Silk particles for use in tissue engineering, and related methods

Abstract

Provided herein is a composition including a microparticle comprising a therapeutic agent embedded therein, wherein the microparticle comprises a silk fibroin polymer matrix and a magnetic nanoparticle.

Description

SILK PARTICLES FOR USE IN TISSUE ENGINEERING, AND RELATED METHODSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to United States Provisional Patent Application No. 63 / 535,695, filed August 31 , 2023, the disclosure of which is herby incorporated by reference in its entirety.STATEMENT REGARDING FEDERAL FUNDING

[0002] This invention was made with government support under HL129066 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Regenerative medicine is generally directed to developing and applying new treatments to heal tissues and organs and restore function lost due to aging, disease, damage, or defects. While the field is emerging, progress is needed in effectively and naturally treating tissues for therapeutic and remodeling purposes.

[0004] Abdominal aortic aneurysm (AAA) is a life-threatening dilation and thinning of the distal aorta wall that affects millions of Americans. Without repair, the AAA can rupture, typically resulting in death of the patient. One significant factor of aortic expansion is the degradation and improper repair of extracellular matrix elements, such as elastin.

[0005] There is therefore a need for compositions and methods useful in vascular engineering and, more generally, in tissue engineering for any application. There is also a need for drug delivery devices that release encapsulated factors in a therapeutically-effective manner.SUMMARY

[0006] Provided herein is a composition including a microparticle comprising a therapeutic agent embedded therein, wherein the microparticle comprises a silk fibroin polymer matrix and a magnetic nanoparticle.

[0007] Also provided herein is a composition including: a microparticle comprising a therapeutic agent embedded therein, wherein the microparticle comprises a bioerodible polymer matrix and a magnetic nanoparticle.

[0008] Also provided herein is a method of delivering a therapeutic agent to a patient, including administering to the patient a composition as described herein and guiding the composition with a magnet to a location in the patient.

[0009] Further non-limiting embodiments are set forth in the following numbered clauses:

[0010] 1. A composition comprising: a microparticle comprising a therapeutic agent embedded therein, wherein the microparticle comprises a silk fibroin polymer matrix and a magnetic nanoparticle.

[0011] 2. The composition of clause 1 , wherein the silk fibroin is obtained from a Bombyx Mori silk cocoon.

[0012] 3. The composition of clause 1 or clause 2, wherein the magnetic nanoparticle is conjugated to the silk fibroin polymer matrix.

[0013] 4. The composition of any of clauses 1 -3, wherein the magnetic nanoparticle is chemically conjugated to the silk fibroin polymer matrix.

[0014] 5. The composition of any of clauses 1 -4, wherein the magnetic nanoparticle comprises an iron oxide nanoparticle.

[0015] 6. The composition of any of clauses 1 -5, wherein the therapeutic agent comprises an extracellular vesicle.

[0016] 7. The composition of any of clauses 1 -6, wherein the extracellular vesicle is obtained from conditioned media of a culture of a multipotent cell.

[0017] 8. The composition of any of clauses 1 -7, wherein the multipotent cell is a mesenchymal stem cell.

[0018] 9. The composition of any of clauses 1 -8, wherein the multipotent cell is an adipose-derived mesenchymal stem cell.

[0019] 10. The composition of any of clauses 1 -9, wherein the media of the conditioned media is serum-free and / or xeno-free.

[0020] 1 1 . The composition of any of clauses 1 -10, wherein the cells are cultured for at least 12 hours in the media to produce the conditioned media.

[0021] 12. The composition of any of clauses 1 -1 1 , wherein the extracellular vesicles are less than 0.22 microns in diameter.

[0022] 13. The composition of any one of clauses 1 -12, wherein cells and cell debris are removed from the conditioned media containing the extracellular vesicles and the conditioned media is filtered through a 0.22 micron filter, with the extracellular vesicles passing through the filter in the conditioned media filtrate.

[0023] 14. The composition of any of clauses 1 -13, wherein the conditioned media filtrate is filtered by tangential flow filtration, with the extracellular vesicles being retained in the retentate by a 300 kDa hollow fiber membrane filter.

[0024] 15. The composition of any of clauses 1 -14, wherein the extracellular vesicles can be in the retentate by a 300 kDa hollow fiber membrane filter using tangential flow filtration at a flow rate in the conditioned media of 27 mL / min.

[0025] 16. The composition of any of clauses 1 -15, wherein the therapeutic agent comprises a cytokine, a growth factor, an active pharmaceutical ingredient, a biologic pharmaceutical ingredient, a polypeptide, and / or a nucleic acid or nucleic acid analog.

[0026] 17. The composition of any of clauses 1 -16, wherein the therapeutic agent is an anti-inflammatory drug, doxycycline, or a TIMP.

[0027] 18. The composition of any of clauses 1 -17, wherein the magnetic particles are conjugated with glutathione.

[0028] 19. The composition of any of clauses 1 -18, wherein the polymer matrix further comprises a bioerodible polymer in addition to the silk fibroin.

[0029] 20. A composition comprising: a microparticle comprising a therapeutic agent embedded therein, wherein the microparticle comprises a bioerodible polymer matrix and a magnetic nanoparticle.

[0030] 21. The composition of clause 20, wherein the magnetic nanoparticle is conjugated to the bioerodible polymer matrix.

[0031] 22. The composition of clause 21 , wherein the magnetic nanoparticle is chemically conjugated to the bioerodible polymer matrix.

[0032] 23. The composition of any of clauses 20-22, wherein the magnetic nanoparticle comprises an iron oxide nanoparticle.

[0033] 24. The composition of any of clauses 20-23, wherein the therapeutic agent comprises an extracellular vesicle.

[0034] 25. The composition of any of clauses 20-24, wherein the extracellular vesicle is obtained from conditioned media of a culture of a multipotent cell.

[0035] 26. The composition of any of clauses 20-25, wherein the multipotent cell is a mesenchymal stem cell.

[0036] 27. The composition of any of clauses 20-26, wherein the multipotent cell is an adipose-derived mesenchymal stem cell.

[0037] 28. The composition of any of clauses 20-27, wherein the media of the conditioned media is serum-free and / or xeno-free.

[0038] 29. The composition of any of clauses 20-28, wherein the cells are cultured for at least 12 hours in the media to produce the conditioned media.

[0039] 30. The composition of any of clauses 20-29, wherein the extracellular vesicles are less than 0.22 microns in diameter.

[0040] 31 . The composition of any of clauses 20-30, wherein cells and cell debris are removed from the conditioned media containing the extracellular vesicles and the conditioned media is filtered through a 0.22 micron filter, with the extracellular vesicles passing through the filter in the conditioned media filtrate.

[0041] 32. The composition of any of clauses 20-31 , wherein the conditioned media filtrate is filtered by tangential flow filtration, with the extracellular vesicles being retained in the retentate by a 300 kDa hollow fiber membrane filter.

[0042] 33. The composition of any of clauses 20-32, wherein the extracellular vesicles can be in the retentate by a 300 kDa hollow fiber membrane filter using tangential flow filtration at a flow rate in the conditioned media of 27 mL / min.

[0043] 34. The composition of any of clauses 20-33, wherein the therapeutic agent comprises a cytokine, a growth factor, an active pharmaceutical ingredient, a biologic pharmaceutical ingredient, a polypeptide, and / or a nucleic acid or nucleic acid analog.

[0044] 35. The composition of any of clauses 20-34, wherein the therapeutic agent is an anti-inflammatory drug, doxycycline, or a TIMP.

[0045] 36. The composition of any of clauses 20-35, wherein the magnetic particles are conjugated with glutathione.

[0046] 37. The composition of any of clauses 20-36, wherein the polymer matrix further comprises a bioerodible polymer in addition to the silk fibroin.

[0047] 38. A method of delivering a therapeutic agent to a patient, comprising administering to the patient a composition according to any of clauses 1 -37, and guiding the composition with a magnet to a location in the patient.

[0048] 39. The method of clause 38, wherein the location is a blood vessel wall of the patient.

[0049] 40. The method of clause 38 or clause 39, wherein the location is an aneurysm in the blood vessel wall.

[0050] 41 . The method of any of clauses 38-40, wherein the blood vessel is the patient’s abdominal aorta.

[0051] 42. The method of any of clauses 38-41 , wherein the magnetic particle is guided by the magnet to adventitia of the blood vessel.

[0052] 43. Use of the composition of any of clauses 1 -37 for treatment of an aneurysm in a patient.

[0053] 44. The use of claim 43, wherein the aneurysm is an abdominal aortic aneurysm.BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1 depicts (schematically) a cross-section structure of an exemplary particle as described herein (not to scale).

[0055] FIGS. 2A and 2B show schematically the deposition of magnetized silk particles as described herein at a site of an aneurysm before (left) and after (right) magnetic deposition of the particles.

[0056] FIG. 3 is a visual representation of a separation assay of Silk MPs and SIMPs with a magnet. Each fraction (magnetic and non-magnetic) was used in a Lowry assay to detect the concentration of protein. These fractions were also characterized by SEM to see individual MPs.

[0057] FIG. 4 shows a comparative Characterization of IONP and IONP-GSH. A) FTIR spectra of GSH, IONP, and IONP-GSH. (b) TGA analysis reveals higher mass loss in IONP-GSH. (c) XPS S2p, (d) XPS N1 s, and I XPS C1 s spectra of GSH, IONP, and IONP-GSH together confirm conjugation, (f, g) SEM images depict uniform particle size and shape in both IONP and IONP-GSH, with slightly enhanced nanoparticle dispersion in IONP-GSH.

[0058] FIG. 5 shows SEM images of MPs prepared using the salting out method. Representative (a) high magnification and (b) low magnification images show Silk MPs without lONPs. Representative (c) high magnification and (d) low magnification images depict SIMPs with lONPs conjugated with GSH. Representative (e) high magnification and (f) low magnification images show SIMPs-N with lONPs that are not conjugated with GSH. (g) SEM image with overlaid rectangles indicating the areas where EDS spectra were gathered. The bar chart presents the average weight percent of Fe measured in these spectra for SIMPs and Si chip regions. The average weight percentage of Fe on the Si chip was consistently measured as zero, and thus no error bars are shown for the blue column. Scale bars: Panels a, c, e, g (1 pm) and panels b, d, f (10 pm).

[0059] FIG. 6 shows stability assessment of SIMPs stored at 4 °C in the refrigerator for 13 and 28 days. SEM images at D13, at (a) high magnification and (b) low magnification, and at D28, at (c) high magnification and (d) low magnification, show that size and morphology remain unchanged over time, (e) Amide I FTIR spectra forone batch of SIMPs at D3, D18, and D27 show minimal changes, indicating stable secondary structure. Scale bars: Panels a, c (1 pm) and panels b, d (10 pm).

[0060] FIG. 7 shows degradation analysis of SIMPs incubated in Protease XIV and PBS. SEM image at (a) D1 of incubation in Protease XIV shows signs of degradation, while SEM image at (b) D7 of incubation in PBS show that SIMPs maintain structural integrity and spherical shape, (c) Amide I FTIR spectra for SIMPs without incubation (DO), at D1 in Protease XIV, and at D7 in PBS indicate minimal changes over 7 days in PBS, while 1 day in Protease XIV induces substantial [3-sheet increase and a-helix reduction, consistent with SEM degradation observations. (d) Representative SEM image and (e) corresponding Fe Ka1_2 EDS map of degraded SIMPs at D1 in Protease XIV support the localization of IONPS within the SIMPs. Scale bars: Panels a, b (1 pm) and panels d, e (10 pm).

[0061] FIG. 8 shows concentration of silk protein is relatively consistent across batches (a) but are significantly different between iron concentrations (b). Three different batches of silk and lONPs conjugated with GSH were mixed at different concentrations of lONPs-GSH. Average concentration of protein across all three batches were not significantly different between batches (p = 0.726, 0.994, and 0.665). However, with iron concentrations higher than 20 mg / mL, there was a significant difference in protein concentration. * = p < 0.05, *** = p < 0.001 .

[0062] FIG. 9 shows increasing IONP concentration increases magnetic moveability of SIMPs. The concentration of protein detected in the magnetic fraction portion of the SIMPs increased at 20 mg / mL of lONPs and plateaued afterward (a). Comparing the ratio of protein in the magnetic fraction to the protein in the non-magnetic fraction showed this plateau effect (b). There was a significant difference in this ratio of protein between no lONPs (0 mg / mL) and 20 mg / mL and higher concentrations of lONPs. *** = p < 0.001.

[0063] FIG. 10 shows representative SEM images of (a, b) Silk MPs, (c, d) SIMPs, and (e, f) SIMPs-N, displaying their magnetic and non-magnetic separated fractions. Unique particle distributions are observed: Silk MPs mostly in non-magnetic fraction, SIMPs predominantly in magnetic fraction, and SIMPs-N with a notably higher nonmagnetic fraction count compared to SIMPs. The scale bars in all panels are 20 pm.

[0064] FIG. 11 shows SIMPs can be magnetically moved through a hydrogel. Silk MPs and SIMPs were loaded into fibrin hydrogels and exposed to a neodymium magnet to see magnetic movement of the particles. The neodymium magnet wasplaced at the top of the fibrin gels. Particles containing lONPs were able to be detected using a Prussian blue stain (indicated with black arrows), with higher concentrations of IONP containing particles (i.e. SIMPs) detected closer to the magnet (above the dashed line), indicating magnetic moveability and detection. The black square shows a zoomed in image of the SIMPs with or without magnetic exposure, qualitatively showing a higher concentration of MPs in gels exposed to a magnet. Scale bar (red) = 1 mm.

[0065] FIG. 12 shows a LIVE / DEAD stain of SMCs co-cultured with Silk MPs and SIMPs incubated in PBS or digested with Protease XIV showed low cytotoxicity of the particles. SMCs were also cultured with water as a negative control. Green channels (a,c,e, and g) showed confluent live cells in all MP treated groups compared to the negative control (i). Red channels (b,d,f, and h) showed minimal amounts of red (dead) cells compared to the negative control (j). The number of live (green) and dead cells (red) were counted across treatments (n = 3) and the percentage of live and dead cells were compared to the negative control. There was a significantly higher percentage of live cells (k) and simultaneously a significantly lower percentage of dead cells (I) in the MP groups compared to the negative control. Scale bar = 100 pm ** = p < 0.01 , *** = p <0.001.

[0066] FIG 13 shows nanoparticle tracking analysis data of RoosterBio Inc. EVs prior to loading into SIPEs. Total concentration (area under the curve) was calculated to be 8.57E9 ± 2.72E8 particles / mL with an average diameter of 144.9 nm ± 47.4 nm.

[0067] FIG. 14 shows flow cytometry of releasates. Releasates from SIPEs (solid dot) and SIMPs (empty triangle) show a larger number of accumulating CD63 binding events from SIPEs as expected. These results imply that SIPEs were able to release EVs over a 7 day period.

[0068] FIG. 15 shows SMCs in fibrin gel constructs take up released EVs from SIPEs. Following 24 hours of incubation, EVs were able to be released and taken up by SMCs, as shown by the increased signal in the Cy5 channel compared to the dye control.

[0069] FIG. 16 shows co-culture of SIPEs with SMCs and U937 cells attenuated degradative activity. SIPEs and EVs (positive control) had smaller bands (hence decreased activity) with both pro and active forms of MMP9 and MMP2.

[0070] FIG. 17 shows the deconvoluted XPS spectra of (a) Fe2p and (b) O1 s for lONPs. In Fe2p spectrum, peaks corresponding to Fe2+ and Fe3+ species, as well assatellite peaks, are labeled. In 01 s spectrum, a peak corresponding to 02- ions in Fe3O4 and a peak corresponding to C=0 bonds are identified.

[0071] FIG. 18 shows EDS analysis of SIMPs-N (a-f) and SIMP (g-l). Red symbols on electron images (a, g) indicate locations of EDS spectra acquisition from particles and silicon chip substrate. Elemental maps show the spatial distribution of Si (b, h), C (c, i), N (d, j), O (e, k), and Fe (f, I). The C, N, and O maps correlate with particle morphology. However, Fe does not correlate well due to lower content and weaker signal. The scale bars in all panels are 2.5 pm.

[0072] FIG. 19 shows amide I FTIR spectra of Silk MPs, SIMPs, and Silk-IONP MPs (SIMPs-N) (B1 , B2, B3 batches). Consistency observed among batches. Increased [3- sheet and [3-turn intensity is observed in SIMPs and SIMPs-N with lONPs.

[0073] FIG. 20 shows stability assessment of Silk MPs and SIMPs-N stored at 4°C in the refrigerator for 13 and 28 days. SEM images at D13, at high magnification (a: Silk MPs; c: SIMPs-N) and low magnification (b: Silk MPs; d: SIMPs-N), and at D28, at high magnification (e: Silk MPs; g: SIMPs-N) and low magnification (f: Silk MPs; h: SIMPs-N), further confirm stability, (i) Amide I FTIR spectra for one batch of Silk MPs and SIMPs-N at D3, D18, and D27 show stable secondary structure. Scale bars: Panels a, c, e, g (1 pm) and panels b, d, f, h (10 pm).

[0074] FIG. 21 shows degradation analysis of Silk MPs incubated in Protease XIV and PBS. SEM image at (a) D1 of incubation in Protease XIV shows signs of degradation, while SEM image at (b) D7 of incubation in PBS show that silk MPs maintain structural integrity and spherical shape. All the scale bars are 1 pm.(c) Amide I FTIR spectra for Silk MPs without incubation (DO), at D1 in Protease XIV, and at D7 in PBS indicate minimal changes over 7 days in PBS, while 1 day in Protease XIV induces substantial [3-sheet increase and a-helix reduction, consistent with SEM degradation observations.

[0075] FIG. 22 shows EDS analysis of degraded SIMPs after D1 of incubation in Protease XIV. Elemental mapping results from EDS confirm the primary localization of lONPs within the microparticles and their association with degraded protein. The scale bars in all panels are 10 pm.

[0076] FIG. 23 shows lONPs-GSH interfered with protein concentration measurements obtained using a Lowry assay. When the concentration of lONPs-GSH was at least 20 mg / mL, the measured concentration of protein also increased. A linear comparison between actual BSA protein concentration and detected BSA proteinconcentration demonstrated a steeper slope as IONP-GSH concentration increased (a). This interference relationship was shown to be linear and calculated by plotting the slope of detected protein / actual protein vs. concentration of lONPs-GSH (b).

[0077] FIG. 24 shows interference with increasing IONP concentration can be seen while GSH concentration does not affect measured protein concentration. Concentrations of GSH, lONPs, and lONPs-GSH were used to test their inference with measured protein. Mainly IONP concentration affected the measured protein concentration.

[0078] FIG. 25 shows three images of the SIMPs + magnet fibrin gels demonstrate magnetic localization. Between the different images for each group, it is shown that SIMPs are stained blue compared to Silk MP controls. Additionally, SIMP groups exposed to the magnet show Prussian Blue stained SIMPs are moved toward the top of the gel, where the magnet was located.DETAILED DESCRIPTION

[0079] Other than in the operating examples, or where otherwise indicated, the use of numerical values in the various ranges specified in this application are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. As used herein “a” and “an” refer to one or more.

[0080] As used herein, the term “comprising” is open-ended and may be synonymous with “including”, “containing”, or “characterized by”. The term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting of” excludes any element, step, or ingredient not specified in the claim. As used herein, embodiments “comprising” one or more stated elements or steps also include, but are not limited to embodiments “consisting essentially of” and “consisting of” these stated elements or steps. For definitions provided herein, those definitions refer to word forms, cognates and grammatical variants of those words or phrases.

[0081] As used herein, the term “polymer composition” is a composition comprising one or more polymers. As a class, “polymers” includes, without limitation, homopolymers, heteropolymers, copolymers, block polymers, block co-polymers and can be both natural and synthetic. Homopolymers contain one type of building block, or monomer, whereas copolymers contain more than one type of monomer.

[0082] A polymer “comprises” or is “derived from” a stated monomer if that monomer is incorporated into the polymer. Thus, the incorporated monomer that the polymer comprises is not the same as the monomer prior to incorporation into the polymer, in that at the very least, during incorporation of the monomer, certain groups, e.g. terminal groups, that are modified during polymerization are changed, removed, and / or relocated, and certain bonds may be added, removed, and / or modified. An incorporated monomer is referred to as a “residue” of that monomer. A polymer is said to comprise a specific type of linkage if that linkage is present in the polymer. Unless otherwise specified, molecular weight for polymer compositions refers to weight average molecular weight (Mw). A “moiety” is a portion of a molecule, compound or composition, and includes a residue or group of residues within a larger polymer.

[0083] A bioerodible polymer is a polymer that degrades in vivo over a time period, which can be tailored to erode over a time period ranging from days to months, and up to two years, for example a polymeric structure, when placed in vivo, will fully degrade within a time period of up to two years. By “bioerodible,” it is meant that a polymer, once implanted and placed in contact with bodily fluids and / or tissues, will degrade either partially or completely through chemical, biochemical and / or enzymatic processes. Non-limiting examples of such chemical reactions include acid / base reactions, hydrolysis reactions, and enzymatic cleavage. The bioerodible polymer may comprise a protein, and in the context of the present invention, silk fibroin. A biodegradable polymer may comprise a homopolymer, a copolymer, and / or a blend comprising, without limitation, one or more of the following monomers: glycolide, lactide, caprolactone, dioxanone, and trimethylene carbonate. The polymer(s) comprise labile chemical moieties, non-limiting examples of which include esters, anhydrides, or polyanhydrides, which can be useful in, for example and without limitation, controlling the degradation rate of the scaffold or particles and / or the release rate of therapeutic agents, such as the conditioned medium, from the scaffold or particles.

[0084] By “biocompatible,” it is meant that a polymer composition and its normal degradation in vivo products are cytocompatible and are substantially non-toxic and non-carcinogenic in a patient within useful, practical and / or acceptable tolerances. By “cytocompatible,” it is meant that the polymer can sustain a population of cells and / or the polymer composition, device, and degradation products thereof are not cytotoxic and / or carcinogenic within useful, practical and / or acceptable tolerances. For example, the polymer when placed in a human epithelial cell culture does not adversely affect the viability, growth, adhesion, and number of cells. The compositions and / or devices described herein may be “biocompatible” to the extent they are acceptable for use in a human or veterinary patient according to applicable regulatory standards in a given jurisdiction. In another example the materials described herein, when administered to a patient, do not cause a substantial adverse reaction or substantial harm to cells and tissues in the body, for instance, the materials or devices do not cause unacceptable inflammation, necrosis, or an infection resulting in harm to tissues from the implanted scaffold. A “patient” is a human or non-human animal.

[0085] Provided herein are compositions that may include a particle formed of a polymer matrix and a magnetic particle. As used herein “particles” do not imply a size, though in one aspect the particles may be less than 1 mm (e.g., may be microparticles), and in another, less than 100 pm. In non-limiting embodiments, particles may be less than 1 pm in size (e.g., may be nanoparticles). Accordingly, it is to be understood that while certain terminology may be used herein for example that the compositions include microparticles that include a polymer matrix with a magnetic nanoparticle embedded therein and / or tethered thereto, the composition may include a particle of any size, and a polymer matrix with a magnetic particle of any size embedded therein and / or tethered thereto.

[0086] In non-limiting embodiments the particles can pass through a mesh having the stated pore size. For example, a particle having a longest measurement of 100 pm can pass through a mesh having a pore size of 100 pm, but not a mesh having a smaller pore size. The particles may be any shape, such as spherical, oblong, irregular, etc., and therefore can be microspheres or nanospheres. The combination of delivered factors and the way in which they are delivered are unique features of the compositions, structures and devices provided herein.

[0087] In non-limiting embodiments, the particle formed of a polymer matrix may be a microparticle. In non-limiting embodiments, the magnetic particle may be ananoparticle. While the particle formed of a polymer matrix will be referred to herein as a microparticle, and the magnetic particle will be referred to herein as a nanoparticle, those of skill in the art will appreciate that those terms are not to be considered limiting.

[0088] The microparticle may, in some embodiments, include a therapeutic agent. In non-limiting embodiments, the magnetic nanoparticles may be attached to and / or embedded in the polymer matrix. The therapeutic agent may in some embodiments be an extracellular vesicle (EV), for example an EV obtained from conditioned media of a culture of a multipotent cell.

[0089] FIG. 1 depicts schematically a structure of an exemplary microparticle 10 as described herein (not to scale). The microparticle 10 may include a polymer matrix 12 into which a therapeutic agent may be embedded. In non-limiting embodiments the polymer matrix is a bioerodible polymer matrix, such as those described herein. In non-limiting embodiments the polymer matrix may be a silk fibroin matrix, for example from a Bombyx Mori silk cocoon. Microparticle 10 may further include a magnetic nanoparticle 16 embedded in and / or conjugated to the polymer matrix 12. In nonlimiting embodiments, the magnetic nanoparticle may be a superparamagnetic, paramagnetic, ferromagnetic, iron oxide (including Ferucarbotran and Ferudextran), magnetite, and / or maghemite particle. In non-limiting embodiments, magnetic nanoparticle 16 is linked to the polymer matrix 12 by a suitable chemical linker 17, such as a thiol-container linker (for example a glutathione linker) or moiety. Although the magnetic nanoparticles 16 are depicted as being tethered to the surface of the polymer matrix 12, they can be attached to or embedded into the polymer matrix 12 as a consequence of the linking method and reagents, for example by conjugation, for example by chemical conjugation as is known in the art. Glutathione, while providing a well-established and simple linking moiety and method, is not in any sense the only compound useful in linking the magnetic beads to the particle, and one of ordinary skill can readily devise alternative methods and linkers to use for that purpose, including through thiol-based linkages (e.g., sulfhydryl moieties) or other sulfur-containing moieties. While covalent linkage of the magnetic particle to the silk may be preferred, non-covalent binding, such as using antibodies and antigens or other binding partners also may be used for the same purpose.

[0090] As described above, microparticles 10 described herein include a polymer matrix material. In non-limiting embodiments, the polymer matrix material is silkfibroin. “Silk fibroin” or “fibroin” refers to protein polymers, which are present in the glands of arthropods such as silkworms, spiders, scorpions, mites, and bees, and then spun into fibers during their metamorphosis. (Qi Y, et al. A Review of Structure Construction of Silk Fibroin Biomaterials from Single Structures to Multi-Level Structures. Int J Mol Sci. 2017 Mar 3; 18(3):237; Sun W, et al. Silk Fibroin as a Functional Biomaterial for Tissue Engineering. Int J Mol Sci. 2021 Feb 2; 22(3):1499; Song W, et al., Magnetic-Silk Core-Shell Nanoparticles as Potential Carriers for Targeted Delivery of Curcumin into Human Breast Cancer Cells. ACS Biomater Sci Eng. 2017 Jun 12; 3(6):1027-1038; Mathur AB, Gupta V. Silk fibroin-derived nanoparticles for biomedical applications. Nanomedicine (Lond). 2010 Jul; 5(5):807- 20; Bari E, etal., Silk Fibroin Bioink for 3D Printing in Tissue Regeneration: Controlled Release of MSC extracellular Vesicles. Pharmaceutics. 2023 Jan 22; 15(2):383; and WO2014021954A2).

[0091] The particles may optionally comprise a second and / or further bioerodible and / or biocompatible polymer, for example in addition to silk fibroin, or replacing silk fibroin (e.g., one or two or more bioerodible polymers which are not silk fibroin. Microparticles 10 may include up to 100% by weight of polymer matrix component of a second polymer to tune degradation rate, content release rate, and enhance biocompatibility, e.g., up to 1 %, up to 5%, up to 10%, up to 20%, up to 25%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 75%, up to 80%, up to 90%, or up to 95%, including any increment therebetween, by weight of a second polymer to tune degradation rate, content release rate, and enhance biocompatibility. Nonlimiting examples of useful bioreodible polymers include: silk fibroin, a polyester, a polyester-containing copolymer, a polyanhydride, a polyanhydride-containing copolymer, a polyorthoester, and a polyorthoester-containing copolymer. In one aspect, the polyester or polyester-containing copolymer is a poly(lactic-co-glycolic) acid (PLGA) copolymer. In another embodiment, the bioerodible polymer is selected from the group consisting of poly(lactic acid) (PLA); poly(trimethylene carbonate) (PTMC); poly(caprolactone) (PCL); poly(glycolic acid) (PGA); poly(glycolide-co- trimethylenecarbonate) (PGTMC); poly(L-lactide-co-glycolide) (PLGA); polyethyleneglycol (PEG-) containing block copolymers; and polyphosphazenes. Additional bioerodible, biocompatible polymers include: a poly(ester urethane) urea (PEUU); poly(ether ester urethane)urea (PEEUU); poly(ester carbonate)urethane urea (PECUU); poly(carbonate)urethane urea (PCUU); a polyurethane; a polyester; apolymer comprising monomers derived from alpha-hydroxy acids such as: polylactide, poly(lactide-co-glycolide), poly(L-lactide-co-caprolactone), polyglycolic acid, poly(dl- lactide-co-glycolide), and / or poly(l-lactide-co-dl-lactide); a polymer comprising monomers derived from esters including polyhydroxybutyrate, polyhydroxyvalerate, polydioxanone, and / or polyglactin; a polymer comprising monomers derived from lactones including polycaprolactone; or a polymer comprising monomers derived from carbonates including polycarbonate, polyglyconate, poly(glycolide-co-trimethylene carbonate), or poly(glycolide-co-trimethylene carbonate-co-dioxanone).

[0092] Non-erodible polymers may be included in the microparticles described herein, which either do not erode substantially in vivo or erode over a time period of greater than two years. Compositions such as, for example and without limitation, PTFE, poly(ethylene-co-vinyl acetate), poly(n-butylmethacrylate), poly(styrene-b- isobutylene-b-styrene) and polyethylene terephthalate are considered to be non- erodable polymers. Other suitable non-erodable polymer compositions are broadly known in the art, for example in stent coating and transdermal reservoir technologies.

[0093] As described above, microparticles as disclosed herein may include a therapeutic agent. In non-limiting embodiments, the therapeutic agent may be an extracellular vesicle (EV). EVs from stem or progenitor cells, such as multipotent cells, such as mesenchymal stem cells (MSCs), for example and without limitation adipose- derived mesenchymal stem cells (ADMSCs), may be included. The stem or progenitor cells, such as the MSCs or ADMSCs may be human or recombinant, and do not have to be isolated from the patient to be treated, and can be allogeneic, recombinant, and even xenogeneic. As a class, stem cells and progenitor cells are cells that are capable of differentiation into another cell type, such as another progenitor cell or a fully differentiated, mature cell, for example and without limitation, a somatic cell, that has characteristic morphology and specialized functions. Stem cells are found in all multicellular organisms. Through mitotic cell division, they have the capacity to self-renew and can differentiate into a diverse range of specialized cell types. Mammalian stem cells include as a class: embryonic stem cells, adult stem cells, and cord blood stem cells. Embryonic stem cells are derived from the inner cell mass of pre-implantation embryos. Adult stem cells are found in adult tissue. Cord blood stem cells are derived from the umbilical cord, which is rich in hematopoietic stem cells - stem cells that can differentiate to form all cellular components of blood. Embryonic stem cells are pluripotent, that is, they are able to differentiate into all of the somatic cell types of thethree primary germ layers: the ectoderm, the mesoderm and the endoderm. Embryonic stem cells differentiate into the different cell types in the adult body. Absent stimulation to differentiate, embryonic Stem cells can expand indefinitely, maintaining pluripotency. Adult stem cells also can self-renew indefinitely; however, unlike embryonic stem cells, are not pluripotent. They are multipotent in that they can differentiate into some, but not all mature cell types that have characteristic morphologies and specialized functions. Recent studies have shown that adult stem cells have a degree of plasticity and can be encouraged to differentiate into other cell lineages, a process also known as transdifferentiation. A non-exhaustive and nonlimiting list of adult stem cells includes: mammary, intestinal, mesenchymal, endothelial, neural, olfactory, neural crest and testicular stem cells (adult germline stem cells).

[0094] Typically, stem cells generate an intermediate cell type or types before they achieve their fully differentiated state. The intermediate cell is referred to herein as a precursor or progenitor cell. Progenitor or precursor cells in fetal or adult tissues are partly differentiated cells that divide and give rise to differentiated cells. Such cells are usually regarded as "committed" to differentiating into one or more further differentiated cell types along a particular cellular development pathway, but there are exceptions. Although they can differentiate into one or more cell types, unlike adult stem cells, adult progenitor cells cannot renew indefinitely, though they typically can retain their multipotency for several rounds of cell division.

[0095] Mesenchymal stem cells (“MSCs”, also referred to as bone marrow stromal stem cells, skeletal stem cells, or mesenchymal stromal cells) are broadly-known, and make up a small proportion of the stromal cell population in the bone marrow. MSCs can generate bone, cartilage, and fat cells that support the formation of blood and fibrous connective tissue. They have been reported to be present in many tissues. Those from bone marrow (bone marrow stromal stem cells, skeletal stem cells) give rise to a variety of cell types: bone cells (osteoblasts and osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and stromal cells that support blood formation. A variety of methods of preparing MSCs are known to those of skill in the art.

[0096] For use in the methods, compositions and products described herein, the cells cultured for use in the production of conditioned media may be healthy, in that the cells may be obtained from a healthy, non-aged human (or in the case of veterinary uses,from a healthy, non-aged animal), e.g., in the case of humans, a non-diabetic, nonaged (e.g., under 45 years old) person.

[0097] Cell growth media or medium is a solution used to support growth and optionally expansion of a cell population in vitro. A large variety of media are available commercially. Often serum, such as fetal bovine serum, is used to promote cell growth. Additional factors, including buffers, co-factors, antimicrobials, salts, cellular extracts and / or amino acids, carbon source(s), among other factors, are included in various media. For different mammalian cell types, different ingredients are present in any given medium, which often is optimized to promote growth and expansion of that given cell type. The end-use of the cells also is taken into consideration when formulating and optimizing a particular medium. For example, to produce cells or conditioned medium for use in human patients, “xeno free” (xenogeneic-free) media may be used, which contains no natural products, e.g., proteins or other potentially antigenic substances, obtained from non-human sources, such as fetal bovine serum, or cell extracts. “Xeno-free” relates to the species in which the conditioned media is to be used, and therefore if the conditioned media is to be used in humans or for propagating human cells, the media contains no non-human products (e.g., proteins or serum), while if the conditioned media is to be used for veterinary purposes, e.g., in dogs or for propagating dog cells, the media may contain no products from a different species. A non-limiting example of a useful, xeno-free, medium for expansion of human mesenchymal stem cells is Rooster Nourish MSC-XF (RoosterBio). Other media for expansion of mesenchymal stem cells are commercially available and / or described in the literature.

[0098] In one embodiment, the microparticles described herein comprise stem cell exosomes, e.g., MSC exosomes (extracellular vesicles, EVs). Such exosomes may be obtained by culture of stem cells, e.g., MSCs, in suitable media, such as xeno-free media, and obtaining conditioned media, which comprises exosomes produced by the cultured MSCs. Current methods for EVs preparation include, for example and without limitation: centrifugation, filtration, tangential flow filtration, differential centrifugation, density gradient ultracentrifugation, size-exclusion chromatography (SEC), precipitation, and immune-based capture methods. Ultrafiltration followed by SEC may produce higher yields with preservation of biophysical and functional properties. EVs are nano-sized membrane-bound vesicles that shuttle important biomolecules between cells to maintain physiological homeostasis and EVs from MSCs (MSC-EV)have regenerative and anti-inflammatory properties. MSC-EV, as an alternative to MSCs, confers several advantages for therapeutic or regenerative use, including higher safety profile, lower immunogenicity, and the ability to cross biological barriers. Their use, as compared to MSCs avoids complications that arise from stem cell- induced ectopic tumor formation, entrapment in lung microvasculature, and immune rejection (see, e.g., Gowen A, et al., Front Cell Dev Biol. 2020 Mar 12;8:149).

[0099] In further details, conditioned media refers to cell culture media in which cells are grown for a sufficient time to release secreted factors, and any other cellular products or other materials into the culture media. For example, and without limitation, conditioned media may be media in which multipotent cells, such as MSCs or ADMSCs, are grown, and which cells release into the cell culture media a variety of secreted factors, such as cytokines, growth factors, and exosomes that may comprise comprising pro-regenerative factors. The cells may be cultured for, for example, from one hour to one week, or more, in media to cause the media to become conditioned. For example and without limitation, fresh cell culture media may be added to cultured cells (at t=0) corresponding to initial addition, the cells are then cultured to “condition” the media, and the conditioned media may be removed at 3, 6, 12, 24, 36, 48, or 72 hours, or after 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, or 14 days, or longer, or any increment therebetween. In one example, cellular secreted factors such as EVs may be obtained by administering fresh culture media to a near confluent flask of cells, such as MSCs, and returning the cells to an incubator to undergo additional culture. During this time, the cells, e.g., MSCs, secrete potent factors including EVs, and thereby “condition” their media. After several days, the now conditioned media is collected and utilized for subsequent encapsulation within the described silk particles.

[0100] The conditioned media may comprise cells and cellular debris, which can be removed by, for example, by one or more of centrifugation, in which the supernatant is preserved and / or filtration steps, in which the filtrate is preserved. Large particles, such as larger vesicles may be removed by filtration, such as by filtration of the conditioned media through a filter that allows the EVs in the conditioned media to pass with the filtrate, such as a 0.22 micron filter, though filters with larger and smaller pore diameters are contemplated. Tangential flow filtration may then be used to retain (in the filtration retentate) the EVs and to pass smaller particles and factors, such as tangential filtration using a 700 kDa or smaller hollow fiber membrane, for example 100-700 kDa, 200-600 kDa, 300-500 kDa, and / or 300 kDa, all values and subrangestherebetween inclusive. In non-limiting embodiments, a 300 kDa membrane is used. Retained (retentate) EVs may then be washed from the membrane into a suitable solution for further processing into the silk particles as described herein. While the described centrifugation and filtration methods are useful in preparing EVs for use in the compositions and methods described herein, and may be preferred in certain instances, they are not intended to be limiting, as there are numerous methods useful for isolating EVs from conditioned culture media.

[0101] In addition to, or as an alternative to the EVs, other active agents may be incorporated into the particles. The other active agents may be growth factors, cytokines chemoattractants, and / or inflammation regulators, used to supplement the conditioned medium to tailor the cellular response, deposition, growth and other aspects of the tissue remodeling process as it occurs on the artificial growth scaffolds described herein. Specific examples of active agents, include: growth factors, small molecule drugs, cytokine drugs, biologies, proteins, or polypeptides (including dipeptides, tripeptides, oligopeptides, and, generally, without limitation, proteins, peptides, and proteinaceous biological active agents, such as antibodies, antibody fragments and analogs or fragments thereof, paratope-containing peptides, ligands, or binding reagents), chemoattractants, binding reagents such as antibodies or antibody fragments, receptors or receptor fragments, ligands, or antigens and / or epitopes.

[0102] Additional active agents that may be incorporated into the composition and / or microparticles include, without limitation, anti-inflammatories, such as, without limitation, NSAIDs (non-steroidal anti-inflammatory drugs) such as salicylic acid, indomethacin, sodium indomethacin trihydrate, salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal, diclofenac, indoprofen sodium salicylamide, antiinflammatory cytokines, and anti-inflammatory proteins or steroidal anti-inflammatory agents); antibiotics; anticlotting factors such as heparin, Pebac, enoxaparin, aspirin, hirudin, plavix, bivalirudin, prasugrel, idraparinux, warfarin, coumadin, clopidogrel, PPACK, GGACK, tissue plasminogen activator, urokinase, and streptokinase; growth factors. Other active agents include, without limitation: (1 ) immunosuppressants; glucocorticoids such as hydrocortisone, betamethasone, dexamethasone, flumethasone, isoflupredone, methylprednisolone, prednisone, prednisolone, and triamcinolone acetonide; (2) antiangiogenics such as fluorouracil, paclitaxel, doxorubicin, cisplatin, methotrexate, cyclophosphamide, etoposide, pegaptanib,lucentis, tryptophanyl-tRNA synthetase, anecortave acetate, CA4P, AdPEDF, VEGF- TRAP-EYE, AG-103958, Avastin, JSM6427, TG100801 , ATG3, OT-551 , endostatin, thalidomide, bevacizumab, neovastat; (3) anti-proliferatives such as sirolimus, paclitaxel, perillyl alcohol, farnesyl transferase inhibitors, FPTIII, L744, antiproliferative factor, Van 10 / 4, doxorubicin, 5-FU, Daunomycin, Mitomycin, dexamethasone, azathioprine, chlorambucil, cyclophosphamide, methotrexate, mofetil, vasoactive intestinal polypeptide, and PACAP; (4) antibodies; drugs acting on immunophilins, such as cyclosporine, zotarolimus, everolimus, tacrolimus and sirolimus (rapamycin), interferons, TNF binding proteins; (5) taxanes, such as paclitaxel and docetaxel; statins, such as atorvastatin, lovastatin, simvastatin, pravastatin, fluvastatin, and rosuvastatin; (6) nitric oxide donors or precursors, such as, without limitation, Angeli's Salt, L-Arginine, Free Base, nitrates, nitrites, Diethylamine NONOate, Diethylamine NONOate / AM, Glyco-SNAP-1 , Glyco-SNAP-2, S-Nitroso-N-acetylpenicillamine, S-Nitrosoglutathione, NOC-5, NOC-7, NOC-9, NOC- 12, NOC-18, NOR-1 , NOR-3, SIN-1 , Hydrochloride, Sodium Nitroprusside, Dihydrate, Spermine NONOate, Streptozotocin; and (7) antibiotics, such as, without limitation: acyclovir, ofloxacin, ampicillin, amphotericin B, atovaquone, azithromycin, ciprofloxacin, clarithromycin, clindamycin, clofazimine, dapsone, diclazuril, doxycycline, erythromycin, ethambutol, fluconazole, fluoroquinolones, foscarnet, ganciclovir, gentamicin, itraconazole, isoniazid, ketoconazole, levofloxacin, lincomycin, miconazole, neomycin, norfloxacin, ofloxacin, paromomycin, penicillin, pentamidine, polymyxin B, pyrazinamide, pyrimethamine, rifabutin, rifampin, sparfloxacin, streptomycin, sulfadiazine, tetracycline, tobramycin, trifluorouridine, trimethoprim sulfate, Zn-pyrithione, and silver salts such as chloride, bromide, iodide, and periodate.

[0103] Further examples of additional active agents include: basic fibroblast growth factor (bFGF or FGF-2), acidic fibroblast growth factor (aFGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factor-beta pleiotrophin protein, midkine protein, platelet-derived growth factor (PDGF), angiopoietin-1 (Ang-1 ), tissue inhibitor of matrix metalloproteinase 1 and 2 (TIMP-1 and TIMP-2)., and regulatory RNA such as microRNA and siRNA. Active agents are included in the delivery system described herein, and are administered in amounts effective to achieve a desired end-point, suchas angiogenesis, tissue growth, inhibition of tissue growth, reduction of degradative activity, reduction in inflammation, or any other desirable end-point.

[0104] As described above, microparticles 10 may include magnetic nanoparticles 16, such as ferromagnetic particles, which may be tethered to the polymer matrix. Magnetic nanoparticles may include iron oxide (ferrite) particles as described herein or clusters of particles, often in the 50-200 nm size range. The iron oxide particles may be in any useful crystal structure, such as maghemite or magnetite and may be superparamagnetic, e.g., if less than 128 nm in size. Particle size may be determined by any useful method. Magnetic nanoparticles may be functionalized with a reactive group, such as glutathione, for linking to a protein, e.g., silk fibroin. Magnetic nanoparticles may be aggregated and / or coated, e.g., with functionalized silica. Iron oxide particles can be readily precipitated from Fe2+ / Fe3+salt solutions in a base, such as ammonium hydroxide, as in the example below. Reaction conditions can dictate particle size and magnetic properties. Other suitable methods exist for synthesis of magnetic nanoparticles useful in the methods and compositions described herein.

[0105] In one aspect of the invention, a drug product, device, drug dosage form, or, more generally a “product” is provided. Provided is a formulation of particles as a system for the controllable delivery of EVs and / or release of other active ingredients in a controlled manner using magnetic fields. The particles described herein also permit slow or delayed release of the EV and / or active agents contained within the silk particle. By “slow” release, “delayed” release, “extended” release, “modified”-release, it is meant that the constituents of the particle are released over time, and not immediately as a bolus, for example, and without limitation, over 1 , 2, 3, 4, 5, 6, 7, 8, 9 ,10, 1 1 , 12, 18, or 24 hours, or over 1 , 2, 3, 4, 5, 6, 7, 14, or 21 days, all values and subranges therebetween inclusive, or even over weeks or months.

[0106] Compositions including microparticles as described herein may be dispersed into, a liquid drug product with any suitable, e.g., pharmaceutically-acceptable, aqueous solution, carrier, or excipient (collectively, aqueous carrier), such as, without limitation: water; buffer solutions; salt solutions, such as saline; buffered salt solutions, such as phosphate-buffered saline; among others as are known in the pharmaceutical and compounding arts. By "pharmaceutically acceptable", it is meant that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof. The term "compatible", as used herein, means that the components of the composition are capable of being commingled withthe subject compound, and with each other, in a manner such that there is no interaction that would substantially reduce the pharmaceutical efficacy of the composition under ordinary use situations. In some embodiments, a pharmaceutical formulation further comprises an excipient. In some embodiments, an excipient can be a chelator. In some embodiments, a chelator can be a fungicidal chelator. In some embodiments, a pharmaceutical formulation further comprises a diluent. In some embodiments, a diluent can be an aqueous acid. In some embodiments, a pharmaceutical formulation further comprises cysteamine. In some embodiments, a pharmaceutical formulation further comprises a surfactant. In some embodiments, a pharmaceutical formulation further comprises a small molecule. In some embodiments, the pharmaceutical composition further comprises one or more additional pharmaceutically acceptable excipients. See, e.g., Remington: The Science and Practice of Pharmacy (Gennaro, 21 st Ed. Mack Pub. Co., Easton, PA (2005) for a list of pharmaceutically acceptable excipients. In some embodiments, the pharmaceutically acceptable excipient is of sufficiently high purity and sufficiently low toxicity to render them suitable for administration preferably to an animal, preferably a mammal, being treated.

[0107] In use, the particles described herein may be delivered as a therapeutic and targeted using an applied magnetic field. For example, as a treatment for an aneurysm, such as an aortic aneurysm, the particles may be delivered locally to the site of the aneurysm, and applying a magnetic field to draw the particles to walls of a blood vessel at a site of the aneurysm, such as an aortic aneurysm. The magnetic field may be applied externally to the patient, or locally, e.g., via a minimally-invasive technique in which a magnet is placed externally to the target site. For example, a magnet may be placed external to an aneurysm site on a blood vessel while the particles are delivered to the wall of the vessel at the site of the aneurysm from the lumen of the vessel. In non-limiting embodiments, the opposite configuration may be utilized (e.g., composition(s) including microparticle(s) as described herein may be applied external to a vessel, and a magnet arranged intraluminally may be used to direct the composition to a target. In non-limiting embodiments, a catheter may be used to feed the particles to the lumen of the blood vessel while the magnetic field is applied. For larger surfaces, the magnet and catheter may be moved external and internal to the blood vessel (lumen), respectively, to target different locations on the vessel. The magnet may be a rare earth magnet or an electromagnet. The magnetmay be permanent or temporary. Permanent magnets include: neodymium iron boron (NdFeB), samarium cobalt (SmCo), alnico, and ceramic or ferrite magnets. Temporary magnets are exemplified by electromagnets or solenoids. Magnets may be (relatively) weak and can be applied locally internally to the patient, while larger (stronger) magnets may be applied external to the patient. A person of ordinary skill can readily determine an appropriate magnetic field source, where to locate the magnet, and where to deliver the particles, to effectively target an aneurysm or other site for delivery of the particles.

[0108] FIG. 2A depicts schematically the deposit of the composition, including microparticles described herein, on a wall of a blood vessel at the site of an aneurysm. A blood vessel 100 is shown having a wall 102 and an aneurysm 104 at a site on the wall 102. In use (left), a catheter 110 is depicted depositing a composition (including microparticles described herein) 1 12 using a needle 1 1 1 at the site of the aneurysm 104, on the adventitial layer of the vessel 100 wall. Magnet 120 is placed in the lumen of the vessel 100. As shown on the right side of FIG. 2A, a magnetic field (dotted lines, which are not intended to depict absolute directionality of the field) is generated by the magnet 120, which draws the composition 1 12a to the adventitial blood vessel wall 102 at the site of the aneurysm. Alternatively, the magnetic silk particles can be deposited intralumenally in the blood vessel, with the magnet outside the vessel, to deliver the magnetic silk particles to the lumen side of the vessel wall.

[0109] FIG. 2B depicts schematically the deposit of the composition 1 12 described herein on a wall of a blood vessel at the site of an aneurysm. A blood vessel 100 is shown having a wall 102 and an aneurysm 104 at a site on the wall 102. In use (left), a catheter 110 is depicted depositing the composition 1 12 at the site of the aneurysm 104, internally in the vessel 100, that is, in the lumen of the vessel 100. Magnet 120 is placed external to the vessel 100. As shown on the right side of FIG. 2B, a magnetic field (dotted lines, which are not intended to depict absolute directionality of the field) is generated by the magnet 120, which draws the composition 112a to the blood vessel wall 102 at the site of the aneurysm.

[0110] While blood vessels and aneurysms are candidates for magnetic deposition of the magnetized silk particles described herein, further uses for the EV-impregnated and / or active agent-impregnated silk particles include:• Drug and EV combinations, such as EVs + anti-inflammatory drugs, EVs + doxycycline, and EVs + TIMPs• Transplant rejection• Myocardial infarction• Tumors (e.g., cancer)• Vascular conditions (e.g., aneurysms, such as thoracic aortic aneurysm and / or abdominal aortic aneurysm)• Joint replacement / osteoarthritis• Pain and inflammation management• Wounds (both civilian and battle)• Gastrointestinal (e.g. diverticulitis)• EndometriosisExampleAbdominal Aortic Aneurysm (AAA)

[0111] AAA is a life-threatening dilation and thinning of the distal aorta wall that affects millions of Americans. Without repair, the AAA can rupture and cause the patient to die. One large factor of aortic expansion is the degradation and improper repair of extracellular matrix elements such as elastin. In order to treat this fatal condition, MSCs have been exploited for their therapeutic effects. In a previous elastase-induced AAA mouse model, MSCs were shown to slow the expansion of the aneurysm along with degradation of elastic lamellae. MSCs also induce biological effects by secreting membrane-bound vesicles called extracellular vesicles. By using MSC-derived extracellular vesicles in place of MSCs, a cell-free therapy can be created to allow for faster translation into clinical trials. By incorporating the extracellular vesicles into silk-iron microparticles, the therapy can be localized to the AAA, providing a controlled method of delivering the regenerative factors. As a general treatment, SIPEs can be moved to the desired tissues to provide a localized regenerative therapy.

[0112] Magnetic substances have been used for a variety of medical applications, specifically drug targeting and delivery. Magnetic movement of substances provides a non-invasive to minimally invasive method for treatment of disease, as an external magnetic field can be used to move the desired magnetic material to the place of interest. These magnetic materials can either be labeled with a drug of choice or incorporated into a material, making said material magnetic. lONPs specifically have been used for a variety of these applications.

[0113] lONPs have been shown to be non-cytotoxic and biocompatible depending on their size, their coating, and their dosage. These characteristics can make them excellent candidates for magnetizing substances for drug delivery if the material attributes are tuned properly. They have also been employed in clinical trials. Typically, lONPs can be cleared and degraded by the reticuloendothelial system, but others have investigated ways to secrete them through the renal system via polymer coatings. As the lONPs can be removed from the body, this further demonstrates the advantages of using them for regenerative purposes.

[0114] Silk fibroin isolated from Bombyx Mori silkworm cocoons has been the source for a variety of biological applications, some of which have been FDA approved. From hydrogels to particles, the versatility of silk fibroin is vast. Silk fibroin is both degradable and biocompatible, only eliciting a low immune response. It also has tunable material characteristics based on how it is fabricated. Given its biocompatibility, it has been used as a platform for drug delivery. Previously, our lab has reported success in the use of silk fibroin to generate tissue engineered vascular grafts. Given its biocompatibility, our goal was to expand the use of silk into microparticle fabrication and eventual magnetic localization.

[0115] Silk fibroin is also commonly processed for biomedical and tissue engineering applications into diverse formats including fibers, films, particles, hydrogels, aerogels, and 3D porous scaffolds. Inorganic nanoparticles, including iron oxide nanoparticles (lONPs) have been added to silk fibroin particles to impart magnetic responsiveness to enable their precise manipulation and tracking.

[0116] While lONPs are typically directly incorporated into the silk fibroin material without specific functionalization or use of chemical bonding agents, magnetic nanoparticles coated with human serum albumin have been reported to enhance cell viability in magnetic silk fibroin scaffolds, indicating that magnetic nanoparticles could improve cellular activity. A common approach is physical blending of the lONPs into RSF solution before processing the final material format. Most studies presume physical embedding of lONPs into the silk and do not perform any direct characterization to confirm chemical bonding between the lONPs and silk fibroin. The primary focus has rather been the characterization of the composite material properties.

[0117] To address this gap, our study aims to enhance lONPs dispersion and retention within silk microparticles (MPs) by utilizing GSH. Thiol bond formationbetween lONPs and GSH aims to facilitate interactions between lONPs and silk fibroin. In this study, we fabricated lONPs with or without a GSH tripeptide and then fabricated conjugated silk-GSH-iron microparticles (SIMPs) or non-conjugated (SIMPs-N) silk-iron microparticles. Characterization, stability, and degradation of these particles were performed and analyzed. Magnetic moveability of the SIMPs was also assessed. The cytotoxicity of the particles was also measured to ensure biocompatibility of the material.Extracellular Vesicles

[0118] Extracellular vesicles (EVs) are bilipid membrane capsules that carry signalling cargo including DNA, RNA, proteins, lipids, and other materials for intercellular communication. These EVs present similar characteristics to their parent cells, thus if the cells are regenerative like mesenchymal stem cells (MSCs), then their EVs will also be regenerative. Our lab and others have utilized MSC EVs for the context of elastin synthesis and immunomodulation related to aneurysm, indicating the utility and potential they have for regenerative medicine.Fabrication Methods for Creating Silk-Iron Packaged Extracellular Vesicles (SIPEs) Cell CultureMaterials:• ADMSCs from RoosterBio• Growth media (supplemented)• Harvest media (EV-collect or EV-collect supplemented with EV boost)• Trypsin• FBS• T175 flasks• Tower flasks• 50 mL conical tubesThese should all be sterile.

[0119] RoosterBio adipose derived mesenchymal stem cells (ASCs, C46001 AD, Lot: 210323) at passage 4 were plated in a T175 flask in 20 mL of growth media (RoosterNourish, SU-022 supplemented with RoosterBooster SU-003). Media was replaced the next day to remove residual DMSO. After 3 days of culture, cells were counted and passaged into two 5-layer tower flasks, 3 million cells per tower flask.Cells were cultured in 75 mL of growth media in the tower flasks for 3 days. At >80% confluency, cells were washed twice with PBS. Then 110 mL of harvest media (RoosterCollect, M2001 ) was added to each tower flask. Cells were cultured in RoosterCollect for 48 hours to be used for extracellular vesicle isolation.EV Isolation - UltracentrifuqationMaterials:• CM from cell culture step• Autoclaved ultracentrifuge tubes• 50 mL conical tubes• 18-G blunt needle• 60 mL syringe• 0.22 pm PES filters• Beckman Coulter, SW28.1 , 1 17.1 gram cases• Beckman Coulter, L8-70M• Sterile drape• Potassium phosphate buffer (2M, pH = 8) These should all be sterile.

[0120] After 48 hours of culture, conditioned media (CM) from ASCs was removed and placed in 50 mL conical tubes. The CM was centrifuged at 250 x g (20 minutes at 4 °C) to remove cells followed by 2,500 x g (20 minutes at 4 °C) to remove cell debris. After centrifugation, the CM was decanted into fresh 50 mL conical tubes. A 60 mL syringe and 18-G blunt needle was used to extract CM from the 50 mL conical tube. The CM was then filtered through 0.22 pm filters into fresh 50 mL conical tubes. The autoclaved ultracentrifuge tubes were placed into the red metal ultracentrifuge cases and filled with 34 mL of filtered CM. CM was ultracentrifuged (Beckman Coulter, SW28 and L8-70M) at 100,000 x g for 70 minutes at 4°C to pellet the EVs. After ultracentrifugation, the supernatant was removed, and the tubes were inverted on a sterile drape to drain any remaining supernatant. Residual droplets containing free proteins and RNA were aspirated from the tubes with a vacuum tip. EV pellets were resuspended in 1 10 pl of 4°C potassium phosphate buffer (2M, pH =8) through pipetting.EV Isolation - Tangential Flow FiltrationMaterials:• CM from cell culture step• Ethylene oxide sterilized filter system for Repligen• 50 mL conical tubes• 0.22 pm bottle top filters• 300 kDa hollow fiber membrane filter (Repligen)• KR2i Jr system from RepligenThese should all be sterile.

[0121] After 48 hours of culture, conditioned media (CM) from ASCs was removed and placed in 50 mL conical tubes. CM was then added to the feed end of the TFF system. CM was used to isolate EVs by flowing media at 100 mL / min through a 300 kDa hollow fiber membrane filter. EVs were obtained in the retentate. Isolated EVs were mixed with potassium phosphate monobasic and dibasic to create a potassium phosphate buffer solution (2M, pH =8) for fabricating the SIPEs.EV DilutionMaterials:• RoosterVial Exosomes• Dulbecco’s phosphate buffered saline (DPBS) These should all be sterile.RoosterVial Exosomes (E42059AD, Lot: EA20240002) were obtained from RoosterBio Inc. and diluted with DPBS to achieve 1 E9 particles / mL. Particles counts were achieved using nanoparticle tracking analysis (NTA).Regenerated silk fibroin (RSF) generationMaterials:• Bombyx Mori silk cocoons• Sodium carbonate• Lithium Bromide• Oven• Dialysis tubing / cassettes• Deionized water

[0122] We followed a common method

[0046] for RSF synthesis. Silk cocoons were degummed in 0.05 M sodium carbonate, washed, and air-dried. About 12.5 g of fibroin was dissolved in 9.6 M lithium bromide at 65 °C for 3.5 hours. The solution was dialyzed with periodic water changes over three days to remove lithium bromide. The RSF solution was filtered through cleanroom wipes (Contec™ AMSI0001 ) and stored at 4 °C. It was used within three days to maintain quality. RSF concentration, ranging from 54 mg / ml to 71 mg / ml, was determined by drying a known volume on a glass slide and re-weighing.Iron oxide nanoparticle (IQNPs) fabricationMaterials:• Round bottom three neck flask• Argon gas• Iron (II) chloride tetrahydrate• Iron (III) hexahydrate• Ethanol

[0123] Magnetic lONPs were synthesized via co-precipitation method, which involves the simultaneous precipitation of Fe2+ and Fe3+ ions in an alkaline solution. This process follows the reaction: Fe2++ 2Fe3++ 8OH’ FesC + 4H2O. Precise control over parameters such as reactant concentration^?], temperature [47, 48], stirring speed

[0049] , and the removal of oxygen from the reaction environment

[0048] is essential to govern the size, shape, morphology, and phase of the particles.

[0124] In a round-bottom flask under argon (Ar) flow, a solution of 1 g (0.05 molar) FeCl2.4H2O and 2.7 g (0.1 molar) FeCl3.6H2O in 100 mL ultrapure water was prepared and stirred for 30 minutes. Subsequently, 100 mL of ammonium hydroxide was added dropwise while stirring at 550 rpm. The solution was stirred at room temperature for 30 minutes, then heated to 70 °C for 30 minutes, and cooled under Ar flow. A neodymium disc magnet (D4020, surface field ~0.53 T, DIYMAG) was used to confine the magnetic nanoparticles during washing with ultrapure water and ethanol. The nanoparticles were dried overnight in a vacuum oven at 45 °C and stored in a desiccator.Conjugation of IQNPs with glutathione (GSH)Materials:• Glutathione• Methanol• Neodymium magnet• Deionized water

[0125] lONPs were conjugated with GSH following a scaled-up version of a procedure previously published. Approximately 16 mg of lONPs was added to 480 pL ultrapure water and 160 pL methanol and sonicated for 20 minutes to uniformly disperse the lONPs. Subsequently, 13 mg of GSH was added and the mixture was sonicated for 2.5 hours with periodic agitation of the vial to ensure uniform sonication. A neodymium magnet (D4020, surface field ~0.53 T, DIYMAG), confined the magnetic nanoparticles during sequential washes with ultrapure water and methanol. The nanoparticles were dried overnight in a vacuum oven at 45 °C and stored in a desiccator.Incorporation of IQNPs into RSF solutionMaterials:• Glutathione conjugated IQNPs• RSF• Deionized water

[0126] As a tripeptide with a highly reactive thiol group, GSH effectively binds to heavy metal ions, forming metal-ion complexes that functionalize IONPs[6, 50, 51 ]. The amine and carboxyl groups of GSH facilitate bonding with carboxyl and amine functional groups on silk fibroin, enhancing interactions between nanoparticles and silk fibroin.

[0127] To achieve a final RSF concentration of 50 mg / ml with or without IQNPs, the original RSF solution was diluted with ultrapure water. For example, 0.794 ml of a 62 mg / ml RSF solution was mixed with 0.206 ml of ultrapure water. Before dilution, 25 mg of IQNPs, with or without GSH conjugation, was sonicated in the required amount of ultrapure water for 20 minutes, then mixed with RSF solution and sonicated foruniform dispersion. The suspensions were frequently agitated until used in microparticle synthesis, as detailed in subsequent sections.Microparticle (MP) FabricationMaterials:• Potassium phosphate buffer (2M, pH = 8)• Silk solution (50 mg / mL)• Silk iron solution• Ultrapure water• Isolated EVs

[0128] The silk solution (50 mg / mL) with or without conjugated iron oxide nanoparticles (100 pL) was mixed with potassium phosphate butter (2M, pH = 8, 1000 pL) with (SIPEs) or without isolated EVs (SIMPs) and incubated at 37°C overnight to form MPs via a salting out method. Isolated vesicles were mixed 1 :1 with 4M potassium phosphate buffer before fabricating SIPEs. Following incubation, MPs were centrifuged at 10,000 x g and 4°C for 20 minutes to pellet the particles. Supernatant was removed and the MPs were resuspended in 1000 pL of ultrapure water. The MPs were subsequently washed 5 times by centrifuging at the same speed and resuspending in the same volume of ultrapure water. MPs (Silk MPs, Silk-GSH-iron MPs (SIMPs), Silk-iron MPs (SIMPs-N), and SIPEs were stored at 4°C until ready for use.Material Characterization MethodsIron oxide nanoparticles characterization

[0129] The lONPs, both with and without GSH conjugation, were dispersed in methanol and drop-cast onto silicon (Si) chips for SEM using a Zeiss SIGMA 500 VP instrument to assess particle size, shape, and morphology variations.

[0130] Thermal gravimetric analysis (TGA) was performed on approximately 4 mg samples of lONPs (with and without GSH) using a Perkin Elmer STA 6000. Samples were heated from 75 °C to 850 °C at 10 °C / min, and mass loss profiles were recorded to evaluate GSH conjugation efficiency.

[0131] X-ray photoelectron spectroscopy (XPS) was utilized to confirm FesO4 synthesis and examine the elemental composition and chemical states of thenanoparticles. The analysis was conducted with an ESCALAB 250Xi instrument using monochromatic Al Ka X-rays and an X-ray spot size of 250 pm.

[0132] FTIR analysis was performed on GSH, lONPs, and GSH-conjugated lONPs using a Thermo Nicolet IS50 FTIR spectrometer. Samples were prepared as KBr pellets and analyzed in the 4000-400 cnr1spectral range with 128 scans and a nominal resolution of 4 cm-1to identify functional groups and chemical bonds.Silk microparticle materials characterization

[0133] Three batches of Silk MPs and SIMPs suspensions in ultrapure water were analyzed shortly after preparation using SEM, energy-dispersive X-ray spectroscopy (EDS), and FTIR.

[0134] For SEM, 25 pl of each suspension was cast onto Si wafer chips, air-dried overnight, and mounted on SEM stubs. A Zeiss SIGMA 500 VP microscope was used to examine morphology, with multiple randomly selected areas imaged for each sample. Particle sizes were measured with Imaged software to obtain average values and standard deviations. EDS analysis was conducted using the same microscope with a voltage > 10 kV, and an 8.5 mm working distance to determine elemental composition.

[0135] For FTIR, 35 pl of each suspension was deposited on the attenuated total reflection (ATR) diamond crystal and dried. FTIR spectra were recorded using a Thermo Nicolet IS50 FTIR spectrometer operating in ATR mode with 128 scans at 4 cm-1resolution in the range of 4000-400 cm-1. The Amide I region of the spectra was analyzed to determine variations in the secondary structure of silk fibroin.Stability of silk microparticles (SEM, EDS, FTIR)

[0136] To assess the degradation of Silk MPs and SIMPs, 44 pL aliquots were incubated in PBS or mixed 1 :1 with Protease XIV (1 U / mL, P5147, Millipore Sigma, St. Louis, MO) at 37°C for 1 , 4, or 7 days. Samples were centrifuged at 10,000 x g and 4°C for 20 minutes, and the supernatant containing potentially degraded protein was removed. The samples were resuspended in equal volume of ultrapure water. To minimize additional degradation from residual protease, the resuspended samples were promptly cast onto Si wafer chips for SEM, EDS, and FTIR analysis as described previous.Degradation of MPs (SEM, EDS, FTIR)Materials:• Phosphate buffered saline• Protease XIV (P5147)

[0137] To assess the degradation of Silk MPs and SIMPs, 44 pL aliquots were incubated in PBS or mixed 1 :1 with Protease XIV (1 U / mL, P5147, Millipore Sigma, St. Louis, MO) at 37°C for 1 , 4, or 7 days. Samples were centrifuged at 10,000 x g and 4 °C for 20 minutes, and the supernatant containing potentially degraded protein was removed. The samples were resuspended in equal volume of ultrapure water. To minimize additional degradation from residual protease, the resuspended samples were promptly cast onto Si wafer chips for SEM, EDS, and FTIR analysis as described previously.Magnetic Moveabilitv Analysis with a Lowry Assay for Protein DetectionMaterials:• Ultrapure Water• Lowry Assay Kit• Magnet• Silk MPs and SIMPs• Plate reader

[0138] Three different batches of RSF and three different batches of lONPs-GSH were mixed together to create the silk-iron solution. This solution was used to generate three batches of SIMPs at varying IONP-GSH concentrations (0-50 mg / mL in 10 mg / mL increments) to find the optimal concentration of lONPs-GSH to magnetically move the SIMPs. The optimal concentration was determined with a separation assay followed by a Lowry assay (23240, ThermoFisher Scientific, Pittsburgh, PA) to measure total protein in the samples (i.e. silk). Two different magnets were used for the different separation assays (protein detection via Lowry assay and physical separation via SEM). As outlined in Figure 3, a 100 pL aliquot of MPs was taken tobe used for separation assays. The aliquot was placed next to a magnet (S2, surface field -0.88T, K&J Magnetics Inc, Jamison, PA) for 3 minutes to attract magnetic MPs to the edge of the tube. The non-magnetic fraction was removed and placed in a separate tube. An equal volume of ultrapure water was added to the original tube to resuspend the magnetic fraction. Then 15 pL of original unseparated MPs, magnetic fraction, or non-magnetic fraction was diluted 1 :10 into 135 pL of ultrapure water for detection in a Lowry assay (23240, ThermoFisher Scientific). For a larger magnet surface for separation of magnetic and non-magnetic fractions for SEM, a larger neodymium magnet (D93, surface field ~0.44T, K&J Magnetics Inc.) was used. By physically separating the particles into their magnetic and non-magnetic fractions, we were able to quantify the concentration of protein moved magnetically and visually see the moved particles with SEM. SEM was performed on aliquots of 25 pL of these separated fractions which were drop cast onto Si chips.

[0139] A Lowry assay (23240, ThermoFisher Scientific) was performed on the samples to determine protein concentration (e.g., silk) as described in the manufacturer’s protocol. For this, 40 pL of each sample or a standard curve (in triplicate) of bovine serum albumin (BSA, 0 to 1500 pg / mL) diluted in ultrapure water was added to 96-well plates and mixed with 200 pL of Lowry reagent. Samples were incubated at room temperature for 10 minutes. Then 20 pL of a phenol reagent was added to each well and incubated for another 30 minutes at room temperature. The plate was read at 750 nm.Interference analysis of GSH and IQNPs on protein concentration readingsMaterials:• Ultrapure Water• Lowry Assay Kit• lONPs with or without GSH• GSH• Plate reader

[0140] Detection of possible interference from GSH, lONPs, and lONPs-GSH on the Lowry assay was performed. Equal ratio of interfering agent to BSA protein was determined based on concentration of GSH or lONPs-GSH to concentration of silk, assuming 25 mg / mL of GSH-IONP per 50 mg / mL of silk, 13 mg of GSH per 16 mg ofIONP and 1 mg of IONP to 1 mg of lONPs-GSH. The standard curve containing potential interfering agents was then analyzed and compared to the actual standard curve using a Lowry assay (23240, Thermofisher) as described previously. To calculate the amount of interference seen with the lONPs-GSH, varying concentrations (0-50 mg / mL in 10 mg / mL increments) were used with the same standard curve procedure. The slopes of these standard curve comparisons were plotted to see the relationship between IONP-GSH concentration and interference on protein readout.Magnetic moveabilitv analysis through a hydrogelMaterials:• Fibrinogen• Thrombin• Silk MPs / SIMPs• DMEM+Glutamax• HEPES Buffered Saline• Calcium Chloride• Magnet• Paraformaldehyde• Prussian Blue Stain

[0141] Fibrin gels were made as previously described. Bovine fibrinogen type 1 (1 .2 mL, 3.7 mg / mL, #8630, Millipore Sigma) and 0.21 U / mL bovine thrombin (300 pL, #T7513, Millipore Sigma) were mixed with 300 pL of Silk MPs or SIMPs resuspended in DMEM + Glutamax media (10564-01 1 , Gibco, Gaithersburg, MD). To create the hydrogel, 200 pL of the gel mixture was added to heat-stamped circular molds created with 7.94 mm (5 / 16”) diameter cork borers imprinted in 24 well plates. For groups exposed to the magnet, neodymium magnets (D93, surface field -0.44T, K&J Magnetics Inc.) were placed next to the gels during the gelation process (both for 10 minutes at room temperature and 45 minutes at 37°C). Control groups without magnetic exposure were incubated for the same period of time. Gels were stored in PBS at 4°C prior to staining.

[0142] Fibrin gels were fixed using 4% paraformaldehyde (ThermoFisher Scientific) followed by 3 PBS washes. The gels were stained for 20 minutes at room temperaturewith a Prussian Blue stain (LC190601 , LabChem Inc., Zelienople, PA) mixed 1 :1 with a 20% concentrated hydrochloric acid solution. Excess stain was removed with 3 ultrapure water washes. Fibrin gels were imaged using an Olympus ZX7 microscope (Olympus, Lake Success, NY).In vitro analysis of SIMPs and SIPEsCytotoxicity of MPsMaterials:• Aortic Vascular Smooth Muscle Cells• Basal Media with Supplement• LIVE / DEAD Stain Kit• Transwells• 24 Well Plates• HBSS• Silk MPs / SIMPs• Nikon Ti2

[0143] Human aortic vascular smooth muscle cells (SMCs) from Cell Applications (354-05a, San Diego, CA) were cultured at 37 °C and 5% CO2 (3530, Isotemp, ThermoFisher Scientific) in supplemented basal media (31 1 K-500, Cell Applications Inc.) between passages 6-10. SMCs were plated at a density of 40,000 cells / well in 1 mL of supplemented basal media in 24 well plates. Cells were cultured overnight to adhere prior to treatment. Before adding transwells, 400 pL of media was removed. Transwells (0.4 pm pores, 662641 , Greiner, Monroe, NC) were placed on top and 150 pL of treatment was added to each transwell. Treatments consisted of Silk MPs or SIMPs resuspended in either PBS or Protease XIV (0.01 U / mL). Cells were incubated with treatment for 24 hours. Cytotoxicity was determined using a LIVE / DEAD stain (R37601 , (488 / 570) Invitrogen, Carlsbad, CA). Media was removed from cells and washed with 1 mL of Hank’s Balanced Salt Solution (HBSS) (Gibco). Filtered ultrapure water (200 pL) was added to one group of wells for 15 minutes to induce hypotonic lysis of the cells (positive control for cell death). The LIVE / DEAD stain was made according to the manufacturer’s protocol by mixing the green LIVE stain 1 :1 with the red DEAD stain; this solution was then mixed 1 :1 with HBSS for the working solution.Then 180 pL of LIVE / DEAD stain was added to each well and incubated for 20 minutes. Cells were imaged with a Nikon Ti2 wide lens fluorescent microscope (Nikon, Melville, NY) with living cells staining green (488 nm) and dead cells staining red (570 nm).

[0144] The percentage of live and dead cells was quantified using Imaged (National Institutes of Health, Bethesda, MD). Images were split into red and green channels and the number of cells were counted in each channel using the Imaged thresholding function. An optimal threshold for green and red cell counts was determined and used for counting the number of cells in all images. The percentage of live cells was calculated by dividing the number of green cells by the number of green and red cells (total cells). Conversely, the percentage of dead cells was calculated by dividing the number of red cells by the total number of cells.Nanoparticle Tracking AnalysisMaterials:• EV isolation• Microparticle releasates• Nanosight (NS300, Malvern Panalytical, NTA 3.4 Build 3.4.4)• Luerslip syringe• Ultrapure water• 0.22 pm filters

[0145] Nanoparticle tracking analysis was used to determine particle counts, i.e EV counts. For EV isolation counts, an aliquot of 40 pL was mixed with 360 pL of Nanosight water (ultrapure water filtered through 0.22 pm filters) to create a 1 :10 dilution. For microparticle releasates, sample was filtered through a 0.22 pm filter to remove large silk debris; releasate samples were also not diluted. For EVs purchased from RoosterBio Inc, an aliquot of 10 pL was mixed with 390 pL of Nanosight water to create a 1 :40 dilution. Sample was added to Luerslip syringe to see particles on the Nanosight. Syringe speed was 50 pL / min to read particle counts. Three 45 second particle recordings were taken and averaged to calculate particle counts.Flow CytometryMaterials:• Microparticle releasates• Lipophilic dye (PKH26)• Attune NyT Flow Cytometer• Antibody Beads (CD63, Ref: 106060)• Phosphate buffered saline

[0146] SIPEs or SIMPs were incubated at 37°C in 500pL of PBS in an end over end turner for 7 days, with collection of releasates daily. To collect releasates, samples were centrifuged at 10,000 x g, 4°C, for 20 minutes to pellet microparticles. The supernatant (releasates) was collected and stored at -80 °C until analysis. For flow cytometry, 40 pL of CD63 magnetic antibody beads were added to individual tubes. These beads were washed with 400 pL of PBS and a magnet was used to separate the supernatant from the beads. Following removal of PBS, 200 pL of releasate sample was mixed with the antibody beads. Samples were incubated overnight at 4 °C for antibody attachment to CD63 in EVs. The following day, the beads were briefly centrifuged and mixed with 300 pL of PBS. Using the magnet, the beads remained in the tube while the supernatant was removed. The beads were mixed again in 400 pL of PBS, and the supernatant was subsequently removed. The antibody beads were resuspended in 600 pL of PBS. Lipophilic PKH26 dye was made by mixing 64 pL of dye with 8 mL of diluent, according to the manufacturer’s protocols. To stain the attached EVs, 200 pL of dye was mixed with 200 pL of resuspended antibody beads in flow cytometry tubes. Staining took place for 1 hour at room temperature. Following staining, 300 pL of PBS was added to each tube to wash the antibody beads. A magnet was used to help remove the supernatant and retain the antibody beads. Antibody beads were washed with another 300 pL of PBS and finally resuspended in a total volume of 1000 pL. In a 96 well plate, 100 pL of sample was added to each well. Analyzed each sample with an Attune NyT Flow Cytometer with the following parameters:• Measurement parameter: FSC (forward scatter), SSC (side scatter), YL1 - AlexaFluor568• Voltages: FSC: 288, SSC: 500• Threshold: AND FSC: 0.1 x 1000, And SSC: 2 x 1000• Measured number of fluorescent events to determine presence of EVs in releasates.EV UptakeMaterials:• Lipophilic dye (CellMask Deep Red)• Extracellular vesicles• Dulbecco’s phosphate buffered saline• Phalloidin-488• Fibrinogen• Thrombin• DMEM+Glutamax• HEPES Buffered Saline• Calcium Chloride• Aortic Vascular Smooth Muscle Cells• Basal Media with Supplement• Transwells

[0147] Human aortic vascular smooth muscle cells (SMCs) from Cell Applications (354-05a, San Diego, CA) were cultured in supplemented basal media (31 1 K-500, Cell Applications Inc.) between passages 6-10. Fibrin gels were made as previously described. Bovine fibrinogen type 1 (2 mL, 3.7 mg / mL, #8630, Millipore Sigma) and 0.21 U / mL bovine thrombin (500 pL, #T7513, Millipore Sigma) were mixed with 500 pL of SMCs (3 million cells / mL) resuspended in DMEM + Glutamax media (10564- 01 1 , Gibco, Gaithersburg, MD). To create the SMC seeded constructs, 200 pL of the gel mixture was added to heat-stamped circular molds created with 7.94 mm (5 / 16”) diameter cork borers imprinted in 24 well plates. Gels were allowed to polymerize for 10 minutes at room temperature and 45 minutes at 37°C before adding 1 mL of supplemented basal media with 12mM s-aminocaproic acid. Cells were incubated overnight to settle before adding EVs.

[0148] For staining EVs, 2500 pL at 1 E9 particles / mL were added to autoclaved ultracentrifuge tubes. EVs were stained with CellMask Deep Red (1 :250, 10 pL, ThermoFisher Scientific, C10046) for 15 minutes at room temperature. To remove excess dye, EVs were subjected to ultracentrifugation at 100,000 x g for 2 hours (Microultracentrifuge: Sorvall Discovery M150 SE, Rotor: S100-AT4). EVs wereresuspended in cold DPBS at half the volume due to EV loss (1250 pL). Potassium phosphate monobasic and dibasic were added to the DPBS to create a 2 M buffer solution (pH = 8) for SIPE fabrication. Silk-iron solution as described previously was added directly to the EVs to form SIPEs.

[0149] For SIPEs, 100 pL was loaded into 0.4 pm transwells for co-culture with SMCs in fibrin gels and were cultured for 24 hours to allow for release from SIPEs and cellular uptake. Following incubation, cells were fixed and stained according to previous protocols. Media was removed and a stripping buffer (500 pM NaCI and 0.5% glacial acetic acid) was added for 45 seconds to remove free floating EVs. Cells were washed three times with PBS and then fixed with 3.33% paraformaldehyde (Thermofisher Scientific) for 20 minutes. Fixation was quenched with 1 % (w / v) BSA in PBS solution for 5 minutes, and then cells were washed three times with PBS. Permeabilizing solution (0.1 % Triton-X 100 in PBS) was added for 1 minute. Cells were then stained with blocking buffer (300 pL, 1 % BSA and 0.1 % T riton-X in PBS) containing Ph al Io id i n - 488 (1 :400, ThermoFisher Scientific, A12379) and Hoechst 33258, pentahydrate (bis- benzimide) (1 :1000, Thermofisher Scientific, H1398) for 1 hour at room temperature. Cells were washed three times with PBS and then mounted on slides for imaging. Imaging was done with an EVOS M7000 fluorescent imaging system.SIPE Co-Culture for Deqradative ActivityMaterials:• Extracellular vesicles• Dulbecco’s phosphate buffered saline• Fibrinogen• Thrombin• DMEM+Glutamax• HEPES Buffered Saline• Calcium Chloride• Aortic Vascular Smooth Muscle Cells• Basal Media with Supplement• Transwells• U937 cells• PMARPMI mediaDMEM

[0150] Human aortic vascular smooth muscle cells (SMCs) from Cell Applications (354-05a, San Diego, CA) were cultured in supplemented basal media (31 1 K-500, Cell Applications Inc.) between passages 6-10. Fibrin gels were made as previously described. Bovine fibrinogen type 1 (2 mL, 3.7 mg / mL, #8630, Millipore Sigma) and 0.21 U / mL bovine thrombin (500 pL, #T7513, Millipore Sigma) were mixed with 500 pL of SMCs (3 million cells / mL) resuspended in DMEM + Glutamax media (10564- 01 1 , Gibco, Gaithersburg, MD). To create the SMC seeded constructs, 200 pL of the gel mixture was added to heat-stamped circular molds created with 7.94 mm (5 / 16”) diameter cork borers imprinted in 24 well plates. Gels were allowed to polymerize for 10 minutes at room temperature and 45 minutes at 37°C before adding 1 mL of DMEM with 1 % FBS, 1 % pen / strep, and 12mM s-aminocaproic acid.

[0151] U937 cells from ATCC (CRL-1593.2, Lot: 70057944) were cultured in Phenol red free RPMI media supplemented with 10% FBS and 1 % pen / strep. Approximately 5 million U937 cells were plated into a T75 flask with 10 mL of supplemented RPMI media. The next day, U937 cells were activated with phorbol 12-myristate-13-acetate (PMA) (10 nM) for 24 hours. Activated cells were passaged and resuspended in the low serum DMEM for culturing SMCs. Approximately 24,000 cells / well of U937 cells were added to the fibrin gel constructs and left overnight to settle before adding treatments.

[0152] U937 cell and SMC co-culture media was changed to treatment media (same low serum media as described previously with lipopolysaccharides (LPS) (20 pg / mL (eBioScience)) and 100 pL treatments were added either directly to media (Free EVs) or in transwells (SIPEs and SIMPs). Media was changed on day 3, 5, and 7 and replenished with fresh EVs each time. Removed media was centrifuged at 2,500 x g for 5 minutes to remove dead cells, and stored at -80 °C.Zymogram for Deqradative ActivityMaterials:• Acrylamide gels• Gelatin• Calcium ChlorideZinc ChlorideConditioned media

[0153] Acrylamide gels (7.5%) with gelatin (1.06 mg / mL) were cast to create zymogram gels. Conditioned media (15 pL) was mixed with Lamelli buffer (5 pL) and incubated at room temperature for 15 minutes. Samples (20 pL) were loaded into wells of the zymogram gel and ran for 60 minutes at constant amperage (25 mA / gel). Gels were removed and washed three times in ultrapure water, two times in 2.5% Triton-X solution, and another three times in ultrapure water. Gels were then incubated overnight (about 18 hours) in development buffer (50 mM Tris-HCI (pH = 8.0), 5 mM CaCI2, and 1 pM ZnCI2) to activate the matrix metalloproteinase (MMPs).

[0154] Following overnight incubation, gels were stained with 25 mL of Coomassie solution (0.1 % w / v Coomassie Brilliant Blue, 10% v / v ethanol, 7.5% v / v acetic acid), rinsed with ultrapure water, and then destained overnight. Images were captured with white bands indicating MMP activity.Results from SIPEs ExperimentsCharacterization of IQNPs (with and without conjugation) using SEM, XPS, TGA, FTIR

[0155] Figure 4 illustrates the characterization of lONPs and GSH-conjugated lONPs (IONP-GSH). The FTIR spectra (Figure 4, panel a) reveal a peak at 564 cm-1for both IONP and IONP-GSH, corresponding to the Fe-0 bond. The IONP-GSH spectrum shows additional peaks at 1400 cm-1(symmetric stretching vibration of COO’), 1520 cm-1(amide bond for GSH), and an enhanced peak at 1640 cm-1. The absence of the S-H bond peak at 2524 cm-1in IONP-GSH further confirms conjugation. The peak around 2360 cm-1in all three samples corresponds to CO2. The broad peak spanning 3000-3600 cm-1in both IONP and IONP-GSH spectra arises from O-H stretching vibrations. The thermal gravimetric analysis (TGA) in Figure 4, panel b shows higher mass loss in IONP-GSH, indicating GSH coverage. The XPS S2p core scan (Figure 4, panel c) shows a peak at 165 eV in IONP-GSH, suggesting thiol bond formation, which is absent in the IONP spectrum. The XPS N1 s spectrum (Figure 4, panel d) shows peaks at 398.5 eV and 404 eV in GSH, and at 400.8 eV in IONP-GSH, confirming changes in the nitrogen environment upon conjugation. The XPS C1 s spectrum (Figure 4, panel e) shows peaks at 286.8 eV and 289.3 eV in IONP-GSH,indicating specific carbon functional groups from GSH. SEM images (Figure 4, panels f-g) reveal uniform particle shape and size, with enhanced dispersion in IONP-GSH.

[0156] XPS analysis was conducted to investigate the oxidation state of the lONPs. The deconvoluted Fe2p spectrum (Figure 17, panel a) shows peaks at 709.7 eV and 722.9 eV, corresponding to the 2p3 / 2 and 2p1 / 2 states of Fe2+, respectively, and peaks at 711.3 eV and 724.9 eV, corresponding to the 2p3 / 2 and 2p1 / 2 states of Fe3+, respectively. Satellite peaks are observed at 717.7 eV and 731.1 eV. These results align with previous reports for Fe3O4. Considering the Fe3+: Fe2+of 2:1 in Fe3O4, the areas of Fe3+peaks were constrained to be twice those of corresponding Fe2+peaks during deconvolution. Peak position, full width at half maximum (FWHM), the percentage of Gaussian-Lorentzian mix, as well as the areas of Fe2+and satellite peaks were used as fitting parameters, resulting in a satisfactory fit (Figure 17, panel a) with parameters listed in Table 1 , below.Table 1

[0157] Additionally, the deconvolution of 01 s spectrum (Figure 17, panel b) shows peaks at 529.5 eV, corresponding to O2’ ions in the FesO4 lattice, and at 531.1 eV, likely due to C = O bonds from surface contamination. Surface hydroxyl groups from adsorbed moisture may also contribute to peaks in this region.Characterization of Silk MPs and SIMPs

[0158] Representative SEM images at high and low magnifications are shown in Figure 35. The MPs, both without (Figure 5, panels a and b) and with (Figure 5, panels c-f) lONPs, exhibited spherical morphology with well-preserved structural integrity. The surfaces of the MPs displayed some porosity both with and withoutlONPs, but the SIMPs had smoother surfaces with less porosity. Average particle sizes were 1 .9 ± 0.8 pm, 1 .63 ± 0.6 pm, and 1 .4 ± 0.7 pm for Silk MPs, SIMPs, and SIMPs-N, respectively. The initial silk concentration is known to strongly influence MP size. Figure 5, panel g shows the area where several EDS spectra were collected from both SIMPs and the Si chip substrate. Blue and red rectangles overlaid on the SEM image indicate the Si chip and SIMPs locations, respectively. Si was detected but excluded from elemental analysis. The weight percent of iron was measured from these spectra, and the average values are presented as a bar chart in Figure 5, panel g

[0159] Figure 18 shows SEM images and EDS elemental maps of Si, C, N, O, and Fe for both SIMPs-N (panels a-f) and SIMP (panels g-l). The elemental maps for C, N, and O closely correspond to the particle morphology seen in the electron images for both SIMPs and SIMPs-N. However, the Fe map is less correlated to the particle morphology because of its lower concentration and weaker signal.

[0160] The amide I region of FTIR spectra for three batches (B1 , B2, and B3) of each sample type-Silk MPs, SIMPs, and SIMPs-N-is shown in Figure 19. The spectra demonstrate good consistency among different batches of the same sample type. All samples exhibit a peak around 1652 cm-1, corresponding to the a-helix structure. Additionally, peaks at approximately 1624 cm1and 1700 cm1, typically associated with [3-sheet secondary structure, are more pronounced in both SIMPs and SIMPs-N, especially in SIMPs. Peaks at about 1683 and 1695 cm-1, associated with [3-turns, also exhibit increased intensity in both SIMPs and SIMPs-N. This enhancement of secondary structures might be due to the presence of lONPs in the MPs and the formation of additional [3-sheets from IONP interference during microparticle formation. It could also be influenced by trace amounts of methanol or ethanol used during the final washing of the lONPs.Stability analysis

[0161] Figure 6 shows SEM images of SIMPs at day 13 (D13) and day 28 (D28), as well as the amide I region of FTIR spectra for one batch of SIMPs at D3, D18, and D27. Additionally, Figure 20 presents SEM images for Silk MPs and SIMPs-N at D13 and D28, and the amide I region of FTIR spectra for one batch at D3, D18, and D27. Comparing these SEM images with those in Figure 5 reveals that particle size and morphology remain unchanged over time, indicating the stability of the MPs. The FTIR spectra also show minimal changes, further confirming the stability of the MPs inultrapure water suspensions. Notably, the increased intensity of [3-sheet and [3-turn peaks in SIMPs and SIMPs-N, as described herein, persists even after prolonged exposure, suggesting the stability of the secondary structure of MPs throughout the exposure period.Degradation analysis

[0162] SEM images of Silk MPs (Figure 21, panels a and b) and SIMPs (Figure 7, panels a and b) at D1 of incubation in Protease XIV and D7 in PBS show degradation signs at D1 in Protease XIV for both materials. However, at D7 in PBS, both retained their structural integrity and spherical shape. Figure 7, panel d shows a representative SEM image of degraded SIMPs at D1 in Protease XIV, and Figure 7, panel e presents the corresponding EDS map for Fe Ka1_2, indicating the spatial distribution of iron within the analyzed area. The significant overlap between the SEM image and EDS map confirms that lONPs were primarily localized within the SIMPs and remained associated with the degraded protein. Full EDS elemental maps are presented in Figure 22.

[0163] The amide I region of FTIR spectra at D1 in Protease XIV and D7 in PBS for both Silk MPs (Figure 21 , panel c) and SIMPs (Figure 7, panel c) shows that this region remained largely unaffected after 7 days in PBS, consistent with the SEM images (Figures 7 and 21 ). However, at D1 in Protease XIV, both Silk MPs and SIMPs exhibited a substantial increase in the [3-sheet secondary structure peak at -1624 cnr1and a significant reduction in the a-helical secondary structure peak at -1652 cm-1. This spectral alteration can be attributed to the selective signal retention of stable structures, which aligns with the accelerated degradation observed in SEM images (Figures 7 and 21), indicating the susceptibility of the MPs to Protease XlV-induced degradation.Magnetic separation assay of SIMPs with Lowry assays and SEM

[0164] Concentration of silk protein in SIMPs before separation was relatively consistent between the three batches (n = 6, 60.1 1 ± 24.15 mg / mL, 49.67 ± 22.79 mg / mL, and 61 .48 ± 23.38 mg / mL, p>0.05) with varying amounts of lONPs (Figure 8, panel a and Table 2, below).Table 2

[0165] However, comparing the concentration of silk protein based on GSH-IONP concentration showed a difference between 0 mg / mL and 30 mg / mL, 40 mg / mL, and 50 mg / mL (n = 3, 92.25 ± 19.08 mg / mL vs. 51 .98 ± 14.64 mg / mL, p = 0.0279, 49.91 ± 14.13 mg / mL, p = 0.0203, and 27.61 ± 13.72 mg / mL, p = 0.0007) (Figure 8, panel b and Table 3, below).Table 3

[0166] Comparing the concentration of protein between the separated magnetic and non-magnetic fractions of SIMPs showed that there were higher concentrations of protein in groups with higher concentrations of lONPs-GSH (Figure 9, panel a andTable 4, below).Table 4

[0167] There was also a significant difference between the ratio of magnetic to nonmagnetic fraction of groups with 0 mg / mL of lONPs-GSH and concentrations of 20 mg / mL and above (Figure 9, panel b and Table 5, below) (n =3, 0.028 ± 0.007 vs. 0.578 ± 0.126, p = 0.0002, 0.61 1 ± 0.005, p = 0.0001 , 0.604 ± 0.077, p = 0.0001 , and 0.722 ± 0.21 1 , p < 0.0001 ).Table 5

[0168] Representative SEM images of the magnetic and non-magnetic fractions of each sample type are shown in Figure 10. For Silk MPs (Figure 10, panels a and b),most particles are in the non-magnetic fraction. In SIMPs, most particles are found in the magnetic fraction. In SIMPs-N, while fewer particles are in the non-magnetic fraction compared to Silk MPs, it is significantly higher than in the non-magnetic fraction of SIMPs. The more efficient magnetic moveability of SIMPs is likely due to the enhanced incorporation of lONPs within the MPs.Interference of IQNPs-GSH with protein concentration measurements

[0169] Interference from the lONPs in the Lowry Protein Assay was detected and shown to increase as the concentration of lONPs in the SIMPs increased (Figure 23, panel a). A relationship between the concentration of lONPs and the slope of all protein / BSA protein was shown to be linear with an r2value of 0.9979 (Figure 23, panel b). Interference was not seen from the GSH alone (Figure 24).Magnetic movement of SIMPs through fibrin gel

[0170] SIMPs loaded into fibrin gels showed blue coloration after being stained with Prussian blue, a specific stain for iron, compared to Silk MPs, which were not stained blue. SIMPs exposed to the magnet appeared more concentrated as they were moved toward the magnet (Figure 11 ). Additional images of magnetic movement through the fibrin gel can be seen in the Supplementary Figure Section (Figure 25).Cytotoxicity of MPs

[0171] Co-culturing SIMPs and Silk MPs with SMCs did not induce cytotoxicity (Figure 12). All combinations of MPs and PBS or Protease XIV had lower cytotoxicity compared to the negative control (Silk MPs + PBS: n =3, 97.2 ± 1.1 % live cells, p = 0.0004, Silk MPs + Protease XIV: 92.3 ± 5.7% live cells, p = 0.0023, SIMPs + PBS: 94.7 ± 5.3% live cells, p = 0.0010, and SIMPs + Protease XIV: 92.6 ± 4.2 % live cells, p = 0.0021 vs. Water: 73.3 ± 5.9 % live cells).Nanoparticle tracking analysis

[0172] Before loading the EVs into SIPEs, NTA was performed to assess EV count and size of the RoosterBio Inc EVs. Total concentration (area under the curve) was calculated to be 8.57E9 ± 2.72E8 particles / mL with an average diameter of 144.9 nm ± 47.4 nm (Figure 13).Flow cytometry of releasates demonstrates EV release from SIPEs

[0173] Comparing number of cumulative CD63+ events between SIPEs and unloaded SIMPs demonstrated relatively higher numbers in SIPE releasates. Over 7 days, there appeared to be continued release of CD63+ EVs from SIPEs (Figure 14).SIPEs can release EVs that can be subsequently taken up by SMCs in 3D fibrin gel constructs

[0174] Following 24 hours of incubation with SMCs, SIPEs were able to release fluorescently labelled EVs. These EVs were then taken up by SMCs. EVs had more fluorescent signal in the Cy5 channel compared to dye controls (Figure 15).SIPEs and EVs attenuate deqradative activity

[0175] SIPEs co-cultured with SMCs and U937 cells had decreased MMP activity (pro-MMP9, MMP9, pro-MMP2, and MMP2) compared to unloaded SIMPs. EVs were used as positive control and also decreased degradative activity (Figure 16).Discussion related to SIMP Characterization

[0176] This study demonstrates the successful synthesis and characterization of lONPs, their conjugation with GSH, and incorporation into silk microparticles. The lONPs, synthesized via co-precipitation, were identified as FesCk by XPS (Figure 17). FTIR analysis (Figure 4, panel a) confirmed GSH conjugation by the absence of the S-H bond peak and the presence of new peaks corresponding to GSH, which were not observed in pure lONPs. The increased mass loss in TGA for IONP-GSH (Figure 4, panel b) also supported GSH attachment. XPS validated thiol bond formation and changes in nitrogen and carbon spectra (Figure 4, panels c-e). Thus, GSH was effectively conjugated to lONPs, improving their dispersion while preserving their morphology and size distribution (Figure 4, panels f and g).

[0177] The incorporation of lONPs-GSH into MPs, fabricated using the salting out technique, resulted in slightly smoother surfaces while maintaining the spherical morphology (Figure 5). FTIR analysis revealed consistent secondary structure across batches, with an increased p-sheet content in SIMPS, suggesting that the interaction between lONPs and silk proteins influences the secondary structure of the silk protein (Figure 19).

[0178] The stability of the MPs assessed by SEM and FTIR over a 28-day period (Figures 6 and 20) indicated that their structural integrity was preserved in ultrapure water suspensions with minimal changes in morphology and secondary structure.

[0179] The degradation analysis revealed the susceptibility of both Silk MPs and SIMPs to Protease XIV, as evidenced by significant morphological changes in SEM images and increased p-sheet content in FTIR spectra (Figures 7, panels a-e and 21, panels a-c). In contrast, minimal degradation was observed in PBS over seven days, suggesting that Protease XIV induces more pronounced degradation comparedto PBS. EDS mapping of degraded SIMPs (Figures 7, panel e and 22) confirmed that lONPs remained associated with the silk matrix even after degradation.

[0180] SIMPs and Silk MPs showed good stability when stored at 4°C (Figure 6), as there was little to no difference between the samples at various time points. This indicates the long-term storage capability of these particles. Both SIMPs and Silk MPs were easily degraded in Protease XIV (Figure 7), indicating they could release any potential cargo relatively easily in a degradative environment. Fabrication of SIMPs demonstrated relatively consistent concentration of silk between batches (Figure 8, panel a), indicating the reproducibility of the salting out technique for MP fabrication. Increasing IONP-GSH concentration showed an initial increase in magnetic moveability up to a threshold of 20 mg / mL (Figure 9, panel b), which could imply a limit of IONP incorporation with the silk. SIMPs were also able to show successful magnetic movement compared to the Silk MPs both in solution (Figures 9 and 10) and through a hydrogel (Figure 11), thus demonstrating that the MPs can be moved through various mediums. Conjugation of the lONPs to the silk fibroin also showed better magnetic separation compared to their non-conjugated counterparts, as indicated by SEM imaging of the magnetic and non-magnetic fractions (Figure 10). This better magnetic separation further indicated the magnetic properties of the SIMPs were enhanced through chemical conjugation. The SIMPs, Silk MPs, Protease XIV, and degradation products were not cytotoxic (Figure 12), meaning that these MPs could be used in cellular assays in the future.

[0181] Typically, carbodiimide chemistry with compounds like EDC (1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) is used to activate carboxylic acid groups to facilitate amide bond formation with amine groups. However, for our small-scale reaction between silk fibroin and lONPs-GSH, employing these toxic coupling reagents presented challenges for removal after the coupling step. Therefore, we developed an alternative, cleaner, conjugation strategy taking advantage of the silk microsphere salting out method. The pH increase to ~8 during microsphere formation inherently activated both the silk carboxyl groups and the GSH amine groups by deprotonation, improving their reactivity with each other without needing problematic coupling agents. With thorough mixing during microsphere formation, sufficient contact between the activated groups was expected to enable amide bond formation in the absence of condensing agents. This approach also enabled simultaneous conjugation and nanoparticle trapping within the microspheresas they formed. By circumventing the use of condensing agents, we aimed to simplify the approach, reduce chemical complications, and optimize biocompatibility of the material for its intended application. Overall, increasing pH during microsphere formation allowed straightforward amide bond formation between silk and nanoparticles without requiring carbodiimide or other coupling agents.

[0182] Prior studies have rarely confirmed or characterized chemical bonding between silk fibroin and lONPs. In our work, we prioritized evaluating the magnetic responsiveness enabled by conjugation. As will be further discussed in the next section, the demonstrated magnetic separation provided evidence that silk-iron binding was achieved. While more in-depth techniques are needed to directly characterize the bond, the magnetic results supported successful conjugation.

[0183] Various researchers have evaluated magnetic properties using techniques such as hysteresis curves. Our study looked at a practical step of magnetic attraction by evaluating the physical magnetic properties of SIMPs. This separation assay was also performed with the magnet on the side, as the particles could potentially settle due to gravity, while other groups placed their samples directly on top of a magnet for separation. Additionally, introducing the SIMPs into a hydrogel provided an additional medium for the particles to move through magnetically.

[0184] Some researchers have utilized magnetic nanoparticles to enhance transfection of cells. They showed that magnetofection could increase the number of cells transfected. They also saw a decrease in uptake time with magnetic localization. To transfect their cells or see uptake of their nanoparticles, they had placed the magnet below their cells. This approach was different than ours as we were interested in magnetic separation that was not due to sedimentation; this group was intending to use both gravity and a magnetic field to pull their magnetic particles to cell surface. Other researchers have also utilized a magnet to enhance tumor spheroid penetration of their magnetic nanoparticles. The magnetic particles alone showed better penetration into the spheroids than the cargo alone, and the penetration of the particles was further enhanced by magnet field exposure.

[0185] Other groups have investigated silk fibroin particles for a variety of drug delivery applications. In a previous study, a 2.5-fold increase in the average particle size (from 0.5 pm to 1 .2 pm) was observed as the silk concentration increased from 0.5 mg / ml to 5 mg / ml. Considering that the silk concentration in our study was 50 mg / ml, the higher average particle size obtained for Silk MPs was expected.Additionally, the reduction in particle size, especially in SIMPs-N, indicated the impact of IONP incorporation on disrupting the silk particle formation, resulting in smaller MP sizes. Other groups have investigated drug release from silk only nanoparticles. The silk nanoparticles alone showed low cytotoxicity (<10%) in MCF-7 cells, similar to our Silk MPs and SIMPs. Only when doxorubicin was added did the cytotoxicity increase, indicating the cargo was cytotoxic and not silk particles. Some groups have used MPs to encapsulate BMP-2, BMP-9, or BMP-14. Even though their MPs were fabricated using ethanol precipitation, these MPs were 2.7 ± 0.3 pm in diameter, a similar size to our Silk MPs and SIMPs. Additionally, others have fabricated silk MPs through a microfluidic device; these particles were 180 pm diameter, much larger than our particles. These particles also demonstrated low cytotoxicity, further indicating the biocompatibility of silk fibroin-based particles.

[0186] Outside of silk MPs, magnetic particles with other biomaterials have also been utilized for localized and targeted treatment. Some groups have chemically conjugated urokinase to magnetic nanoparticles to induce targeted clot breakdown. These lONPs did not induce toxicity in vivo in mice, further indicating that lONPs are biocompatible. Another group encapsulated lONPs into poly-lactic-co-glycolic acid (PLGA) MPs for treatment of bone defects. They tested the magnetic attraction of their particles by placing the particles on a magnet and inverting the magnet. PLGA MPs containing lONPs were able to stay adhered to the magnet while plain PLGA MPs could not remain adhered. Their particles also had low cytotoxicity (<2%) when incubating them with bone marrow derived mesenchymal stem cells.

[0187] Magnetic properties such as magnetic guidance and magnetic responsivity are desirable characteristics for clinical drug delivery systems. One in vitro study used an alternating magnetic field to move magnetic silk fibroin particles deeper into the skin. The alternating magnetic field mixed with a stationary magnetic field allowed for deeper skin penetration than non-magnet exposed controls. Another group used an external magnetic field to induce hyperthermia for tumor eradication in a mouse model. Specifically, their magnetic particle imaging technology allowed for the hyperthermia to be effective and localized to the tumor of interest, with negligible damage to surrounding organs. This study demonstrated that magnetic particles can be localized to a site of interest and reduce the off-target effects on the treatment. Loading SIMPs with a drug of interest could have a similar outcome as they were also magnetically moved. Magnetic particles are also a big pull for cancer treatment. An in vivo studyinvolving magnetic localization showed that systemic delivery of silk iron nanoparticles could be localized to a tumor in a mouse model. In their study, they used fluorescent imaging to track where their doxorubicin loaded silk iron particles accumulated; magnetically localized particles were more concentrated at the tumor site. Magnetic particle technology has also been used to separate cells from human blood. One group created a NanoOctopus that combined magnetic particles and DNA strands to capture cancer cells from patient blood. With this NanoOctopus, they were able to magnetically separate PTK7-expressing cancer cells from leukemia patients. The versatility of magnetic particles has clear clinical applications from theranostics to drug delivery. Discussion related to SIPEs

[0188] EVs can be loaded into SIPEs (Figure 13) and released to be detected (Figure 14) via flow cytometry and to be taken up by SMCs in 3D fibrin gel constructs (Figure 15). These released EVs (and thus SIPEs) appeared to maintain their biological activity of the encapsulated EVs (Figure 16). These results demonstrate promise that cargo loaded into the SIMPs (EVs for SIPEs or other biologies) can be released and maintain their original properties.Conclusion

[0189] In summary, this study synthesized lONPs, conjugated them with GSH, and incorporated them into RSF solutions to fabricate SIMPs. SIPEs were also able to encapsulate EVs and release them. These EVs could be detected, taken up by cells, and maintained their biological function. Characterization using SEM, XPS, FTIR, and TGA showed successful GSH conjugation to the lONPs, and successful integration of GSH-conjugated lONPs with silk fibroin. FTIR analysis revealed consistent secondary structure across various batches. The smoother surface as well as increased p-sheet content for SIMPs indicates improved interactions between GSH-conjugated lONPs and silk fibroin. All sample types demonstrated stability, indicating their biodegradability. These SIMPs were able to be magnetically moved through solution and hydrogel, indicating that they are localizable. Additionally, the SIMPs and their degraded bioproducts did not induce cytotoxicity. Thus, SIMPs could potentially be loaded with bioactive cargo that can be magnetically localized / directed for treatment of a variety of diseases.

[0190] Having described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range ofconditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof.

Claims

CLAIMS:

1. A composition comprising: a microparticle comprising a therapeutic agent embedded therein, wherein the microparticle comprises a silk fibroin polymer matrix and a magnetic nanoparticle.

2. The composition of claim 1 , wherein the silk fibroin is obtained from a Bombyx Mori silk cocoon.

3. The composition of claim 1 or claim 2, wherein the magnetic nanoparticle is conjugated to the silk fibroin polymer matrix.

4. The composition of claim 3, wherein the magnetic nanoparticle is chemically conjugated to the silk fibroin polymer matrix.

5. The composition of any one of claims 1 -4, wherein the magnetic nanoparticle comprises an iron oxide nanoparticle.

6. The composition of any one of claims 1 -5, wherein the therapeutic agent comprises an extracellular vesicle.

7. The composition of claim 6, wherein the extracellular vesicle is obtained from conditioned media of a culture of a multipotent cell.

8. The composition of claim 7, wherein the multipotent cell is a mesenchymal stem cell.

9. The composition of claim 7, wherein the multipotent cell is an adipose-derived mesenchymal stem cell.

10. The composition of any one of claims 7-9, wherein the media of the conditioned media is serum-free and / or xeno-free.1 1 . The composition of any one of claims 7-10, wherein the cells are cultured for at least 12 hours in the media to produce the conditioned media.

12. The composition of any one of claims 7-1 1 , wherein the extracellular vesicles are less than 0.22 microns in diameter.

13. The composition of any one of claims 7-12, wherein cells and cell debris are removed from the conditioned media containing the extracellular vesicles and the conditioned media is filtered through a 0.22 micron filter, with the extracellular vesicles passing through the filter in the conditioned media filtrate.

14. The composition of claim 13, wherein the conditioned media filtrate is filtered by tangential flow filtration, with the extracellular vesicles being retained in the retentate by a 300 kDa hollow fiber membrane filter.

15. The composition of any one of claims 7-14, wherein the extracellular vesicles can be in the retentate by a 300 kDa hollow fiber membrane filter using tangential flow filtration at a flow rate in the conditioned media of 27 mL / min.

16. The composition of any one of claims 1 -15, wherein the therapeutic agent comprises a cytokine, a growth factor, an active pharmaceutical ingredient, a biologic pharmaceutical ingredient, a polypeptide, and / or a nucleic acid or nucleic acid analog.

17. The composition of any one of claims 1 -16, wherein the therapeutic agent is an anti-inflammatory drug, doxycycline, or a TIMP.

18. The composition of any one of claims 1 -17, wherein the magnetic particles are conjugated with glutathione.

19. The composition of any one of claims 1 -18, wherein the polymer matrix further comprises a bioerodible polymer in addition to the silk fibroin.

20. A composition comprising: a microparticle comprising a therapeutic agent embedded therein, wherein the microparticle comprises a bioerodible polymer matrix and a magnetic nanoparticle.

21. The composition of claim 20, wherein the magnetic nanoparticle is conjugated to the bioerodible polymer matrix.

22. The composition of claim 21 , wherein the magnetic nanoparticle is chemically conjugated to the bioerodible polymer matrix.

23. The composition of any one of claims 20-22, wherein the magnetic nanoparticle comprises an iron oxide nanoparticle.

24. The composition of any one of claims 20-23, wherein the therapeutic agent comprises an extracellular vesicle.

25. The composition of claim 24, wherein the extracellular vesicle is obtained from conditioned media of a culture of a multipotent cell.

26. The composition of claim 25, wherein the multipotent cell is a mesenchymal stem cell.

27. The composition of claim 25, wherein the multipotent cell is an adipose-derived mesenchymal stem cell.

28. The composition of any one of claims 25-27, wherein the media of the conditioned media is serum-free and / or xeno-free.

29. The composition of any one of claims 25-28, wherein the cells are cultured for at least 12 hours in the media to produce the conditioned media.

30. The composition of any one of claims 25-29, wherein the extracellular vesicles are less than 0.22 microns in diameter.

31. The composition of any one of claims 25-30, wherein cells and cell debris are removed from the conditioned media containing the extracellular vesicles and the conditioned media is filtered through a 0.22 micron filter, with the extracellular vesicles passing through the filter in the conditioned media filtrate.

32. The composition of claim 31 , wherein the conditioned media filtrate is filtered by tangential flow filtration, with the extracellular vesicles being retained in the retentate by a 300 kDa hollow fiber membrane filter.

33. The composition of any one of claims 25-32, wherein the extracellular vesicles can be in the retentate by a 300 kDa hollow fiber membrane filter using tangential flow filtration at a flow rate in the conditioned media of 27 mL / min.

34. The composition of any one of claims 20-33, wherein the therapeutic agent comprises a cytokine, a growth factor, an active pharmaceutical ingredient, a biologic pharmaceutical ingredient, a polypeptide, and / or a nucleic acid or nucleic acid analog.

35. The composition of any one of claims 20-34, wherein the therapeutic agent is an anti-inflammatory drug, doxycycline, or a TIMP.

36. The composition of any one of claims 20-35, wherein the magnetic particles are conjugated with glutathione.

37. The composition of any one of claims 20-36, wherein the polymer matrix further comprises a bioerodible polymer in addition to the silk fibroin.

38. A method of delivering a therapeutic agent to a patient, comprising administering to the patient a composition according to any one of claims 1 -37, and guiding the composition with a magnet to a location in the patient.

39. The method of claim 38, wherein the location is a blood vessel wall of the patient.

40. The method of claim 38 or claim 39, wherein the location is an aneurysm in the blood vessel wall.41 . The method of claim 39 or claim 40, wherein the blood vessel is the patient’s abdominal aorta.

42. The method of any one of claims 38-41 , wherein the magnetic particle is guided by the magnet to adventitia of the blood vessel.

43. Use of the composition of any one of claims 1 -37 for treatment of an aneurysm in a patient.

44. The use of claim 43, wherein the aneurysm is an abdominal aortic aneurysm.

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