage lymphocytes (CD45+ / F480-), and macrophages (CD45+ / F480+)) positive for MPs 7 days after implantation of scaffolds with MP loaded macrophages.

[0051] FIG. 29 presents a graphical representation of data related to the percentage of host cell populations (non-myeloid cells (CD45-), non-macrophage lymphocytes (CD45+ / F480-), and macrophages (CD45+ / F480+)) positive for MPs 7 days after implantation of scaffolds with MP loaded macrophages.

[0052] FIG. 30 presents a graphical representation of data related to the influence of administration of unloaded macrophages (BMDM) or macrophages loaded with 1.5 pm or 2.5 pm size blank or Dexamethasone loaded microparticles in a dose of 100 pg or 250 pg on host macrophage phenotype. Mean fluorescence intensity (MFI) values are shown. As a control, a scaffold vehicle without cells was implanted.

[0053] FIG. 31 A-FIG. 3 IF presents schematics, data, and results related to microparticle uptake by macrophages and effects thereof. FIG. 31A presents a schematic representation of an experimental set-up. For all experiments, bone marrow-derived cells (BMCs) were cultivated for 24h before incubation with blank or dexamethasone encapsulated (Dex) MPs with varying doses (10 pg / 106 cells, 20 pg / 106 cells, 40 pg / 106 cells, 100 pg / 106 cells) for Ih, 4h or 24h. Then, cells were washed to remove unphagocytosed particles and analyzed at 2 or 7 days following removal of MPs. FIG. 3 IB presents a graphical representation of the percentage of macrophages (F4 / 80+) and monocytes (Ly6c+,CD1 lb+) of leukocytes (CD45+) in BMCs immediately (day 0), 2 days, and 7 days after MP treatment . FIG. 31C presents graphical representations of the percentage of MP-positive macrophages immediately (day 0), 2 days, and 7 days after incubation with dexamethasone encapsulated (Dex) and blank MPs. FIG. 3 ID presents a graphical representation of the percentage of MP-positive monocytes directly (dO) after incubation with Dex and blank MPs. FIG. 3 IE and FIG. 3 IF present graphical representations of the percentage of dead (fixable dead stain+) (FIG. 3 IE) and apoptotic cells (Annexin V+) (FIG. 3 IF) cells directly (dO), 2 and 7 days (only e) after incubation with Dex and blank MPs. n = 3. Two-way ANOVA with Tukey post hoc, *p < 0.05,**p < 0.01, ***p < 0.001, bp < 0.05 compared to respective blank MP group, cp < 0.05 compared to control.

[0054] FIG. 32A-FIG. 32C presents schematics and results related to a murine model of pulmonary fibrosis that was induced by influenza infection to analyze the phenotype maintenance in vivo during either the inflammatory phase (10 days after infection) or the fibrotic phase (27 days after infection). FIG. 32A presents an overview of a study related to biomaterial mediated intracellular control of macrophage phenotype. Dexamethasone-loaded microparticles are phagocytosed by macrophages. Over time, dexamethasone is intracellularly released and promotes an anti-inflammatory, anti-fibrotic phenotype in vitro and in vivo. FIG. 32B presents representative image of macrophages (stained with Celltracker in blue) loaded with dexamethasone-loaded MPs (false-colored in green). Scale bar: 20 pm. FIG. 32 C presents a graphical representation of the percentage of macrophages that contain microparticles (MPs) (blank or dexamethasone-loaded) 7 days after administration of the MPs for 24h n=3. Two-way ANOVA with Tukey post hoc, *p < 0.05,**p < 0.01, ***p < 0.001,bp < 0.05 compared to respective blank MP group.

[0055] FIG. 33A-FIG. 33J presents schematics, data and results related to analysis of the effects of microparticle dosing on macrophage phenotype. FIG. 33A presents a schematic representation of an experimental set-up. Bone marrow-derived cells (BMCs) were cultivated for 24h before blank or Dexamethasone encapsulated (Dex) MPs with varying doses (10 pg / 106cells, 20 pg / 106cells, 40 pg / 106cells, 100 pg / 106cells) were administered to the cells for 24h. Then, MPs were removed, and cells were cultivated for up to 7 days following removal of excess MPs. FIG. 33B presents a graphical representation of Umap cluster analysis based on macrophage treatment groups 2 days and 7 days after incubation with MPs presenting differences between groups. FIG. 33C and FIG. 33D present graphical representations of protein expression analysis via flow cytometry 2 days (FIG 33C) and 7 days (FIG. 33D) after incubation with MPs. One-way ANOVA with Tukey post hoc, *p < 0.05, **p < 0.01, ***p < 0.001,bp < 0.05 compared to respective blank MP group,cp < 0.05 compared to control. Statistics were calculated based on Log2 of unnormalized data. FIG. 33E-FIG. 33H present multidimensional analysis of protein marker expression 7 days after incubation with blank and Dex MPs. Cluster analysis was performed via FlowSOM (Flowjo) after the cluster number was determined via Phenograph. FIG. 33E presents distribution of treatment groups in each cluster, ^indicates significances of p < 0.05 between treatment groups. Two-way ANOVA with Tukey post hoc. FIG. 33F presents heatmap showing MFI values of each cluster and tested marker. FIG. 33G and FIG. 33H present bar graphs showing MFI Z-score for each marker in clusters with the greatest differences between treatment groups of Dex-MP dominant (FIG. 33G) and untreated and blank MP dominant cluster (FIG. 33H). FIG. 331 and FIG. 33J present phenograph single-cell cluster analysis based on MP content per cell (MFI of TRITC-labeled MPs) 7 days after incubation with blank MPs (FIG. 331) and Dex MPs (FIG. 33J). n = 3. For simplification, only the 20pg dose is shown, with other doses shown in FIG. 40, FIG. 41 and FIG. 42.

[0056] FIG. 34 presents graphical representations of the efficiency of a washing procedure used to remove free floating particles from cells that were treated with 20 pg of Dex MPs for 4h.

[0057] FIG. 35A-FIG. 35F present schematics, data, and results related to analysis of the influence of MP dose and content per cell on phagocytic capacity of macrophages. FIG. 35A presents a schematic representation of an experimental set-up. Bone marrow-derived cells (BMCs) were cultivated for 24h before blank or dexamethasone encapsulated (Dex) MPs with varying doses (10 pg / 106cells, 20 pg / 106cells, 40 pg / 106cells, 100 pg / 106cells) were administered to the cells for 24h followed by removal of excess MPs. After 2 days cells were incubated with fluorescent polystyrene (PS) beads (10 beads / cell) for up to 4h and uptake was analyzed via flow cytometry and live cell imaging. FIG. 35B presents representative images of nontreated control, blank MP treated and Dex MP -treated macrophages (blue with false-colored green MPs indicated by green arrows) after incubation with PS beads (false-colored in red indicated by white arrows) for 4h. Scale bar: 20 pm. FIG. 35C presents a graphical representation of the percentage of PS bead-positive cells after incubation for 30 minutes, 2h and 4h. FIG. 35D presents a graphical representation of MFI values of PS beads in cells after incubation for 30 minutes, 2h, and 4h. FIG. 35E and FIG. 35F present phenograph single-cell cluster analysis based on MP content per cell (MFI of Trite-labeled MPs) of blank MP (Fig. 35E) and Dex MP (FIG. 35F) treated cells 30 min, 2h and 4h after incubation with PS beads. Z-scores represented in the heatmap were calculated within bead incubation time point groups. Two-way ANOVA with Tukey post hoc, *p < 0.05, **p < 0.01, ***p < 0.001,bp < 0.05 compared to respective blank MP group,ep < 0.05 compared to control. n=3. For simplification, only the 20pg MP dose is shown, whereas other doses are shown in FIG. 43.

[0058] FIG. 36A-FIG. 36B present graphical representations of the percentage of dead (FIG. 36A) and apoptotic cells (FIG. 36B) of untreated and MP -treated populations separated by MP- negative and positive cells.

[0059] FIG. 37A-FIG. 37F present schematics, data, and results related to related to microenvironmental stimuli challenge of macrophage phenotypes in vitro. FIG. 37A presents a schematic representation of an experimental set-up. Bone marrow-derived cells (BMCs) were cultivated for 24h before 20 pg / 106cells blank or dexamethasone encapsulated (Dex) MPs or soluble dexamethasone (s.Dex, 2.5 pg / 106cells) were administered to the cells for 24h. Then, cells were washed, transferred into new media containing stimuli that were pro-inflammatory (LPS+IFNg), Th2 / pro-fibrotic (IL4+IL13), immunosuppressive (IL10), or lhl7 / fibro- inflammatory (IL17a), and cultivated for up to 7 days. FIG. 37B presents hierarchical clustering of samples and genes; in the heatmap represents Z-score of relative gene expression 2 days after transfer to media supplemented with MCSF (M0) or additional stimuli. FIG. 37C presents a graphical representation of single marker presentation of relative gene expression of macrophages 2 days after transfer to environmental stimuli supplemented media. FIG. 37D presents a graphical representation MMP8 secretion analyzed by ELISA ages 2 and 7 days after transfer to media supplemented with MCSF (M0) or supplemented additionally with LPS / IFNg or IL4 / IL13. n = 3. FIG. 37E presents representative images of untreated control macrophages, blank MP -loaded and Dex-MP-loaded macrophages 2 and 7 days following incubation with MPs after 24-hour cultivation on DQ-collagen I-containing collagen I coatings. The nuclei of macrophages are stained with Hoechst 33342 in blue. Green fluorescent areas indicate collagen I degradation. FIG. 37F presents graphical representations of quantitative image analysis of the DQ collagen I signal for area and intensity after 24-hour macrophage cultivation on DQ collagen-containing collagen coatings 2 and 7 days after MP incubation. For area calculations, total area per image was divided by the number of nuclei per image. For intensity calculations, the average intensity per DQ fluorescent event was calculated per image. For each sample, the average of 4 images are presented. Two-way ANOVA with Tukey post hoc, *p < 0.05,**p < 0.01, ***p < 0.001, Op < 0.05 compared to M0 Ctrl.

[0060] FIG. 38A-FIG. 38B present graphical representations of results related to the Effect of MP dose on macrophage phenotype, displayed as percentage of F4 / 80+ cells positive for CD163, CD206, CD301b, CD9, MHC2, or CD86, two (FIG. 38A) and seven (FIG. 38B) days after MP treatment. One-way ANOVA with Tukey post hoc, *p < 0.05,**p < 0.01, ***p < 0.001, bp < 0.05 compared to respective blank MP group, cp < 0.05 compared to control.

[0061] FIG. 39A-FIG. 39G present schematics, data, and results related to phenotype analysis of macrophages administered to inflamed and fibrotic lungs. FIG. 39A presents a schematic representation of an experimental set-up Bone marrow-derived cells (BMCs) were cultivated for 24h before blank or dexamethasone encapsulated (Dex) MPs at a dose of 20 pg / 106cells were administered to the cells for 24h. Then, cells were washed and either incubated in vitro or administered to inflamed or fibrotic lungs of mice for 2 days. FIG 39B and FIG 38C present graphical representations of protein marker expression of GFP+ administered macrophages that was analyzed via flow cytometry 2 days after administration into inflamed (FIG. 39B) and fibrotic (FIG. 39C) lungs. FIG. 39D and FIG. 39F present hierarchical clustering based on MFI values of protein expression of in vitro cultivated vs. GFP+ macrophages administered into inflamed (FIG. 39D) or fibrotic lungs (FIG. 39F). FIG. 39E and FIG. 39G present hierarchical clustering based on MFI values of administered GFP+ macrophages vs. host macrophages under inflamed (FIG. 39 E) or fibrotic (FIG. 39 G) conditions. Two-way ANOVA with Tukey post hoc, n = 3. *p < 0.05,**p < 0.01, ***p < 0.001.

[0062] FIG. 40A-FIG. 40G present schematics, data, and results related to multidimensional analysis of protein marker expression 2 days after incubation with blank and Dex MPs. Cluster analysis was performed via FlowSOM (Flowjo). FIG. 40A presents a graphical representation of the distribution of treatment groups in each cluster. *indicates significances of p < 0.05 between treatment groups. Two-way ANOVA with Tukey post hoc. FIG. 40B presents a heatmap showing MFI values of each cluster and tested marker. FIG. 40C and FIG. 40D present pie graphs showing the distribution of cells and bar graphs showing MFI Z-score for each marker in Dex-MP dominant (FIG. 40C) and Untreated / Blank MP dominant (FIG. 40D) clusters that were identified as significantly different between treatment groups in FIG. 40C. FIG. 40E presents Umap cluster analysis based live cells / singlets / F480+, showing significant clusters identified in (f) presenting similarity between clusters. FIG. 40F and FIG. 40G present phenograph single-cell cluster analysis based on MP loading per cell (MFI of TRITC-labeled MPs) 2 days after incubation with blank MPs (FIG. 40F) and Dex MPs (FIG. 40G).

[0063] FIG. 41A-FIG. 41G present schematics, data, and results related to multidimensional analysis of protein marker expression 7 days after incubation with blank and Dex MPs. Cluster analysis was performed via FlowSOM (Flowjo). FIG. 41A presents a graphical representation of the distribution of treatment groups in each cluster. *indicates significances of p < 0.05 between treatment groups. Two-way ANOVA with Tukey post hoc. FIG. 41B presents a heatmap showing MFI values of each cluster and tested marker FIG. 41C and FIG 41D present pie graphs showing the distribution of cells and bar graphs showing MFI Z-score for each marker in Dex- MP dominant (FIG. 41C) and Untreated / Blank MP dominant (FIG. 4 ID) clusters that were identified as significantly different between treatment groups in (FIG. 41C). FIG. 4 IE presents Umap cluster analysis based live cells / singlets / F480+, showing significant clusters identified in (f) presenting similarity between clusters. FIG. 41F and FIG. 41G present phenograph single-cell cluster analysis based on MP loading per cell (MFI of TRITC-labeled MPs) 7 days after incubation with blank MPs (FIG. 4 IF) and Dex MPs (FIG. 41G).

[0064] FIG. 42A-FIG. 42D present data and results related to multidimension analysis of protein marker expression 7 days after incubation with blank and Dex MPs with reduced cluster number. Cluster analysis was performed via FlowSOM (Flowjo) with 7 clusters. FIG. 42A presents a graphical representation of the distribution of treatment groups in each cluster, ^indicates significances of p < 0.05 between treatment groups. Two-way ANOVA with Tukey post hoc. FIG. 42B presents a heatmap showing MFI values of each cluster and tested marker. FIG. 42C and FIG. 42D present bar graphs showing MFI Z-score for each marker in Dex-MP dominant (FIG. 42C) and Untreated / Blank MP dominant (FIG. 42D) clusters that were identified as significantly different between treatment groups in (FIG. 42C) and included at least 5% of all cells. FIG. 43A-FIG. 43D present data and results related to the influence of MP dose and content per cell on phagocytic capacity of macrophages. FIG. 43A presents a graphical representation of the percentage of PS bead-positive cells after incubation for 30 minutes, 2h and 4h. FIG. 43B presents a graphical representation of MFI values of PS beads in cells after incubation for 30 minutes, 2h, and 4h. FIG. 43C and FIG. 43D present phenograph single-cell cluster analysis based on MP content per cell (MFI of TRITC-labeled MPs) of blank MP (FIG. 43C) and Dex MP (FIG. 43D) treated cells 30 min, 2h and 4h after incubation with PS beads. Z- scores represented in the heatmap were calculated within bead incubation time point groups. Two-way ANOVA with Tukey post hoc, *p < 0.05,**p < 0.01, ***p < 0.001,bp < 0.05 compared to respective blank MP group,cp < 0.05 compared to control. n=3.

[0065] FIG. 44A-FIG. 44E present data and results related to microparticle characterization. FIG. 44A presents representative images of microparticles loaded with the fluorescent dye TRITC (blank MPs) and TRITC plus Dexamethasone (Dex MPs). FIG. 44B presents a graphical representation of MP size distribution based on Fiji particle analyzation of each 5 randomly taken images of blank and Dex MPs as seen in FIG. 44A. FIG. 44C presents scanning electron microscopic images of blank MPs before and after one hour UV sterilization. FIG. 44D presents a graphical representation of cumulative Dexamethasone release from 20 pg of Dex-loaded PLGA microparticles (56 % w / w) of freshly prepared and 3, 9, or 12-month-old samples that were stored at -80C. n=3 individual batches. FIG. 44E presents a table of fabrication parameters.

[0066] FIG. 45A-FIG. 45B present data and results related to the influence of Dexamethasone on phagocytic capacity of macrophages. FIG. 45A and FIG. 45B present graphical representations of the percentage (FIG. 45 A) and MFI values (FIG. 45B) of PS bead-positive cells 2 days after incubation with Dex MPs or the respective amount of soluble Dex released within 24h for 24h. PS bead uptake was analyzed after 4h incubation. One-way ANOVA with Tukey post hoc, ***p < 0.001, sp < 0.05 compared to respective soluble Dex group. n=3.

[0067] FIG. 46A-FIG. 46C present data and results related to gene expression analysis of macrophages loaded with 20 pg of MPs challenged with environmental stimuli in vitro. FIG. 46A presents hierarchical clustering of samples and genes (MMP8, TNF , CD86, ILlb, LRP1, CD206, CD 163, IL 10, CD9, TIMP1) in the heatmap represents Z-score of relative gene expression 7 days after incubation with MPs and transfer to media supplemented with MCSF (M0) or additional LPS / IFNg; IL4 / IL13; IL10; IL17a. FIG. 46B presents graphical representations of single marker presentation of relative gene expression of macrophages 7 days after incubation with MPs and transfer to environmental stimuli supplemented media. FIG. 46C presents graphical representations of MFI values of protein expression (CD163, CD206, CD301b, CD9, MHC2, CD86) analyzed via Flow Cytometry 2 and 7 days after incubation with MPs and transfer to media supplemented with MCSF (MO) or supplemented additionally with LPS / IFNg or IL4 / IL13. n=3 Two-way ANOVA with Tukey post hoc, *p < 0.05,**p < 0.01, ***p < 0.001.

[0068] FIG. 47A-FIG. 47C present data and results related to macrophage analysis. FIG. 47A presents data related to a gating strategy used for GFP+ cells. After exclusion of cell clusters and dead cells, cells were gated on CD45, followed by F480. Afterward, cells were gated on GFP+ and negative for analysis. FIG. 47B presents graphical representations of the percentage of recovered GFP positive cells in relation to all cells and macrophages. FIG. 47C presents graphical representations of the percentage of total recovered GFP positive cells. Calculations based on average yield of 40xl06total recovered cells from lung tissue, 4xl06administered GFP+ cells and recovered percentage from (B, all cells).

[0069] FIG. 48A-FIG. 48F present data and results related to analysis of macrophage administration. FIG. 48A and FIG. 48D present graphical representations of protein marker expression, which are presented as MFI values of GFP+ administered macrophages and host macrophages (F4 / 80+) 2 days after administration into inflamed (FIG. 48A) and fibrotic (FIG. 48D) lungs. FIG. 48B and FIG. 48E present principal component analysis based on MFI values of protein expression of in vitro cultivated vs. GFP+ macrophages administered into inflamed (FIG. 48B) or fibrotic lungs (FIG. 48E). FIG. 48C and FIG. 48F present principal component analysis based on MFI values of administered GFP+ macrophages vs. host macrophages under inflamed (FIG. 48C) or fibrotic (FIG. 48F) conditions. Two-way ANOVA with Tukey post hoc, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0070] FIG. 49A-FIG. 49B present data and results related to phenotype analysis of host macrophages and macrophages administered to inflamed and fibrotic lungs after influenza infection. FIG. 49A and FIG. 49B present graphical representations of protein marker expression of GFP+ administered macrophages and host macrophages (F4 / 80+) that was analyzed via flow cytometry 2 days after administration into inflamed (FIG. 49A) and fibrotic (FIG. 49B) lungs. Two-way ANOVA with Tukey post hoc, n = 3. *p < 0.05,**p < 0 01, ***p < 0.001. FIG. 50 presents graphical representations of host macrophage subtypes. Cell number of Leukocytes (CD45+) and macrophage subtypes (alveolar macrophages, monocyte-derived alveolar macrophages, interstitial macrophages) in healthy as well as inflamed and fibrotic lungs after influenza infection and two days after administering untreated, blank MP or Dex MP loaded macrophages or PBS. The arrow indicates the occurrence of macrophage subtypes during homeostasis and fibrosis based on A.L. McCubbrey, et al.„ Am J Respir Cell Mol Biol 58(1) (2018) 66-78.

[0071] FIG. 51 presents a schematic representation of an experimental setup for a model of pulmonary fibrosis. Progressive, non-reversible fibrosis was established by three Bleomycin doses administered intratracheally bi-weekly. Six weeks after the last Bleomycin dose mice, progressive pulmonary fibrosis was established, and treatments were administered intratracheally. After 1-14 days, microparticle distribution, uptake as well as macrophage phenotype and fibrosis reversal were analyzed.

[0072] FIG. 52 presents a graphical representation of the biodistribution of microparticles in whole organs 24 hrs after treatment administration.

[0073] FIG. 53 presents representative images of whole organ microparticle distribution after 24 hours.

[0074] FIG. 54 presents a graphical representation of the biodistribution of microparticles in whole organs 48 hours after treatment administration.

[0075] FIG. 55 presents representative images of whole organ microparticle distribution after 48 hours.

[0076] FIG. 56 presents a graphical representation of Dexamethasone content in blood plasma 24 and 48 hrs after treatment administration.

[0077] FIG. 57 presents a graphical representation of Dexamethasone content in bronchoalveolar lavage fluid 24 and 48 hrs after treatment administration.

[0078] FIG. 58 presents a graphical representation of the percentage of host macrophages positive for microparticles 2, 7 and 14 days after treatment administration - pulmonary fibrosis model

[0079] FIG. 59 presents a graphical representation of the number of host macrophages positive for microparticles 2, 7 and 14 days after treatment administration - pulmonary fibrosis model. FIG. 60 presents a graphical representation of the percentage of administered (GFP+) macrophages positive for microparticles 2, 7 and 14 days after treatment administration - pulmonary fibrosis model.

[0080] FIG. 61 presents a schematic representation of experimental set-up. 5 days after isolation of GFP+ cells, mature macrophages were treated with 2.5 pm size blank or Dexamethasone loaded microparticles in a dose of 250 pg per IxlO6macrophages for 4 hours. A control group of macrophages was left untreated. After 4 hours cells were detached, and free-floating MPs were removed and cells were incubated until the next morning. 2 hours before the surgery, 500k cells were seeded onto a collagen scaffold (5 mmx2mm). 7 days after implantation, scaffolds were digested and host and administered cells were analyzed via flow cytometry.

[0081] FIG. 62 presents a graphical representation of the percentage of host macrophages in subcutaneous implantation model positive for MPs 14 days after scaffold implantation.

[0082] FIG. 63 presents a graphical representation of the percentage of host CD45 negative, nonleukocytes in subcutaneous implantation model positive for MPs 14 days after scaffold implantation.

[0083] FIG. 64 presents graphical representations of analysis of the phenotype of administered (GFP+) macrophages and host macrophages 7 days after treatment administration as measured by MFI.

[0084] FIG. 65 presents a graphical representation of the total collagen content of lungs 7 days after treatment administration compared to naive, healthy lungs and baseline (fibrotic lungs at the time point of treatment administration).

[0085] FIG. 66 presents a graphical representation of total collagen content of lungs 14 days after treatment administration compared to naive, healthy lungs and baseline (fibrotic lungs at the time point of treatment administration).

[0086] FIG. 67 presents a graphical representation of total collagen content of lungs 7 days after treatment administration compared to naive, healthy lungs and baseline (fibrotic lungs at the time point of treatment administration).

[0087] FIG. 68 presents a graphical representation of total collagen content of lungs 14 days after treatment administration compared to naive, healthy lungs and baseline (fibrotic lungs at the time point of treatment administration). FIG. 69 presents 3D reconstructions of micro CT image analysis of fibrotic lungs, 14 days after treatment administration compared to Naive, healthy lungs.

[0088] FIG. 70 presents graphical representations of the phenotype of implanted (GFP+) macrophages and host macrophages 7 days after implantation of scaffolds either unloaded (SF) or loaded with macrophages (M), blank MP macrophages (MB) or Dex-MP-macrophages MD), measured by MFI.

[0089] FIG. 71 presents a graphical representation of the percentage of endothelial cells of the non-leukocyte cell population 14 days after subcutaneous implantation of scaffolds either unloaded (SF) or loaded with macrophages (M), blank MP macrophages (MB) or Dex-MP- macrophages MD.

[0090] FIG. 72 presents a graphical representation of the number of endothelial cells 14 days after subcutaneous implantation of scaffolds either unloaded (SF) or loaded with macrophages (M), blank MP macrophages (MB) or Dex-MP-macrophages MD).

[0091] FIG. 73 presents a graphical representation of the percentage of microparticle positive host cells in a model of volumetric muscle loss 1 day after implantation.

[0092] FIG. 74 presents a graphical representation of the number of microparticle positive host cells based on 5000 live cells of in a model of volumetric muscle loss 1 day after implantation.

[0093] FIG. 75 presents a graphical representation of the percentage of microparticle-positive host cells in a model of volumetric muscle loss 7 days after implantation.

[0094] FIG. 76 presents a graphical representation of the number of microparticle positive host cells based on 5000 live cells of in a model of volumetric muscle loss 7 days after implantation.

[0095] FIG. 77 presents graphical representations of the phenotype of implanted (GFP+) macrophages and host macrophages 7 days after treatment administration as measured by MFI. Mice were either untreated, or treated with surgifoam (SF) scaffold, SF scaffold loaded with macrophages (BMDM), SF loaded with BMDM loaded with blank MPs or SF loaded with BMDM loaded with Dex MP.

[0096] DETAILED DESCRIPTION

[0097] DEFINITIONS Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0098] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and nucleic acid hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0099] That the disclosure may be more readily understood, select terms are defined below.

[0100] As used herein, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. By way of example, “an element” means one element or more than one element. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

[0101] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0102] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 and so forth, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0103] 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.

[0104] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by any degree of suppression, remission, or eradication of a disease state.

[0105] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0106] As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0107] The terms “patient”, “subject”, and “individual” are used interchangeably and are intended to include living organisms that may be subjected to treatment for a given disease, e.g., mammals. A “subject”, “patient”, or “individual”, as used herein, can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals, as well as simian and non-human primate mammals. Preferably, the subject is human.

[0108] As used herein, the terms “effective amount” and “therapeutically effective amount” are used interchangeably and refer to the amount required to reduce or improve at least one symptom or change in a clinical marker of a disease relative to an untreated patient The effective amount of the treatment used for therapeutic treatment of the disease varies depending upon the manner of the specific disorder, condition or disease, extent of the disorder, condition or disease, and administration of the cells, as well as the age, body weight, and general health of the subject. The effective amount is capable of achieving a particular desired biological result and / or provides a therapeutic or prophylactic benefit.

[0109] As used herein, the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0110] As used herein, the term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0111] As used herein, the term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0112] As used herein, the term “identity” refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0113] As used herein, the terms "conservative variation" or "conservative substitution" generally refers to the replacement of an amino acid residue by another, biologically similar residue. Conservative variations or substitutions are not likely to change the shape of the peptide chain. Examples of conservative variations, or substitutions, include the replacement of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.

[0114] As used herein, the term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not “isolated,” but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0115] An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids that 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 that 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 that is part of a hybrid gene encoding additional polypeptide sequence.

[0116] The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using recombinant DNA methods. The term "recombinant DNA" as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[0117] "Variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A variant of a nucleic acid or peptide may be a naturally occurring such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

[0118] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0119] As used herein, the term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (z.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T.”

[0120] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0121] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c ), intravenous (i.v ), intramuscular (i.m ), or intrasternal injection, or infusion techniques

[0122] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, “nucleic acid” and “polynucleotide” as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides” and which comprise one or more “nucleotide sequence(s)”. The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences (he., “nucleotide sequences”) which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0123] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0124] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like

[0125] The term “biological” or “biological sample” refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. Frequently the sample will be a “clinical sample” which is a sample derived from a patient. Such samples include, but are not limited to, bone marrow, cardiac tissue, sputum, blood, lymphatic fluid, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes

[0126] As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and poly adenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. There are numerous expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art that may be used in the compositions of the invention. “Operably linked” should be construed to include RNA expression and control sequences in addition to DNA expression and control sequences.

[0127] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0128] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence, which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements, which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0129] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0130] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0131] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with other chemical components, such as carriers, stabilizers, diluents, adjuvants, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism Multiple techniques of administering a compound exist in the art including, but not limited to: intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0132] The language “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.

[0133] The term “MO macrophage” as used herein refers to a subtype of macrophages that are resting or unactivated (unpolarized).

[0134] The term “Ml macrophage” as used herein refers to a subtype of macrophages that are classically activated or exhibit an inflammatory macrophage phenotype. Ml macrophages may be activated by LPS and IFN-y, or other pro-inflammatory stimuli, and secrete high levels of IL- 1-beta, TNF-alpha, and IL-12. Ml macrophages also include macrophages that exhibit a hybrid phenotype that is predominantly the Ml phenotype.

[0135] The term “M2A macrophage” as used herein refers to a macrophage subtype of prohealing macrophages. They are activated by IL-4 and IL-13 and secrete high levels of CCL18 and CCL22. M2A macrophages also include macrophages that exhibit a hybrid phenotype that is predominantly the M2A phenotype.

[0136] The term “M2C macrophage” as used herein refers to a macrophage subtype of proremodeling macrophages. M2C macrophages are involved in matrix remodeling and tissue repair. They are activated by IL- 10 and secrete high levels of MMPs, in particular MMP9 M2C macrophages also include macrophages that exhibit a hybrid phenotype that is predominantly the M2C phenotype.

[0137] The term “M2” broadly refers to macrophages that function in constructive processes like wound healing and tissue repair, although an excessive amount of tissue repair may lead to fibrosis.

[0138] “Hydrogel microsphere,” “hydrogel microspheres” and “microspheres” as used herein refer to a three dimensional structure on the order of about 500nm to about 999 microns made of polymers, such as natural polymers, synthetic polymers, or combinations of both, and liquid, such as water.

[0139] As used herein, the term “monocyte” means cells with the potential to differentiate into macrophages.

[0140] As used herein, the term “microparticle” refers to any synthetic structure that can be phagocytosed by a macrophage, where the synthetic structure is less than about 20 pm in its largest dimension. Microparticles include nanoparticles, that is, particles ranging from 1-999 nm in their largest dimension. Microparticles can be assembled and / or aggregated, e g , through covalent, physical, and / or ionic bonds. Examples of microparticles include but are not limited to structures comprising hydrophobic or hydrophilic polymers, charged or uncharged polymers, metals such as gold or iron, liposomes, vesicles, and / or carbon structures such as nanotubes or nanodiamonds. In some instances, a microparticle is biodegradable. In some instances, a microparticle is non-biodegradable. In some aspects, a microparticle comprises one or more polymers, such as one or more of the polymers described herein. In some aspects, a microparticle comprises a hydrogel.

[0141] As used herein, the terms “conditioned macrophage” and “loaded macrophage” are used interchangeably to refer to a macrophage that has internalized a microparticle. Conditioned macrophages can be any type of macrophage, such as, for example, an MO, an Ml, and / or an M2 macrophage. The conditioned macrophage can be present in any environment, such as in vivo or in vitro.

[0142] As used herein, the term “unconditioned macrophage” refers to a macrophage that has not internalized a microparticle. Unconditioned macrophages can include, for instance, host or bystander macrophages that have not internalized a microparticle. Unconditioned macrophages can be any type of macrophage. Unconditioned macrophages can be present in any environment, such as in vivo or in vitro.

[0143] As used herein, the term “agent” refers to a molecule intended to have a desired biological, physiological, chemical, and / or mechanical effect. In some instances, the agent is a biological molecule, such as a nucleic acid and / or a polypeptide. In some instances, the agent is a chemical entity, such as a drug molecule. In some aspects, the agent is a therapeutic agent. In some aspects, the agent is a pro-reparative agent. In some aspects, the agent is a pro- inflammatory agent. In some aspects, the agent is an anti-inflammatory agent. In some aspects, the agent is an anti-fibrotic agent

[0144] As used herein, the term “functionalize” refers to any means of modification that results in attachment or association of a group or moiety, such as attachment or association of an agent to a microparticle. For instance, a microparticle can be functionalized with one or more agents. Agents can be attached to the microparticles by any means known in the art and any means needed to attach the agent to the microparticle. In some instances, the attachment is reversible and / or degradable. In some instances, the attachment comprises a covalent bond. In some instances, the attachment comprises an ionic bond. In some instances, the attachment comprises physical entrapment within the polymer microparticle.

[0145] COMPOSITIONS AND METHODS

[0146] As discussed herein, in some aspects, the present invention describes compositions and methods comprising conditioned macrophages. In some aspects, the present invention relates to methods of treating a disease or condition in a subject in need thereof, wherein the method comprises macrophage-based therapy with conditioned macrophages.

[0147] As described herein and as presented in the results of the working examples, drug-loaded polymeric microparticles can be engineered to control their macrophage-mediated transfer of microparticles from conditioned macrophages to unconditioned macrophages such as host macrophages or to other cell types such as fibroblasts. Such control can be influenced, for instance, by altering macrophage loading conditions, drug formulation, size, charge, polymer type, and / or exposure to soluble stimuli. As discussed herein, microparticle-carrying macrophages can be engineered to retain certain microparticles but efficiently transfer others, thereby enabling a therapeutic macrophage cell therapy strategy in which the adoptively transferred macrophages are intracellularly controlled by one formulation of microparticles while another formulation of microparticles are transferred to surrounding cell types. Such microparticle-transferring macrophage formulations can be used to treat pathological conditions characterized by impaired cellular behavior, particularly because macrophages can home to sites of inflammation and infiltrate dense tissues. Microparticle-carrying macrophages can also be engineered to transfer certain microparticles to unconditioned macrophages such as host macrophages and other microparticles to other cell types, such as fibroblasts.

[0148] Compositions

[0149] In one aspect, the present disclosure generally relates to a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise a) a plurality of macrophages comprising one or more microparticles designed to promote microparticle transfer; and / or b) a plurality of macrophages comprising one or more microparticles designed to inhibit microparticle transfer. In some aspects, the one or more microparticles designed to promote microparticle transfer can be of any size, amount, composition, state of functionalization, degree of functionalization, and / or of any biophysical property, such as those described herein, that promotes microparticle transfer. In some aspects, the one or more microparticles designed to inhibit microparticle transfer can be of any size, amount, composition, state of functionalization, degree of functionalization, and / or of any biophysical property, such as those described herein, that inhibits microparticle transfer. As discussed further infra., in some aspects, microparticles designed to inhibit microparticle transfer are designed as such that, relative to microparticles designed to promote transfer, microparticles designed to inhibit transfer will transfer at a slower rate as compared to those designed to promote transfer. A person of skill in the art in possession of this disclosure will appreciate that due to trends related to biophysical properties that promote or inhibit microparticle transfer, a given biophysical property does not in an absolute sense in every case determine whether the microparticle is such that transfer is promoted or inhibited but may in various aspects be a relative term in which one set of conditioned macrophages comprises microparticles which will have a relatively lower frequency of transfer to other cells and a separate set of conditioned macrophages comprises microparticles with a relatively higher frequency of transfer to other cells. In this way, in various aspects and embodiments, a skilled person can achieve a desired frequency of transfer for each of two or more populations of microparticles within conditioned macrophages. Further examples of properties that increase microparticle transfer and properties that inhibit microparticle transfer are discussed throughout the application and are also presented in the table below.

[0150] TABLE OF PROPERTIES THAT INFLUENCE MICROPARTICLE TRANSFER

[0151] In one aspect, the present disclosure generally relates to a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising microparticles sized to promote transfer; and / or b) a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer. In one aspect, the present disclosure generally relates to a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising an amount of microparticles that promotes microparticle transfer; and b) a plurality of conditioned macrophages comprising an amount of microparticles that inhibits microparticle transfer.

[0152] Microparticles

[0153] In some aspects, the microparticle sized to promote transfer and / or the microparticle sized to inhibit transfer and / or the microparticle, e g., a microparticle designed to promote transfer, e.g., a microparticle designed to inhibit transfer, comprises poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly(caprolactone) (PCL), or polystyrene. In some aspects, the microparticle sized to promote transfer and / or the microparticle sized to inhibit transfer and / or the microparticle comprises poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL) In some aspects, the microparticle sized to promote transfer and / or the microparticle sized to inhibit transfer and / or the microparticle comprises hydrophobic or hydrophilic polymers, charged or uncharged polymers, metals such as gold or iron, liposomes, vesicles, and / or carbon structures such as nanotubes or nanodiamonds. In some aspects, the microparticle sized to promote transfer and / or the microparticle sized to inhibit transfer and / or the microparticle comprises a biodegradable microparticle. In some aspects, the microparticle sized to promote transfer and / or the microparticle sized to inhibit transfer and / or the microparticle comprises a non-biodegradable microparticle. In some aspects, the microparticle sized to promote transfer and / or the microparticle sized to inhibit transfer and / or the microparticle comprises a hydrogel microparticle. In some aspects, the microparticle sized to promote transfer and / or the microparticle sized to inhibit transfer and / or the microparticle comprises one or more natural polymers, one or more synthetic polymers, or combinations of both natural and synthetic polymers. In some aspects, natural polymers include polymers such as anionic polymers (for example, hyaluronic acid, alginic acid, pectin, carrageenan, chondroitin sulfate, dextran sulfate), cationic polymers (for example, chitosan and polylysine), amphipathic polymers (such as collagen, gelatin, carboxymethyl chitin and fibrin) and neutral polymers (for example, dextran, agarose and pullulan), and their derivatives or neutral polymers known in the art. In some aspects, synthetic polymers include, but are not limited to, polymers such as polyesters, poly(ethylene glycol), poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(hydroxyl butyrate); polypropylene fumerate-co-ethylene glycol)±acrylate end groups, poly(poly(ethylene glycol) / poly(butylene oxide)terephthalate), and any derivatives thereof or known in the art. In some aspects, synthetic polymers include, for example, other polymers such as: poly(ethylene glycol)-bis-(poly(lactic acid)-acrylate); poly(ethylene glycol)±cyclodextrins; poly(ethylene glycol)-g-poly(acrylamide-co-Vamine); polyacrylamide; poly(N-isopropyl acrylamide-co-acrylic acid); poly(N-isopropyl acrylamide-co- ethyl methacrylate); poly(vinyl acetate) / poly(vinyl alcohol); poly(N-vinyl pyrrolidone); poly(methyl methacrylate-co-hydroxy ethyl methacrylate); polyacrylonitrile-co-allyl sulfonate); poly(biscarboxy-phenoxy-phosphazene); poly(glucosylethyl methacrylate-sulfate), and derivatives thereof or synthetic polymers known in the art. In some aspects, combinations of natural and synthetic polymers include polymers such as poly(polyethylene glycol-co-peptides), alginate g-(polyethylene oxide-polypropylene oxide-polyethylene oxide), poly(polylactic-co- glycolic acid-co-serine), col lagen-acry late, alginate-acrylate, poly(hydroxyethly methacyrlate-g- peptide), poly(hydroxyethyl methacyrlate / collagen), hyaluronic acid-g-N-isopropyl acrylamide), or any combination of natural and synthetic polymers described here or known in the art.

[0154] In some aspects, the microparticles sized to promote transfer are smaller in diameter relative to the diameter of the microparticles sized to inhibit transfer. Without wishing to be bound by theory, it is posited that microparticles of smaller diameter promote microparticle transfer relative to microparticles of a larger diameter. A person of skill in the art in possession of this disclosure will appreciate that due to this trend, the diameter of the microparticles does not in an absolute sense in every case determine whether the microparticle is sized to promote or inhibit transfer but may in various embodiments be a relative term in which one set of conditioned macrophages comprises relatively larger microparticles which will have a relatively lower frequency of transfer to other cells and a separate set of conditioned macrophages comprises relatively smaller microparticles with a relatively higher frequency of transfer to other cells. In this way, in various aspects and embodiments, a skilled person can achieve a desired frequency of transfer for each of two or more populations of microparticles within conditioned macrophages. In some aspects, the microparticles sized to promote transfer are about 0.001 pm, about 0.005 pm, about 0.01 pm, about 0.02 pm, about 0.03 pm, about 0.04 pm, about 0.05 pm, about 0.06 pm, about 0.07 pm, about 0.08 pm, about 0.09 pm, about 0.1 pm, about 0.2 pm, about 0.30 pm, about 0.40 pm, about 0.50 pm, about 0.60 pm, about 0.70 pm, about 0.80 pm, about 0.90 pm, about 1.00 pm, about 1.25 pm, about 1.50 pm, about 1.75 pm, about 2.00 pm, about 2.25 pm, about 2.50 pm, about 2.75 pm, about 3.00 pm, about 3.25 pm, about 3.50 pm, about 3.75 pm, about 4.00 pm, about 4.25 pm, about 4.75 pm, about 5.0 pm, about 6.00 pm, about 7.00 pm, about 8.00 pm, about 9.00 pm, about 10.0 pm , about 12.5 pm , about 15.0 pm, about 17.5 pm, or about 20.0 pm in diameter, or any value therebetween.

[0155] In some aspects, the microparticles sized to inhibit transfer are larger in diameter relative to the diameter of the microparticles sized to promote transfer. Without wishing to be bound by theory, it is posited that microparticles of larger diameter inhibit microparticle transfer relative to microparticles of a smaller diameter. In some aspects, the microparticles sized to inhibit transfer are about 0.001 pm, about 0.005 pm, about 0.01 pm, about 0.02 pm, about 0.03 pm, about 0.04 pm, about 0.05 pm, about 0.06 pm, about 0.07 pm, about 0.08 pm, about 0.09 pm, about 0.1 pm, about 0.2 pm, about 0.30 pm, about 0.40 pm, about 0.50 pm, about 0.60 pm, about 0.70 pm, about 0.80 pm, about 0.90 pm, about 1.00 pm, about 1.25 pm, about 1.50 pm, about 1.75 pm, about 2.00 pm, about 2.25 pm, about 2.50 pm, about 2.75 pm, about 3.00 pm, about 3.25 pm, about 3.50 pm, about 3.75 pm, about 4.00 pm, about 4.25 pm, about 4.75 pm, about 5.0 pm, about 6.00 pm, about 7.00 pm, about 8.00 pm, about 9.00 pm, about 10.0 pm , about 12.5 pm , about 15.0 pm, about 17.5 pm, or about 20.0 pm in diameter, or any value therebetween.

[0156] In some aspects, the microparticle sized to promote transfer and / or the microparticle sized to inhibit transfer and / or the microparticle is functionalized with an agent.

[0157] In some aspects, the composition comprises: a) one or more microparticles designed to promote microparticle transfer comprising one or more microparticles comprising biophysical properties designed to promote microparticle transfer; and / or b) one or more microparticles designed to inhibit microparticle transfer comprising one or more microparticles comprising biophysical properties designed to inhibit microparticle transfer. In some aspects, the biophysical property can be any known biophysical property In some aspects, the biophysical property includes size, charge, and / or hydrophobicity.

[0158] In some aspects, the composition comprises the composition comprises: a) one or more microparticles designed to promote microparticle transfer comprising one or more microparticles comprising a molecular weight designed to promote microparticle transfer; and / or b) one or more microparticles designed to inhibit microparticle transfer comprising one or more microparticles comprising a molecular weight designed to inhibit microparticle transfer. In some aspects, microparticles comprising a molecular weight designed to promote microparticle transfer have a molecular weight of about 5 kDa, about 7 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, or about 100 kDa. In some aspects, microparticles comprising a molecular weight designed to inhibit microparticle transfer have a molecular weight of about 5 kDa, about 7 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, or about 100 kDa. In some aspects, microparticles of a lower relative molecular weight have a relatively shorter degradation time, which further promotes transfer. In some aspects, microparticles of a higher molecule weight have a relatively longer degradation time, which further inhibits microparticle transfer.

[0159] Agents

[0160] In some aspects, the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer and / or the microparticles are functionalized with an agent. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is an anti-fibrotic agent, an anti-inflammatory agent, a pro-reparative agent, a pro-regenerative agent, or a chemotherapeutic agent. In some aspects, the agent, e.g., therapeutic agent, comprises a nucleic acid. In some aspects, the nucleic acid comprises DNA and / or RNA. In some aspects, the nucleic acid is a synthetic nucleic acid. In some aspects, the nucleic acid comprises a vector, such as an expression vector. In some aspects, the nucleic acid encodes for a therapeutic molecule, such as a therapeutic polypeptide. In some aspects, the nucleic acid encodes for an IL- 10 polypeptide. In some aspects, the nucleic acid encodes for a matrix metalloprotease such as MMP12.

[0161] In some aspects, the agent, e.g., therapeutic agent, comprises a drug molecule. In some aspects, the agent comprises dexamethasone. In some aspects, the agent comprises anti-fibrotic agents such as nitedanib, pirfenidone, metformin, belapectin, colchicine, verteporfin, TP0427736, CBT-295, UNC2250, sitravatinib, and theophylline. In some aspects, the agent comprises a chemotherapeutic or anti-cancer agent such as doxorubicin, paclitaxel, 5- fluorouracil, cisplatin, carboplatin, tooendanin, pi3k gamma inhibitor ipi-549, entrectinib. In some aspects, the agent comprises an anti-inflammatory agent such as prednisone, minocycline, etanercept, tranilast, atorvastatin, rosuvastatin, simvastatin, lovastatin, pravastatin, aspirin, and non-steroidal anti-inflammatory drugs. In some aspects, the agent comprises a tolerogenic agent such as rapamycin, vitamin D3, LF 15-0195, IL-2. In some aspects, the therapeutic agent comprises a drug that promotes over-expression of PINK 1, a drug that promotes over-expression of extracellular vesicle transport mechanisms, and / or a drug that increases metabolic activity. In some aspects, the therapeutic agent comprises Prostaglandin E2 (PGE2).

[0162] In some aspects, the agent, e.g., therapeutic agent, comprises a polypeptide. In some aspects, the polypeptide comprises IL-10, IL-4, IL-13, IL-31, or IL-33. In some aspects, the therapeutic agent comprises antibodies or inhibitors of these proteins.

[0163] In some aspects, the agent comprises a pro-reparative agent. In some aspects, the proreparative agent is curcumin, celastrol, rosiglitazone, troglitazone, alpha-ketoglutarate, simvastatin. In some aspects, the agent comprises a pro-inflammatory agent. In some aspects, the pro-inflammatory agent is lipopolysaccharide, other toll like receptor agonists, STING agonists, or pro-inflammatory proteins like tumor necrosis factor-alpha, ILl-beta, and interferon-gamma. Toll like receptor agonists include HSP, HMGB1, uric acid, fibronectin, poly(I:C), beta-defensin, snapin, tenascin C, CpG, zymosan, lipoteichoic acid, flagellin, ssRNA, poly GIO, poly G3, MPLA, imiquimod, BCG, complete Freund’s adjuvant.

[0164] In some aspects, the agent comprises an anti-microbial agent, or an agent that increases antimicrobial activity of macrophages, like (R)-roscovitine, azithromycin, resolving DI, bedaquiline.

[0165] In some aspects, the microparticles sized to promote transfer and / or the microparticles are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is an anti-fibrotic agent or a chemotherapeutic agent. In some aspects, the anti-fibrotic agent is a TGFB pathway inhibitor. In some aspects, the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof. In some aspects, the anti-fibrotic agent is an SPP1 inhibitor. In some aspects, the anti-fibrotic agent is a TREM2 inhibitor.

[0166] In some aspects, the microparticles sized to inhibit transfer and / or the microparticles are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is dexamethasone.

[0167] In some aspects, the therapeutic agent comprises any molecule that has a desired therapeutic effect.

[0168] In one aspect, the present disclosure relates to a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising a microparticle functionalized with an agent designed to promote microparticle transfer; and / or b) a plurality of conditioned macrophages comprising a microparticle functionalized with an agent designed to inhibit microparticle transfer. In some aspects, the agent of a) and / or b) comprises any of the agents described herein. In some aspects, the agent designed to inhibit microparticle transfer is dexamethasone. In some aspects, the agent designed to promote microparticle transfer is a drug that promotes over-expression of PINK1, a drug that promotes over-expression of extracellular vesicle transport mechanisms, and / or a drug that increases glycolysis or metabolic activity.

[0169] Conditioned Macrophages

[0170] In some aspects, a conditioned macrophage is prepared by introducing a microparticle or more than one, i.e., multiple, microparticles into a macrophage. For instance, a macrophage can internalize a microparticle through phagocytosis of a microparticle that is in close enough proximity to a macrophage. In some aspects, the conditioned macrophage is prepared by incubating the macrophage with a loading dose of microparticles of from about 10 pg / 106cells to about 1000 pg / 106cells. In some aspects, the conditioned macrophage is prepared by incubating the macrophage with a loading dose of microparticles of from about 100 jj.g / 106cells to about 250 pg / 106cells. In some aspects, the conditioned macrophage is prepared by incubating the macrophage with a loading dose of microparticles of from about 10 pg / 106cells to about 500 pg / 106cells. In some aspects, the conditioned macrophage is prepared by incubating the macrophage with a loading dose of microparticles of about 100 pg / 106cells. In some aspects, the conditioned macrophage is prepared by incubating the macrophage with a loading dose of microparticles of about 250 pg / 106cells. In some aspects, the conditioned macrophage is prepared by incubating the macrophage with a loading dose of microparticles of about 1 pg / 106cells, about 5 pg / 106cells, about 10 pg / 106cells, about 25 pg / 106cells, about 50 pg / 106cells, about 75 pg / 106cells, about 100 pg / 106cells, about 125 pg / 106cells, about 150 pg / 106cells, about 175 pg / 106cells, about 200 pg / 106cells, about 225 pg / 106cells, about 250 pg / 106cells, about 275 pg / 106cells, about 300 pg / 106cells, about 350 pg / 106cells, about 400 pg / 106cells, about 450 pg / 106cells, about 500 pg / 106cells, about 600 pg / 106cells, about 700 pg / 106cells, about 800 pg / 106cells, about 900 pg / 106cells, or about 1000 pg / 106cells. In some aspects, the conditioned macrophage is prepared by exposing the macrophage to a pro-reparative agent and / or by exposing the macrophage to a pro-inflammatory agent.

[0171] In some aspects, the amount of microparticles that promotes microparticle transfer is between about 10 pg / 106cells to about 100 pg / 106cells. In some aspects, the amount of microparticles that promotes microparticle transfer is about 1 pg / 106cells, about 5 pg / 106cells, about 10 pg / 106cells, about 25 pg / 106cells, about 50 pg / 106cells, about 75 pg / 106cells, about 100 pg / 106cells, about 125 pg / 106cells, about 150 pg / 106cells, about 175 pg / 106cells, about 200 pg / 106cells, about 225 pg / 106cells, about 250 pg / 106cells, about 275 pg / 106cells, about 300 pg / 106cells, about 350 pg / 106cells, about 400 pg / 106cells, about 450 pg / 106cells, about 500 pg / 106cells, about 600 pg / 106cells, about 700 pg / 106cells, about 800 pg / 106cells, about 900 pg / 106cells, or about 1000 pg / 106cells.

[0172] In some aspects, the amount of microparticles that inhibits microparticle transfer is from about 250 pg / 106cells to about 1000 pg / 106cells In some aspects, the amount of microparticles that inhibits microparticle transfer is about 1 pg / 106cells, about 5 pg / 106cells, about 10 pg / 106cells, about 25 pg / 106cells, about 50 pg / 106cells, about 75 pg / 106cells, about 100 pg / 106cells, about 125 pg / 106cells, about 150 pg / 106cells, about 175 pg / 106cells, about 200 pg / 106cells, about 225 pg / 106cells, about 250 pg / 106cells, about 275 pg / 106cells, about 300 pg / 106cells, about 350 pg / 106cells, about 400 pg / 106cells, about 450 pg / 106cells, about 500 pg / 106cells, about 600 pg / 106cells, about 700 pg / 106cells, about 800 pg / 106cells, about 900 pg / 106cells, or about 1000 pg / 106cells.

[0173] In some aspects, a conditioned macrophage comprises an exogenous macrophage, an allogenic macrophage, and / or an autologous macrophage. In some aspects, the macrophage, e g., the conditioned macrophage, is derived from a monocyte. In some aspects, the macrophage is derived from a tissue-resident macrophage population, such as alveolar macrophages in the lung, Kupffer cells in the liver, or osteoclasts in bone. In some aspects, a conditioned macrophage at least partly maintains its phenotype. In some aspects, a conditioned macrophage is prepared by contacting a macrophage with microparticle comprising a lipid nanoparticle. In some aspects, a conditioned macrophage is derived from induced pluripotent derived stem cells.

[0174] Pharmaceutical Compositions and Formulations

[0175] The compositions of the invention may be formulated as a pharmaceutical composition. Such a pharmaceutical composition may be in a form suitable for administration to a subject (i.e. mammal), or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The various components of the pharmaceutical composition may be present in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0176] Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous, or another route of administration. The route(s) of administration is readily apparent to the skilled artisan and depends upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[0177] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it is understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation.

[0178] Administration / Dosing The regimen of administration of the compositions of the invention can affect what constitutes an effective amount. For instance, the composition of the invention may be administered to the subject (i.e. mammal) in a single dose, in several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0179] Administration of the composition of the invention to a subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat the disease in the subject. An effective amount of the composition necessary to achieve the intended result will vary and will depend on factors such as the disease to be treated or prevented, the age, sex, weight, condition, general health, and prior medical history of the subject being treated, and like factors well-known in the medical arts. In particular embodiments, it is especially advantageous to formulate the composition in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the composition, and the particular therapeutic effect to be achieved.

[0180] One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Routes of administration of any of the compositions of the invention include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0181] Kits

[0182] In some aspects a kit is provided for treating, preventing, or ameliorating a given disease, disorder or condition, such as fibrotic disease or condition, or a symptom thereof, as described herein wherein the kit comprises: a) a composition as described herein; and optionally b) an additional agent or therapy. The kit can further include instructions or a label for using the kit to treat, prevent, or ameliorate the disease, disorder or condition. In some aspects, the invention extends to kits assays for a given disease, disorder or condition, or a symptom thereof, as described herein.

[0183] Methods of Treatment

[0184] In one aspect, the present disclosure relates to a method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of a composition as described herein, such, as, for instance, a composition comprising a) a plurality of conditioned macrophages comprising microparticles sized to promote transfer; and b) a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer, to the subject in need thereof. In some aspects, the microparticles sized to promote transfer are transferred to a cell of the subject. In some aspects, the cell is a macrophage, a fibroblast, a neuron, a tumor cell, a non-macrophage lymphocyte, a neutrophil, an epithelial cell, an endothelial cell, a progenitor cell, and / or a non-myeloid cell.

[0185] In one aspect, the present disclosure relates to a method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles sized to promote transfer. In some aspects, the method further comprises administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer. In some aspects, the disease or condition comprises pulmonary fibrosis. In some aspects, the microparticles sized to promote transfer are transferred to a cell of the subject. In some aspects, the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, an epithelial cell, an endothelial cell, a progenitor cell, a non-myeloid cell, and / or a fibroblast. In some aspects, the microparticles sized to promote transfer are functionalized with an agent, such as any of the agents described herein. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is an anti-fibrotic agent. In some aspects, the anti- fibrotic agent is pirfenidone, nintedanib, or a combination thereof. In some aspects, the microparticles sized to inhibit transfer are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is dexamethasone. In some aspects, the microparticles sized to promote transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL). In some aspects, the microparticles sized to promote transfer are 3 pm or less in diameter. In some aspects, the microparticles sized to promote transfer are from about 0.1 pm to about 1.0 pm in diameter. In some aspects, the conditioned macrophages comprising microparticles sized to promote transfer are derived from the subject’s monocytes. In some aspects, the conditioned macrophages comprising microparticles sized to promote transfer comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. In some aspects, the microparticles sized to inhibit transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL) In some aspects, the microparticles sized to inhibit transfer are 5 pm or more in diameter. In some aspects, the conditioned macrophages comprising microparticles sized to inhibit transfer are derived from the subject’s monocytes. In some aspects, the conditioned macrophages comprising microparticles sized to inhibit transfer comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. In some aspects, expression of CD163, CD301, CD206, CD9 and / or CD38 is increased relative to an untreated subject, and / or expression of CD86 decreased relative to an untreated subject.

[0186] In one aspect, the present disclosure relates to a method of treating a pulmonary fibrosis in a subject in need thereof, comprising administering to the subject: a) an effective amount of a plurality of conditioned macrophages comprising microparticles sized to promote transfer; and b) an effective amount of a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer. In some aspects, the microparticles sized to promote transfer are transferred to a cell of the subject. In some aspects, the cell of the subject in need thereof is a macrophage, a nonmacrophage lymphocyte, a neutrophil, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast. In some aspects, the microparticles sized to promote transfer are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is an anti-fibrotic agent. In some aspects, the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof. In some aspects, the microparticles sized to inhibit transfer are functionalized with an agent. In some aspects, the agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is dexamethasone. In some aspects, the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL). In some aspects, the conditioned macrophages of (a) and / or the conditioned macrophages of (b) are derived from a subject’s monocytes. In some aspects, the conditioned macrophages of (a) and / or the conditioned macrophages of (b) comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. In some aspects, expression of CD 163, CD301, CD206, CD9 and / or CD38 is increased relative to an untreated subject, and / or expression of CD86 is decreased relative to an untreated subject.

[0187] In one aspect, the present disclosure generally relates to a method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of a composition as described herein, such as a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising an amount of microparticles that promotes microparticle transfer; and b) a plurality of conditioned macrophages comprising an amount of microparticles that inhibits microparticle transfer. In some aspects, the microparticles are transferred to a cell of the subject. In some aspects, the cell is a macrophage, a fibroblast, a neuron, a tumor cell, a non-macrophage lymphocyte, a neutrophil, an epithelial cell, an endothelial cell, a progenitor cell, and / or a non- myeloid cell.

[0188] In one aspect, the present disclosure generally relates to a method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a composition as described herein, such as a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising an amount of microparticles that promotes microparticle transfer; and b) a plurality of conditioned macrophages comprising an amount of microparticles that inhibits microparticle transfer. In some aspects, the disease or condition comprises pulmonary fibrosis. In some aspects, the microparticles are transferred to a cell of the subject. In some aspects, the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast. In one aspect, the present disclosure generally relates to a method of treating a pulmonary fibrosis in a subject in need thereof, comprising administering to the subject an effective amount of a composition as described herein, such as a composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising an amount of microparticles that promotes microparticle transfer; and b) a plurality of conditioned macrophages comprising an amount of microparticles that inhibits microparticle transfer. In some aspects, the microparticles are transferred to a cell of the subject. In some aspects, the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast.

[0189] In one aspect, the present disclosure generally relates to a method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles functionalized with at least one agent, wherein the agent comprises dexamethasone. In some aspects, the microparticles comprise microparticles designed to promote transfer. In some aspects, the microparticles are sized to promote transfer. In some aspects, the microparticles comprise an amount of microparticles that promotes microparticle transfer. In some aspects, the microparticles are further functionalized with an agent that promotes microparticle transfer. In some aspects, the fibrotic disease or condition is pulmonary fibrosis or is muscle fibrosis. In some aspects, the fibrotic disease or condition is pulmonary fibrosis. In some aspects, the conditioned macrophages accumulate in the lungs of the subject. In some aspects, the microparticles are transferred to a cell of the subject. In some aspects, the cell of the subject is a macrophage, a non-macrophage lymphocyte, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, a neutrophil, and / or a fibroblast In some aspects, the treatment decreases expression of CD206, MERTK and CD301 in host macrophages of the subject. In some aspects, the treatment decreases the total collagen content of the lungs of the subject. In some aspects, the treatment decreases the collagen content of the lungs of the subject relative to the lungs of an untreated subject having pulmonary fibrosis. In some aspects, the treatment decreases the total amount of fibrotic lung tissue of the subject. In some aspects, the treatment decreases the amount of fibrotic lung tissue of the subject relative to an untreated subject having pulmonary fibrosis. In some aspects, the treatment increases the number of endothelial cells in the subject as compared to an untreated subject. Tn some aspects, the conditioned macrophages are derived from the subject’s monocytes. In some aspects, the conditioned macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. In some aspects, the microparticles comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

[0190] In one aspect, the present disclosure generally relates to a method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of: a) a plurality of conditioned macrophages comprising microparticles functionalized with a first agent, wherein the first agent comprises dexamethasone; and b) a plurality of conditioned macrophages comprising microparticles functionalized with a second agent. In some aspects, the second agent comprises a nucleic acid and / or a polypeptide. In some aspects, the agent is a therapeutic agent. In some aspects, the therapeutic agent is an anti-fibrotic agent. In some aspects, the anti-fibrotic agent is a TGFB pathway inhibitor. In some aspects, the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof. In some aspects, the anti- fibrotic agent is an SPP1 inhibitor. In some aspects, the anti-fibrotic agent is a TREM2 inhibitor. In some aspects, the fibrotic disease or condition is pulmonary fibrosis or is muscle fibrosis. In some aspects, the fibrotic disease or condition is pulmonary fibrosis. In some aspects, the conditioned macrophages accumulate in the lungs of the subject. In some aspects, the microparticles are transferred to a cell of the subject. In some aspects, the cell of the subject is a macrophage, a non-macrophage lymphocyte, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, a neutrophil, and / or a fibroblast In some aspects, the treatment decreases expression of CD206, MERTK and CD301 in host macrophages of the subject. In some aspects, the treatment decreases the total collagen content of the lungs of the subject. In some aspects, the treatment decreases the collagen content of the lungs of the subject relative to the lungs of an untreated subject having pulmonary fibrosis. In some aspects, the treatment decreases the total amount of fibrotic lung tissue of the subject. In some aspects, the treatment decreases the amount of fibrotic lung tissue of the subject relative to an untreated subject having pulmonary fibrosis. In some aspects, the treatment increases the number of endothelial cells in the subject as compared to an untreated subject. In some aspects, the conditioned macrophages are derived from the subject’s monocytes. In some aspects, the conditioned macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. In some aspects, the microparticles comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL). In some aspects, the microparticles of (a) and / or the microparticles of (b) comprise microparticles designed to promote transfer. In some aspects, the microparticles of (a) and / or the microparticles of (b) are sized to promote transfer. In some aspects, the microparticles of (a) and / or the microparticles of (b) comprise an amount of microparticles that promotes microparticle transfer. In some aspects, the microparticles of (a) and / or the microparticles of (b) are further functionalized with an agent that promotes microparticle transfer.

[0191] EXAMPLES

[0192] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0193] Example 1: Macrophage Microparticle Transfer

[0194] The present example describes macrophage microparticle transfer, in particular transfer of microparticles from conditioned macrophage to bystander macrophage. A schematic representation of the assay is presented in FIG. 1. Briefly, to evaluate whether bone marrow- derived macrophages loaded with microparticles (MPs) can transfer microparticles to unloaded bystander macrophages, a direct coculture with bystander macrophages was performed over 7 days in vitro. The transfer of non-degradable polystyrene beads with a size of 1 pm from GFP+ fully mature macrophages (BMDMs) (5 days of culture before MP loading) and early bone marrow cells (BMCs), consisting of a mix of monocytes and macrophages (1 day of culture before MP loading) was analyzed. Both cell populations were cultivated with 20 polystyrene beads / cell for 4 hours before excess beads were removed. MP-loaded cells were cocultured with bystander macrophages in different ratios (20:80, 10:90, 1 :99%) for 48h and 7 days in basal media and the MP transfer was analyzed via flow cytometry. GFP+ MP loaded macrophages and bystander macrophages were distinguished by GFP positivity. Referring to FIG. 2A-FIG. 2B, FIG. 2A-FIG. 2B present graphical representations of data related to microparticle transfer to bystander macrophages. FIG. 2A presents a graphical representation of the ratio of the percent GFP negative / PS beads positive cells for various ratios of % GFP to % bystander cells for either a coculture with BMDM or a coculture with BMC. The results demonstrated that BMDMs transfer particles to a higher percentage of bystander macrophages at a more rapid rate as compared to BMCs. FIG. 2B presents a graphical representation of the ratio of the percent GFP negative / PS beads positive cells for various ratios of % GFP to % bystander cells for either a coculture with BMDM or a coculture with BMC. The results demonstrated that less GFP+ MP loaded cells were positive for microparticles after coculture, thereby demonstrating particle transfer to bystander macrophages. n=3 for each of FIG. 2 A and FIG. 2B.

[0195] Referring to FIG. 3A-FIG. 3B, FIG. 3A-FIG. 3B present graphical representations of data related to microparticle transfer to bystander macrophages by analyzing the MFI of PS beads after coculture. FIG. 3A presents a graphical representation of data related to the MFI of PS beads of bystander macrophages for various different ratios of % GFP to % bystanders for either a coculture with BMDM or a coculture with BMC. The results demonstrated that BMDMs transfer more particles at a more rapid rate as compared to BMCs. FIG. 3B presents a graphical representation of data related to the MFI for PS beads of GFP positive cells for various different ratios of % GFP to % bystanders for either a coculture with BMDM or a coculture with BMC. The results demonstrated that GFP+ MP loaded cells had less microparticles per cell after coculture, thereby demonstrating particle transfer to bystander macrophages. n=3 for each of FIG. 3 A and FIG. 3B.

[0196] Example 2: Macrophage Microparticle Transfer

[0197] The present example describes macrophage microparticle transfer, in particular transfer of microparticles from conditioned macrophage to bystander macrophage. A schematic representation of the assay is presented in FIG. 4. Briefly, in the assay, the transfer of PLGA microparticles that were either blank microparticles (+TRITC (red fluorescent dye)) with an average size of 2.5 pm and a loading dose of either 250 pg or 500 pg / 106cells, Dex MP (56%)(+TRITC) with an average size of 2.5 pm and a loading dose of either 250 pg or 500 pg / 106cells, or commercially available PLGA MPs (Degradex MP) with sizes of 0.1 pm, 0.5 pm, 1 m in a concentration of 250 pg / / 106cells, to bystander macrophages, was analyzed. GFP+ BMDMs were treated with MPs for 4h before excess MPs were removed and coculture was started. MP loaded macrophages and bystander macrophages were cocultured for 48h in a 1 MP-loaded macrophage to 5 bystander macrophages ratio. The transfer rate was analyzed via flow cytometry.

[0198] Referring now to FIG. 5, FIG. 5 presents a graphical representation of data related to microparticle transfer to bystander macrophages after 48 hours of co-culture. The results of FIG. 5 demonstrate that degradable PLGA microparticles were transferred to bystander macrophages. The results of FIG. 5 also indicated that the transfer was influenced by microparticle size and microparticle content per cell.

[0199] Example 3: Macrophage Microparticle Transfer

[0200] The present example describes macrophage microparticle transfer, in particular transfer of microparticles from conditioned macrophage to bystander macrophage in different environments (media supplemented with IL4 / IL13 as a pro-reparative / pro-fibrotic environment, media supplemented with LPS / INFg as a pro-inflammatory environment, and basal media (MO)) using GFP+ BMCs. BMCs were treated with MPs in a concentration of 20 pg / 106cells for 24h before removing excess free-floating MPs and start of co-culture. The transfer of PLGA MPs was tested via flow cytometry for both blank and Dexamethasone encapsulated MPs after 48h and 7 days of cocultivation with bystander macrophages in a ratio of 1 MP loaded macrophages to 8 bystander macrophages. A schematic representation of the assay is presented in FIG. 6.

[0201] Referring now to FIG. 7A-FIG. 7B, FIG. 7A-FIG. 7B present graphical representations of data related to microparticle transfer from BMCs to bystander macrophages in different conditions. FIG. 7A presents a graphical representation of the transfer of MPs to bystander macrophages for MO, LPS / IFNg, and IL4 / IL13 groups at 48 h and at 7 days. FIG. 7B presents a graphical representation of the viability of the total cells for each of the MO, LPS / IFNg, and IL4 / IL13 groups at 48 h and at 7 days. It is noted that 48h LPS / IFNg was removed from analysis in FIG. 7A and FIG. 7B as almost all cells were dead due to experimental issues. The results of FIG. 7A-FIG. 7B indicated that the environmental stimuli effected transfer independent of cell death, and that increased transfer was observed when cells were loaded with Dex MPs. The highest transfer rate was observed in basal media after seven days, followed by media supplemented with LPS / IFNg. The lowest transfer was observed in media with IL4 / IL13. The results indicated that in basal media and media with IL4 / IL13 Dex MP were more transferred compared to blank MPs by BMCs.

[0202] Example 4: Phagocytosis / Efferocytosis

[0203] The present example describes the process of phagocytosis / efferocytosis that was recorded by live cell imaging via Incucyte microscopy to track the transfer of red fluorescent (Trite-labeled) MPs from GFP+ (green fluorescent) to unstained bystander macrophages. GFP+ macrophages were treated with MPs in a concentration of 100 pg / 106cells for 4h before excess MPs were removed and a co-culture was started in a ratio of 1 GFP+ macrophage to 5 bystander macrophages. Cells were cultured in basal media.

[0204] Referring now to FIG. 8, FIG. 8 presents an image of phagocytosis / efferocytosis at the indicated timepoints, indicating that phagocytosis / efferocytosis of MP conditioned macrophages by bystander macrophages can transfer MPs to bystander macrophages.

[0205] Example 5: Macrophage Microparticle Transfer

[0206] The present example describes macrophage microparticle transfer, in particular transfer of microparticles from conditioned macrophage to bystander macrophage. The transfer of biodegradable PLGA microparticles (size range: 1-3 pm) between MP -loaded BMDMs and bystander macrophages was analyzed. Live cell imaging via Incucyte microscopy was used to track the transfer of red fluorescent (Trite-labeled) MPs from GFP+ (green fluorescent) MP- loaded macrophages to unstained bystander macrophages (FIG. 9). GFP+ macrophages were treated with 100 pg / 106MPs for 4h before excess MPs were removed and a co-culture was started in a ratio of 1 GFP+ macrophage to 5 bystander macrophages. Cells were cultured in basal media.

[0207] In multiple areas of the time-lapse images, transfer of MPs was detected (FIGs. 10-13). In the observed transfer events, direct contact of MP-loaded and bystander macrophages occurred. Furthermore, red fluorescent MPs were detected in cellular extensions, sometimes referred to as tunneling nanotubes, between GFP+ MP-loaded macrophages (green) and bystander macrophages stained in blue via confocal microscopy (FIG. 14). In summary, the results demonstrated that both non-degradable and degradable microparticles with a size ranging from 1-3 pm were transferred from MP-loaded macrophages to bystander macrophages.

[0208] Example 6: Macrophage Phenotype Modulation

[0209] In the present example, the effects of coculture of bystander macrophages with MP- loaded macrophages was analyzed, in particular the effects on the phenotype of the bystander macrophages. For this assay, GFP+ BMDMs were treated with 100 pg / 106cells MPs for 4h before excess MPs were removed. Bystander macrophages were cultivated for 3 days with both GFP+ blank MP-loaded macrophages and Dex MP-loaded macrophages in a ratio of 1 MP loaded macrophage to 5 bystander macrophages and the phenotype of bystander macrophages was analyzed via flow cytometry To further analyze if the size of the MP has an influence, MPs with two different sizes were fabricated: 1-3 pm and < 1 pm (FIG. 15). The phenotype of bystander macrophages was affected by cocultivation with Dex MPs with upregulation of proreparative markers CD 163, CD206, CD301 and Argl as well as downregulation of the pro- fibrotic marker CD9 compared to the bystander control and bystander macrophages cocultured with blank MP loaded macrophages. Additionally, the expression of these markers was affected by cocultivation with MP loaded macrophages that were treated with smaller-sized MPs.

[0210] Example 7: Macrophage Phenotype Modulation In Vivo in a Subcutaneous Implantation Model

[0211] In the present example, the ability of macrophage-transferring MPs to modulate host macrophages / bystander macrophage behavior in vivo was analyzed by utilizing a subcutaneous implantation model in mice. GFP+ BMDMs were treated with 100 pg / 105cells MPs for 4 h before excess MPs were removed. One day later 500k MP loaded cells or unloaded macrophages were seeded onto a porous collagen scaffold (5 mm x 2 mm) and implanted in the subcutaneous space on the back of mice. After seven days, host cells were analyzed on MP positivity (FIG. 16). The results indicated that MPs were transferred to host cells. It was observed that around 10- 15 % of host cells found in scaffolds loaded with blank MP and Dex MP-loaded macrophages were positive for MPs, with the greatest MP-positive population being host macrophages (CD45+ / F480+), with ~20% of all host macrophages receiving MPs (FIG. 17). Furthermore, the effects of implantation ofMP-loaded cells on the phenotype of host macrophages was analyzed (FIG. 18). As a control, scaffolds with unloaded macrophages and scaffolds without macrophages were implanted. After seven days, host macrophages found in the scaffold with Dex MP -loaded macrophages showed the greatest change in expression pattern with upregulation of the pro-reparative marker CD163, CD206 and CD301. Additionally, compared to the scaffold alone, treatment groups that included macrophages resulted in a lower expression of the pro-inflammatory marker CD86. The effects of the MP uptake by host macrophages on the phenotype of macrophages was analyzed by gating on MP -positive (+) and MP-negative (-) macrophages (FIG. 19). The phenotype of host macrophages differed when they were positive for MPs compared to negative populations While CD163 and CD301 did not seem to be directly influenced by MP uptake, the markers CD206, Mertk, CD38 and CD9 were upregulated in all MP+ populations and, additionally, CD86 in Dex MP+ host macrophages, which indicated the potency of transferred MPs from administered macrophages to modulate bystander macrophages. In conclusion, the adoptive transfer of Dex MP macrophages into the subcutaneous space led to changes in the host macrophage phenotype influenced by MP transfer.

[0212] Finally, whether microparticle-transferring macrophages could transfer microparticles to non-myeloid cells was analyzed. It was found that MPs were transferred to CD45 negative non- myeloid cells with up to 15 % positive for MPs (FIG. 16, FIG. 20). These results were particularly surprising because they demonstrated that the microparticle transfer effect was not simply a result of macrophages regurgitating large particles which are then taken up by other phagocytes, but rather the macrophages were actively transferring the particles to non-phagocytic cells.

[0213] Example 8: Microparticles and Macrophage Phagocytosis

[0214] In the present example, macrophage phagocytic capacity was analyzed. In particular, the uptake of 1 um polystyrene (PS) beads by macrophages was analyzed 48 hours after treatment with PLGA microparticles. A schematic representation of the assay is presented in FIG. 21. The experiment was performed with BMCs that were loaded with blank or Dex MPs at a particle concentration of 20 pg / 106cells for 24h. 48 hours after MP treatment, 1 um polystyrene (PS) beads were given to the cells and the uptake was analyzed after 30 minutes. Referring to FIG. 22, FIG. 22 presents confocal images of control, blank MP, and Dex MP macrophages 48h after treatment with PLGA microparticles and 30 minutes following incubation with PS beads. Referring to FIG. 23, FIG. 23 presents a graphical representation of the % of PS positive macrophages for each group after a 30 minute incubation time with PS beads. The results of FIG. 22 and FIG. 23 indicated that Dex-microparticles increased macrophage phagocytotic capacity. Moreover, Dex MP treated macrophages even overcame the reduced phagocytic capacity of macrophages treated with blank MPs compared to untreated macrophages.

[0215] Example 9: Microparticles and Fibrosis

[0216] In the present example, the anti-fibrotic potential of macrophages comprising microparticles was analyzed by Nanostring gene expression analysis. BMDMs were cultivated in basal media and either left untreated or treated with either 100 ug / 106Dex MPs, 100 pg / 106blank MPs or 12.5 pg / 106soluble Dexamethasone for 4 hours before excess microparticles were removed or media was changed. After 48h, cells were lysed, RNA isolated and prepared for Nanostring analysis.

[0217] Referring now to FIG. 24A-FIG. 24B, the expression of both genes related to inflammation, regeneration, fibrosis, and angiogenesis was analyzed for untreated, blank MPs, Dex MPs, and soluble Dex groups. Macrophages treated with Dex MPs exhibited a anti-fibrotic phenotype by upregulation of the anti-fibrotic genes MMP8, TGFBR2, and TGFBR3 genes were upregulated. Moreover, the pro-fibrotic genes CD9, TREM2, S100A4, SPP1, VIM, CD69, FN1, HGF, IGF1, TFGB1, TIMP1, and TIMP2 were downregulated.

[0218] Example 10: Microparticle-loaded Macrophage-based Therapy

[0219] In the present example, microparticle-loaded macrophage-based therapy for pulmonary fibrosis was analyzed. In particular, dexamethasone-loaded macrophage cell therapy for pulmonary fibrosis was analyzed A schematic representation of the assay for dexamethasone- loaded macrophage cell therapy for pulmonary fibrosis is presented in FIG. 25. To investigate the effects of pro-fibrotic external stimuli on macrophage phenotype retention in vivo, 4 million BMCs (unloaded or loaded with MPs (20 pg / 106cells)) were administered to fibrotic lungs 27days after influenza infection. BMCs isolated from GFP+ mice were used to distinguish administered from host macrophages, and also directly compared the phenotype of administered macrophages to controls tested in parallel in vitro under basal conditions (i.e., no pro- inflammatory or pro-fibrotic stimuli). The phenotype was analyzed 48h after administration by analyzing the marker expression of pro-reparative markers (CD301b, CD163, CD206, Argl), the pro-inflammatory marker CD86, the pro-fibrotic marker CD9 and the antigen presenting related marker MHC2 via flow cytometry.

[0220] Referring to FIG. 26, FIG. 26 presents graphical representations of data related to the effects of transplantation of macrophages to fibrotic lungs. The results indicated that dex microparticle loaded macrophages partly maintained phenotype in vivo. In fibrotic lungs, Dex MP-loaded macrophages expressed higher levels of CD206. Expression levels (MFI) of MHC2, CD86, and CD 163 were lower in Dex-MP-loaded macrophages compared to untreated macrophages.

[0221] Example 11: Effects of Size, Dose, and Drug Loading of Microparticles on Microparticle Transfer

[0222] In the present example, the effects of size, dose, and drug loading of microparticles on microparticle transfer to host cell populations was analyzed. A schematic representation of the assay is presented in FIG. 27. 5 days after isolation of GFP+ cells, mature macrophages were treated with 1.5 pm or 2.5 pm size blank or Dexamethasone loaded microparticles in a dose of 100 pg or 250 pg per 1x106macrophages for 4 hours. A control group of macrophages was left untreated After 4 hours cells were detached, and free-floating MPs were removed. Cells were frozen and stored at -80°C for 10 days. One day before the surgery, cells were thawed and seeded into a well plate. 2 hours before the surgery, 500k cells were seeded onto a collagen scaffold (5 mm x 2 mm). 7 days after implantation, scaffolds were digested and host and administered cells were analyzed via flow cytometry.

[0223] Referring now to FIG. 28 and FIG. 29, seven days after implantation, microparticles of all groups were transferred to myeloid (CD45+) and non-myeloid (CD45-) host cell populations, however, at different rates depending on size, dose and drug loading. Overall, blank MPs were transferred to host cells at a higher rate than Dex MPs of the respective group with the same size or dose. The phenomenon was especially pronounced with 1.5 pm sized MPs, resulting in over 60% positive non-myeloid cells and macrophages (CD45+ / F4 / 80+) when administered macrophages were loaded with 1 .5 pm sized blank MPs with a dose of 250 pg, while Dex MPs with the same size and dose resulted only in around 30% MP positive non-myeloid cells and macrophages. These results indicated that Dex influenced the transfer of microparticles, potentially in part due to the phenotypical effects on macrophages. For blank MPs, size and dose strongly influenced the transfer to host macrophages and non-myeloid cells. An increase in dose led to a higher number and percentage of MP-positive host cells, which was further increased when 1 .5 pm particles were used, indicating the potential to modify the transfer rate by size and dose depending on the application and whether a higher or lower transfer rate would be beneficial. The lowest transfer was detected for 2.5 pm Dex MPs at a dose of 100 pg, resulting in 20% MP-positive non-myeloid cells and 30% MP-positive host macrophages. These results indicated that transfer rate of Dex MPs could be influenced by size and dose.

[0224] Example 12: Analysis of Microparticle Size, Dose, and Loading on Host Macrophage Phenotype using Implantation of Loaded Macrophages

[0225] In the present example, the effects of microparticle size, dose, and loading on host macrophage phenotype when implanting loaded macrophages was analyzed. A schematic representation of the assay is presented in FIG. 27.

[0226] Referring now to FIG. 30, administration of MP-loaded macrophages influenced the phenotype of host macrophages in vivo depending on size and dose of loading. Treatment with macrophages loaded with 1.5 pm or 2.5 pm Dex MPs resulted in host macrophages with different marker expressions. While host macrophages from mice treated with macrophages loaded with 2.5 pm Dex MPs showed upregulation of the pro-reparative marker CD 163 and downregulation of the pro-reparative marker CD86, 1.5 pm Dex MPs led to upregulation of the pro-reparative marker CD301, indicating the potential to influence host macrophage phenotype by particle size. Administration of macrophages loaded with 1.5 pm blank MPs at a higher dose led to the highest upregulation of CD206 and CD9 compared to the control groups. The same group resulted in the highest transfer to host cell populations (FIG. 28 and FIG. 29), indicating that increased transfer affects the host macrophage phenotype. Overall, higher MP loading doses caused more substantial changes in expression compared to the control groups (scaffold alone and unloaded macrophages) than the respective group with a lower loading dose. Additionally, 2.5 pm blank and Dex MPs resulted in the upregulation of CD38 compared to the control groups and groups with 1 .5 pm MPs. It is noted that CD38 is a marker related to mitochondria transfer via tunneling nanotubes.

[0227] Materials and Methods (Examples 13-17)

[0228] Microparticle Fabrication and Characterization

[0229] PLGA microparticles loaded with or without dexamethasone were prepared using a standard single emulsion technique as described previously (K.L. Wofford, et al., Acta Biomater 101 (2020) 237-248. The organic phase was prepared with 20 mg of PLGA Resomer 504H (Sigma Aldrich) and 25 mg Dexamethasone dissolved in 1 mL of 9:1 Dichloromethane (DCM; Acros Organics): Trifluoroethanol (TFE; Thermo Fisher). The internal aqueous phase of 4 ml of 2% polyvinyl alcohol (PVA; Sigma Aldrich) (15 KDa) was added to the organic phase. This emulsion was homogenized for 1 min at 20,000 rpm. Then, 5 mL of 2% PVA was added and the emulsion was stirred for 4 h at 1200 rpm to evaporate the DCM: TFE The MPs were collected by centrifugation and washed 3 times with deionized (DI) water. For in vitro MP uptake studies, 50 pg tetramethyl rhodamine (TRITC) was added to the organic phase. Size and polydispersity index of microparticles were quantified via dynamic light scattering on a Malvern Zetasizer. Microparticles were frozen, lyophilized, sterilized with UV light for Ih in the biosafety cabinet at 254 nm, resuspended in sterile lx phosphate-buffered saline (PBS), and stored at -80°C until use. Microparticle size was determined using confocal fluorescent images of TRITC-loaded blank microparticles and Dex-loaded microparticles (LSM 700). For each sample, 5 randomly chosen images were taken, and the particle diameter was measured using the Fiji particle analyzer tool (FIG. 44A, FIG. 44B). Scanning electron microscopy images were taken on Apreo 2S (Thermo Fisher Scientific) before and after UV sterilization (FIG. 44C).

[0230] Characterizing Dex Release Kinetics

[0231] Dex release was quantified from 20pg of freshly fabricated microparticles, or microparticles that had been frozen at -80C for 3 months, 9 months and 12 months, by resuspending in lx PBS incubated at 37°C while gently shaking inside tubes containing a 200 nm membrane. At each time point, the microparticle-containing solution was separated from the microparticles by centrifugation and the flow-thru solution was quantified UV spectrophotometrically for absorbance at 242 nm, and fresh IxPBS was added to the microparticles (FIG. 44D). Encapsulation efficiency was calculated based on the total dexamethasone released from 20 pg MPs within 7 days divided by 56% (% w / w of Dex to PLGA) of 20 pg (11.2 pg) of MPs (FIG. 44E).

[0232] Cell culture

[0233] Primary bone marrow derived monocytes and macrophages (Bone marrow cells - BMCs)

[0234] Male C57BL / 6 mice (Charles River Laboratories) were used to harvest bone marrow. Hindlimbs were collected from mice after euthanasia via carbon dioxide asphyxiation and bones were removed and placed into PBS on ice. After removing both ends, a 26.5G needle was used to flush RPMI 1640 media (Glibco) through the bones. The bone marrow cells cultured on 100 mm2non-tissue culture-treated polystyrene Petri dishes in complete media (RPMI 1640), supplemented with 10% heat-inactivated fetal bovine serum (FBS; VWR), 1% penicillin / streptomycin (Fisher Scientific), and 20 ng / mL murine macrophage colony-stimulating factor (M-CSF; Peprotech)) for 1 day before microparticle or soluble Dexamethasone (2.5 pg / ml) treatment and up to 7 days after. For experiments involving treatment with additional soluble factors, either LPS (100 ng / ml) and IFNg (100 ng / ml); IL4 (20 ng / ml) and IL13 (20 ng / ml); IL 10 (40 ng / ml) or IL 17a (10 ng / ml) were added to complete media.

[0235] For in vivo studies of exogenous cell administration, GFP + BMCs were collected and cultured in the same manner, but the hindlimbs were harvested from C57BL / 6-Tg (UBC- GFP)30Scha / J male mice (Jackson Laboratories). When preparing the GFP + BMCs for injection, after MP treatment, the cells were removed from the dishes using TrypLE Express (Thermo Fisher) for 5 minutes following gentle cell scraping.

[0236] Microparticle Treatment

[0237] One day after cell isolation blank or dexamethasone encapsulated (56 w / w %), microparticles (MPs) were added to the cell culture in a 10-100 pg / 106 concentration. For experiments involving different doses of MPs, only the amount given to the cells changed not the microparticle composition. After 1 hour, 4 hours or 24 hours, excess MPs were removed by aspirating media and washing dishes twice with RPMI 1640 media without supplements before adding fresh complete media. Figure 34 shows the efficiency of washing for removal of free- floating MPs.

[0238] Flow Cytometry

[0239] For analysis of cells collected from in vitro experiments, cells were incubated for 10 min in TruStain FcX (Biolegend cat #101320) at 1 :200 to block Fc receptors, then incubated with Aqua Live / Dead (Fisher Scientific cat #L34965) at 1 :500, AF700 CD45 (Biolegend, Cat # 103127), APC-Cy7 F4 / 80 (Biolegend Cat# 157315), Brilliant Violet 421 CD206 (Biolegend, Cat# 141717), APC CD163 (Biolegend, Cat# 155306), PE-Cy7 CD301b (Biolegend, Cat# 146808), SuperBright 600 CD9 (ThermoFisherScientific, Cat# 63-0091-82), PE-Dazzle 594 MHC2 (Biolegend, Cat # 107648), PE-Cy5 CD86 (Biolegend, Cat # 105016) at 1 :200 for 15 min at 4°C and then washed twice. Cells were resuspended in fixation / permeabilization solution (BD) at 4°C for 20 min. Cells were then washed twice and resuspended in FACS buffer (lx PBS, 1 % HEPES, 0.5 % FBS).

[0240] For analysis of cells isolated from in vivo experiments, the single cell suspensions were incubated for 10 min in TruStain FcX at 1 :200 to block Fc receptors, with Aqua Live / Dead (Fisher Scientific) at 1 :500, AF700 CD45 (Biolegend, Cat # 103127), APC-Cy7 F4 / 80 (Biolegend, Cat# 157315), Brilliant Violet 421 CD206 (Biolegend, Cat# 141717), APC CD163 (Biolegend, Cat # 155305), PE-Cy7 CD301b (Biolegend, Cat# 146808), SuperBright 600 CD9 (ThermoFisherScientific, Cat# 63-0091-82), PE-Dazzle 594 MHC2 (Biolegend, Cat # 107648), PE-Cy5 CD86 (Biolegend, Cat # 105016) for 15 min at 4°C and then washed twice. For all antibodies, a concentration of 1:200 was used. Cells were resuspended in fixation / permeabilization solution from the BD CytoFix / CytoPerm Kit at 4°C for 20 min. Next, cells were washed twice and stained with specified intracellular marker APC Arginase 1 (Argl) (ThermoFisherScientific, Cat# 17-3697-82) for 30 min at 4°C. Cells were then washed twice and resuspended in FACS buffer. The gating strategy for GFP-positive cells is shown in Figure 47A. Separately, to stain for alveolar macrophages, interstitial macrophages and monocyte-derived macrophages, the cell suspension was stained with Brilliant Violet 605 CD45 (Biolegend, Cat # 103139), APC-Cy7 F4 / 80 (Biolgend, Cat# 157315), PE CD 11b (Biolegend, Cat # 101207), APC CDl lc (Biolegend, Cat # 117309), Brilliant Violent 421 Siglec-F (Biolegend, Cat # 155509). Cells were analyzed using a BD LSRII flow cytometer. For flow cytometry analysis, only samples that were run at the same time with the same settings and compensation were analyzed together. Gene expression analysis via qPCR

[0241] Cellular RNA of macrophages was isolated using RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher) per the manufacturer's protocol. According to the manufacturer's manual, cDNA was generated with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The mRNA expression levels of macrophages were analyzed via quantitative Real-Time PCR (qPCR) (qTower3; Analytic Jena) with SYBR™ Select Master Mix (Thermo Fisher Scientific). Each 10 pl qPCR reaction comprised 5 ng of cDNA and 200 nM primer sequences (Thermo Fisher, Table 1). For all reactions, an annealing / extend temperature of 60°C was used. For each cDNA sample, the threshold cycle (Ct) value of each target sequence was subtracted from the Ct value of the housekeeping mRNA Hnrnpab to derive ACt. Gene expression changes were calculated by the 2-AACt method. A control group of macrophages (no MPs or soluble factors), as indicated in the figure legends, was used for normalization.

[0242] TABLE 1 - PRIMER SEQUENCES

[0243] Phagocytosis assay

[0244] Two days after treatment with MPs and soluble dexamethasone, 1 pm polystyrene beads (yellow-green fluorescent, Fluoresbrite) were added to cell culture media in 10 beads / cell doses. Cells were incubated for 30 minutes, 2h hours and 4 hours before the uptake was analyzed via Flow cytometry or confocal imaging (LSM 700). For confocal imaging, cells were stained with Celltracker CMAC blue (Thermo Fisher) for 45 minutes before treatment with beads.

[0245] MMP8 secretion MMP8 secretion was measured in supernatants of macrophages via the mouse MMP8

[0246] ELISA Kit (Abeam) according to the manufacturer's protocol. All samples were diluted 1 : 10. Individual samples were used for day 2 and day 7. For normalization, after the media was collected, cells were detached and counted.

[0247] Collagen I degradation and MMP activity were tested using DQ™ Collagen, type I From Bovine Skin, Fluorescein Conjugate (Thermo Fisher Scientific) Macrophages were seeded on a collagen I coating containing 25 pg / ml DQ Collagen. For the collagen I coating, 4.4 ml Collagen I solution (3.4 mg / ml)(Gibco) was mixed gently with 0.5 ml l Ox PBS and 1 10 .1 IN NaOH. For each sample, 250 pl of Collagen I / DQ mixture was spread on a 35 mm FluoroDish Cell culture dish (World Precision Instruments) and solidified for Ih at 37°C. For each sample, 4 individual images were taken for analysis. DQ-signal and number of nuclei were analyzed using the particle analyzer tool in Fiji. The area of DQ fluorescent signal was calculated by dividing the total area per image by the number of nuclei per image. The intensity for each image was calculated by using the average intensity obtained per DQ fluorescence event.

[0248] Animal Model

[0249] Influenza lung injury

[0250] Adult male mice were used at 6 to 8 weeks old. Mice were housed in Animal Biosafety Level 2 facility under the standard dark / light, ambient temperature, and humidity. Mice were anesthetized with isoflurane and influenza virus A / H1N1 / PR / 8 was administered intranasally at 25 to 45 TCID50 units of median tissue culture infectious dose to mice (20 to 25 g, 35 U; 25 to 30 g, 45 U) dissolved in 30 pl of phosphate buffer saline (PBS) as previously described (A. I. Weiner et al., Cell Rep 41, 111805 (2022); G. Zhao et al., Science Advances 6, eabc4493 (2020)). Mice that lost >15% of their initial body weight by day 9 were deemed adequately infected and used for all experiments involving influenza infection. Control mice were administered the same volume of PBS. Mice were weighed regularly and euthanized at the specified time points for tissue harvest. All animal experiments followed all National Institutes of Health (NIH) Office of Laboratory Animal Welfare regulations.

[0251] Cell administration

[0252] The mice were anesthetized using isoflurane and the 4xl06GFP+ cells were resuspended in 30 uL of PBS and administered intranasally 10 or 27 days after influenza infection. Recovery of administered cells and the phenotype of administered and host macrophages was analyzed after 48h via Flow Cytometry.

[0253] Lung tissue digestion for Flow Cytometry

[0254] Lungs were harvested from mice, and single-cell suspensions were prepared as previously described (A. I. Weiner et al., NPJ Regen Med 4, 17 (2019)). Briefly, the lungs were thoroughly perfused with cold PBS via the right ventricle to remove residual blood in the vasculature. Lung lobes were separated, collected, minced using scissors and chemically digested with 2 ml Collagenase Type II (5mg / ml) (Thermo Fisher Scientific) in PBS for 45 min at 37°C and mechanically dissociated by pipetting in FACS buffer. Next, cell suspensions were filtered through a 40-pm cell strainer (Thermo Fisher Scientific) and treated with ACK red blood cell lysis buffer (Thermo Fisher Scientific) for 5 min and resuspended with FACS buffer.

[0255] Statistical analysis

[0256] Statistical analysis was performed in GraphPad Prism (GraphPad Software Inc., La Jolla, CA). All data are represented as means ± SEM. To determine the effects of MP dose on macrophage phenotype via flow cytometry, and quantitative analyses of Collagen I degradation, one-way ANOVA with Tukey’s post hoc multiple comparisons test was performed. For all other datasets, data were analyzed by two-way ANOVA with Tukey’s post hoc multiple comparisons test. For all analyses, P < 0.05 was considered significant.

[0257] Example 13: Microparticle dose and treatment time affect uptake rate and cell viability

[0258] PLGA microparticles (MPs) were chosen as the drug delivery vehicle in part because PLGA is used in several FDA approved therapies. Dexamethasone (Dex) was selected for encapsulation into the PLGA MPs.. Dex presented an appropriate choice to study the effects on uptake rate, phenotype, and phenotype retention in inflamed and fibrotic lungs in a mouse model after influenza infection. For the present study, murine bone marrow cells (BMCs) were utilized, which were composed predominantly of monocytes (75%) at day 0, with a transition to a total macrophage population by two days after MP treatment (FIG. 31 A and FIG. 3 IB). The use of immature monocytes as compared to fully mature bone marrow-derived macrophages (BMDMs) may be desirable as monocytes are known to home to sites of inflammation and injury, which could be beneficial for numerous applications. It was decided to work with murine cells to compare effects in vitro to in vivo studies directly.

[0259] First, it was investigated whether the uptake of MPs depends on the dose of MPs or their incubation time with the cells prior to removal of any unphagocytosed MPs (efficiency of free- floating MP removal procedure is shown in FIG. 34). BMCs were cultured for 24 hours after harvest and then treated with 10 pg, 20 pg, 40 pg, or 100 pg drug-free blank or Dex MPs per 106 cells for either 1 hour, 4 hours, or 24 hours prior to washing (FIG. 32A). For all experiments involving different doses of MPs only the amount of MPs given to the cells changed not the MP composition itself. Increasing dose of MP caused increased uptake by macrophages and monocytes, independently of the MP incubation time (FIG. 32C). After 1 hour of incubation, almost 100% of macrophages in the culture were positive for MPs by flow cytometry when incubated with the highest dose of MPs, whereas less than 50% of macrophages had taken up MPs when incubated with the lowest dose of MPs (FIG. 32C). However, a longer incubation time with MPs further increased the uptake rate and the number of MPs per cell (FIG. 32C). Moreover, higher uptake was observed when BMCs were treated with blank compared to Dex MPs, with more pronounced effects for shorter incubation times, and macrophages generally phagocytosed more MPs than monocytes for both MP types (FIG. 32D). Two days (d2) after MP treatment, a decrease in MP-positive macrophages was observed for the 1 hour MP incubation time, however not for the 4 hour and 24 hour incubation times (FIG. 32C). By 7 days after MP treatment, the proportion of macrophages containing MPs ranged from 75 to 100%, with MP dose-dependent increases (FIG. 32B, FIG. 32C) These results indicate that MP loading of macrophages can be controlled via MP dose and incubation time, that immature monocytes are more difficult to load than mature macrophages within the heterogenous population, and that macrophages contain MPs for at least 7 days following MP administration.

[0260] Next, it was examined how MP uptake affects cell viability. Immediately (day 0) and two days after MP incubation, there were slight but statistically significant increases in cell death in most treatment groups compared to the untreated control, although viability remained over 90% for all groups by day 2 except for the highest loading of Dex MPs, in which viability was around 85% (FIG. 32E). However, by 7 days, the percentage of dead cells increased in all groups, with higher levels with increasing MP doses and reaching more than 50% for the highest Dex MP doses (FIG. 32E). While immediately after MP treatment, a higher percentage of apoptotic cells was observed compared to the untreated cells, after two days, higher levels were only detected for blank MPs with longer incubation time, which were those cell populations with higher MP uptake rates (FIG. 32F). MP -negative cells showed overall lower levels of dead and apoptotic cells than MP-positive cells, indicating that the uptake process of MPs, especially at higher doses, can induce cell death. Example 14: Dex MPs modulate macrophage phenotype in a dose-dependent manner

[0261] Next, how MP dosing affects macrophage phenotype was studied Based on the results shown in FIG. 32A-FIG. 32C, it was decided to proceed only with the 24 hour MP incubation time. The time points of analysis, day 2 and day 7, were chosen as early and late time points to study changes in phenotype over time. In general, macrophages loaded with Dex MPs showed higher expression of the reparative markers CD163, CD206, and CD301b after 2 and 7 days, although the effect on CD301b expression was only significant at 2 days when considering the percentage of CD301b+ cells and not mean fluorescent intensity (MFI) (FIG. 33B, FIG. 38A- FIG. 38B). Dex MP-loaded macrophages also expressed lower levels of the pro-fibrotic marker CD9 compared to macrophages loaded with blank MPs (FIG. 33B, FIG. 38). These effects were influenced by the dose of Dex MPs, in that increasing the dose of Dex MPs caused decreased expression of the reparative markers CD163 and CD301b on days 2 and 7 after MP treatment but increased CD206 expression at day 2. The fibrosis-related marker CD9 increased with increasing doses of Dex MPs on day 2 but remained lower than with blank MPs. MHC2 and CD86, receptors involved in antigen presentation, were upregulated with increasing doses of Dex MPs at day 2, but generally remained lower than for blank MPs. At day 7, increasing Dex MP dose increased MHC2 expression but decreased CD86 expression. In contrast, blank MP-loaded macrophages showed dose-dependent effects only on MHC2 expression, which was upregulated with increasing dose. Overall the results suggest that the main dose-dependent effects resulted from increasing the dose of dexamethasone rather than MPs.

[0262] Analysis of each marker individually showed that Dex MP-loaded macrophages exhibited a distinct phenotype compared to both untreated and blank MP -treated cells (FIG. 33C, FIG. 33D) with increased reparative marker expression and decreased CD9 and MHC2 expression. This finding was further confirmed by multidimensional analysis of subpopulations (FIG. 33E- FIG. 33G, FIG. 40A-FIG. 40G, FIG. 41A-FIG. 41G, FIG. 42A-FIG. 42D), revealing distinct clusters dominated by either Dex MP treated groups or untreated and blank MP-treated groups. By 7 days following MP treatment, major differences were observed in the number of cells comprising 4 clusters in particular, clusters 14, 20, 4 and 1, which together comprised 67% of all cells included in the analysis (FIG. 33E). Dex MP treatment increased the number of cells in clusters 14 and 20 compared to blank MPs or untreated cells; these clusters were characterized by higher expression of the reparative markers CD163, CD206, and CD301b, higher expression of the antigen presentation marker CD86, and lower levels of the fibrotic marker CD9 and the antigen presentation marker MHC2 (FIG. 33F, FIG. 33G). Similarly, Dex MP treatment decreased the number of cells in clusters 4 and 1, which were characterized by lower expression of CD163, CD206, CD301b, and CD86, and higher expression of CD9 and MHC2 (FIG. 33F, FIG. 33G). Similar results were found across all doses of MPs and also on day 2 after treatment (FIG. 40-41). Finally, similar results were found when the clustering analysis was conducted with only 7 clusters, showing that over-clusterization did not influence the results (FIG. 42A- FIG. 42D).

[0263] Based on the dose-dependent expression patterns, it was investigated if heterogeneity in MP uptake per cell would influence marker expression. Therefore, marker expression on the single cell level based on level of MP content was analyzed (FIG. 331, FIG. 33J, FIG. 41F, FIG. 41G). Higher levels of MP content per cell, as measured by the fluorescent intensity of TRITC, caused increased expression of MHC2 and lower expression of CD86 in Dex MP -treated cells (FIG.. 331, FIG. 33 J), regardless of MP dose (FIG. 40F, FIG. 40G, FIG. 4 IF, FIG. 41G). On the other hand, expression of CD163, CD206, and CD301b were unaffected by MP content per cell in both treatment groups (FIG. 331, FIG. 33 J), indicating an environmental effect of MPs on the population as opposed to individual cells. Additionally, extracellular release of dexamethasone could also occur, which would influence the population more than single cells in the Dex MP group.

[0264] Example 15: Particle loading influences phagocytic capacity

[0265] Phagocytosis of pathogens and apoptotic cells is an essential function of macrophages and a vital part of the resolution of inflammation, and is impaired in many inflammatory diseases. Soluble dexamethasone has been previously shown to increase phagocytic capacity of macrophages. To determine if MP loading within macrophages affects their phagocytotic activity, untreated, blank, and Dex MP-loaded macrophages were incubated with fluorescent polystyrene (PS) beads (1 pm) at 2 days after removal of excess MPs (FIG. 35A, FIG. 35B). Day 2 was chosen for the experiment because all cells were mature macrophages at this time (FIG. 3 IB). Over 4 hours of incubation with PS beads, 80% of untreated macrophages showed uptake of PS beads (FIG. 35C, FIG. 43 A). Loading macrophages with blank MPs caused significantly less uptake of PS beads compared to untreated controls, while loading macrophages with Dex MPs increased their uptake of PS beads Within each treatment group, increasing the dose of MPs led to decreasing uptake of PS beads. Since we used the 24 hour MP incubation time, it was reasoned that any soluble dexamethasone released by the microparticles during this time frame prior to their phagocytosis could affect the results. Thus, Dex MP loaded macrophages were compared to macrophages treated with soluble Dex for 24 hours, simulating the maximum amounts of Dex that could have been released during macrophage uptake of MPs (based on release kinetics shown in FIG. 44A-FIG. 44E). Dex MP-treated macrophages showed a similar percentage of phagocytic active cells for doses between 10 pg-20 pg (FIG. 45 A) but phagocytosed fewer beads overall (FIG. 45B). While increasing dose of soluble Dex further enhanced the uptake of PS beads, a dose of 40-100 pg Dex MP decreased the phagocytic capacity, indicating an MP dose-dependent influence on the phagocytic capacity overriding the effect of Dex.

[0266] Next, it was sought to understand if the MP content per cell directly influences the capability for phagocytosis. Surprisingly, MP content in blank MP-treated cells did not directly influence the uptake of PS beads (FIG. 35E), although cells treated with higher doses of MPs generally showed less uptake of PS beads (regardless of MP content per cell) compared to cells treated with lower doses (FIG. 43C). For Dex-MP -loaded macrophages, MP content per cell did not have strong effects on PS bead uptake, although a subtle trend was evident that cells with either very low or very high MP content phagocytosed fewer PS beads compared to cells with intermediate levels of MPs (FIG. 35F, FIG. 43D).

[0267] Example 16: Dex MP-loaded macrophages maintain phenotype in vitro despite being challenged with various stimuli and show anti-fibrotic properties

[0268] As highly plastic cells, macrophages respond to microenvironmental cues by changing their phenotype and associated functions. For a successful macrophage cell therapy, the phenotype must be maintained despite conflicting external cues in order to fulfill the desired functions To evaluate whether MP-loaded macrophages could withstand various microenvironmental stimuli, loaded cells were transferred into media containing either pro- inflammatory stimuli (LPS / IFNg), pro-reparative but also pro-fibrotic cytokines (IL4 / IL13), an immunosuppressive cytokine (IL10), or IL17a, which is associated with inflammatory fibrotic diseases, for 2 or 7 days (FIG. 37A). It was only proceeded with an MP dose of 20 pg / 106cells as this dose resulted in Dex -MP macrophages that were around 80% positive for MPs (FIG. 32C) and exhibited a phenotype characterized by high CD 163, CD206 and CD301b and low CD9 and MH2 expression (FIG. 33-FIG. 33J) while maintaining high phagocytotic capacity (FIG. 35A- FIG. 35F) To investigate possible effects of soluble Dex on phenotype maintenance, a control group was included in which BMCs were treated with 2.5 ug soluble dexamethasone, which is the amount released in the first 24h by 20 pg Dex MPs in PBS in vitro (FIG. 44A-FIG. 44E). Gene expression analysis was used to analyze a panel of 10 genes associated with macrophage phenotype and fibrosis in the four different microenvironmental conditions.

[0269] Hierarchical clustering of relative gene expression of tested markers demonstrated that all Dex-treated (soluble and MP-loaded) macrophages clustered together and far from untreated and blank MP-loaded cells despite being challenged with the different external stimuli (FIG. 37B, 7 day time point shown in FIG. 46B). In contrast, untreated and blank MP-loaded cells clustered together in groups that separated more by external stimuli, indicating phenotypic shifts in response to those cues. Dex -treated macrophages exhibited a phenotype that would be considered more regenerative based on upregulation of CD 163 and CD206, anti-inflammatory based on downregulation of TNFa, IL lb, and CD86, and anti-fibrotic based on downregulation of CD9 and upregulation of the ECM-degradative gene MMP8. Moreover, the immunosuppressive marker IL 10 and the pro-fibrotic Tissue Inhibitor of Metalloproteinases (TIMP1) showed a trend of being downregulated by Dex and Dex MPs in most environments, while LRP1, a marker involved in collagen degradation in pulmonary fibrosis, was upregulated, although these trends differed based on the external stimuli. Although soluble Dex appeared to have equal potential to maintain the phenotype as Dex-MPs based on gene expression (FIG. 37C), protein expression analysis by flow cytometry indicated superior performance of Dex-MPs under pro-inflammatory and pro-reparative / fibrotic conditions (FIG. 46C). Macrophages loaded with Dex-MPs showed enhanced upregulation of reparative markers (CD 163, CD206) and downregulation of pro-inflammatory (CD86), pro-fibrotic (CD9) compared to soluble Dex treatment, blank loaded and untreated cells. These effects were partly maintained for 7 days, suggesting a protective effect of Dex release from intracellular MPs on macrophage phenotype. While the expression pattern of CD 163 and CD9 was maintained over seven days in all tested conditions for Dex MP-treated macrophages, CD86 was only downregulated in media with pro- inflammatory stimuli. No difference between groups was detected for CD206 at 7 days, while Dex MPs upregulated CD301b in basal media and caused lower expression of MHC2 in all conditions compared to the control groups (FIG. 46A-FIG. 46C).

[0270] Based on the findings that Dex-MPs increased macrophages’ phagocytic capacity and expression of MMP8, a major collagenase, it was examined how Dex MP treatment affected collagen I degradation and uptake by macrophages. First, it was confirmed that secretion of MMP8 was increased in basal, pro-inflammatory, or pro-fibrotic conditions by Dex MPs on the protein level (FIG. 37D). To assess collagen degradation, untreated macrophages and macrophages loaded with blank or Dex MPs were cultured for 24 hours on a collagen I coating containing DQ collagen type I, which fluoresces after cleavage by MMPs (FIG. 37E, FIG 37F). Compared to the control groups, Dex MP macrophages caused greater cleavage and uptake of collagen I, as shown by the green fluorescent area and intensity of the signal per cell, suggesting the potential for these macrophages to remove excess collagen during fibrosis.

[0271] Example 17: Dex MP loaded macrophages partially maintain phenotype in vivo

[0272] It was sought to determine whether intracellular loaded of Dex MPs could modulate macrophage phenotype in vivo. To continue to investigate the effects of various external stimuli, a lung fibrosis model was utilized, in which influenza-infected mice develop inflamed lungs at early time points (7-10 days post-infection), although the virus is fully cleared by this time point, and then transition to a more fibrotic environment at later time points (25-30 days post-infection) (J. Qiao et al., Respiratory Research 10, 107 (2009)). Thus, to investigate the effects of pro- inflammatory or pro-fibrotic external stimuli on macrophage phenotype retention in vivo, the macrophage cell therapy was administered at either 10 days or 27 days after infection. BMCs isolated from GFP+ mice were used to distinguish administered from host macrophages, and the phenotype of administered macrophages was directly compared to controls tested in parallel in vitro under basal conditions (i.e., no pro-inflammatory or pro-fibrotic stimuli). Day 2 was chosen as analysis time point to detect early changes in phenotype after administration and to be able to compare the results to in vitro data. Two days after administration to inflamed lungs and fibrotic lungs, around 2.5% and 1.25 % of administered macrophages could be recovered for analysis (FIG. 47A-FIG. 47C). After 2 days in the inflamed lungs, administered macrophages containing intracellular Dex MPs expressed higher levels of the reparative marker CD206 compared to untreated and blank MP-containing macrophages (FIG. 39B, FIG. 48A). However, expression of the other reparative markers (CD301b, Argl , CD163) did not differ between groups, unlike in vitro controls. Furthermore, the pro-fibrotic factor CD9 was not downregulated in vivo as much as it was in vitro

[0273] Greater phenotype control was achieved in Dex MP-loaded macrophages administered to the fibrotic lungs (FIG. 39C). In fibrotic lungs, Dex MP-loaded macrophages expressed higher levels of both CD206 (both CD206+ cells and MFI) and CD301b (as measured by CD301b+ cells but not MFI, FIG. 48D). Expression levels (MFI) of MHC2, CD86, and CD 163 were lower in Dex-MP -loaded macrophages compared to untreated macrophages (FIG. 39C), although the number of MHC2+ and CD163+ cells did not differ between groups and the number of CD86+ cells actually increased with Dex MP treatment (FIG. 48D). Therefore, the potential for intracellular Dex-MPs to modulate administered macrophage phenotype in vivo appeared stronger in the fibrotic environment compared to the inflammatory environment. Moreover, even though the phenotype was partially maintained, the macrophages were substantially different compared to the in vitro cultured cells, indicating a strong influence of microenvironmental cues in vivo. These results are in agreement with principal component analysis (PCA) and hierarchical clustering, which showed that administered macrophages clustered apart from in vitro cultivated cells at either baseline (day 0) or day 2 (FIG. 39D, FIG. 39F, 48B). Hierarchical clustering revealed greater similarities between administered and host macrophages in inflamed lung (FIG. 39E, FIG 48C) compared with fibrotic lung (FilG. 39G, FIG. 48F), particularly in the expression of Argl, MHC2 (MFI values, FIG. 49A, FIG. 49B), CD163, and CD86 (percentages, FIG. 48A, FIG. 49D), although this effect was less evident in principal component analysis (FIG. 48C, FIG. 49F).

[0274] Macrophages represent a substantial population of cells in the lung with distinct subpopulations that change in response to injury, inflammation, and fibrosis. While alveolar macrophages are the primary type in healthy lungs with homeostatic functions, monocyte- derived macrophages and interstitial macrophages increase in inflammatory and fibrotic conditions, respectively. Concordantly, this change was observed in the macrophage subtype population after influenza infection, which was unaffected by the administration of untreated or MP -treated macrophages (FIG. 50). However, administration of macrophages into inflamed lungs led to a slight effect on host macrophages with significant downregulation of CD86 MFI (FIG. 49A). No change in host macrophage phenotype was observed in MP-treated groups compared to the PBS-treated group when macrophages were administered into fibrotic lungs (FIG. 48A, FIG. 48D, FIG. 49A-FIG. 49B).

[0275] Collectively, these results indicate that intracellular Dex MPs hold the potential to control the phenotype of macrophage cell therapies in vivo, but that more optimization will be required particularly for use in more inflammatory environments.

[0276] Example 18: Microparticle distribution in vivo

[0277] The biodistribution of microparticles (MPs) was analyzed 24 and 48 hours after administering macrophages loaded with blank MPs, Dex MPs or administration of free Dex MPs to fibrotic lungs. For all groups, the majority of the MPs accumulated in the lungs after 24 and 48 hours (see FIGs. 52-55). Free administration of Dex MPs resulted in accumulation in the thymus after 24 hours, which was significantly lower when MPs were loaded into macrophages before administration. While the accumulation of MPs in the thymus from macrophages loaded with blank and Dex MPs increased slightly after 48 hours, free Dex MP still accumulated at a higher level. In all groups, there was no accumulation observed in the liver, and only a slight level of accumulation was noted in the spleen. All groups accumulated MPs in the kidney, with an overall reduction after 48 hours.

[0278] Furthermore, freely administered Dex MPs resulted in higher concentrations of released Dex in blood plasma and bronchial fluid after 24 and 48 hours compared to Dex MPs loaded into macrophages (see FIGs. 56-57).

[0279] Example 19: Analysis of Residence time of Transferred Microparticles in Host Cells

[0280] Administration of MP-loaded macrophages to fibrotic lungs resulted in the transfer of MPs to host macrophages (FIG. 58, FIG. 59). While no significant difference was observed between groups, the percentage of MP-positive host macrophages increased over time from approximately 1% on day 2 to about 3% on day 14 (FIG. 58). However, the cell number indicated only an increase of MPs from day 2 to day 7, which was followed by a slight decrease after 14 days (FIG. 59 / Fig. 9). No MP transfer to other non-leukocyte cells like fibroblasts was observed. Furthermore, the percentage of MP-positive administered (GFP+), MP-loaded macrophages decreased over time (FIG. 60), which further indicated a transfer of MPs to host cells. Similarly, also in the subcutaneous implantation model (FIG. 61) MPs were still found in host cells after 14 days, both in host macrophages (FIG. 62) and host, CD45 negative, nonleukocyte (FIG. 63). Around 20 % of host macrophages and 4-6 % of host non-leukocyte (fibroblast) were positive for microparticles. Without wishing to be bound by theory, it was postulated that the difference in MP transfer to host non-leukocyte cells between pulmonary fibrosis and subcutaneous implantation model could indicate a difference in cell contact between administered macrophages and fibroblasts with an increased direct cell-cell contact in the subcutaneous model.

[0281] Example 20: Analysis of Host Macrophage Phenotype Modulation and Lung Fibrosis Resolution

[0282] Administration of MP-loaded macrophages influenced the phenotype of administered and host macrophages (FIG. 64). Macrophages in fibrotic lungs upregulated the marker CD206, MERTK and CD301, which are associated with fibrotic diseases, compared to naive animals. Seven days after administration, GFP+ Dex-MP macrophages decreased the expression of these markers compared to unloaded and blank MP-loaded macrophages. Also, host macrophages of animals treated with Dex-MP-macrophages downregulated the expression of all three markers. This effect was not observed when free Dex MPs were administered, indicating the importance of administered macrophages.

[0283] Furthermore, administration of Dex-MP-macs decreased the total collagen content in fibrotic lungs after 7 and 14 days (FIGs. 65-68) compared to untreated fibrotic lungs (PBS). Additionally, the total collagen amount after Dex-MP macrophage treatment was significantly decreased compared to baseline animals representing the fibrotic state at the time of treatment administration, indicating that Dex-MP macrophages were able to reverse fibrosis. Dex-MP macrophages reduced the total collagen amount compared to baseline and untreated control mice by about 20% and 19%, after 7 and 14 days, respectively. That Dex-MP-macrophages were able to decrease fibrotic matrix in the lung was further confirmed by micro CT imaging (FIG. 69), showing less fibrotic tissue when mice were treated with Dex-MP macrophages as compared to controls. Example 21: Analysis of Dex-MP macrophages influence on macrophage phenotype and presence of endothelial cells after subcutaneous implantation

[0284] In contrast to the findings in the pulmonary fibrosis model, the phenotype of implanted and host macrophages was differently influenced in the subcutaneous model. Both administered (GFP+) and host macrophages of mice treated with Dex-MP macrophages showed upregulation of the pro-reparative marker CD163, CD206 and CD301 (FIG. 70). While these markers are related to fibrosis in tissue fibrosis, these markers are also related and important to wound healing, which is necessary after implantation of biomaterials. Therefore, without wishing to be bound by theory, it is postulated that Dex-MPs loaded in macrophages could play a protective role that supports the appropriate regulation of the host environment to facilitate tissue repair.

[0285] Fourteen days after subcutaneous implantation of scaffolds unloaded or loaded with macrophages (FIG. 61), a difference in endothelial percentage (FIG. 71) and cell number (FIG. 72) was observed with an increase after implantation of scaffolds loaded with Dex-MP macrophages. This increase in endothelial cells indicated enhanced angiogenesis compared to the controls, either promoted by the recruitment of endothelial cells or by a reduction of fibrosis, thereby allowing easier endothelial cell infiltration.

[0286] Example 22: Analysis of MP-loaded macrophage transfer of MPs to host cells and influence on macrophage phenotype in volumetric muscle loss model

[0287] In a model of volumetric muscle loss, MP loaded macrophages were implanted inside a collage-based sponge, surgifoam, into the injury side. Both blank MP-loaded macrophages and Dex-MP-loaded macrophages transferred particles to host macrophages, neutrophils, and CD45- negative non-myeloid cells, such as fibroblasts. While no difference was observed on day one after implantation (FIGs. 73-74), seven days after implantation, a higher percentage of CD45 negative cells and neutrophils were positive for MPs in mice treated with blank MP loaded macrophages compared to mice treated with Dex-MP loaded macrophages (FIGs. 75-76). Furthermore, in both groups, an increase in MP-positive macrophages from around 10% to 20% could be observed, indicating that MPs were increasingly transferred over time.

[0288] Additionally, the different treatments influenced the macrophage phenotype of both implanted (GFP+) and host macrophages (FIG. 77). Seven days after implantation, implanted blank MP-loaded macrophages showed downregulation of CD301 and upregulation of CD9. Additionally, both blank MP and Dex MP loaded macrophages showed slight upregulation of CXCR4 compared to unloaded macrophages. Host macrophages from mice treated with MP- loaded macrophages showed lower expression of CD206. In addition, host macrophages from mice treated with blank MP-loaded macrophages further showed downregulation of PDL1 and CD38 compared to controls. Overall, these results indicated that MP-loaded macrophages had the ability to modulate the host macrophages in a muscle fibrosis model, potentially due to the transfer of MPs to host cells.

[0289] Enumerated Embodiments

[0290] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0291] Embodiment 1 : A composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of macrophages comprising one or more microparticles designed to promote microparticle transfer; and / or b) a plurality of macrophages comprising one or more microparticles designed to inhibit microparticle transfer.

[0292] Embodiment 2: The composition of embodiment 1, wherein: a) the one or more microparticles designed to promote microparticle transfer comprises one or more microparticles comprising biophysical properties designed to promote microparticle transfer; and / or b) the one or more microparticles designed to inhibit microparticle transfer comprises one or more microparticles comprising biophysical properties designed to inhibit microparticle transfer.

[0293] Embodiment 3: A composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising microparticles sized to promote transfer; and / or b) a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer.

[0294] Embodiment 4: The composition of embodiment 3, wherein composition comprises both a) and b).

[0295] Embodiment 5: The composition of embodiment 3, wherein the microparticles sized to promote transfer are smaller in diameter relative to the diameter of the microparticles sized to inhibit transfer.

[0296] Embodiment 6: The composition of any one of the preceding embodiments, wherein the microparticles sized to promote transfer are functionalized with an agent. Embodiment 7: The composition of embodiment 6, wherein the agent comprises a nucleic acid and / or a polypeptide.

[0297] Embodiment 8: The composition of embodiment 6 or embodiment 7, wherein the agent is a therapeutic agent.

[0298] Embodiment 9: The composition of embodiment 8, wherein the therapeutic agent is an anti-fibrotic agent, an anti-inflammatory agent, a pro-reparative agent, a pro-regenerative agent, a chemotherapeutic agent, or an anti-cancer agent.

[0299] Embodiment 10: The composition of embodiment 9, wherein the anti-fibrotic agent is a TGFB pathway inhibitor.

[0300] Embodiment 11 : The composition of embodiment 9, wherein the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof.

[0301] Embodiment 12: The composition of any one of the preceding embodiments, wherein the microparticles sized to inhibit transfer are functionalized with an agent.

[0302] Embodiment 13: The composition of embodiment 12, wherein the agent comprises a nucleic acid and / or a polypeptide.

[0303] Embodiment 14: The composition of embodiment 12 or embodiment 13, wherein the agent is a therapeutic agent.

[0304] Embodiment 15: The composition of embodiment 14, wherein the therapeutic agent is dexamethasone.

[0305] Embodiment 16: The composition of any one of the preceding embodiments, wherein the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL)

[0306] Embodiment 17: The composition of any one of the preceding embodiments, wherein the conditioned macrophages of (a) and / or the conditioned macrophages of (b) are derived from a subject’s monocytes.

[0307] Embodiment 18: The composition of any one of the preceding embodiments, wherein the conditioned macrophages of (a) and / or the conditioned macrophages of (b) comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. Embodiment 19: A method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of the composition of any one of embodiments 1- 18 to the subject in need thereof.

[0308] Embodiment 20: The method of embodiment 19, wherein the microparticles sized to promote transfer are transferred to a cell of the subject.

[0309] Embodiment 21 : The method of embodiment 20, wherein the cell is a macrophage, a fibroblast, a neuron, a tumor cell, a non-macrophage lymphocyte, a neutrophil, an epithelial cell, an endothelial cell, a progenitor cell, and / or a non-myeloid cell.

[0310] Embodiment 22: A method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles sized to promote transfer.

[0311] Embodiment 23: The method of embodiment 22, further comprising administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer.

[0312] Embodiment 24: The method of embodiment 22 or embodiment 23, wherein the disease or condition comprises pulmonary fibrosis.

[0313] Embodiment 25: The method of any one of embodiments 22-24, wherein the microparticles sized to promote transfer are transferred to a cell of the subject.

[0314] Embodiment 26: The method of embodiment 25, wherein the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast.

[0315] Embodiment 27: The method of any one of embodiments 22-26, wherein the microparticles sized to promote transfer are functionalized with an agent.

[0316] Embodiment 28: The method of embodiment 27, wherein the agent comprises a nucleic acid and / or a polypeptide.

[0317] Embodiment 29: The method of embodiment 27 or embodiment 28, wherein the agent is a therapeutic agent.

[0318] Embodiment 30: The method of embodiment 29, wherein the therapeutic agent is an anti- fibrotic agent.

[0319] Embodiment 31 : The method of embodiment 30, wherein the anti -fibrotic agent is a TGFB pathway inhibitor Embodiment 32: The method of embodiment 30, wherein the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof.

[0320] Embodiment 33 : The method of any one embodiments 23-32, wherein the microparticles sized to inhibit transfer are functionalized with an agent.

[0321] Embodiment 34: The method of embodiment 33, wherein the agent comprises a nucleic acid and / or a polypeptide.

[0322] Embodiment 35: The method of embodiment 33 or embodiment 34, wherein the agent is a therapeutic agent.

[0323] Embodiment 36: The method of embodiment 35, wherein the therapeutic agent is dexamethasone.

[0324] Embodiment 37: The method of any one of embodiments 22-36, wherein the microparticles sized to promote transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

[0325] Embodiment 38: The method of any one of embodiments 22-37, wherein the microparticles sized to promote transfer are smaller in diameter relative to the diameter of the microparticles sized to inhibit transfer.

[0326] Embodiment 39: The method of any one of embodiments 22-38, wherein the conditioned macrophages comprising microparticles sized to promote transfer are derived from a subject’s monocytes.

[0327] Embodiment 40: The method of any one of embodiments 22-39, wherein the conditioned macrophages comprising microparticles sized to promote transfer comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

[0328] Embodiment 41 : The method of any one of embodiments 23-40, wherein the microparticles sized to inhibit transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

[0329] Embodiment 42: The method of any one of embodiments 23-41, wherein the conditioned macrophages comprising microparticles sized to inhibit transfer are derived from a subject’s monocytes.

[0330] Embodiment 43 : The method of any one of embodiments 23-42, wherein the conditioned macrophages comprising microparticles sized to inhibit transfer comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. Embodiment 44: A method of treating a pulmonary fibrosis in a subject in need thereof, comprising administering to the subject: a) an effective amount of a plurality of conditioned macrophages comprising microparticles sized to promote transfer; and b) an effective amount of a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer.

[0331] Embodiment 45: The method of embodiment 44, wherein the microparticles sized to promote transfer are transferred to a cell of the subject.

[0332] Embodiment 46: The method of embodiment 45, wherein the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a neuron, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast.

[0333] Embodiment 47: The method of any one of embodiments 44-46, wherein the microparticles sized to promote transfer are functionalized with an agent.

[0334] Embodiment 48: The method of embodiment 47, wherein the agent comprises a nucleic acid and / or a polypeptide.

[0335] Embodiment 49: The method of embodiment 47 or embodiment 48, wherein the agent is a therapeutic agent.

[0336] Embodiment 50: The method of embodiment 49, wherein the therapeutic agent is an anti- fibrotic agent.

[0337] Embodiment 51 : The method of embodiment 50, wherein the anti -fibrotic agent is a TGFB pathway inhibitor.

[0338] Embodiment 52: The method of embodiment 50, wherein the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof.

[0339] Embodiment 53 : The method of any one embodiments 44-52, wherein the microparticles sized to inhibit transfer are functionalized with an agent.

[0340] Embodiment 54: The method of embodiment 53, wherein the agent comprises a nucleic acid and / or a polypeptide.

[0341] Embodiment 55: The method of embodiment 53 or embodiment 54, wherein the agent is a therapeutic agent.

[0342] Embodiment 56: The method of embodiment 55, wherein the therapeutic agent is dexamethasone.

[0343] Embodiment 57: The method of any one of embodiments 44-56, wherein the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly (caprolactone) (PCL).

[0344] Embodiment 58: The method of any one of embodiments 44-57, wherein the conditioned macrophages of (a) and / or the conditioned macrophages of (b) are derived from a subject’s monocytes.

[0345] Embodiment 59: The method of any one of embodiments 44-58, wherein the conditioned macrophages of (a) and / or the conditioned macrophages of (b) comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

[0346] Embodiment 60: A composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising an amount of microparticles, wherein the amount of microparticles is an amount that promotes microparticle transfer; and / or a plurality of conditioned macrophages comprising an amount of microparticles, wherein the amount of microparticles is an amount that inhibits microparticle transfer.

[0347] Embodiment 61 : The composition of embodiment 60, wherein the amount of microparticles that inhibits microparticle transfer is between about 10 pg / 106cells to about 100 pg / 106cells.

[0348] Embodiment 62: The composition of embodiment 60 or embodiment 61, wherein the amount of microparticles that promotes microparticle transfer is from about 250 pg / 106cells to about 1000 pg / 106cells.

[0349] Embodiment 63 : The composition of any one of embodiments 60-62, wherein the microparticles are from about 0.01 pm to about 20.0 pm in diameter.

[0350] Embodiment 64: The composition of any one of embodiments 60-63, wherein the microparticles are functionalized with an agent.

[0351] Embodiment 65: The composition of embodiment 64, wherein the agent comprises a nucleic acid and / or a polypeptide.

[0352] Embodiment 66: The composition of embodiment 64 or embodiment 65, wherein the agent is a therapeutic agent.

[0353] Embodiment 67: The composition of embodiment 66, wherein the therapeutic agent is an anti-fibrotic agent, an anti-inflammatory agent, a pro-reparative agent, a pro-regenerative agent, a chemotherapeutic agent, or an anti -cancer agent. Embodiment 68: The composition of embodiment 67, wherein the anti-fibrotic agent is a TGFB pathway inhibitor.

[0354] Embodiment 69: The composition of embodiment 67, wherein the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof.

[0355] Embodiment 70: The composition of any one of embodiment 66, wherein the therapeutic agent is dexamethasone.

[0356] Embodiment 71 : The composition of any one of embodiments 60-70, wherein the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

[0357] Embodiment 72: The composition of any one of embodiments 60-71, wherein the macrophages are derived from a subject’s monocytes.

[0358] Embodiment 73 : The composition of any one of embodiments 60-72, wherein the macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

[0359] Embodiment 74: A method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of the composition of any one of embodiments 60- 73 to the subject in need thereof.

[0360] Embodiment 75: The method of embodiment 74, wherein the microparticles are transferred to a cell of the subject.

[0361] Embodiment 76: The method of embodiment 75, wherein the cell is a macrophage, a fibroblast, a neuron a tumor cell, a non-macrophage lymphocyte, a neutrophil, an epithelial cell, an endothelial cell, a progenitor cell, and / or a non-myeloid cell.

[0362] Embodiment 77: A method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of the composition of any one of embodiments 60-73 to the subject in need thereof.

[0363] Embodiment 78: The method of embodiment 77, wherein the disease or condition comprises pulmonary fibrosis.

[0364] Embodiment 79: The method of embodiment 77 or embodiment 78, wherein the microparticles are transferred to a cell of the subject. Embodiment 80: The method of embodiment 79, wherein the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a neuron, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast.

[0365] Embodiment 81 : A method of treating a pulmonary fibrosis in a subject in need thereof, comprising administering to the subject an effective amount of the composition of any one of embodiments 60-73 to the subject in need thereof

[0366] Embodiment 82: The method of embodiment 81, wherein the microparticles are transferred to a cell of the subj ect.

[0367] Embodiment 83: The method of embodiment 82, wherein the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a neuron, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast.

[0368] Embodiment 84: A composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising a microparticle functionalized with an agent designed to promote microparticle transfer; and / or b) a plurality of conditioned macrophages comprising a microparticle functionalized with an agent designed to inhibit microparticle transfer.

[0369] Embodiment 85: A method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of the composition of embodiment 84 to the subject in need thereof.

[0370] Embodiment 86: A method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles functionalized with at least one agent, wherein the agent comprises dexamethasone.

[0371] Embodiment 87: The method of embodiment 86, wherein the microparticles comprise microparticles designed to promote transfer.

[0372] Embodiment 88: The method of embodiment 86, wherein the microparticles are sized to promote transfer

[0373] Embodiment 89: The method of embodiment 86 or embodiment 87, wherein the microparticles comprise an amount of microparticles that promotes microparticle transfer.

[0374] Embodiment 90: The method of any one of embodiments 86-89, wherein the microparticles are further functionalized with an agent that promotes microparticle transfer. Embodiment 91 : The method of any one of embodiments 86-90, wherein the fibrotic disease or condition is pulmonary fibrosis or is muscle fibrosis.

[0375] Embodiment 92: The method of embodiment 91, wherein the fibrotic disease or condition is pulmonary fibrosis.

[0376] Embodiment 93 : The method of any one of embodiments 86-92, the conditioned macrophages accumulate in the lungs of the subject.

[0377] Embodiment 94: The method of any one of embodiments 86-93, wherein the microparticles are transferred to a cell of the subject.

[0378] Embodiment 95: The method of embodiment 94, wherein the cell of the subject is a macrophage, a non-macrophage lymphocyte, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, a neutrophil, and / or a fibroblast

[0379] Embodiment 96: The method of any one of embodiments 86-95, wherein the treatment decreases expression of CD206, MERTK and CD301 in host macrophages of the subject.

[0380] Embodiment 97: The method of any one of embodiments 86-96, wherein the treatment decreases the total collagen content of the lungs of the subject.

[0381] Embodiment 98: The method of any one of embodiments 86-97, wherein the treatment decreases the collagen content of the lungs of the subject relative to the lungs of an untreated subject having pulmonary fibrosis.

[0382] Embodiment 99: The method of any one of embodiments 86-98, wherein the treatment decreases the total amount of fibrotic lung tissue of the subject.

[0383] Embodiment 100: The method of any one of embodiments 86-99, wherein the treatment decreases the amount of fibrotic lung tissue of the subject relative to an untreated subject having pulmonary fibrosis.

[0384] Embodiment 101 : The method of any one of embodiments 86-100, wherein the treatment increases the number of endothelial cells in the subject as compared to an untreated subject.

[0385] Embodiment 102: The method of any one of embodiments 86-101, wherein the conditioned macrophages are derived from the subject’s monocytes.

[0386] Embodiment 103 : The method of any one of embodiments 86-102, wherein the conditioned macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages. Embodiment 104: The method of any one of embodiments 86-103, wherein the microparticles comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL)

[0387] Embodiment 105: A method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of: (a) a plurality of conditioned macrophages comprising microparticles functionalized with a first agent, wherein the first agent comprises dexamethasone; and (b) a plurality of conditioned macrophages comprising microparticles functionalized with a second agent.

[0388] Embodiment 106: The method of embodiment 105, wherein the second agent comprises a nucleic acid and / or a polypeptide.

[0389] Embodiment 107: The method of embodiment 105 or embodiment 106, wherein the agent is a therapeutic agent.

[0390] Embodiment 108: The method of embodiment 107, wherein the therapeutic agent is an anti-fibrotic agent.

[0391] Embodiment 109: The method of embodiment 108, wherein the anti-fibrotic agent is a TGFB pathway inhibitor.

[0392] Embodiment 110: The method of embodiment 108, wherein the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof.

[0393] Embodiment 111 : The method of any one of embodiments 105-110, wherein the fibrotic disease or condition is pulmonary fibrosis or is muscle fibrosis.

[0394] Embodiment 112: The method of embodiment 111, wherein the fibrotic disease or condition is pulmonary fibrosis.

[0395] Embodiment 113: The method of any one of embodiments 105-112, the conditioned macrophages accumulate in the lungs of the subject.

[0396] Embodiment 114: The method of any one of embodiments 105-113, wherein the microparticles are transferred to a cell of the subject.

[0397] Embodiment 115: The method of embodiment 114, wherein the cell of the subject is a macrophage, a non-macrophage lymphocyte, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, a neutrophil, and / or a fibroblast Embodiment 1 16: The method of any one of embodiments 105-1 15, wherein the treatment decreases expression of CD206, MERTK and CD301 in host macrophages of the subject.

[0398] Embodiment 117: The method of any one of embodiments 105-116, wherein the treatment decreases the total collagen content of the lungs of the subject.

[0399] Embodiment 118: The method of any one of embodiments 105-117, wherein the treatment decreases the collagen content of the lungs of the subject relative to the lungs of an untreated subject having pulmonary fibrosis.

[0400] Embodiment 119: The method of any one of embodiments 105-118, wherein the treatment decreases the total amount of fibrotic lung tissue of the subject.

[0401] Embodiment 120: The method of any one of embodiments 105-119, wherein the treatment decreases the amount of fibrotic lung tissue of the subject relative to an untreated subject having pulmonary fibrosis.

[0402] Embodiment 121 : The method of any one of embodiments 105-120, wherein the treatment increases the number of endothelial cells in the subject as compared to an untreated subject.

[0403] Embodiment 122: The method of any one of embodiments 105-121, wherein the conditioned macrophages are derived from a subject’s monocytes.

[0404] Embodiment 123 : The method of any one of embodiments 105-122, wherein the conditioned macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

[0405] Embodiment 124: The method of any one of embodiments 105-123, wherein the microparticles comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL)

[0406] Embodiment 125: The method of any one of embodiments 105-124, wherein the microparticles of (a) and / or the microparticles of (b) comprise microparticles designed to promote transfer

[0407] Embodiment 126: The method of embodiment 125, wherein the microparticles of (a) and / or the microparticles of (b) are sized to promote transfer. Embodiment 127: The method of embodiment 125 or embodiment 126, wherein the microparticles of (a) and / or the microparticles of (b) comprise an amount of microparticles that promotes microparticle transfer.

[0408] Embodiment 128: The method of any one of embodiments 125-127, wherein the microparticles of (a) and / or the microparticles of (b) are further functionalized with an agent that promotes microparticle transfer.

[0409] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

[0410] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0411] In sum, while this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:1 . A composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of macrophages comprising one or more microparticles designed to promote microparticle transfer; and / or b) a plurality of macrophages comprising one or more microparticles designed to inhibit microparticle transfer.

2. The composition of claim 1, wherein: a) the one or more microparticles designed to promote microparticle transfer comprises one or more microparticles comprising biophysical properties designed to promote microparticle transfer; and / or b) the one or more microparticles designed to inhibit microparticle transfer comprises one or more microparticles comprising biophysical properties designed to inhibit microparticle transfer.

3. A composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising microparticles sized to promote transfer; and / or b) a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer.

4. The composition of claim 3, wherein composition comprises both a) and b).

5. The composition of claim 3, wherein the microparticles sized to promote transfer are smaller in diameter relative to the diameter of the microparticles sized to inhibit transfer.

6. The composition of any one of the preceding claims, wherein the microparticles sized to promote transfer are functionalized with an agent.

7. The composition of claim 6, wherein the agent comprises a nucleic acid and / or a polypeptide.

8. The composition of claim 6 or claim 7, wherein the agent is a therapeutic agent.

9. The composition of claim 8, wherein the therapeutic agent is an anti-fibrotic agent, an anti-inflammatory agent, a pro-reparative agent, a pro-regenerative agent, a chemotherapeutic agent, or an anti-cancer agent10. The composition of claim 9, wherein the anti -fibrotic agent is a TGFB pathway inhibitor11. The composition of claim 9, wherein anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof.

12. The composition of any one of the preceding claims, wherein the microparticles sized to inhibit transfer are functionalized with an agent.

13. The composition of claim 12, wherein the agent comprises a nucleic acid and / or a polypeptide.

14. The composition of claim 12 or claim 13, wherein the agent is a therapeutic agent.15 The composition of claim 14, wherein the therapeutic agent is dexamethasone.

16. The composition of any one of the preceding claims, wherein the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer comprise poly(lactic- co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

17. The composition of any one of the preceding claims, wherein the conditioned macrophages of (a) and / or the conditioned macrophages of (b) are derived from a subject’s monocytes.

18. The composition of any one of the preceding claims, wherein the conditioned macrophages of (a) and / or the conditioned macrophages of (b) comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.19 A method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of the composition of any one of claims 1-18 to the subject in need thereof20. The method of claim 19, wherein the microparticles sized to promote transfer are transferred to a cell of the subj ect.

21. The method of claim 20, wherein the cell is a macrophage, a fibroblast, a neuron, a tumor cell, a non-macrophage lymphocyte, a neutrophil, an epithelial cell, an endothelial cell, a progenitor cell, and / or a non-myeloid cell.

22. A method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles sized to promote transfer23. The method of claim 22, further comprising administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer.

24. The method of claim 22 or claim 23, wherein the disease or condition comprises pulmonary fibrosis.

25. The method of any one of claims 22-24, wherein the microparticles sized to promote transfer are transferred to a cell of the subject.

26. The method of claim 25, wherein the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, a neutrophil, and / or a fibroblast27. The method of any one of claims 22-26, wherein the microparticles sized to promote transfer are functionalized with an agent.

28. The method of claim 27, wherein the agent comprises a nucleic acid and / or a polypeptide.

29. The method of claim 27 or claim 28, wherein the agent is a therapeutic agent.

30. The method of claim 29, wherein the therapeutic agent is an anti-fibrotic agent.31 . The method of claim 30, wherein the anti-fibrotic agent is a TGFB pathway inhibitor.

32. The method of claim 30, wherein the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof.

33. The method of any one claims 23-32, wherein the microparticles sized to inhibit transfer are functionalized with an agent.

34. The method of claim 33, wherein the agent comprises a nucleic acid and / or a polypeptide.

35. The method of claim 33 or claim 34, wherein the agent is a therapeutic agent.

36. The method of claim 35, wherein the therapeutic agent is dexamethasone.37 The method of any one of claims 22-36, wherein the microparticles sized to promote transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

38. The method of any one of claims 22-37, wherein the microparticles sized to promote transfer are smaller in diameter relative to the diameter of the microparticles sized to inhibit transfer.

39. The method of any one of claims 22-38, wherein the conditioned macrophages comprising microparticles sized to promote transfer are derived from a subject’s monocytes.

40. The method of any one of claims 22-39, wherein the conditioned macrophages comprising microparticles sized to promote transfer comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.41 . The method of any one of claims 23-40, wherein the microparticles sized to inhibit transfer comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).42 The method of any one of claims 23-41, wherein the conditioned macrophages comprising microparticles sized to inhibit transfer are derived from a subject’s monocytes.

43. The method of any one of claims 23-42, wherein the conditioned macrophages comprising microparticles sized to inhibit transfer comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

44. A method of treating a pulmonary fibrosis in a subject in need thereof, comprising administering to the subject: a) an effective amount of a plurality of conditioned macrophages comprising microparticles sized to promote transfer, and b) an effective amount of a plurality of conditioned macrophages comprising microparticles sized to inhibit transfer.

45. The method of claim 44, wherein the microparticles sized to promote transfer are transferred to a cell of the subj ect.

46. The method of claim 45, wherein the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast.

47. The method of any one of claims 44-46, wherein the microparticles sized to promote transfer are functionalized with an agent.

48. The method of claim 47, wherein the agent comprises a nucleic acid and / or a polypeptide.

49. The method of claim 47 or claim 48, wherein the agent is a therapeutic agent.

50. The method of claim 49, wherein the therapeutic agent is an anti-fibrotic agent.51 . The method of claim 50, wherein the anti-fibrotic agent is a TGFB pathway inhibitor.

52. The method of claim 50, wherein the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof.

53. The method of any one claims 44-52, wherein the microparticles sized to inhibit transfer are functionalized with an agent.

54. The method of claim 53, wherein the agent comprises a nucleic acid and / or a polypeptide.

55. The method of claim 53 or claim 54, wherein the agent is a therapeutic agent.56 The method of claim 55, wherein the therapeutic agent is dexamethasone.

57. The method of any one of claims 44-56, wherein the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer comprise poly(lactic-co- glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

58. The method of any one of claims 44-57, wherein the conditioned macrophages of (a) and / or the conditioned macrophages of (b) are derived from a subject’s monocytes.

59. The method of any one of claims 44-58, wherein the conditioned macrophages of (a) and / or the conditioned macrophages of (b) comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages60 A composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising an amount of microparticles, wherein the amount of microparticles is an amount that promotes microparticle transfer; and b) a plurality of conditioned macrophages comprising an amount of microparticles, wherein the amount of microparticles is an amount that inhibits microparticle transfer.61 . The composition of claim 60, wherein the amount of microparticles that inhibits microparticle transfer is between about 10 pg / 106cells to about 100 pg / 106cells.

62. The composition of claim 60 or claim 61, wherein the amount of microparticles that promotes microparticle transfer is from about 250 pg / 106cells to about 1000 pg / 106cells.

63. The composition of any one of claims 60-62, wherein the microparticles are from about 0.01 pm to about 20.0 pm in diameter.

64. The composition of any one of claims 60-63, wherein the microparticles are functionalized with an agent65. The composition of claim 64, wherein the agent comprises a nucleic acid and / or a polypeptide.

66. The composition of claim 64 or claim 65, wherein the agent is a therapeutic agent.

67. The composition of claim 66, wherein the therapeutic agent is an anti-fibrotic agent, an anti-inflammatory agent, a pro-reparative agent, a pro-regenerative agent, a chemotherapeutic agent, or an anti-cancer agent.

68. The composition of claim 67, wherein the anti -fibrotic agent is a TGFB pathway inhibitor.69 The composition of claim 67, wherein the anti-fibrotic agent is pirfenidone, nintedanib, or a combination thereof.

70. The composition of any one of claim 66, wherein the therapeutic agent is dexamethasone.

71. The composition of any one of claims 60-70, wherein the microparticles sized to promote transfer and / or the microparticles sized to inhibit transfer comprise poly(lactic-co- glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

72. The composition of any one of claims 60-71, wherein the macrophages are derived from a subject’s monocytes.

73. The composition of any one of claims 60-72, wherein the macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

74. A method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of the composition of any one of claims 60-73 to the subject in need thereof.

75. The method of claim 74, wherein the microparticles are transferred to a cell of the subject.76 The method of claim 75, wherein the cell is a macrophage, a fibroblast, a neuron, a tumor cell, a non-macrophage lymphocyte, a neutrophil, an epithelial cell, an endothelial cell, a progenitor cell, and / or a non-myeloid cell.

77. A method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of the composition of any one of claims 60-73 to the subject in need thereof78. The method of claim 77, wherein the disease or condition comprises pulmonary fibrosis.

79. The method of claim 77 or claim 78, wherein the microparticles are transferred to a cell of the subject.

80. The method of claim 79, wherein the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast.

81. A method of treating a pulmonary fibrosis in a subject in need thereof, comprising administering to the subject an effective amount of the composition of any one of claims 60-73 to the subject in need thereof.

82. The method of claim 81, wherein the microparticles are transferred to a cell of the subject.

83. The method of claim 82, wherein the cell of the subject in need thereof is a macrophage, a non-macrophage lymphocyte, a neutrophil, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, and / or a fibroblast.84 A composition comprising conditioned macrophages, wherein the conditioned macrophages comprise: a) a plurality of conditioned macrophages comprising a microparticle functionalized with an agent designed to promote microparticle transfer; and / or b) a plurality of conditioned macrophages comprising a microparticle functionalized with an agent designed to inhibit microparticle transfer.

85. A method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of the composition of claim 84 to the subject in need thereof.86 A method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a plurality of conditioned macrophages comprising microparticles functionalized with at least one agent, wherein the agent comprises dexamethasone.

87. The method of claim 86, wherein the microparticles comprise microparticles designed to promote transfer.

88. The method of claim 86, wherein the microparticles are sized to promote transfer.

89. The method of claim 86 or claim 87, wherein the microparticles comprise an amount of microparticles that promotes microparticle transfer.

90. The method of any one of claims 86-89, wherein the microparticles are further functionalized with an agent that promotes microparticle transfer.

91. The method of any one of claims 86-90, wherein the fibrotic disease or condition is pulmonary fibrosis or is muscle fibrosis.

92. The method of claim 91, wherein the fibrotic disease or condition is pulmonary fibrosis.

93. The method of any one of claims 86-92, the conditioned macrophages accumulate in the lungs of the subject.

94. The method of any one of claims 86-93, wherein the microparticles are transferred to a cell of the subject.

95. The method of claim 94, wherein the cell of the subject is a macrophage, a nonmacrophage lymphocyte, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, a neutrophil, and / or a fibroblast.96 The method of any one of claims 86-95, wherein the treatment decreases expression of CD206, MERTK and CD301 in host macrophages of the subject.

97. The method of any one of claims 86-96, wherein the treatment decreases the total collagen content of the lungs of the subject.

98. The method of any one of claims 86-97, wherein the treatment decreases the collagen content of the lungs of the subject relative to the lungs of an untreated subject having pulmonary fibrosis.

99. The method of any one of claims 86-98, wherein the treatment decreases the total amount of fibrotic lung tissue of the subject.

100. The method of any one of claims 86-99, wherein the treatment decreases the amount of fibrotic lung tissue of the subject relative to an untreated subject having pulmonary fibrosis.

101. The method of any one of claims 86-100, wherein the treatment increases the number of endothelial cells in the subject as compared to an untreated subject.

102. The method of any one of claims 86-101, wherein the conditioned macrophages are derived from the subject’s monocytes.

103. The method of any one of claims 86-102, wherein the conditioned macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

104. The method of any one of claims 86-103, wherein the microparticles comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

105. A method of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of: a) a plurality of conditioned macrophages comprising microparticles functionalized with a first agent, wherein the first agent comprises dexamethasone; and b) a plurality of conditioned macrophages comprising microparticles functionalized with a second agent.

106. The method of claim 105, wherein the second agent comprises a nucleic acid and / or a polypeptide.

107. The method of claim 105 or claim 106, wherein the agent is a therapeutic agent.

108. The method of claim 107, wherein the therapeutic agent is an anti-fibrotic agent.

109. The method of claim 108, wherein the anti -fibrotic agent is a TGFB pathway inhibitor.

110. The method of claim 108, wherein the anti -fibrotic agent is pirfenidone, nintedanib, or a combination thereof111. The method of any one of claims 105-110, wherein the fibrotic disease or condition is pulmonary fibrosis or is muscle fibrosis.

112. The method of claim 111, wherein the fibrotic disease or condition is pulmonary fibrosis.

113. The method of any one of claims 105-112, the conditioned macrophages accumulate in the lungs of the subject.

114. The method of any one of claims 105-113, wherein the microparticles are transferred to a cell of the subj ect.1 15. The method of claim 1 14, wherein the cell of the subject is a macrophage, a nonmacrophage lymphocyte, a non-myeloid cell, an epithelial cell, an endothelial cell, a progenitor cell, a neutrophil, and / or a fibroblast.

116. The method of any one of claims 105-115, wherein the treatment decreases expression of CD206, MERTK and CD301 in host macrophages of the subject.

117. The method of any one of claims 105-116, wherein the treatment decreases the total collagen content of the lungs of the subj ect.118 The method of any one of claims 105-117, wherein the treatment decreases the collagen content of the lungs of the subject relative to the lungs of an untreated subject having pulmonary fibrosis.

119. The method of any one of claims 105-118, wherein the treatment decreases the total amount of fibrotic lung tissue of the subject.

120. The method of any one of claims 105-119, wherein the treatment decreases the amount of fibrotic lung tissue of the subject relative to an untreated subject having pulmonary fibrosis.

121. The method of any one of claims 105-120, wherein the treatment increases the number of endothelial cells in the subject as compared to an untreated subject.

122. The method of any one of claims 105-121, wherein the conditioned macrophages are derived from a subject’s monocytes.

123. The method of any one of claims 105-122, wherein the conditioned macrophages comprise exogenous macrophages, allogenic macrophages, and / or an autologous macrophages.

124. The method of any one of claims 105-123, wherein the microparticles comprise poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(caprolactone) (PCL).

125. The method of any one of claims 105-124, wherein the microparticles of (a) and / or the microparticles of (b) comprise microparticles designed to promote transfer.

126. The method of claim 125, wherein the microparticles of (a) and / or the microparticles of (b) are sized to promote transfer.

127. The method of claim 125 or claim 126, wherein the microparticles of (a) and / or the microparticles of (b) comprise an amount of microparticles that promotes microparticle transfer.128 The method of any one of claims 125-127, wherein the microparticles of (a) and / or the microparticles of (b) are further functionalized with an agent that promotes microparticle transfer.

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