Use of solid synthetic particles for amplification and composition control of phagocyte-generated extracellular vesicles

Abstract

The present invention relates to a solid synthetic particle (SSP). The SSP may comprise a synthetic polymer, which may form a crosslinked structure. The SSP may have a diameter of 0.2 - 20 µm. A method for producing extracellular vesicles (EVs) by host cells is provided, comprising (a) exposing host cells to a plurality of the SSPs; internalizing the SSPs by the host cells through phagocytosis; and (c) secreting EVs by the host cells. Also provided are host cells comprising the SSPs, and EVs produced by the host cells. Further provided is a method for modulating a biological activity of target cells, for example, immune cells.

Description

[0001] USE OF SOLID SYNTHETIC PARTICLES FOR AMPLIFICATION AND COMPOSITION

[0002] CONTROL OF PHAGOCYTE-GENERATED EXTRACELLULAR VESICLES

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims priority to United States Provisional Application No. 63 / 677,438, filed July 31, 2024, the contents of which are incorporated herein by reference in their entireties for all purposes.

[0005] REFERENCE TO U.S. GOVERNMENT SUPPORT

[0006] This invention was made with government support under grant number R35GM142866A awarded by National Institute of Health. The United States has certain rights in the invention.

[0007] FIELD OF THE INVENTION

[0008] The invention relates to extracellular vesicles (EVs) and modulation of EV production.

[0009] BACKGROUND OF THE INVENTION

[0010] Extracellular vesicles (EVs) are lipid bilayer-enclosed vesicles actively secreted by cells that carry bioactive cargo such as proteins, RNAs, and lipids to host cells. In phagocytes such as macrophages, EVs influence immune responses, tissue repair, and disease progression. Phagocyte-derived EVs are increasingly recognized as key players in a range of conditions including cancer, chronic inflammation, and fibrosis. Depending on their source and context, they can act as pathogenic mediators that amplify disease signals or as therapeutic agents that promote immunoregulation, infection defense, and tissue remodeling.

[0011] Although EVs hold great promise as therapeutic modifiers, there are few effective strategies to produce EVs of controlled properties, and those that exist face significant limitations. Existing approaches that aim to generate EVs in situ within the body lack control over cargo, function, and quantity. Meanwhile, ex vivo manufacturing from cultured cells often depends on genetic engineering or labor-intensive protocols that are difficult to scale and reproduce. Both methods yield heterogeneous EV populations with poorly defined biological activity.

[0012] There remains a clear need for non-genetic, scalable methods that allow precise control over EV production, composition, and function.

[0013] SUMMARY OF THE INVENTION

[0014] The present invention relates to solid synthetic particle (SSP). The present invention is based on the inventors' surprising discovery of a tunable SSP chemistry to control over extracellular vesicles (EVs) secretion by host cells, cargo loading, and composition in tandem. The present invention provides a solid synthetic particle (SSP). The SSP comprises a synthetic polymer. The polymer forms a crosslinked structure. The SSP has a diameter of 0.2 - 20 pm.

[0015] The polymer may comprise poly(ethylene glycol) diacrylate (PEGDA), polystyrene, poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) dithiol, 2- carboxyethyl acrylate (CEA), 2-aminoethyl methacrylate, nitrobenzyl photolabile molecules, hyaluronic acid, gelatin, chitosan, alginate, or a combination thereof.

[0016] The polymer may comprise a monomer backbone, a charge-carrying comonomer, a stimuli-responsive co-monomer, a covalently-linked fluorescent probe, or a combination of thereof. The stimuli-responsive co-monomer may be selected from the group consisting of a photoresponsive, redox reactive, ester labile, enzymatically degradable, and a combination of thereof.

[0017] The crosslinked structure may have a mechanical stiffness (Young's modulus) of 100 kPa to 3.6 GPa.

[0018] The SSP may further comprise an agent. The agent may be a bioactive molecule.

[0019] A method for producing extracellular vesicles (EVs) by host cells is provided. The EV production method comprises: (a) exposing host cells to a plurality of the SSPs of the present invention; (b) internalizing the SSPs by the host cells through phagocytosis; and (c) secreting EVs by the host cells, whereby the EVs are produced.

[0020] The EVs may be smallEVs.

[0021] The host cells may be exposed to the SSPs in an amount of 60-800 pg per cell. The host cells may reside in the tissue of a subject. The host cells may be in a cell culture. The host cells may comprise eukaryotic phagocytes.

[0022] The EV production method may further comprise measuring a first EV number of the EVs produced by the host cells before being exposed to the SSPs, and measuring a second EV number of the EVs produced by the host cells after being exposed to the SSPs, wherein the second EV number is greater than the first EV number.

[0023] The EVs may carry a cargo. The cargo may be attached to the surface of the SSPs or encapsulated within the SSPs.

[0024] The EV production method may further comprise collecting the EVs.

[0025] For each EV production method of the present invention, extracellular vesicles (EVs) are provided. The EVs are produced by host cells according to the EV production method.

[0026] A method for modulating a biological activity of target cells is provided. The modulation method comprises administering to target cells an effective amount of the EVs of the present invention, whereby a biological activity of the target cells is modulated. The target cells may be immune cells, barrier cells, or stem cells. The biological activity may be selected from the group consisting of target cell proliferation, survival, activation, cytokine secretion, barrier function, and differentiation. For each SSP of the present invention, host cells are provided. The host cells comprises the SSP. The SSP may have been internalized by the host cells through active phagocytosis.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 illustrates the use of solid synthetic particles (SSPs) for amplified secretion of phagocyte-generated extracellular vesicles (EVs).

[0029] FIGS. 2A-C show that SSP internalization drives a dose dependent release of extracellular vesicles in culture conditions. Nanoparticle tracking analysis (NTA) determines concentrations of ultracentrifuge isolated EVs as a variable of dosed SSP for various cell types dosed in vitro. Results are shown for SSPs of poly(ethylene glycol) diacrylate (PEGDA) formulated with 2-carboxyethyl acrylate (CEA) resulting in anionic SSPs of ~300 nm in diameter. For each panel, left graph shows EV production for each treatment group 72 hours after SSP dosing, and the right graph shows the size distribution of collected EV sample. A) RAW 264.7 macrophages immortalized (originally isolated from BALB / c mice), B) THP-1 immortalized human monocytes terminally differentiated into macrophages, C) ex vivo primary bone marrow derived macrophages (BMM) isolated from c57bl / 6 mice (N = 3, SD error bars), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 using Ordinary one-way ANOVA tests.

[0030] FIG. 3 shows that SSP internalization drives an increase of extracellular vesicles following in vivo delivery. Left graph shows that murine alveolar macrophages internalized SSP following orotracheal delivery (N = 4, SD error bars, ****p<0.0001 using student's T-test). Middle graph shows EVs collected from the bronchoalveolar lavage fluid (BALF) 48 hours after SSP dosing are increased following SSP treatment (p = 0.043 using student's t-test) and the right graph shows the size distribution of collected EV samples via NTA. Results are shown for SSPs of PEGDA formulated with CEA ~300 nm in diameter.

[0031] FIG. 4 shows that SSP properties regulate EV secretion in BMMs. Left: NTA analysis of ultracentrifuge-isolated EVs collected 72 hours following dosing with 100 pg / mL of various conditions including: anionic CEA PEGDA SSP (300 nm in diameter), cationic PEGDA SSP (300 nm in diameter), polystyrene SSP (190 nm in diameter), polystyrene SSP microparticle MP (1 pm in diameter), and lipopolysaccharide (N = 3, SD error bars), *p<0.05, ***p<0.001, using Brown-Forsythe and Welch ANOVA tests, i, ii, ill show the size distribution of collected EV samples via NTA for each labeled group. FIG. 5 provides SEM and TEM imaging of SSP dosed ev vivo BMMs showing generation of smallEVs, supported by formation of multivesicle (MVB) bodies and colocalization of lysosomal compartment containing internalized SSPs and MVBs after 24 hours. Results are shown for SSPs of PEGDA formulated with CEA ~300 nm in diameter. Top 3 rows are SEM images. Bottom 3 rows are TEM images. Conditions include untreated BMM (column 1) and highlighted sections (I. and II.). Both sets of images show expected morphology for external (SEM) and internal (TEM) structure of a BMM. The SEM images do not show abundance of budding in any conditions, ruling out microvesicle (a subclass of EV) generation. PEGDA CEA (column 2) and AEM (column 3) SSP-dosed BMM show internalization of SSPs in lysosomal compartment along with formation of nearby MVB. Dosing with polystyrene SSP microparticles (column 4) shows individual internalization of SSP in lysosomal compartment along with colocalization of MVB to nearby internalized MPs shown in highlight sections (I. and II.) FIGS. 6A-F show enrichment for metabolic activity and membrane-bounded organelles based on proteomic analysis of SSP-dosed BMMs after 24 hours. Results are shown for SSPs of PEGDA formulated with CEA ~300 nm in diameter. Significantly differentiated proteins for proteomic analysis of A) whole cell proteome and B) secreted proteome shows identification of 1051 and 213 hits respectively (Student's T-test, FDR = 0.1). Hierarchical clustering of significantly differentiated proteins for C) whole cell proteomics and D) secreted proteomics. Performed gene enrichment analysis of clusters with increased presence in SSP dosed conditions. E) Gene enrichment analysis using GO cellular component shows significant increase for membrane-bounded organelles, i.e. EVs. F) Enrichment analysis performed with GO biological function as the database shows increase in metabolic process following dosing with SSPs.

[0032] FIGS. 7A-C show increased survival and metabolic activity of ex vivo BMM following dosing with A) CEA PEGDA SSPs (~300 nm diameter) and B) EVs derived from SSP treated BMMs. A) BMM dosed with varying concentration of SSP show increase in population half-life as modeled through a one-phase decay equation (N = 4, SEM error bars), *p<0.05, **p<0.01, using Brown-Forsythe and Welch ANOVA tests. B) BMMs dosed with ultracentrifuged isolated SSP-derived EVs show an increase in population half-life fitted with a one-phase decay model equation with bands representing 95% confidence interval (N = 4, SEM error bars), *p<0.05, ****p<0.0001, using Ordinary one-way ANOVA. C) Metabolic activity of both BMM cultures after 4 days of dosing with SSPs and SSP-derived EVs as measured through Cell Titer gio 2.0 (N = 4, SD error bars) ****p<0.0001, using Brown-Forsythe and Welch ANOVA tests. FIGS. 8A-E show encapsulation and transfer of SSP material to SSP-derived EVs. Results are shown for SSPs of PEGDA formulated with CEA and maleimide-Cy5 ~300 nm in diameter. A) EVs collected from ex vivo BMM cultures dosed with SSPs containing Cy5 cargo were captured with Exoquick anti-CD63 magnetic beads and show encapsulation of Cy5 material derived from SSP. EVs collected from ex vivo BMM cultures dosed with SSPs containing Cy5 cargo show no significant increase in expression of B) pro-inflammatory marker CD40 (N=3, SD error bars) and C) proantigen presentation marker MHCii (N = 3, SD error bars). D) Ex vivo BMM cultures dosed with SSPs show transfer of SSP material through SSP-Cy5 fragments, while untreated and LPS controls do not (N = 3, SD error bars). E) Confocal microscopy of SSP-dosed BMMs and SSP-Cy5-derived EV-dosed BMMs show presence of Cy5 fluorescence for both cell populations (Scale bar 10 pm).

[0033] FIGS. 9A-D show that host cells having different phenotypes yield distinct EV profiles when dosed with SSPs and that these EVs can transfer that profile to other cells. Results are shown for SSPs of PEGDA formulated with CEA and maleimide-Cy5 ~300 nm in diameter. SmallEVs are characterized from host cells with distinct phenotypes through magnetic bead capture and flow cytometry. A) Relative MFI for antigen presentation protein MHCii on SSP-derived EVs shows distinct expression between EVs from different host phenotype cells, with smallEVs from host cells dosed with pro-inflammatory soluble factor IFN-y and SSP showing the highest increase in MHCii expression. B) Relative MFI for transferred SSP material on SSP-derived EVs, indicated by changes in relative MFI for fluorescent cargo Cy5, shows distinct rate of encapsulation for smallEVs from host cells with different phenotypes. SmallEVs from host cells dosed with both pro-inflammatory soluble factor IFN-y and SSPs show the highest transfer of SSP material to resultant EVs. C) Relative MFI for antigen presentation protein MHCii shows increase for naive macrophages dosed with SSP- derived EVs from host cells dosed with pro-inflammatory soluble factor, indicating potential transfer of protein from smallEVs to dosed cells. D) Relative MFI of Cy5 molecule in naive macrophages dosed with SSP-derived EVs shows transfer of SSP material to dosed macrophages for every culture that received SSP amplified smallEVs.

[0034] FIG. 10 shows that SSPs of tunable functionalization change the host cell phenotype. Varying the amount of CpG on SSPs yields differential changes in activation marker expression on the dosed macrophage cells. Results are shown for SSPs of PEGDA formulated with AEM and maleimide-acrylate that was functionalized with thiolated CpG ~300 nm in diameter.

[0035] FIG. 11 shows BMM EV secretion as a function of SSPs dosing is dependent on the total mass of SSPs delivered, not the total number of SSP internalization events. Results are shown for SSPs of PEGDA formulated with CEA and maleimide-Cy5 ~300 nm in diameter. Two densities of SSP were fabricated and dosed to BMMs at equivalent total mass. 50 wt% SSPs are less dense than 75 wt% SSPs; when dosed to BMMs at equivalent mass, 50 wt% SSPs will have almost two times more SSP number (1.25el0 particles / mL compared to 0.64el0 particles / mL for the 75 wt%). Concentrations of produced EVs shows that SSPs dosed at equivalent mass dosing yielded ~3.5 fold increase of EV secretion from both SSP treated groups.

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention relates to solid synthetic particles (SSPs) and uses thereof for producing extracellular vesicles (EVs) by host cells. The present invention is based on the inventors' surprising discovery of a tunable SSP chemistry to control phagocyte EV secretion, cargo loading, and composition in tandem. In particular, the interaction of the SSP size, density / modulus, and composition plays a key role in delivering a critical mass and bioactive signals to the cell that ensures sufficient phagocytosis, amplifies EV biogenesis, and regulates EV phenotype. The inventors have taken advantage of a newly uncovered natural mechanism by which phagocytes increase EV output in response to the phagocytic uptake of SSP. By varying SSP attributes such as size, stiffness, surface chemistry, or degradability, the inventors could influence EV yield, cargo composition, and immune phenotype. This discovery offers a new platform for directing EV function through rational design of the SSP materials presented to phagocytes, enabling new applications in therapy, diagnostics, and vaccine development.

[0038] The present invention provides methods and SSP compositions for modulating the secretion and functional characteristics of EVs produced by phagocytic immune cells, including but not limited to macrophages, monocytes, neutrophils, and dendritic cells. Taking advantage of an endogenous pathway, the inventors have successfully manipulated the SSP materials to engineer the output phenotype of secreted EVs with unique and desirable properties. These SSPs serve as programmable triggers that dictate downstream EV products, thereby providing a novel and highly modular route to engineer EVs in a predictable and scalable manner.

[0039] As illustrated in FIG. 1, phagocytosis of internalized SSP leads to intracellular signaling that regulates the host cell phenotype and directs the cell to process the SSP. SSPs are shuttled into the lysosome as part of the degradation process, inducing an increase in the formation of intracellular multivesicle bodies, where SSP-material is transferred during the EV biogenesis process. Newly created EVs contained in multivesicle bodies are exocytosed, releasing EVs containing SSP material from the host cell. The inventors have developed methods for stimulating and directing EV secretion, compositions of synthetic particles (SSPs) with defined physical and chemical properties, and compositions of the EVs produced as a result of these SSP-cell interactions.

[0040] The present invention enables a non-genetic, modular, and scalable approach to producing defined EVs with programmable function, establishing a new modality in immune engineering and translational medicine.

[0041] The term "solid synthetic particles (SSP)" as used herein refers to particulates made from synthesized material such as polymers or metal with regulated physical properties engineered for desirable cellular interactions. The particulates can act as carriers for biologic agents such as peptides, proteins, and oligonucleotides.

[0042] The term "extracellular vesicles (EVs)" as used herein refers to a heterogeneous group of cell-derived membrane-bound particles released into an extracellular environment by cells. They may play a significant role in intercellular communication by transporting various cellular components, including proteins, lipids, and nucleic acids. EVs may be isolated from all bodily fluids and are secreted by nearly all cell types.

[0043] The term "small extracellular vesicles (smallEVs or sEVs)" as used herein refers to EVs with a diameter less than 200 nm and larger than 30 nm, in accordance with the latest guidelines from the International Society for Extracellular Vesicles (MISEV2018).

[0044] The term "exosomes" as used herein refers to a specific subtype of sEVs or smallEVs that originate from the endosomal system, specifically from multivesicular bodies (MVBs) that fuse with the plasma membrane to release their intraluminal vesicles (ILVs). Exosomes typically have a diameter ranging from 30 nm to 150 nm. Exosomes may carry a diverse set of molecules, including nucleic acids, proteins, and lipids, reflecting their cell of origin. Exosomes are involved in various physiological and pathological processes, including immune responses, viral pathogenicity, and cancer progression.

[0045] The term "polymer" as used herein refers to a molecular structure consisting of a large number of similar units covalently bonded together. The polymer may be crosslinked by a chemical bond that connects two or more polymer chains, effectively linking them together to form a three-dimensional (3D) network, which is also referred to as "crosslinked structure." The length of the polymer chains and how they interact with each other in the 3D network define the mass, density, and mechanical properties of the crosslinked structure.

[0046] The term "synthetic polymer" as used herein refers to polymers generated outside of a host cell through controlled chemical reactions that connect monomer base units and polymer chains for a defined structure. They are engineered to have specific controlled properties. The term "EV yield" as used herein refers to the total amount of EVs produced by host cells upon exposure of SSPs. The EV yield may be amplified, as measured by nanoparticle tracking analysis (NTA).

[0047] The term "EV cargo composition" as used herein refers to a composition of one or more agents carried by an EV.

[0048] The term "EV surface phenotype" as used herein refers to the surface characteristics of an EV. Examples of EV surface phenotype include expression of CD63, MHC molecules, costimulatory proteins, or proteins involved in survival signaling.

[0049] The term "EV functional effects" as used herein refers to phenotypical or immunological changes induced by EVs on target cells. The EVs may have been produced by host cells that are different from the target cells. The EV functional effects may include modulation of the target cells (e.g., T cell or macrophage activation, polarization, survival), and triggered cytokine release.

[0050] The term "phagocytosis" as used herein refers to the mechanism of cellular internalization of particulates larger than 200 nm from the extracellular space. The phagocytosis may be actin-mediated or receptor-mediated phagocytosis. The receptor- mediated phagocytosis may be Fc-mediated, scavenger receptor-mediated, complement-mediated, or LC3-mediated phagocytosis. The phagocytosis is active when involving the restructuring of cellular membrane to facilitate internalization.

[0051] The term "primary cells" as used herein refers to viable cells that have been isolated from a subject but have not been cultured for more than 1 passage.

[0052] The term "subject" as used herein refers to an animal, preferably a mammal. Examples of the subjects include human, murine, rodent, pig, sheep, dog, and nonhuman primates.

[0053] The term "immune cells" as used herein refers to a class of cells called leukocytes that are part of the immune system and are responsible for defending the body against foreign invaders.

[0054] The present invention provides a solid synthetic particle (SSP). The SSP may comprise one or more polymeric, metallic, or hybrid materials, including but not limited to PEGDA, polystyrene, PLGA, silica, other hydrogels, and metallic or hybrid composites.

[0055] In one embodiment, the SSP comprises a synthetic polymer. The polymer forms a crosslinked structure. The polymer may comprise a monomer backbone, a chargecarrying co-monomer, a stimuli-responsive co-monomer, a covalently-linked fluorescent probe, or a combination of thereof. The stimuli-responsive co-monomer may be selected from the group consisting of a photoresponsive, redox reactive, ester labile, enzymatically degradable, and a combination of thereof. Examples of the polymer may include poly(ethylene glycol) diacrylate (PEGDA), polystyrene, poly(lactic- co-glycolic acid) (PLGA), poly(ethylene glycol) dithiol, 2-carboxyethyl acrylate (CEA), 2- aminoethyl methacrylate, nitrobenzyl photolabile molecules, hyaluronic acid, gelatin, chitosan, alginate, or a combination thereof.

[0056] The size of the SSPs may range from 200 nanometers to 20 micrometers, encompassing both nano- and microscale particles. The SSP may have a diameter of about 0.2-1, 0.2-10, 0.2-20, 0.2-100, 0.5-1, 0.5-10, 0.5-20, 0.5-100, 1-10, 1-20, 1- 100, or 10-20 pm. For example, the SSP may have a diameter of about 0.2 - 10 pm.

[0057] The mechanical stiffness of the SSPs may range from soft (<10 kPa) to rigid (>1 MPa), and may be modulated through material selection or crosslinking density. Rigid particles are generally preferred to ensure efficient phagocytic uptake. The crosslinked structure may have a mechanical stiffness (Young's modulus) from about from about 10 kPa to about 1 Gpa, from about 10 kPa to about 5 Gpa, from about 10 kPa to about 10 Gpa, from about 100 kPa to about 2 Gpa, from about 100 kPa to about 3 Gpa, from about 100 kPa to about 3.5 Gpa, from about 100 kPa to about 3.6 Gpa, from about 100 kPa to about 4 Gpa, from about 100 kPa to about 5 Gpa, from about 100 kPa to about 10 Gpa, from about 500 kPa to about 1 Gpa, from about 500 kPa to about 5 Gpa, from about 500 kPa to about 10 Gpa. The mechanical stiffness of the crosslinked structure may be from about 100 kPa to about 3.6 GPa.

[0058] The surface of the SSP may also be functionalized for, for example, charge, ligand presentation, protein adsorption. The SSP may be functionalized with a bioactive molecule (e.g., antigen, cytokine, or pathogen-associated molecular patterns (PAMP)) to further modulate host cell response or enhance EV bioactivity.

[0059] The degradability of the SSP may be through polymer chemistry, crosslinking, or responsive linkers, for example, photoresponsive or redox reactive linkers. The degradation of the SSP may further regulate the generation of EVs by host cells.

[0060] The SSP may further comprise an agent. The agent may be attached to the surface of the SSP or encapsulated within the SSP. The agent may comprise a chemical compound, a biological molecule or a combination thereof. The agent may carry a charge. The agent may be a bioactive stimulatory ligand. The agent may be selected from the group consisting of antigens, cytokines, and pathogen-associated molecular patterns (PAMPs). The bioactive molecule may stimulate a response, for example, inflammatory, anti-inflammatory, or anti-apoptotic signaling cascades, by a host cell. The agent may be a bioactive molecule.

[0061] A composition, also referred to as a formulation, comprising a plurality of the SSPs is provided. The composition may be designed to control EV production by host cells upon exposure to the SSPs. These SSPs may serve as stimuli to phagocytic cells. The present invention also provides methods of inducing and / or modulating EV secretion from host cells, for example, phagocytic cells. These methods involve exposing the host cells to a plurality of the SSPs under defined conditions such that the SSPs are internalized through phagocytosis, thereby activating the natural EV secretion pathway in the cells. A fundamental aspect of this method is that the input properties of the SSPs may predictably control the output characteristics of the resulting EVs. These EV production methods may be performed in vitro (e.g., in tissue culture or bioreactor) or in vivo, with the potential to direct EV production endogenously for therapeutic or diagnostic purposes.

[0062] In one embodiment, a method for producing extracellular vesicles (EVs) by host cells comprises exposing host cells to a plurality of the SSPs of the present invention. The EV production method also comprises internalizing the SSPs by the host cells through phagocytosis. The phagocytosis may be actin-mediated or receptor-mediated phagocytosis. The phagocytosis may be Fc-mediated, scavenger receptor-mediated, complement-mediated, or LC3-mediated. The EV production method further comprises secreting EVs by the host cells. As a result, the EVs are produced. The EVs may be smallEVs.

[0063] The host cells may be eukaryotic phagocytes. The host cells may be mammalian phagocytes, for example, monocytes, macrophages, dendritic cells, osteoclasts, neutrophils, and eosinophils. The host cells may be non-professional phagocytes such as mesenchymal stem cells that undergo phagocytosis of SSPs. The host cells may be non-mammalian phagocytes, for example, hemocytes, plasmatocytes, coelomocytes, and macrophages derived from avian, fish, and amphibian hosts.

[0064] The SSPs may be taken up into the host cells via active phagocytosis. The SSPs that are too small to trigger phagocytosis or are internalized via other mechanisms (e.g., endocytosis) do not elicit the same EV modulation effects. Therefore, the SSP size, density, and surface properties, such as surface material coating, conjugated internalization signals, and charge, may be tailored to ensure internalization by phagocytosis.

[0065] According to the EV production method of the present invention, the mass of SSPs delivered per cell ("SSP-to-cell ratio") is a key determinant of both EV yield and composition. The host cells may be exposed to the SSPs in an amount of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 pg per cell. The SSP-to-cell ratio may be about 10-100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 50-100, 50-200, 50-300, 50-400, 50-500, 50-600, 50-700, 50-800, 50-900, 50-1000, 60-100, 60-200, 60-300, 60-400, 60-500, 60-600, 60-700, 60-800, 60-900, 60-1000, 100-200, 100-300, 100-400, 100- 500, 100-600, 100-700, 100-800, 100-900, 100-1000, 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 200-900, 200-1000, 500-600, 500-700, 500-800, 500- 900, 500-1000, 600-700, 600-800, 600-1000, 700-800, 700-900, 700-1000, 800-900, 800-1000, or 900-1000, pg of SSP per cell, depending on SSP size and cell type. For example, the SSP-to-cell ratio is 60-800 or 200-800 pg of SSP per cell when the SSP size is about 250-5000 nm and host cells are 10 to 30 pm in size.

[0066] The EV production method may be implemented in vitro in a cell culture. The cell culture may be an adherent or suspension culture. The host cells may be primary cells or from a cell line. The cell culture may be scaled in flasks, well plates, or dynamic bioreactor systems. Serum-free or EV-depleted media may be used during harvest to avoid contamination. The inclusion of soluble factors (e.g., cytokines, small molecules) or sequential dosing with distinct SSP formulations may be used to further modulate EV content and function. Soluble factors can regulate the initial host cell phenotype, which will alter the EV phenotype. SSPs dosed to these phenotype controlled cells will then amplify production of the controlled EV phenotype. This may be performed by repeated dosing of the soluble factor and / or SSPs for continuous processing. The SSP may be designed to replicate the stimulatory effect of the soluble factor, requiring only the SSP to be dosed to regulate the phenotype. SSPs of varying compositions (with desired cargo, stimulatory ligand, tuned degradation rates) may be dosed sequentially to the same cells to control EV generation over extended periods. The generated EVs may then be purified and formulated for therapeutic delivery.

[0067] The EV production method may be performed in vivo by direct administration of SSPs via traditional routes of administration and localization to phagocytic immune cells. EVs may be generated locally by the host cells and reflect the stimuli provided by the SSPs. The host cells may be in the tissue of a subject. The subject may be healthy, inflamed, or immunosuppressed. The SSPs may be dosed sequentially or repeatedly for continued local EV production. SSPs of varied compositions (with desired cargo, stimulatory ligand, tuned degradation rates) may be dosed sequentially via the same route of administration to the same cells to control EV generation over extended periods. The generated EVs may then transfer material to other cells to regulate a desired therapeutic or prophylactic response.

[0068] The EV production method may be accomplished in one step. EVs may be secreted with characteristics that reflect the engineered properties of the SSPs, enabling a direct link between SSP design and EV characteristics and function. SSP amplified EVs show encapsulation of SSP carried material following SSP lysosomal trafficking and degradation. Thus, by altering input parameters of the SSPs, such as size, cargo, degradation rate, and surface properties, along with delivery of a critical mass through the manipulation of particulate density, different EV output profiles, for example, EV yield, EV cargo composition, EV surface phenotype, and EV functional effects, may be obtained.

[0069] The EVs may carry a cargo. The EV cargo composition may include one or more fluorescent agents, proteomic agents, RNAs, and / or lipids molecules having profiles that contain distinct communication signals. The cargo may be expressed by the host cells. The cargo may comprise CD63, MHC / HLA molecules, costimulatory proteins, 14- 3-3 associated proteins, antigens, or a combination thereof. The cargo may be derived from the SSP. The cargo may have been attached to the surface of the SSPs or encapsulated within the SSPs.

[0070] The EV production method may further comprise measuring a first EV number of the EVs produced by the host cells before being exposed to the SSPs, and measuring a second EV number of the EVs produced by the host cells after being exposed to the SSPs. The first EV number and the second EV number may be different. The second EV number may be greater than the first EV number. The second EV number may be less than the first EV number.

[0071] The EV production method may further comprise collecting the EVs. EVs may be collected within about 24, 48,72, or 96 hours after the SSP exposure. For example, the EVs may be collected between about 0.5-24, 0.5-48, 0.5-72, 0.5-96, 1-24, 1-48, 1-72, 1-96, 12-24, 12-48, 12-72, 12-96, 24-48, 24-72, 24-96, 48-72, 48-96, or 72-96 hours after the SSP exposure. The collection time may go longer depending on how long it takes the SSPs to degrade and how long the host cells live for. The collection time may be longer with sequential or repeated dosages of SSPs. A standard window of 72 hours post-single SSP exposure may provide robust EV production while preserving cell viability.

[0072] The present invention provides compositions of EVs that are produced in response to exposure to the SSPs of the present invention.

[0073] For each EV production method of the present invention, EVs are provided. The EVs are produced by host cells according to the EV production method.

[0074] The EVs may be derived from SSP-stimulated phagocytic cells under defined culture conditions. The defined culture conditions include cultures on cell treated plastics, cultures on glass, and culture on hydrogels using complete or exosome-free traditional cell media such as RPMI or DMEM.

[0075] The EVs may include a cargo. The cargo may be enriched or depleted based on the input of the SSPs. The SSPs may be characterized by, for example, mass spectrometry, RNA-seq, or surface marker profiling. The SSP exposure may enrich or deplete EV loading from endogenous host cell sources to alter the composition of EVs produced by the host cells. SSPs may carry desirable loading materials (e.g., small molecules, RIMA, and proteins), which may be transferred to the EVs as a cargo.

[0076] The functional phenotype of the EVs may be characterized by their activity in biological assays such as T cell proliferation / cytokine secretion, macrophage polarization, macrophage survival, and / or macrophage cytokine secretion.

[0077] The EVs produced according to the method of the present invention may be tailored for specific applications. For example, EVs may be useful for therapeutic uses such as anti-inflammatory or pro-regenerative EVs, immune activation or suppression, or tissue-specific repair. The PEGDA formulation of SSP may amplify EV production without polarizing macrophages to generate anti-inflammatory EVs. The EVs may also be useful for vaccination and immunotherapy. EVs may be enriched in MHC or costimulatory molecules for antigen presentation or immune priming following SSP treatment. The EVs may further be useful for diagnostics and biosensing. EVs carrying markers indicative of cell state, inflammatory state, disease pathologies, or exposure to inflammatory agonists extracellular foreign particulates, bacteria or viruses, may be amplified following SSP interactions.

[0078] The EVs may further be useful for targeted delivery. For example, EVs may serve as natural carriers for therapeutic RNA, proteins, or small molecules, with surface markers optimized for tissue-specific uptake.

[0079] The present invention further provides a method for modulating a biological activity of target cells. The modulation method comprises administering to target cells an effective amount of the EVs of the present invention. As a result, a biological activity of the target cells is modulated. Where the EVs carry a cargo, the modulation method may further comprise transferring the cargo from the EVs to the target cells. The target cells may be immune cells, barrier cells, or stem cells. The biological activity may be selected from the group consisting of proliferation, survival, activation, cytokine secretion, barrier function, and differentiation.

[0080] According to the modulation method, the target cells may be immune cells. The immune cells may be dendritic cells, T cells, or macrophages. The biological activity of the immune cells may be T cell proliferation, T cell survival, T cell activation, cytokine secretion, macrophage polarization, macrophage survival, macrophage cytokine secretion, dendritic cell polarization, dendritic cell survival, dendritic cell cytokine secretion, or dendritic cell antigen presentation.

[0081] According to the modulation method, the target cells may be non-immune cells. The non-immune cells may be barrier cells such as epithelial or endothelial cells, and their functionality, cytokine secretion, barrier properties, proliferation / survival, and phenotype may be modulated. The non-immune cells may be local stem and / or progenitor cells, and their proliferation, differentiation, and / or phenotype may be modulated.

[0082] For each SSP of the present invention, the present invention provides host cells comprising the SSP. The SSP may have been internalized by the host cells through active phagocytosis.

[0083] The term "about" as used herein when referring to a measurable value such as an amount, a percentage, 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.

[0084] Example 1. SSP Internalization amplifies in vitro EV production

[0085] A study was carried out to characterize release of extracellular vesicles in culture conditions driven by SSP internalization.

[0086] Materials & Methods

[0087] RAW264.7 murine cells were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, United States). All cells were cultured between passages 2- 10 in complete media, Dulbecco's Modified Eagle Medium (DMEM) (Corning) supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco), and 1% penicillin-streptomycin (Cytiva). THP-1 human monocyte cells were purchased from ATCC. All cells were cultured between passages 2-10 in complete media, RPMI 1640 Medium (ThermoFisher Scientific) supplemented with 10% heat inactivated FBS, and 1% penicillin-streptomycin. Before experiments, THP-1 monocytes were terminally differentiated into macrophages by adding 200 nM Phorbol 12-myristate 13-acetate (PMA) for 24 hours, followed by a PBS wash and a 48 hr resting period.

[0088] Primary cells were extracted from female wild type C57BL / 6 mice (Jackson Laboratories) that were kept in a pathogen free facility at the University of Delaware, with free access to chow. Mice ages were controlled, as the bone marrow cell age can influence the rate of cell viability; thus, all experiments use cells from aged-matched cultures. Previously reported standard protocols were used to generate BMMs isolated from mice. (See Marim et aL, PLOS ONE, 2010, 5, el5263; Weischenfeldt and Porse, CSH Protoc, 2008, pdb.prot5080). In summary, bone marrow was extracted from the femurs and tibias of euthanized mice and cultured in L929 fibroblast-conditioned media containing macrophage colony-stimulating factor (M-CSF). The collected bone marrow was strained and cells were isolated using red blood cell lysis buffer. The bone marrow cells were then plated in 10 mL of differentiation Dulbecco's modified eagle's medium (DMEM) without L-glutamate and sodium pyruvate media (20% Fetal Bovine Serum- FBS, 30% L929 conditioned media, and 1% Penicillin-Streptomycin-P / S) at l*105cells / mL, and on day 3, the cells were supplemented with an additional 5 mL of differentiation DMEM. Any excess bone marrow that was not plated on the day of collection (day 0) was then frozen down in freezing media, 90% FBS and 10% DMSO. Following day 6, the cells were ready for experiments. Experimental cultures used complete DMEM media (10% FBS and 1% P / S). CDllb marker was used to determine differentiated BMMs.

[0089] Cells were administered PEGDA CEA SSPs. PEGDA CEA SSPs were fabricated via reverse emulsion polymerization. Pre-polymer solution to form 50 wt% solids poly(ethylene)-glycol diacrylate (PEGDA; Sigma Aldrich)-based hydrogel SSPs was composed of 88.8 wt% PEGDA, 10 wt% charge-establishing co-monomer, 1 wt% diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (TPO; Sigma Aldrich), and 0.2 wt% Cy5 Maleimide (Fluoroprobes) in deionized water. 2-carboxyethyl acrylate (CEA, Sigma Aldrich) was used as the anionic PEGDA charge carrying co-monomer. Polar, prepolymer solution and non-polar, Silicon Oil AP1000 (Sigma Aldrich) were homogenized at roughly 1 mL total volume using a high-speed, benchtop vortex by placing the tube at an angle and applying pressure continuously along the tube to mix the non-polar and polar solutions. This was followed by tip sonication for 30 secs at 30% amplification. Polymerization via UV irradiation was performed using APM LED UV Cube (A = 365 nm, distance from light source = ~28 cm, intensity = ~5-10 mW / cm2) for ~30 secs. Hexanes (Sigma Aldrich) was added to the polymerized mix to break up the remaining oil in solution. SSPs were then washed twice in ethanol by centrifugation at 13,200 rpm for 10 mins and stored in ethanol till use. Thermogravimetric Analysis (TGA) using TGA 550 (TA instruments) was used to determine bulk stock concentration of SSP solutions prior to dosing. Dynamic Light Scattering (DLS) was used to measure particle size and performed using a Malvern Zetasizer Nano S Instrument (Malvern Instruments). Hydrodynamic diameter (Dh), polydispersity index (PDI), and zeta potential were measured at room temperature from 0.1 mg / ml solutions of SSPs in water. PEGDA CEA SSPs had an average diameter of ~300 nm.

[0090] EVs were collected from supernatants from culture conditions and isolated via differential centrifugation. Live cells were removed from media by centrifugation at 180 g for 4 minutes. Cellular debris and apoptotic bodies were removed by centrifugation at 2000 g for 10 minutes. Larger microparticles and remaining PEGDA SSPs were removed by centrifugation at 16,000 g for 45 minutes. Following this last centrifugal step, the resulting supernatants were filtered through sterile 0.2 pm filters and centrifuged twice at 100,000 g for 90 minutes using a TLA 55 rotor and Beckman Coulter Optima MAX Ultracentrifuge. Isolated EVs were then stored at 4°C for further characterization and usage within a week. EVs concentrations were measured using NTA on a NanoSight300. Results

[0091] RAW 264.7 in vitro cultures demonstrated an increase in secreted EV following dosing with 50 to 400 pg / mL of SSPs (FIG. 2A). Cell cultured dosed with 100 pg / mL of CEA PEGDA SSP showed a 5 fold increase in EV concentration, while the RAW cell cultures that received the highest dose (400 pg / mL) showed approximately a 30 fold increase in EV concentration. This increase in EV secretion was found to be significant using an Ordinary one-way ANOVA (*p<0.5, ****p<0.0001). Highlighted as an example, EVs from RAW 267.4 macrophage cultures dosed with 100 pg / mL have a mode and mean (91.4 ± 1.1 nm and 103.1 ± 1.1 nm respectively), characteristic of where we would expect of smallEVs. These results point to a direct relationship between RAW 264.7 macrophage EVs and dosed concentration of SSPs.

[0092] THP-1 monocytes were terminally differentiated into macrophage before performing a similar study. As shown in FIG. 2B, THP-1 macrophage in vitro cultures were dosed with 10 to 200 pg / mL of SSPs, with a smaller concentration of SSPs chosen due to their terminal differentiation. Cultures that received 100 pg / mL approximately a 2 fold increase in EV secretion compared to untreated cultures; while THP-1 macrophage cultures that received the highest dose (200 pg / mL) showed around a 2.5 increase in EV secretion. Despite the reduced magnitude, the increase in EV secretion was shown to be significant through Ordinary one-way ANOVA (*p<0.05, ***p=0.0001). The size distribution for EVs from THP-1 cultures dosed with 100 pg / mL CEA PEGDA SSP show a similar size to EVs from RAW 264.7 dosed cultures and literature results, with a mode of 91.4 ± 1.9 nm and a mean of 109.4 ± 1.6 nm.

[0093] BMMs were dosed with 10 to 200 pg / mL of SSPs, with again a smaller concentration than that of RAW 264.7 due to their terminal differentiation (FIG. 2C). BMM cultures that received 100 pg / mL showed around an 8-fold increase in EVs compared to UT, while the BMM cultures that received the high of 200 pg / mL showed a 14 fold increase in EV secretion. This increase in EV secretion was confirmed to be significant using Ordinary one-way ANOVA (**p<0.01, ****p<0.0001). Furthermore, the mode and mean of the 100 pg / mL dosed BMM cultures again mirrored those found in literature despite being slightly larger than the cell line examples (100.3 ± 4.7 nm and 114.2 ± 2.8 nm respectively).

[0094] Conclusions

[0095] SSPs increased EV production in a range of phagocytic cell types and culture conditions. The effect is dose-dependent, and the ratio of SSP: cell matters. The size of the produced EVs was about 100 nm, suggesting they are smallEVs.

[0096] Example 2. EV production in vivo driven by SSP internalization A study was conducted to characterize release of extracellular vesicles in vivo driven by SSP internalization for potential in situ localized EV release application.

[0097] Materials & Methods

[0098] All studies involving animals were performed in accordance with National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Delaware. Female C57BL / 6 of 2 to 4 months were housed in a pathogen- free facility at the University of Delaware and given unrestricted access to chow and water. Mice were dosed via orotracheal instillation following a standard procedure by suspension via their incisors and gently grabbing and moving the tongue aside to block the esophagus, allowing direct delivery to the trachea and minimal dosage to the gastrointestinal track. SSP dosed mice were administered 100 pg CEA PEGDA dosage in sterile 50 pL IX PBS under anesthesia with isoflurane. CEA PEGDA SSPs were made and characterized as in example 1. Alveolar macrophage uptake and EV BALF secretion analysis had a 72 hour endpoint. Terminal bronchoalveolar lavage fluid (BALF) was obtained via cannulation of the trachea restrained by a suture tie, using 1 mL of sterile IX PBS dispensed into the lungs to withdraw and store for further analysis.

[0099] After collecting, BALF was centrifuged at 500 ref for 5 minutes. The resulting supernatant underwent differential centrifugation for further isolation of EVs, while the pellet was resuspended in 500 pL red blood cell (RBC) lysis buffer (Fisher Scientific) and pipetted for 30—45 seconds. The suspended cells were then washed three times with IX PBS at 500 ref for minutes. Primary antibody staining was applied to the samples, with antibody solution diluted in FACS buffer (2% FBS in IX PBS), and incubated for 45 minutes on ice. Following staining step, samples were washed twice with 200 pL of FACS before running with appropriate fluorophore channels continuously on the Novocyte Flow Cytometer (Agilent Technologies). Cells vs debris gate was determined by gating samples with high forwards scatter area (FSC-A) and side-scatter area (SSC-A). Singlets were isolated as having a 1 : 1 ratio of SSC-A and side-scatter height (SSC-H), as previously shown in literature. Alveolar macrophages were identified as SiglecF+population. SSP uptake was determined as %SSP+based on the percentage of events greater in fluorescence intensity than 1% of untreated population in the SSP+channel.

[0100] The initial sample supernatant intended for the isolation of BALF EVs proceeded to the previously outline differential centrifugation process. Cellular debris and apoptotic bodies were removed by centrifugation at 2000 g for 10 minutes. Larger microparticles and remaining PEGDA SSPs were removed by centrifugation at 16,000 g for 45 minutes. Following this last centrifugal step, the resulting supernatants were filtered through sterile 0.2 pm filters and centrifuged twice at 100,000 g for 90 minutes using a TLA 55 rotor and Beckman Coulter Optima MAX Ultracentrifuge. Isolated EVs were then stored at 4°C for further characterization and usage within a week. BMM EVs concentrations were measured using NTA on a NanoSight300.

[0101] Results

[0102] Using orotracheal delivery, we dosed C57BL / 6 mice with PBS or 100 pg of CEA PEGDA SSP and collected their BALF to measure changes in present EV concentration. Using Siglec-F as a marker for isolated alveolar macrophages in collected BALF samples, we show in FIG. 3, left, Siglec-F positive populations show a significant increase in Cy5 for mice dosed with SSPs, determined with unpaired Student's T-test (****p<0.0001). This result confirmed successful dosing of SSPs to alveolar macrophages through orotracheal delivery. Furthermore, measuring EVs isolated from BALF samples show an increase in EVs present in the BALF of mice that received our SSPs (FIG. 3, middle), determined through unpaired Student's T-test with a p-value of 0.043. Looking at the size distribution, we report a mode of 99.0 ± 3.3 nm and 114.9 ± 3.3 nm for EVs isolated from SSP dosed mice (FIG. 3, right), showing sizing similar to that reported in literature. These results indicate that the link between dosed SSPs and EV secretion is not only present in in vitro macrophages cultures but is persistent in vivo when dosing with our SSPs.

[0103] Conclusions

[0104] The experimental results show that SSPs increased EV phagocyte production when dosed in vivo for localized production. The EVs produced had a size of about 100 nm, indicating that they were smallEVs.

[0105] Example 3. Regulation of EV secretion in BMMs from varied SSPs

[0106] This study was carried out to determine if SSP size and chemistry would have any impact on EV secretion in BMMs.

[0107] Materials & Methods

[0108] BMMs were seeded in T-25 cm flask (3*106cells / flask) and allowed to adhere for at least 4 hr. Flasks were dosed with 100 pg / ml of various particle conditions : AEM PEGDA SSPs, CEA PEGDA SSPs, PS SSPs (190 nm), and PS MP (1 micron). CEA PEGDA CEA SSPs were made and characterized as in example 1. AEM PEGDA SSPs were made and characterized as in example 1, replacing CEA with 2-aminoethyl methacrylate hydrochloride (AEM, Sigma Aldrich) to generate cationic PEGDA SSPs. Non-PEGDA particles, 0.2 pm and 1 pm carboxylated PS particles were obtained from Millipore Sigma. Following dosing, BMMs were allowed to incubate for 72 hr at 37C and 5% CO2. After 72 hrs, BMM derived EVs were isolated from the culture using differential centrifugation. Live cells were removed from media by centrifugation at 180 g for 4 minutes. Cellular debris and apoptotic bodies were removed by centrifugation at 2000 g for 10 minutes. Larger microparticles and remaining SSPs were removed by centrifugation at 16,000 g for 45 minutes. Following this last centrifugal step, the resulting supernatants were filtered through sterile 0.2 pm filters and centrifuged at 100,000 g for 90 minutes using a SW55 Ti rotor and Beckman Coulter ultracentrifuge L-90K. Pelleted EVs were resuspended in PBS and washed once more with PBS using a TLA 55 rotor and Beckman Coulter Optima MAX Ultracentrifuge. Isolated EVs were then stored at 4°C for further characterization and usage within a week. BMM EVs concentrations were measured using NTA on a NanoSight300.

[0109] Results

[0110] BMM cultures secreted more EVs following dosing with negative charged (CEA) PEGDA SSPs, positive charged (AEM) SSPs and negative charged polystyrene (PS) microparticles (MPs) as shown in FIG. 4. These increases in EV secretion were determined to be significant through Ordinary one-way ANOVA (*p<0.05, ***p<0.001) The EVs derived by distinct particles had similar size distribution: modes of 107.3 ± 2.7 nm (FIG. 4.i), 92.6 ± 1.8 nm (FIG. 4.ii), and 100.3 ± 1.7 nm (FIG. 4iii) respectively. Interestingly, negatively charged PS nanoparticles, the smallest particle size tested at 190 nm, did not result in an increase in secreted EVs. Lipid polysaccharide (LPS), used here to induce macrophage activation as a control, showed no increase in secreted BMM EVs. These results indicate that this increase in EV secretion as a response to SSP internalization is not dependent on the charge or chemistry of the particle. However, there might be a role size of SSP has on the EV secretion response, as indicated by the difference response the distinctly sized PS particles induced.

[0111] Conclusions

[0112] The experimental results of this study show that different chemistries of SSPs could increase EV production (polystytene and PEGDA; PEGDA of two charges). The PS nanoparticles having a particle size of 190 nm did not generate this effect but those having a particle size of 300 nm - 1 micron did. The PS MPs have a particle size less than 200 nm would be expected to proceed by endocytosis not phagocytosis. The experimental results in this example demonstrate the importance of active phagocytosis in the generation mechanism and suggest that the size of the SSP has an impact on the EV production.

[0113] Example 4. Generation of smallEVs from SSP administration

[0114] This study was performed to characterize the biological mechanism by which SSP drive smallEV production through the identification of critical cellular structures.

[0115] Materials & Methods BMMs were seeded in ibiTreat Ibidi p-Dish35 mm'highGrid-500 (Ibidi, Munchen, Germany) at l*106cells / dish, and allowed to adhere for at least 4 hrs. Samples were dosed with 100 pg / ml of various particle conditions: AEM PEGDA SSPs, CEA PEGDA SSPs, and PS SSP (1 micron diameter). SSPs were obtained, fabricated as in Example 3. After 24 hours, samples were washed twice with IX PBS and fixed using 2% EM grade glutaraldehyde (diluted from 8% Glutaraldehyde, Electron Microscopy Sciences) for a minimum of 1 hour followed by three rinses in lx PBS. Cells were dehydrated in an ethanol dilution series of increasing concentrations (25%, 50%, 75%, 95%, 100%, 100%). After dehydration the cells were dried using an increasing dilution series of EthanokHexamethyldisilazane (HMDS). Dilutions were as follows 2: 1, 1 :1, 1 :2, 100% HMDS. Following the 100% HMDS step the samples were allowed to air dry in a fume hood and then mounted to SEM stubs. Stubs were coated with 6nm of platinum prior to being imaged at 2.0 kV on a Thermo Fisher Scientific Apreo Volumescope SEM.

[0116] BMMs were seeded in Thermanox™ Coverslips (ThermoFisher Scientific) at 3*105cell / dish and allowed to adhere for at least 4 hrs. Samples were dosed with 100 pg / ml of various particle conditions: AEM PEGDA SSPs, CEA PEGDA SSPs, and PS SSP (1 micron) as in Example 3. After 24 hours, samples were washed twice with IX PBS and fixed using 2% EM grade glutaraldehyde, 2% EM grade paraformaldehyde in 0.1M sodium cacodylate buffer pH 7.4 (Electron Microscopy Sciences) for a minimum of 1 hour followed by three rinses in lx PBS.

[0117] Results

[0118] We performed SEM imaging 24 hours following dosing. SEM imaging allowed for capturing of the cell surface during and after internalization process, shown in FIG. 5. Arrows indicate potential cell membrane blebbing, which would lead to the formation of microvesicles. Across the multiple conditions (untreated, CEA SSP, AEM SSP, and PS SSP microparticle), we did not observe a significant increase in the amount of surface budding. This lack of increase in surface budding indicates that the increased secretion of EV due to particle internalization was not due to the formation of microvesicles. The PS SSP images in particular stand out due to capturing the moment of internalization for the PS particles, as indicated by the arrows. Based on these SEM images, we concluded that the mechanisms for the increase release of EVs following particle design lie in the internal cellular structures instead of membrane budding.

[0119] We performed TEM imaging of untreated, negatively charged (CEA) PEGDA SSPs, positively charged (AEM) PEGDA SSPs, and PS SSP MPs 24 hours after dosing. FIG. 5 bottom 3 rows shows PEGDA and PS SSPs internalized into the lysosomal compartment of the cells, indicated by the arrows labeled as NP for nanoparticle. The lysosomal trafficking of these SSPs points to the phagocytosis of these particles. Furthermore, when comparing the presence of MVBs, distinguished by arrows labels as such, a critical pre-curser to the release of smallEVs, TEM imaging shows an increase presence of their formation in particle dosed conditioned when compared to untreated culture conditions. Interestingly, the formation of these MVBs appear co-localized to internalized particles. As such, image analysis points to the role MVBs have in the SSP- driven EV secretion.

[0120] Conclusions

[0121] SSP dosing yielded smallEVs generated via MVB, making them fit the definition of "exosomes" or "SmallEVs." Lysosomal retention is critical for MVB loading and smallEV generation (implicating benefit for slowly degrading SSPs).

[0122] Example 5. Proteomics analysis of CEA PEGDA dosed BMM

[0123] This study was carried out to understand the impact CEA PEGDA SSP had on the metabolic activity and abundance of membrane-bounded organelles in dosed BMM.

[0124] Materials & Methods

[0125] BMMs were seeded in 24-well plates (3*105cells / well, 3 wells / repeat) and allowed to adhere for at least 4 hrs. Wells were dosed with PEGDA SSPs at 400 pg / cell. After a 24 hr incubation, cells were washed twice with PBS and incubated in incomplete DMEM for 48 hrs (no FBS). After this second incubation period, samples were collected, spun down at 500 g to remove cells, spun through an Amicon® Ultra - 4 containing an Ultracel® - 10K filter (10,000 NMWCO) at 5000 g to concentrate secreted proteins in a 1 mL of incomplete DMEM media. A BSA protein assay (Bio-Rad) was then used to measure sample protein concentration prior to proteomics sample prep.

[0126] Shotgun proteomics was completed according to a previously established method. Shotgun proteomics uses high-pressure liquid chromatography (HPLC) in tandem with mass spectrometry and database searching algorithms to identify proteins present in complex samples. Briefly, samples were loaded and spun through a Protifi™ S-Trap column at 2000 rpm for 2 min. Afterwards, 200 pL of 90% methanol in 100 mM triethylammonium (TEAM) was spun twice through the column at 2000 rpm for 2 minutes as a binding / wash solution. Samples were then incubated overnight at 37 °C with 200 pL of TEAB 1.0 M stock solution and sequencing grade modified trypsin (Promega) in 50 mM ammonium bicarbonate at a 50:1 (wt / wt) relating mass of protein to mass of trypsin. After overnight incubation, samples were spun down at 2000 rpm for 2 minutes before opening the S-Trap column and eluted from column using a series of elution steps. Frist, 200 pL of 50 mM TEAB was added to the column and spun down at 2000 rpm. Following the first elution step, 200 pL of 0.2% formic acid in H2O was added and the column was spun down at 200 rpm. Finally, 200 pL of 0.2% formic acid in 50% acetonitrile / 50% H2O was added, and the column was spun at 2000 rpm. All eluents were pooled into a single clean tube and dried using a SpeedVac.

[0127] After drying, samples were desalted using a STageTip, two layers of Empore C18 extraction disk (CDS / 3M) membrane packed into a 200 pL woodpecker low-binding tip. Dried samples were redissolved in 200 pL of 0.5% acetic acid in water buffer and vortexed for 10~15 minutes, followed by spinning down at 13,000 rpm for 3-5 minutes. While samples were vortexed and spun down, STageTip filters were prepped and activated for de-salting steps. First, 200 pL of methanol was loaded onto the STageTip and spun through the membrane at 4000 rpm for 1~2 minutes while the tip was held in a 2 mL Axygen Maxymum Recovery tube. Following this first activation step, 200 pL of 80% ACN / 0.5% HAc was loaded into the STageTip, followed by a spindown step at 4000 rpm for 1~2 minutes. After activation, STageTip were equilibrated by loading 200 pL H2O / 0.5% Hac and spinning the tip down for 1~2 minutes. Once tips were equilibrated and samples were dissolved, 200 pL of the sample was loaded into the STageTip and spun through at 4000 rpm for 1~2 minutes to load present peptides into the C18 membrane. Following this loading step, 200pL of H2O0.5% Hac was loaded into the STageTips and spun through at 4000 rpm for 2~3 minutes as a washing step. This step was repeated 2 to 3 times. After the last wash, STageTips were transferred to clean collection tubes for final elution steps. First, 200 pL of 60% ACN / 0.5% HAc was loaded and spun through at 4000 rpm for ~2 minutes. Following this first elution step, 200 pL of 80% ACN / 0.5% HAc was loaded into the STageTip and spun through at 4000 rpm for ~2 minutes as a final elution step. Samples were then dried using a SpeedVac machine. Dried samples were stored at 4 C until they were run in electrospray liquid chromatography-tandem MS (LC-MS / MS) using a linear ion trap-orbitrap instrument (LTQ-Orbitrap) in an Orbitrap Nano Eclipse. Proteins were identified using Uniprot database.

[0128] Upon obtaining proteomics analysis results, we used Maxquant Persues 2.0.11 software for further data analysis. Of the total identified proteins, proteins that appeared in only 2 or less of the sample repeats were filtered out. Afterwards, imputations were added for any missing values within the remaining proteins. Values were determined using a normal distribution, with a width of 0.3 and a downshift of 1.8 around the identified proteins mean expression. Having filtered scarcely present proteins and filled in for missing sample values, a two-sample student's T-test was used to determine significantly different levels of protein expression, with a p-value <0.05 as the set threshold. Z-score values of the significant proteins were generated for future use in a hierarchical clustering heat map.

[0129] Results Proteomic profiling was conducted to assess the impact of PEGDA SSP exposure 24hrs after dosing on bone marrow-derived macrophages (BMMs). Hierarchical clustering of significantly altered features identified 1051 significantly different proteins in the whole cell proteome and 213 significantly different in the secreted proteome (Student's t-test, FDR = 0.1) (FIG. 6A-B). Despite the overall modest number of changes between SSP-treated and untreated BMMs, gene enrichment analysis of upregulated clusters in SSP-treated conditions revealed consistent signatures related to extracellular vesicle (EV) biology. GO cellular component analysis showed significant enrichment for membrane-bounded organelles (FIG. 6D), while GO biological process analysis highlighted an increase in metabolic processes (FIG. 6E). Notably, no activation signatures were observed following the SSP dosing to BMMs, highlighting the inert background provided by the PEGDA SSP chemistry. Together, these data indicate that PEGDA SSPs exert minimal broad transcriptional or proteomic shifts in BMMs but selectively influence EV-associated pathways.

[0130] Conclusions

[0131] Proteomics supports the PEGDA SSP composition does not activate cells. PEGDA SSP composition upregulates proteins involved in smallEV production, found in the cell and the secreted supernatant (containing the smallEVs). Example 6 Ex vivo BMMs with SSPs or SSP-derived EVs

[0132] This study was carried out to study survival and metabolic activity of ex vivo BMM dosed with SSPs directly or EVs derived from SSP-treated BMMs.

[0133] Materials & Methods

[0134] BMMs were seeded in 96-well plates (5*104cells / well) and allowed to adhere for at least 4 hrs prior to SSP treatment. CEA PEGDA SSPs were fabricated and characterized as in example 1. We administered SSPs to cells at the following dosing schemes: 200 pg / ml, 100 pg / ml, 50 pg / ml, and 25 pg / ml. After 24 hrs incubation, cells were washed with PBS to remove unbound SSPs and fresh culture media was supplied. The uptake of SSPs at the various dosing concentrations was determined by measurement of Cy-5 labelled SSPs at 24 hrs after dosing. Cells cultures were then measured daily for cell counts, using BioTek Cytation 5 Multimode Imager. Whole well images were captured using a 3 by 3 image grid at 4x magnification. Built-in software was then used to identify and count cells the attached cells in the well, setting minimum object size to 14 pm and maximum object size to 60 pm.

[0135] BMMs were seeded in 96-well plates (5*104cells / well) and allowed to adhere for at least 4 hrs prior to dosing with EVs. We administered the following SSP-derived EV dosing schemes: 5 x 109, 1 x 109, 5 x 108, 1 x 108, 1 x 107particles / mL. SSP-derived EVs were generated and characterized as in example 1 prior to dosing. Cells cultures were then measured daily for cell counts, using BioTek Cytation 5 Multimode Imager. Whole well images were captured using a 3 by 3 image grid at 4x magnification. Built- in software was then used to identify and count cells the attached cells in the well, setting minimum object size to 14 pm and maximum object size to 60 pm.

[0136] After daily cells counts were collected in all experiments, wells were normalized to their day 0 cell count measurement. The resulting survival percentages were then plotted using GraphPad Prism 9 and analyzed using non-linear regression. We chose to model the population dynamics as a one-phase equation: where Yo is initial value, Piat is final plateau value, K is rate of decay, and x is time. For the generated models, we set the following constraints: Yo = 100 and Piat = 0. Following generation of model parameter values, half-lives values were calculated, using the following equation:

[0137] .. 2) (2)

[0138] / Si - 7 A for each well replicate and plotted. Comparison of fits test were used to determine that distinct conditions had significantly different values for K.

[0139] Results

[0140] A single dose of PEGDA SSPs yielded increased BMM survival dependent on SSP concentrations. Using these normalized daily cell counts, we then fitted into a one- phase exponential decay model Eq 1. to describe population dynamics (generated parameters of the fit and their 95% confidence interval. Over the course of the 30 days, we recorded an increase in the % survival of initial cells seeded for SSP-dosed BMMs when compared to the untreated cultures the initial 10 days shown in FIG. 7A, left. The normalized cell counts showed an increased in survival which we could characterize using as one-phase decays model to generate BMM culture half-lives. Average half-lives are plotted for each dosing condition in FIG. 7A, right. A Bartlett's test— a test design to test if k samples have equal variance which is needed for use of an ordinary one-way ANOVA— reported significant difference in standard deviation (SD) (P = 0.0378). As such, we used a Brown-Forsythe and Welch ANOVA test. The results show a significant improvement in population half-life that increased at higher concentrations (*p<0.05) when compared to the half-life of the UT condition. Noticeably, at high enough concentrations, the variance of the population increases significantly.

[0141] We dosed these purified EVs to BMM cultures and tracked their cell counts for a week. As shown in FIG. 7B, left, cell counts were normalized to the wells initial cell counts; half-lives from the fitted one-phase decay are plotted in FIG. 7, right. BMM populations that received purified EVs at a concentration of l*109and 5*109particles / mL showed an increase in population half-life when compared to untreated population. Furthermore, when performing a Cell Titer Gio assay to measure changes in BMM metabolic activity following treatment, both PEGDA SSP-dosed cultures and EV- dosed cultures showed increased metabolic activity compared to the untreated cultures, as shown in FIG. 7C. These results point to macrophages releasing EVs that increases the metabolic activity and survival of other macrophages following SSP dosing.

[0142] Conclusions

[0143] The supernatants containing smallEVs from SSP-dosed BMMs contained unique functionality not present in SSP-free smallEVs. Specifically, SSP-generated smallEVs impart a prosurvival phenotype to other cells, pointing to their unique composition.

[0144] Example 7. SSP-derived EVs contain and transfer SSP-derived cargo

[0145] This study was carried out to characterize delivery of SSP-derived material via SSP-derived EVs.

[0146] Materials & Methods

[0147] BMMs were plated on 24-well plates (3*105cells / well) and given at least 4 hrs to adhere. After adherence, cells were dosed at 100 pg / ml of SSPs, fabricated and characterized as example 1. After a 24 hrs incubation period, cells were detached using 0.25% Trypsin-EDTA (Corning) and washed twice with PBS supplemented with 2% FBS. The cells were then surface-stained for 30 minutes. BMMs were stained with CDllb-Pacific Blue, CD40-FITC, I-A / I-E-Brillian Violet 785 (all from Biolegend). CDllb are chosen are markers of BMMs. After staining, samples were washed twice with FACS and analyzed using ACEA NovoCyte Flow Cytometer. Fluorophore median fluorescent intensity (MFI) was recorded via flow cytometry as a measure of marker expression. In studies, 20 ng / mL LPS from Escherichia coli Olli : B4 (Millipore Sigma) was used as a positive control for macrophage cellular activation.

[0148] BMMs were seeded in a 24-well plate (3*105cells / well), and allowed to adhere for at least 4 hours. After adherence, cells were dosed with 100 pg / mL SSPs or l*109particles / mL SSP-derived EVs and incubated for 24 hrs. Following incubation, BMMs were fixed using 4% paraformaldehyde (PFA) prior to staining with Molecular Probe™ Wheat Germ Agglutinin Conjugates (WGA) (ThermoFisher Scientific) according to manufacturer guidelines. WGA was used to stain the cellular membrane through the presence of N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues. Briefly, wells were washed with PBS and then incubated in WGA working solution (5 pg / mL) for 10 minutes.

[0149] Results We looked for transfer of the Cy5 fluorescent tag added into the SSP. In FIG. 8, we used anti-CD63 magnetic beads to capture SSP-derived EVs and measured Cy5 fluorescence using flow cytometry. We report a significant increase in Cy5 fluorescence in the SSP-derived EVs relative to the capture antibodies, indicating the presence of SSP material in the isolated EVs, determined using a student's t test (****p<0.0001).

[0150] We show expression of CD40 (FIG 8B) and MHCII (FIG 8C), classic markers for inflammatory macrophage phenotypes, on captured SSP-derived EVs as measured through flow cytometry. Our results show a statistically insignificant increase in either marker when BMMs were dosed with le9 particles / mL of SSP-derived EVs, the same concentration we determined to induce a change in metabolic activity in the BMMs. For both studies, LPS was used as a positive control and significance was determined through Ordinary one-way ANOVA (****p<0.0001). This indicates that CEA PEGDA SSP delivery does not alter the EV phenotype from the host cell phenotype. FIG. 8D shows just over a 1.5-fold increase is Cy5 for macrophages dosed with l*109particles / mL SSP-derived EVs relative to UT BMMs, indicating the transfer of SSP material through the secreted EVs.

[0151] We performed confocal microscopy to image BMMs after dosing with 100 pg / mL of SSPs and l*109particles / mL of SSP-derived EVs. FIG. 8E shows how when SSPs are dosed to BMMs, the SSPs are localized in lysosomal compartments, forming large dots of fluorescence within the cells. However, when SSP-derived EVs were delivered, fluorescent cargo from the SSP was packaged into the EVs and transferred into the SSP-free cell.

[0152] Conclusions

[0153] SSPs containing fluorescent cargos could transfer the cargos into the secreted smallEVs, which can then transfer their cargo to another cell.

[0154] Example 8. EV profiles from cells of diverse phenotypes

[0155] This study was carreied to charaterize EV profiles produced by host cells of varied phenotypes when dosed with SSPs.

[0156] Materials & Methods

[0157] Magnetic Bead Capture of Macrophage EVs and Flow Cytometry Analysis. Following RAW 264.7 EV isolation, we used a Basic Exo-Flow Capture Kit (System Biosciences, Palo Alto, CA) for the further characterization of our SSP amplified EVs using fluorescent flow cytometry. Samples were processed as instructed in Kit manual, with separately purchased biotinylated antibodies for conjugation onto streptavidin magnetic beads and capture of EV samples. Biotin anti-mouse CD63 antibody (Biolegend) were incubated for 2 hours with streptavidin magnetic beads for 2 hours to form EV capture beads. After 2 washes, RAW 264.7 EVs were incubated overnight with capture antibodies to ensure isolation. After overnight incubation, samples were washed 2 and incubated with fluorescent stains and antibodies for characterization: I- A / I-E-Brillian Violet 785 (Biolegend) and Exo-FITC exosome FACS stain (System Biosciences). After staining, samples were washed twice and analyzed using ACEA NovoCyte Flow Cytometer. Fluorophore median fluorescent intensity (MFI) was recorded via flow cytometry as a measure.

[0158] EV dosing and RAW 264.7 Flow Cytometry Characterization. RAW 264.7s were plated on 24-well plates (5*105cells / well) and given at least 4 hrs to adhere. After adherence, cells were dosed at 5e9 particles / mL of appropriate EV stock with source cell phenotype (Ml, M2, or MO). After a 24 hrs incubation period, cells were detached using 0.25% Trypsin-EDTA (Corning) and washed twice with PBS supplemented with 2% FBS. The cells were then stained for 30 minutes. BMMs were stained with I-A / I-E- Brillian Violet 785 (Biolegend). After staining, samples were washed twice with FACS and analyzed using ACEA NovoCyte Flow Cytometer. Fluorophore median fluorescent intensity (MFI) was recorded via flow cytometry as a measure of marker expression.

[0159] Results

[0160] Prior to EV isolation, source RAW 264.7 cells were dosed with PEGDA SSP and either IFN-y (Ml) or IL13 / 4 (M2). In FIG 9A and FIG9B, we characterize the resultant phenotype (A) and SSP cargo transfer (B) to the secreted SSP-derived EVs 3-days after SSP dosing through bead capture and flow cytometry. Critically, EVs from RAW 264.7 cells dosed with IFN-y showed a significant increase in MHCii FIG9A and Cy5 FIG9B. MHCii is an antigen presentation protein that is often expressed following dosing with IFN-y and it has been reported to be found in phagocyte sourced EVs. Cy5 encapsulated within isolated EVs stems from the dosed PEGDA SSP and indicates the transfer of material from dosed phagocyte to secreted EVs.

[0161] We dosed the same isolated EVs from phenotypically active and SSP-dosed BMMs to naive RAW 264.7 to characterize changes in the cell protein expression. In FIG9C, the cells dosed with SSP-derived EVs from IFN-y activated BMMs showed an increase expression of MHCii. Thus, the presence of MHCii in the isolated SSP-derived EV seems to have resulted in a subsequent increase in the dosed cells. In FIG9D, all cells that received SSP-derived EVs showed significant increase in Cy5 presence compared to UT conditions, regardless of the variation in cargo observed in SSP- derived EVs (FIG9B). These results indicate the encapsulation and transfer of SSP material to distinct cells.

[0162] Conclusions

[0163] SmallEVs generated from SSP-treated groups have altered composition including cargo transferred from the SSP and upregulation of antigen presentation proteins (e.g., MHCII). Host cells having different phenotypes yielded different smallEV compositions.

[0164] Example 9. SSPs can regulate host cell phenotypes

[0165] This study was carried out to demonstrate that SSPs can be designed to alter the phenotyes of host cells, for example, activation marker expression on the dosed macrophage cells, dosed by PEGDA SSPs having various amounts of CpG.

[0166] Materials & Methods

[0167] PEGDA SSPs were synthesized as in example 1 and included a PEG-maleimide acrylate for further functionalization. Thiolated CpG was reduced in DTT for 3 hr and separated in a desalting column, then reacted with PEGDA-maleimide SSPs at room temperature in water for 2 hours. CpG was added at variable amounts to control the final CpG concentration. Conjugation amounts were determined by measuring the unreacted CpG in the supernatant following two water wash steps using an absorbance reading at 260 nm. CpG-PEGDA SSPs were dosed to BMMs at an equivalent SSP amount of 100 ug / mL; 24 hours after dosing, flow cytometry was used to measure relative activation states of SSP-receiving BMMs.

[0168] Results

[0169] CpG was successfully added to PEGDA SSPs at varying concentrations, ranging from 10 ug CpG / mg SSP ("SSP-low") to 80 ug CpG / mg SSP ("SSP-high"). CpG-PEGDA SSPs successfully activated BMMs 24 hours after dosing, as demonstrated by increases in CD86, MHCII, CD83, and CD80 activation and co-stimulatory surface markers (FIG. 10). This is a dose dependent response; as CpG amounts increased from SSP-low to SSP-high, higher increases in all four surface markers were observed.

[0170] Conclusions

[0171] SSPs could alter host cell phenotype through specific chemistry modifications; paired with the experimental results in Example 8, host cell phenotype changes yield differential smallEV phenotype expression.

[0172] Example 10. SSP amplification of smallEVs dependent on a critical minimum mass.

[0173] This study was carried out to determine whether internalized SSP number or internalized SSP mass was criticial for induction of SSP smallEV amplficiation .

[0174] Materials & Methods

[0175] Polymeric Particles. PEGDA SSPs were synthesized and characterized as in example 1; 75 wt% CEA PEGDA SSPs were fabricated by increasing the total weight % solids in the preparticleff monomer solution to 75%. Both SSP formulations were characterized with TGA and NTA as in example 1. Both 50 wt% and 75 wt% CEA PEGDA SSPs were dosed to BMMs as in example 1 at a fixed mass of 50 pg / mL. Results

[0176] Using TGA and NTA characterization of our generated PEGDA SSPs, we confirmed the capacity to alter particle density through changes in MeOH to monomer mix ratios. The PEGDA SSPs generated with a 1 :1 monomer to MeOH mix (50 wt%) resulted in a density of 57.14 kg / m3; while the 1 :0.33 monomer to MeOH mix (75 wt%) resulted in PEGDA SSPs with a density of 117.25 kg / m3, a 2-fold increase (FIG. 11). Using this difference in SSP density, we delivered an equal mass of SSPs, 50 pg / mL, to determine the effect different delivered particle quantity might have on the SSP amplification of EVs. As shown in the figure above, BMMs dosed with 50 pg / mL of the 50 wt% chemistry received approximately 1.25el0 particles / mL. BMMs dosed with 50 pg / mL of the 75 wt% chemistry received approximately half the number of particles at 0.64el0 particles / mL. Despite this difference, both the 50 wt% and 75 wt% had a similar boost to the secretion of BMM EVs, with a 3.3-fold increase relative to UT and a 3.8-fold increase relative to untreated, respectively. These results point to the importance delivered mass has over the phagocytic events I internalized particles.

[0177] Conclusions

[0178] Delivered mass is critical to the amplification of BMM EVs, rather than instances of SSP internalization.

[0179] All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and / or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

1. WHAT IS CLAIMED:

1. A solid synthetic particle (SSP) comprising a synthetic polymer, wherein the polymer forms a crosslinked structure, wherein the SSP has a diameter of 0.2 - 20 pm.

2. The SSP of claim 1, wherein the polymer comprises poly(ethylene glycol) diacrylate (PEGDA), polystyrene, poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) dithiol, 2-carboxyethyl acrylate (CEA), 2-aminoethyl methacrylate, nitrobenzyl photolabile molecules, hyaluronic acid, gelatin, chitosan, alginate, or a combination thereof.

3. The SSP of claim 1, wherein the crosslinked structure has a mechanical stiffness (Young's modulus) of 100 kPa to 3.6 GPa.

4. The SSP of claim 1, further comprising an agent.

5. The SSP of claim 1, wherein the polymer comprises a monomer backbone, a charge-carrying co-monomer, a stimuli-responsive co-monomer, a covalently-linked fluorescent probe, or a combination of thereof.

6. The SSP of claim 5, wherein the stimuli-responsive co-monomer is selected from the group consisting of a photoresponsive, redox reactive, ester labile, enzymatically degradable, and a combination of thereof.

7. The SSP of claim 4, wherein the agent is a bioactive molecule.

8. A method for producing extracellular vesicles (EVs) by host cells, comprising :(a) exposing host cells to a plurality of the solid synthetic particles (SSPs) of any one of claims 1-7;(b) internalizing the SSPs by the host cells through phagocytosis; and(c) secreting extracellular vesicles (EVs) by the host cells, whereby the EVs are produced.

9. The method of claim 8, wherein the EVs are smallEVs.

10. The method of claim 8, wherein the host cells are exposed to the SSPs in an amount of 60-800 pg per cell.

11. The method of claim 8, wherein the host cells reside in the tissue of a subject.

12. The method of claim 8, wherein the host cells are in a cell culture.

13. The method of claim 8, wherein the host cells comprise eukaryotic phagocytes.

14. The method of claim 8, further comprising measuring a first EV number of the EVs produced by the host cells before being exposed to the SSPs, and measuringa second EV number of the EVs produced by the host cells after being exposed to the SSPs, wherein the second EV number is greater than the first EV number.

15. The method of claim 8, wherein the EVs carry a cargo.

16. The method of claim 15, wherein the cargo is attached to the surface of the SSPs or encapsulated within the SSPs.

17. The method of claim 8, further comprising collecting the EVs.

18. Extracellular vesicles (EVs) produced by host cells according to the method of claim 8.

19. A method for modulating a biological activity of target cells, comprising administering to target cells an effective amount of the EVs of claim 18, whereby a biological activity of the target cells is modulated.

20. The method of claim 18, wherein the target cells are immune cells, barrier cells, or stem cells.

21. The method of claim 19, wherein biological activity is selected from the group consisting of proliferation, survival, activation, cytokine secretion, barrier function, and differentiation.

22. Host cells comprising the solid synthetic particle (SSP) of claim 1.

23. The host cells of claim 22, wherein the SSP has been internalized by the host cells through active phagocytosis.

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