Thermoresponsive hydrogel platforms for controlled extracellular vesicle release for preventing activation of NLRP3, il-18, il-1β and inflammasomes

Abstract

The present invention describes a composition comprising a hydrogel such as a chitosan hydrogel, extracellular vesicles and optionally a thermoresponsive gelation agent such as β-glycerophosphate, as well as methods for administering or delivery system with said compositions for inhibiting, preventing or reducing NLRP3 activation, IL-18 production, IL-1β production or inflammasome activation, that promotes treatment of atrial fibrillation, inflammation, and / or fibrosis.

Description

THERMORESPONSIVE HYDROGEL PLATFORMS FOR CONTROLLED EXTRACELLULAR VESICLE RELEASE FOR PREVENTING ACTIVATION OF NLRP3, IL-18, IL-ip AND INFLAMMASOMESFIELD OF INVENTION

[0001] The present invention relates generally to the inactivation ofNLRP3, IL-18 and IL-ip for the treatment or prevention of arrythmias, inflammation, and / or fibrosis. More specifically, the present invention relates to compositions comprising a hydrogel such as chitosan hydrogel and extracellular vesicles, and uses and methods for treatment of atrial fibrillation, inflammation, and / or fibrosis.BACKGROUND OF THE INVENTION

[0002] Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia globally. For 80% of affected individuals, AF significantly disrupts daily quality of life, manifesting symptoms such as dizziness, light-headedness, palpitations, and fainting. Although not life-threatening in itself, AF increases the risk of blood clots and strokes. Despite advances in preventive healthcare, the societal and medical burdens of AF are expected to increase in the near future, including an increase in related hospitalizations and stroke events.

[0003] Existing treatments for AF are suboptimal. Antiarrhythmic drugs target nonspecific ion channels and offer limited efficacy, frequently accompanied by significant side effects. The success rates for catheter ablation, another treatment option, vary widely across institutions, and patients often require multiple procedures. These limitations highlight the need for innovative biological therapies that address AF's root causes rather than merely alleviating symptoms.

[0004] To tackle this challenge, the potential of extracellular vesicles (EVs) for AF prevention has been investigated.[1]EVs are small, membrane-bound particles secreted by almost all cell types. They contain nucleic acids and proteins that enable intracellular signaling. For instance, EVs derived from heart cells are rich in antiinflammatory and anti -fibrotic microRNAs and proteins.[2]A single intra-myocardial injection of these EVs during open-chest surgery markedly reduced the acute inflammation and fibrosis induced by pericardial inflammation.[lb]Follow-up workhas shown that increasing the dose of suspended EVs correlates with progressive reductions in pro-inflammatory cytokine levels, inflammatory cell infiltration, oxidative stress, and atrial fibrosis.[la]

[0005] However, injection of a large dose of suspended EVs has some significant drawbacks. For instance, the EVs are injected into the moving, perfused heart tissue, causing EV clearance to be quite rapid, which may negatively impact atrial repair. Moreover, administering a high dose of EVs may increase the risk of off-target effects or unwanted immune modulation. Excess EVs could saturate cellular uptake pathways, leading to their accumulation in non-target tissues, such as the liver, spleen, or kidneys, potentially resulting in unintended biological effects. This could include altering the function of immune cells or inducing a systemic response that counteracts the intended therapeutic benefits. More efficient delivery of EVs is needed to effectively treat atrial arrhythmias, inflammation, and fibrosis.SUMMARY OF THE INVENTION

[0006] Existing treatments for arrythmias, especially AF, including the use of antiarrhythmic drugs, offer limited efficacy and are often accompanied by significant side effects. Furthermore, the success rates for catheter ablation in the treatment of AF vary widely. These limitations highlight the need for innovative biological therapies that address the root causes of arrythmias, particularly AF, rather than merely alleviating symptoms.

[0007] To tackle this challenge, the inventors recently examined the potential of extracellular vesicles (EVs) for AF prevention)11A single intra-myocardial injection of heart EVs during open-chest surgery markedly reduced the acute inflammation and fibrosis induced by pericardial inflammation.[lb]Follow-up work has shown that increasing the dose of suspended EVs correlates with progressive reductions in pro- inflammatory cytokine levels, inflammatory cell infiltration, oxidative stress, and atrial fibrosis.[la]

[0008] While a single, large dose of suspended EVs prevented atrial damage when administered preemptively, the inventors investigated whether a reduced dosage (i.e., fewer EVs) would be sufficient if the retention of the EVs at the injection site could be prolonged. Compared to simple injection into the moving, perfused heart tissue,this strategy could reduce EV clearance and potentially improve outcomes in atrial repair.

[0009] To this end, the inventors developed a thermal-sensitive, bioresorbable hydrogel to extend EV release. Polymer hydrogels, such as chitosan hydrogels are 3D networks of crosslinked hydrophilic polymers that are biocompatible and biodegradable, making them ideal for tissue engineering and drug delivery applications.[3]The synergistic application of EVs and polymer hydrogels has been explored in pre-clinical cardiac injury models, predominantly in cases of ischemic ventricular injury. These studies demonstrated improvement in cardiac function and a significant reduction in ventricular scarring when compared to treatments solely based on EVs.[4]

[0010] In an embodiment of the invention, a composition is provided comprising a hydrogel (such as chitosan) and a therapeutically effective amount of extracellular vesicles (EVs), and optionally a thermoresponsive gelation agent (such as f>-glycerophosphate).

[0011] a method is provided for inhibiting NLRP3 activation comprising administering the composition described herein to a subject in need thereof. In certain embodiments, a method is provided for inhibiting or reducing the production of IL- 18 or IL- 10 for a period of about 1 hour to about 96 hours comprising administering the composition described herein to a subject in need thereof. In a further embodiment, a method is provided for preventing or reducing inflammasome activation comprising administering the composition described herein to a subj ect in need thereof.

[0012] In an embodiment of the invention, a method is provided for treating or reducing inflammation in a patient either pre-surgery, at the time of surgery, or postsurgery comprising administering the composition described herein to a subject in need thereof. In certain embodiments, a method is provided for treating or preventing atrial fibrillation, atrial fibrosis, or inflammation comprising administering the composition described herein to a subject in need thereof.

[0013] In an embodiment of the invention, an atrial target tissue delivery system is provided comprising: a catheter configured to dispense a composition onto a targetatrial surface; a guidance system configured to provide imaging and / or mapping guidance to localize the catheter relative to a predefined mechanical landmark on the target atrial surface; and a composition adapted to remain flowable prior to delivery and to undergo in situ gelation upon exposure to physiological temperature to form a depot at the target atrial surface.

[0014] This summary of the invention does not necessarily describe all features of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0016] FIGURE 1 shows the study schematic. Schematic showing the comparison between low and medium molecular weight chitosan on retention of extracellular vesicles within the hydrogel. Also shown are representative images depicting the thermoresponsive nature of the chitosan hydrogel: liquid form at room temperature (25°C), gelation observed after maintaining the solution at 37°C for 10 minutes.

[0017] FIGURE 2 shows the effect of [3-glycerophosphate on the physicochemical properties of chitosan hydrogel. A. Viscosity analysis of chitosan hydrogel with varying concentrations of [3-glycerophosphate ( -GP), displayed as mean ± standard deviation. B. Swelling and degradation analysis of chitosan hydrogel with varying concentrations of [3-GP, presented as mean ± standard error of the mean. C. The left panel shows the viable cell density of primary rat atrial fibroblasts incubated with supernatant collected from chitosan hydrogel with varying concentrations of [3-GP, assessed by manual cell counting after 24 and 48 hours of cell culture. The right panel displays the metabolic activity of atrial fibroblasts after exposure to chitosan hydrogel supernatant with varying [3-GP concentrations (5.5-8.5% w / v), measured using a colorimetric assay of dehydrogenase activity after 24 and 48 hours of incubation. *P<0.05 vs. 8.5% [3-GP, **P<0.05 vs. 7.5% [3-GP. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. Data are represented as mean ± standard deviation. D. Quantification of live and dead cell imaging of primary rat atrial fibroblasts incubated with supernatant fromchitosan hydrogel with varying concentrations of -GP, stained with calcein-AM (live cells, green) and ethidium homodimer (dead cells, red) after 48 hours of incubation. The left panel shows the average number of live cells per field of view (FOV) calculated using ImageJ quantification, and the right panel shows the average number of dead cells per FOV using ImageJ quantification after 48 hours of cell culture. P<0.05. Data are represented as mean ± standard deviation. E. In vitro cumulative release of EVs from chitosan hydrogels with varying concentrations of f>-GP, displayed as mean ± standard deviation.

[0018] FIGURE 3 shows the characterization and comparison of medium and high molecular weight chitosan hydrogels. A. Viscosity analysis of medium molecular weight (MMW; 1% w / v chitosan, 8.5% w / v [3-glycerophosphate ( -GP)) and high molecular weight (HMW; 1.5% w / v chitosan, 8.5% w / v -GP) chitosan hydrogels using a rheometer. Data are presented as mean ± standard deviation. B. In vitro cumulative release of extracellular vesicles from chitosan hydrogels of varying molecular weights. Data are presented as mean ± standard deviation. C. The left panel shows the viable cell density of primary rat atrial fibroblasts incubated with supernatant collected from chitosan hydrogels after 24 and 48 hours, assessed by manual cell counting. Data are presented as mean ± standard deviation. The right panel displays the metabolic activity of atrial fibroblasts after exposure to chitosan hydrogel supernatant collected after 24 and 48 hours, measured using a colorimetric assay of dehydrogenase activity. Data are presented as mean ± standard deviation. D. Swelling and degradation analysis of MMW (1% w / v chitosan, 8.5% w / v -GP) and HMW (1.5% w / v chitosan, 8.5% w / v -GP) chitosan hydrogels. Data are presented as mean ± standard deviation.

[0019] FIGURE 4 shows the impact of high molecular weight chitosan hydrogel-encapsulated extracellular vesicles (EVs) on inflammation and fibroblast proliferation. A. Comparative effects of EVs and hydrogel-encapsulated EVs on the proliferation of rat primary atrial fibroblasts induced by transforming growth factor [3 (TGF-[31), assessed via manual cell counting and cell metabolic activity analysis (CCK-8 assay). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test; P<0.05 vs. TGF-treated cells. B. Influence of EVs alone and hydrogel-encapsulated EVs on the activation of interleukin- 18 (IL-18) secretion following lipopolysaccharide (LPS) and nigericin treatment. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test; P<0.05 vs. TGF-treated cells. All data are represented as mean ± standard deviation. C. Effect of delayed release of EVs fromhydrogel over 48 hours on IL-18 secretion activation following LPS and nigericin treatment, compared to freshly suspended EVs. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test; P<0.05 vs. cells treated with LPS + Nigericin. All data are represented as mean ± standard deviation. D. Effect of delayed release of EVs from hydrogel over 96 hours on interleukin- 1 (IL-10) secretion activation following LPS and nigericin treatment, compared to freshly suspended EVs. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test; P<0.05 vs. cells treated with LPS + Nigericin. All data are represented as mean ± standard deviation. E. Effect of delayed release of EVs fromhydrogel over 96 hours on human caspase- 1 secretion activation following LPS and nigericin treatment, compared to freshly suspended EVs. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (mean of all samples with positive control); P<0.05 vs. cells treated with LPS + Nigericin. All data are represented as mean ± standard deviation.

[0020] FIGURE 5 shows in vivo studies showing retention of trans-epicardially injected fluorescently labeled hydrogel. A. Representative images showing histological analysis of the left atrium of rats 1 hour after intramyocardial injection of chitosan-rhodamine hydrogel during open-chest surgery, highlighting the presence of hydrogel in the left atrium. B. Representative images displaying fluorescent imaging of the left atrium of rats 1 hour post-sacrifice following administration of chitosan-rhodamine hydrogel during open-chest surgery. Scale bar: 50 pm. C. Intramyocardial injection of high molecular weight encapsulated extracellular vesicles (EVs) and suspended EVs reduces fibrosis in a rat model of sterile pericarditis. Comparison of different concentrations of suspended EVs versus high molecular weight encapsulated EVs in reducing fibrosis in a sterile model of talc-induced pericarditis, estimated by hydroxyproline content (n=3). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test; P<0.05 vs. Talc + Vehicle. All data are represented as mean ± standard deviation.

[0021] FIGURE 6 shows live dead cell imaging of primary rat atrial fibroblast incubated with supernatant collected from chitosan hydrogel, stained using calcein- AM (live cells, green) and Ethidium homodimer (dead cells, red) after 48 hours of incubation.

[0022] FIGURE 7 shows three-dimensional electroanatomic mapping images illustrating catheter positioning in the right and left atrial appendages and localization of an epicardial delivery catheter relative to atrial anatomy, consistent with mapping-guided surface administration.

[0023] FIGURE 8 shows representative images demonstrating use of a mechanical marker (green dot) attached to a pericardial wire to localize the epicardial delivery site, complementing imaging / mapping-based localization.

[0024] FIGURE 9 shows fluorescence image showing epicardial delivery and in situ gelation of a labeled hydrogel on the left atrial surface following pericardial catheter-based infusion. The hydrogel adheres to the left atrial epicardial surface and remains localized, as confirmed by fluorescence imaging, analogous to prior fluorescence confirmation approaches described herein.DETAILED DESCRIPTION

[0025] Existing treatments for arrythmias, and in particular atrial fibrillation (AF), have limited efficacy and are accompanied by significant side effects. These limitations highlight the need for innovative biological therapies that address the root causes of arrythmias rather than merely alleviating symptoms. To this end, the inventors developed a thermal-sensitive, bioresorbable chitosan hydrogel that encapsulates heart extracellular vesicles (EVs). These hydrogels release EVs over an extended period and provide an effective treatment of AF, inflammation, and fibrosis.

[0026] As used herein, “patient” or “subject” may encompass any vertebrate organism or cell including mammals, but not limited to humans, non-human primates, rats, dogs, pigs and mice. In a preferred embodiment, the mammal may be a human. Further, a patient or subject ‘in need thereof may encompass any of the above organisms diagnosed with atrial arrhythmia, experiencing symptoms of atrialarrhythmia, and / or diagnosed or experiencing symptoms associated with a condition benefiting from the administration of a hydrogel composition of extracellular vesicles described herein. In a preferred embodiment the atrial arrhythmia is atrial fibrillation.

[0027] As used herein, “thermoresponsive” refers to a material whose physical properties change as a function of temperature, including, without limitation, viscosity, solubility, phase state, or network structure, and that exhibits a reversible or irreversible transition near a defined temperature threshold (e.g., lower or upper critical solution temperature) enabling temperature-mediated control of flow, gelation, or release. The term “thermoresponsive” in view of the polymer or hydrogel composition means that the composition remains flowable in a liquid state at room temperature (about 18-25 °C) and undergoes a temperature-triggered sol -gel transition to a solid or semi-solid depot at or near physiological temperature (approximately 37 °C).

[0028] As used herein, the term “thermosensitive” or “thermosensitive gelling system / gelling agent” or “thermosensitive gelation agent” means a component that, when combined with a polymer, confers temperature-dependent sol-gel behavior by setting or modulating the temperature at which the mixture transitions from a flowable state to a gel. Such an agent operates through mechanisms including, without limitation, modulation of pH, ionic strength, counterion pairing, hydrogen bonding, or hydration state, and is selected in identity and concentration to maintain injectability at handling temperatures (about 18-25 °C) and to induce gelation at or near physiological temperature (approximately 37 °C) within a clinically acceptable timeframe, while also influencing gelation time, viscosity during delivery, and postgel mechanical properties. The term thermosensitive and thermoresponsive are used interchangeably in the context of gelling agent / gelation agent.

[0029] Herein ‘about 1% to about 100%’ may encompass any values defining a range therein, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0030] Described herein are compositions comprising chitosan hydrogel, extracellular vesicles and P-glycerophosphate, and uses and methods thereof. It will be appreciated that embodiments and examples are provided herein for illustrative purposes intended for those skilled in the art, and are not meant to be limiting in any way.

[0031] A person of skill in the art will recognize that different methods to obtain extracellular vesicles from primary cell lines may be possible and said methods impact aspects of the extracellular vesicles. The skilled person having regard to the teachings herein will be able to select a suitable method for a given application. In certain embodiments, it is contemplated that extracellular vesicles may be derived from human heart cells by tangential flow filtration. It may also be desirable to isolate and use non-heart EVs.

[0032] In certain embodiments of any of the compositions, the extracellular vesicles may be a poly disperse population that comprises an average diameter of about 10 nm to about 500 nm, any values defining a range therein, including, for example, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm, 90 nm, 91 nm, 92 nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 nm, 100 nm, 101 nm, 102 nm, 103 nm, 104 nm, 105 nm, 106 nm, 107 nm, 108 nm, 109 nm, 110 nm, 111 nm, 112 nm, 113 nm, 114 nm, 115 nm, 116 nm, 117 nm, 118 nm, 119 nm, 120 nm, 121 nm, 122 nm, 123 nm, 124 nm, 125 nm, 126 nm, 127 nm, 128 nm, 129 nm, 130 nm, 131 nm, 132 nm, 133 nm, 134 nm, 135 nm, 136 nm, 137 nm, 138 nm, 139 nm, 140 nm, 141 nm, 142 nm, 143 nm, 144 nm, 145 nm, 146 nm, 147 nm, 148 nm, 149 nm, 150 nm, 151 nm, 152 nm, 153 nm, 154 nm, 155nm, 156 nm, 157 nm, 158 nm, 159 nm, 160 nm, 161 nm, 162 nm, 163 nm, 164 nm, 165 nm, 166 nm, 167 nm, 168 nm, 169 nm, 170 nm, 171 nm, 172 nm, 173 nm, 174 nm, 175 nm, 176 nm, 177 nm, 178 nm, 179 nm, 180 nm, 181 nm, 182 nm, 183 nm, 184 nm, 185 nm, 186 nm, 187 nm, 188 nm, 189 nm, 190 nm, 191 nm, 192 nm, 193 nm, 194 nm, 195 nm, 196 nm, 197 nm, 198 nm, 199 nm, 200 nm, 201 nm, 202 nm, 203 nm, 204 nm, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, 215 nm, 216 nm, 217 nm, 218 nm, 219 nm, 220 nm, 221 nm, 222 nm, 223 nm, 224 nm, 225 nm, 226 nm, 227 nm, 228 nm, 229 nm, 230 nm, 231 nm, 232 nm, 233 nm, 234 nm, 235 nm, 236 nm, 237 nm, 238 nm, 239 nm, 240 nm, 241 nm, 242 nm, 243 nm, 244 nm, 245 nm, 246 nm, 247 nm, 248 nm, 249 nm, 250 nm, 251 nm, 252 nm, 253 nm, 254 nm, 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, 261 nm, 262 nm, 263 nm, 264 nm, 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, 270 nm, 271 nm, 272 nm, 273 nm, 274 nm, 275 nm, 276 nm, 277 nm, 278 nm, 279 nm, 280 nm, 281 nm, 282 nm, 283 nm, 284 nm, 285 nm, 286 nm, 287 nm, 288 nm, 289 nm, 290 nm, 291 nm, 292 nm, 293 nm, 294 nm, 295 nm, 296 nm, 297 nm, 298 nm, 299 nm, 300 nm, 301 nm, 302 nm, 303 nm, 304 nm, 305 nm, 306 nm, 307 nm, 308 nm, 309 nm, 310 nm, 311 nm, 312 nm, 313 nm, 314 nm, 315 nm, 316 nm, 317 nm, 318 nm, 319 nm, 320 nm, 321 nm, 322 nm, 323 nm, 324 nm, 325 nm, 326 nm, 327 nm, 328 nm, 329 nm, 330 nm, 331 nm, 332 nm, 333 nm, 334 nm, 335 nm, 336 nm, 337 nm, 338 nm, 339 nm, 340 nm, 341 nm, 342 nm, 343 nm, 344 nm, 345 nm, 346 nm, 347 nm, 348 nm, 349 nm, 350 nm, 351 nm, 352 nm, 353 nm, 354 nm, 355 nm, 356 nm, 357 nm, 358 nm, 359 nm, 360 nm, 361 nm, 362 nm, 363 nm, 364 nm, 365 nm, 366 nm, 367 nm, 368 nm, 369 nm, 370 nm, 371 nm, 372 nm, 373 nm, 374 nm, 375 nm, 376 nm, 377 nm, 378 nm, 379 nm, 380 nm, 381 nm, 382 nm, 383 nm, 384 nm, 385 nm, 386 nm, 387 nm, 388 nm, 389 nm, 390 nm, 391 nm, 392 nm, 393 nm, 394 nm, 395 nm, 396 nm, 397 nm, 398 nm, 399 nm, 400 nm, 401 nm, 402 nm, 403 nm, 404 nm, 405 nm, 406 nm, 407 nm, 408 nm, 409 nm, 410 nm, 411 nm, 412 nm, 413 nm, 414 nm, 415 nm, 416 nm, 417 nm, 418 nm, 419 nm, 420 nm, 421 nm, 422 nm, 423 nm, 424 nm, 425 nm, 426 nm, 427 nm, 428 nm, 429 nm, 430 nm, 431 nm, 432 nm, 433 nm, 434 nm, 435 nm, 436 nm, 437 nm, 438 nm, 439 nm, 440 nm, 441 nm, 442 nm, 443 nm, 444 nm, 445 nm, 446 nm, 447 nm, 448 nm, 449 nm, 450 nm, 451 nm, 452 nm, 453 nm, 454 nm, 455 nm, 456 nm, 457 nm, 458 nm, 459 nm, 460 nm, 461 nm, 462 nm, 463 nm, 464 nm, 465 nm, 466 nm, 467 nm, 468 nm, 469 nm, 470 nm, 471 nm, 472 nm, 473 nm, 474 nm, 475 nm, 476 nm, 477 nm, 478nm, 479 nm, 480 nm, 481 nm, 482 nm, 483 nm, 484 nm, 485 nm, 486 nm, 487 nm, 488 nm, 489 nm, 490 nm, 491 nm, 492 nm, 493 nm, 494 nm, 495 nm, 496 nm, 497 nm, 498 nm, 499 nm, and 500 nm.

[0033] As taught in (Chiang and Chen 2019, "Toward characterizing extracellular vesicles at a single-particle level." J Biomed Sci 26(1): 9), extracellular vesicles from a single source are heterogenous and have a distribution of sizes, which characterize the population. A person of skill in the art will recognize that different methods to measure extracellular vesicle size and number may be possible. Such methods including, but not limited to, nanoparticle tracking analysis, high-resolution flow cytometry, standard flow cytometry, resistive pulse sensing, atomic force microscopy, impedance-based detection, laser tweezers Raman spectroscopy, dark field microscopy, electron microscopy, transmission electron microscopy, and cryoelectron microscopy. The skilled person having regard to the teachings herein will be able to select a suitable method for a given application. The person of skill in the art will also recognize that extracellular vesicle number may be indirectly measured with such methods including total protein amount, total lipid amount, total RNA, acetylcholinesterase activity or quantification of specific molecules. In one embodiment, extracellular vesicle particle size and number may be measured using Nanoparticle Tracking Analysis. In certain embodiments, extracellular vesicle particle numbers may be measured using FluoroCet Exosome Quantitation kit.

[0034] The mammalian heart is divided into four chambers consisting of two ventricles (lower chambers) and two atria (upper chambers). The heart chambers are composed of five major cell types including cardiac fibroblasts, cardiomyocytes, cardiac precursor cells, smooth muscle cells and endothelial cells. Herein, all said cells may be encompassed by the expression ‘human heart cell’. In some embodiments, the human heart cells may be derived from a heart biopsy. In certain embodiments, the human heart cells may be derived from a heart biopsy containing any combination of myocardial, epicardial or endocardial tissue. In a further embodiment, the heart biopsy may be an atrial appendage. In some embodiments, the human heart cells may be derived from ventricular tissue. In certain embodiments, the human heart cells may be derived from an entire heart. In a preferred embodiment, the heart biopsy may be a left atrial appendage. In a most preferredembodiment, the right atrial appendage biopsy contains cardiomyocytes and cardiac precursor cells. The skilled person will recognize that in certain embodiments, atrial appendages may be surgically removed at the time of open heart surgery, for example, and ventricular or atrial biopsies may be obtained by guiding a catheter into the heart and taking small bites from the heart tissue, for example. The skilled person will be aware of suitable techniques for obtaining a suitable biopsy or appendage. In some embodiments, heart-derived explant cells may be derived from the heart biopsy. In certain embodiments, the human heart cells may be derived from ventricular tissue. In certain embodiments, the human heart cells may be derived from an entire heart. The person of skill in the art will further recognize that heart explant derived cells may represent a collection of different cell populations which express markers of endothelial, mesenchymal, and stem cell identity. Without wishing to be bound by theory, such a cell may likely be considered a multipotent cell, or a stem cell, which has been at least partially differentiated to heart tissue.

[0035] In certain embodiments, the human heart cells may be allogenic to the recipient of the composition. In certain other embodiments, the human heart cells may be autologous to the recipient of the composition.

[0036] The International Society for Extracellular Vesicles (ISEV) teaches the minimal information for studies of extracellular vesicles (Thery, Witwer et al. 2018; "Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular V esicles and update oftheMISEV2014 guidelines." J Extracell Vesicles 7(1): 1535750). As taught at the time of application, a consensus has not emerged on specific markers of extracellular vesicle subtypes. As such, it is further taught that operational terms for extracellular vesicle subtypes that refer to physical characteristics such as size, biochemical composition such as protein content, conditions of origin or cells of origin are advised. The ISEV further teaches that three categories of broad markers may be analyzed to demonstrate the presence of extracellular vesicles (Categories 1 and 2) and assess their purity from contamination (Category 3). The ISEV also teaches of markers generally restricted to small extracellular vesicles (Category 4) and markers of potential functional activity (Category 5).

[0037] Category 1 is taught to contain transmembrane or GPI-anchored protein, as their presence demonstrates the lipid-bilayer structure specific to extracellular vesicles. Said proteins may comprise the following: tetraspanins (CD63, CD81, CD82); other multi-pass membrane proteins (CD47, GNA); MHC class I (HLA-A / B / C, H2-K / D / Q); integrins (ITGA, ITGB); transferrin receptor (TFR2); LAMP1 / 2, heparan sulfate proteoglycans (SDC); complement-binding proteins (CD55, CD59); MHC class II (HLA-DR / DP / DQ, H2-A); BSG, ADAMI 0, CD73, SHH, TSPAN, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, ICAM, GYP A, CD14, CD3, AChE-S, AChE-E, A , APP, and ABCC1.

[0038] Category 2 is taught to contain cytosolic proteins, as their presence demonstrates the preparations ability to enclose intracellular material. Said proteins may comprise the following: ESCRT-I / II / III and accessory proteins (TSG101, CHMP): caveolins (CAV); enzymes (GAPDH); actin (ACT), tubulin (TUB); ; annexins (ANXA); Heat shock proteins (HSC70, HSP70, HSPA1AHSPA8, HSP84, HSP90AB1); PDCD6IP, VPS4A / B; ARRDC1, FLOT1 / 2, EHD, RHOA, ARF6, SDCBP, and MAPT. It is further taught that such cytosolic protein constituents of extracellular vesicles vary significantly, and therefore extracellular vesicles may contain additional cytosolic proteins.

[0039] Category 3 is taught to contain maj or constituents of non-extracellular vesicle structures often co-isolated with extracellular vesicles. Said proteins may comprise: lipoproteins (APOA1 / 2, APOB, APOB100, ALB) protein and protein / nucleic acid aggregates, UMOD and ribosomal proteins. It is further taught that evaluating molecules of Category 3 assesses the purity of the extracellular vesicle preparation.

[0040] Category 4 is taught to contain larger structures that, due to their size, are substantially lacking or devoid from smaller extracellular vesicles. Said markers may comprise the following: nucleus (including histones, LMNA); mitochondria (including IMMT, CYC1, TOMM20); endoplasmic reticulum (including CANX, HSP90B1, HSPA5); Golgi apparatus (including GM130); autophagosomes (including ATG9A); and cytoskeleton (including ACTN1 / 4, KRT18). A person of ordinary skill will recognize that organelles and large sub-cellular structures may be identified with other markers. The person of skill in the art will also recognizedetermination criteria for substantially lacking or devoid based on a given application or methodology.

[0041] Category 5 is taught to contain functional components used to determine the mode of association with extracellular vesicles. Said proteins may comprise: cytokines (including IFNG, IL); growth factors (including VEGFA, FGF1 / 2, PDGF, EGF, TGFB1 / 2); adhesion and extracellular matrix proteins (including FN1, COL, MFGE8, LG ALS3BP, CD5L, AHSG).

[0042] A person of skill in the art will recognize that many protein markers are members of families with similar or homologous proteins, which may be capable of performing the same function. Therefore, a person of skill in the art would recognize that protein markers may or may not apply to all proteins across a family or class. As a non-limiting example, ITGB broadly refers to the entire class of integrin beta-sub unit proteins, in humans comprising ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, and ITGB8. A person of skill of the art will recognize that homologues of said proteins may or may not have the same function.

[0043] ISEV further teaches that analytical approaches such as Western blots, high resolution flow cytometry, proteomic array or global proteomic analysis using mass spectrometry techniques can be used to identify proteins of Categories 1-5. A person of skill in the art would recognize that additional methods may be used to determine the presence, absence, or level of a given protein in an extracellular vesicle composition.

[0044] The ISEV teaches the non-limiting nature of such foregoing lists, and recommends they be used as a guide to define extracellular vesicles, as not all constituents are present in all populations of vesicles nor are they absent. The ISEV further teaches that extracellular vesicle constituents not listed in any of Categories 1-5 may be present in extracellular vesicles depending on factors including, but not limited to, tissue type, cell type, derivation methodology and physiological conditions. In light of the foregoing and the common general knowledge, a person of skill in the art would recognize that not all markers will be present or absent on extracellular vesicles from a given cell or tissue type, and that said differences in thepresence, absence or levels of certain constituents may act as a defining feature of the extracellular vesicles derived.

[0045] It is understood that certain sub-types of ribonucleic acids (RNA), including, but not limited to, micro RNA (miRNA) and messenger RNA (mRNA) make up a portion of the constituents of extracellular vesicles. miRNA function by regulating post-transcriptional gene expression, generally through binding to complementary sequences on mRNA transcripts. The binding of miRNA to a complementary sequence can result in translational repression, as well as mRNA degradation and / or gene silencing. The activity of miRNA may influence the heart in certain disease states, for example, US 9,828,603 teaches that increasing the level of mir-146a decreases the infarct region in mice following myocardial infarction when compared to animals treated with a control mimic miRNA. US 9,828,603 further teaches that increasing the concentration of mir-210 transfected into cardiomyocytes increased their viability 10-fold following exposure to hydrogen peroxide. The miRNA profile of extracellular vesicles may be determined by reverse transcription polymerase chain reaction (qRT-PCR), miRNA microarray, multiplex fluorescent oligonucleotide-based miRNA detection and RNA sequencing. In one embodiment, the miRNA profile of an extracellular vesicle composition derived from human heart cells may be determined with RNA sequencing. In certain embodiments, the extracellular vesicles contain about 1 to about 1000 unique miRNA transcripts, any values defining a range therein, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, , 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, , 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, , 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, , 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, , 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, , 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, , 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, , 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, , 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, , 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, , 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, , 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, , 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, , 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, , 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, , 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, , 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, , 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, , 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, , 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, , 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, , 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, , 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, , 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, , 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, , 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, , 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, , 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, , 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, , 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, , 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, , 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812,813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000.

[0046] In some embodiments of any of the compositions or methods described herein, the extracellular vesicles include a variety of biomolecules, such as nucleic acids and proteins. Extracellular vesicles contain different types of RNA molecules, as taught in (Zimta, Sigurjonsson et al. 2020; " The Malignant Role of Exosomes as Nanocarriers of Rare RNA Species." Int J Mol Sci 21(16)), and different types of deoxyribonucleic acid (DNA) molecules, as taught in (Hur and Lee 2021; "Characteristics and Clinical Application of Extracellular Vesicle-Derived DNA." Cancers (Basel) 13(15)). In certain embodiments, the extracellular vesicles contain DNA, DNA fragments, DNA plasmids, mRNA, tRNA, snRNA, piRNA, saRNA, miRNA, rRNA, ribozymes, double stranded RNA, other non-coding and coding RNA. In some embodiments, the extracellular vesicles contain non-coding RNAs (ncRNAs), such as, but not limited to, long non-coding RNAs (IncRNAs), microRNAs (miRNAs), long intergenic non-coding RNA (lincRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) and Y RNA fragments.

[0047] It is important to note that the constituents inside extracellular vesicles may be different or present in varied amounts depending on the cell type from which the EVs are derived. Tables 1-7 are taken from W02023082012A1 and reproduced below illustrate this fact.

[0048] TABLE 1 shows exemplary miRNA identified using multiplex fluorescent oligonucleotide-based miRNA detection within extracellular vesicles derived from heart explant derived cells, hsa, homo sapiens

[0049] TABLE 2 shows a list of exemplary differentially expressed miRNA cargo in heart-derived cells (HDC) vs bone marrow derived mesenchymal stromal cells (BM-MSC) EVs. hsa, homo sapiensTABLE 3 shows a list of exemplary differentially expressed miRNA cargo in BM-MSC vs umbilical cord (UC)-MSC EVs. hsa, homo sapiensTABLE 4 shows a list of exemplary differentially expressed miRNA cargo in HDC vs UC-MSC EVs. hsa, homo sapiensTABLE 5 shows a list of exemplary differentially expressed protein cargo in HDC vs BM-MSC EVsTABLE 6 shows a list of exemplary differentially expressed cargo proteins in BM-MSC vs UC-MSC EVsTABLE 7 shows a list of exemplary differentially expressed cargo proteins in HDC vs UC-MSC EVs

[0050] As used herein, the expression “differentially expressed” may refer to a change in expression or level for a given factor, such as, but not limited to, a protein,an RNA, an mRNA, a miRNA, biomolecule or any bioactive cellular component from one sample in comparison to another sample. In certain embodiments, the expression “differentially expressed” may refer to a significant increase or decrease in expression in a cellular component, such as but not limited to, a protein, an RNA, a miRNA, an mRNA, or any equivalent bioactive component known to a person of skill in the art. In certain embodiments, the expression “differentially expressed” may refer to a log2 fold > or <1.5 and p-value < 0.05 increase or decrease in expression in a bioactive cellular component, such as but not limited to, a protein, an RNA, a miRNA, an mRNA, or any component known to a person of skill in the art.

[0051] In certain embodiments, a differentially expressed miRNA may also comprise mRNA target(s) of the differentially expressed miRNA. As would be known to a person of skill, miRNA function by regulating post-transcriptional gene expression, generally through binding to complementary sequences on mRNA transcripts. As such, it is contemplated that in certain embodiments described herein, “differentially expressed miRNA” may include mRNA that may have a putative or predicted miRNA binding site or consensus site for the miRNA(s) that is differentially expressed, and may also encompass protein encoded by mRNA which may contain a putative or predicted miRNA binding site of a differentially expressed miRNA. A person of skill in the art, in light of the teachings herein, would be able to select an appropriate technique or algorithm, such as but not limited to miRWalk (Sticht, De La Torre, et al. 2018), that may provide a list of mRNA target sequences for a given miRNA. In certain embodiments, an algorithm or technique to determine mRNA targets of differentially expressed miRNA may comprise analysis of the 3’UTR and 5’ UTR of mRNAs for miRNA consensus sites or binding sites. In certain embodiments, an algorithm or technique to determine mRNA targets of differentially expressed miRNA may comprise analysis of the 3’UTR of mRNA for miRNA consensus sites or binding sites. In certain embodiments, differentially expressed miRNA may be provided to miRWalk to determine differentially expressed mRNA(s).Inflammation, Fibrosis, Arrhythmia, and other Medical Conditions:

[0052] Inflammation can be a protective response by an immune system to defend against harmful stimuli; however, prolonged inflammation may lead to detrimentalconditions. Inflammation can be regulated by immune cells, said immune cells capable of infiltrating tissues, secreting factors and destroying perceived detrimental material. The infiltration of inflammatory immune cells into atrial tissue may be observed in certain subjects with atrial arrhythmias (as discussed in, for example, (Zhou and Dudley 2020; "Evidence for Inflammation as a Driver of Atrial Fibrillation." Front Cardiovasc Med 7: 62)), therefore the presence of inflammatory immune cells in a tissue may indicate inflammation. A person of skill would recognize that immune cells can infiltrate the heart. In certain embodiments, the phrase “human heart cells” may also comprise immune cells. Inflammasomes are receptors, sometimes referred to as sensors, which regulate the inflammatory response and can serve as indicators of potential inflammation with markers of the inflammasome, including, but not limited to caspase- 1 activation. The infiltration of inflammatory immune cells into the atrial tissue of a subject with atrial arrhythmia presents the possibility that immune cells may absorb extracellular vesicles. Immune cells may bind and / or secrete molecules including, but not limited to, cytokines, interleukins, interferons, chemokines, complement protein, or any equivalent, to induce an inflammatory response in the surrounding tissue. Increases in immune cell infiltration, secreted immune molecules and inflammasome activation may indicate an increase in inflammation. A person of skill in the art will recognize that many techniques are possible to determine inflammation. The skilled person having regard to the teachings herein will be able to select a suitable method for a given application.

[0053] Fibrosis may result in the thickening and / or scarring of the afflicted tissue. Fibrosis may occur due to excess deposition of extracellular matrix components, including, but not limited to, collagen, fibronectin and fibrin, from fibroblast cells and may result from long-term inflammation (Wynn 2008; "Cellular and molecular mechanisms offibrosis." J Pathol 214(2): 199-210). It is further thought that fibrosis may disrupt the electrical activity of the heart leading to conditions, including, but not limited to, atrial arrhythmias. A person of skill in the art will recognize that many techniques are possible to determine fibrosis, including but not limited to, for example, hydroxyproline and relative tissue mass. The skilled person having regard to the teachings herein will be able to select a suitable method for a given application.

[0054] Arrhythmia can be detected by analysis of an electrocardiogram, which is a test monitoring the electrical activity of the heart, which provides a graph of voltage versus time of the electrical activity of the heart that detects the small electrical changes in the heart that are a consequence of cardiac muscle depolarization followed by repolarization during each cardiac cycle (heartbeat)). Electrocardiograms can be performed at a medical clinic or can be sampled continuously (i.e., smart watch or inpatient telemetry). A person of skill will recognize that electrocardiogram may be performed as an invasive or non-invasive procedure. The skilled person having regard to the teachings herein will be able to select a suitable method for a given application. Additional complementary tests including, but not limited to, electrocardiogram, blood pressure machine, Holter monitor, event monitor, blood tests, echocardiogram, stress test, inpatient telemetry, chest x-ray, smart watch, smart ring, any wearable technology capable of determining heart rate, any equivalent techniques or any combination thereof may be used for diagnosis and / or differential diagnosis of atrial arrhythmia.

[0055] Atrial arrhythmias, as taught herein, are a unique class of arrhythmia, which is distinct from other arrhythmias in many ways, such as those arising from the ventricle, including, but not limited to, etiology, pathology, symptoms, treatment options, patient outcomes and patient susceptibility, as discussed in, for example, (Ludhwani, Goyal et al. 2021; "Ventricular Fibrillation.") and (Nesheiwat, Goyal et al. 2021; "Atrial Fibrillation."). Drugs that slow electrical conduction to prevent atrial fibrillation (including but not limited to, for example, flecainide) are contraindicated in patients with ventricular arrhythmias because they are pro-arrhythmic and increase the risk of death (Ledan 2020; "Antiarrhythmic Treatment in Atrial Fibrillation." US Pharm. 45(2):24-27). Furthermore, based on the teachings of (Rizvi, DeFranco et al. 2016; "Chamber-specific differences in human cardiac fibroblast proliferation and responsiveness toward simvastatin." Am J Physiol Cell Physiol 311(2): C330-339), a person of skill would recognize chamber-specific differences exist in the mammalian heart, as atrial fibroblasts are different from ventricular fibroblasts.

[0056] Atrial arrhythmias may include, but are not limited to, atrial fibrillation, postoperative atrial fibrillation, post-infarction atrial fibrillation, thyrotoxicosis, post-viral atrial fibrillation, alcohol-associated atrial fibrillation, drug-induced atrial fibrillation, viral atrial fibrillation, post-viral atrial fibrillation, COVID-19 atrial fibrillation, post-COVID-19 atrial fibrillation, paroxysmal atrial fibrillation, permanent atrial fibrillation, persistent atrial fibrillation, long-term persistent atrial fibrillation, atrial tachycardia, atrial flutter, familial atrial fibrillation, idiopathic atrial fibrillation, lone atrial fibrillation, and any orphan atrial arrhythmia. Atrial arrhythmias may produce symptoms including, but not limited to, general fatigue, rapid heartbeat, irregular heartbeat, dizziness, shortness of breath, anxiety, syncope, heart failure, neck pounding, weakness, confusion, faintness, sweating, chest pain, chest pressure, chest fluttering, or any combination thereof. In certain embodiments of any of the compositions or methods described herein, said compositions of extracellular vesicles improve one or more symptoms of atrial arrhythmias. In certain embodiments, said composition of extracellular vesicles may reduce the duration of atrial fibrillation, wherein duration may refer to the length of time of a single episode of atrial fibrillation, comprising a reduction of 1 s or more, any values defining a range therein, for example, 1 sec, 2 sec, 3 sec, 4 sec, 5 sec, 6 sec, 7 sec, 8 sec, 9 sec, 10 sec, 11 sec, 12 sec, 13 sec, 14 sec, 15 sec, 16 sec, 17 sec, 18 sec, 19 sec, 20 sec, 21 sec, 22 sec, 23 sec, 24 sec, 25 sec, 26 sec, 27 sec, 28 sec, 29 sec, 30 sec, 31 sec, 32 sec, 33 sec, 34 sec, 35 sec, 36 sec, 37 sec, 38 sec, 39 sec, 40 sec, 41 sec, 42 sec, 43 sec, 44 sec, 45 sec, 46 sec, 47 sec, 48 sec, 49 sec, 50 sec, 51 sec, 52 sec, 53 sec, 54 sec, 55 sec, 56 sec, 57 sec, 58 sec, 59 sec, 60 sec, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, 24 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years or more. In certain embodiments, said composition of extracellular vesicles may reduce the incidence of atrial fibrillation, wherein incidence may refer to the length of time of a between episodes of atrialfibrillation, comprising a reduction of 1 s or more, any values defining a range therein for example, 1 sec, 2 sec, 3 sec, 4 sec, 5 sec, 6 sec, 7 sec, 8 sec, 9 sec, 10 sec, 11 sec, 12 sec, 13 sec, 14 sec, 15 sec, 16 sec, 17 sec, 18 sec, 19 sec, 20 sec, 21 sec, 22 sec, 23 sec, 24 sec, 25 sec, 26 sec, 27 sec, 28 sec, 29 sec, 30 sec, 31 sec, 32 sec, 33 sec, 34 sec, 35 sec, 36 sec, 37 sec, 38 sec, 39 sec, 40 sec, 41 sec, 42 sec, 43 sec, 44 sec, 45 sec, 46 sec, 47 sec, 48 sec, 49 sec, 50 sec, 51 sec, 52 sec, 53 sec, 54 sec, 55 sec, 56 sec, 57 sec, 58 sec, 59 sec, 60 sec, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, 24 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years or more.

[0057] In certain embodiments, the compositions described herein can be used to treat or prevent atrial arrythmias, ventricular arrhythmias, inflammation, inflammasome inactivation, atrial or fibrillation.

[0058] In another embodiment, the compositions as described herein may be used to inhibit or treat conditions in other tissues and / or systems, unrelated to the heart, for example, but not wishing to be limiting, preventing, inhibiting or treating inflammation in non-cardiac cells, tissues, organs or systems or a combination thereof.

[0059] In an embodiment of the present invention, there is provided a composition comprising a hydrogel and extracellular vesicles. In certain embodiments of the present invention, there is provided a composition comprising a polymer hydrogel and extracellular vesicles, and / or a gelation agent, optionally wherein the polymer hydrogel and the gelation agent are thermoresponsive / thermosensitive.

[0060] Hydrogels

[0061] Hydrogels are 3D networks composed of hydrophobic polymers made by crosslinking water-soluble polymers. Hydrogels are able retain a substantial amount of water within their network without disturbing their original structure. This allows the hydrogels to be flexible and swell. Moreover, hydrogels also contain a large number of polar functional groups such as amines, amides, carboxylic acids, hydroxyl groups, and / or sulphonic acid groups. Among the factors that enhance the swelling ability of hydrogels are ionic strength, electric field, pH, and temperature. Hydrogels can be made from natural or synthetic polymers. Hydrogels are also porous and resemble living tissue in this way.

[0062] Chitosan, derived from the deacetylation of chitin, is an abundant natural biopolymer. Chitosan contains a number of reactive functional groups, such as amino and hydroxyl groups, which facilitate the synthesis of 3D hydrogels. Chitosan hydrogels typically possess good mechanical strength and low toxicity, making them highly biocompatible. In the exemplary embodiment of the present invention, the composition comprises a chitosan hydrogel, however in other embodiments alternative natural biopolymers may be used such as alginate, starch, cellulose, carboxymethylcellulose, fibrin, gelatin, collagen, and hyaluronic acid, without being limiting. The use of natural biopolymers offers the advantage of being biocompatible, non-toxic, and biodegradable. Alternatively, biocompatible synthetic polymers may be used such as poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA), poly(glycolic acid) (PGA), poly(3-hydroxybutyrate)-co-3-hydroxyvalerate (PHBV), poly(hydroxybutyrate) (PHB), or other families, such as poly(cyanoacrylates), poly(p-dioxanone) (PPDO), poly(acrylic acid) (PAA), poly(amides) (PA), poly(anhydrides), poly(ethylene glycol) (PEG), poly(ortho esters) (PES), poly(vinyl alcohol) (PVA), and PLGA-PEG-PLGA triblocks without being limiting. Synthetic polymers have the advantages of typically being stronger and longer lasting than natural polymers. They also can be produced with high purity and reproducibility. There is also the possibility of using semi-synthetic polymers, which are a mix of natural and synthetic polymers, and provide some of the advantages of each group.

[0063] In certain embodiments, the composition of the present invention comprises a polymer hydrogel of chitosan, alginate, starch, cellulose, carboxymethylcellulose, fibrin, gelatin, collagen, hyaluronic acid, poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA), poly(glycolic acid) (PGA), poly(3-hydroxybutyrate)-co-3-hydroxyvalerate (PHBV), poly(hydroxybutyrate) (PHB), poly(cyanoacrylates), poly(p-dioxanone) (PPDO), poly(acrylic acid) (PAA), poly(amides) (PA), poly(anhydrides), poly(ethylene glycol) (PEG), poly(ortho esters) (PES), poly(vinyl alcohol) (PVA), PLGA-PEG-PLGA blocks, or a combination thereof.

[0064] In certain embodiments, the composition of the present invention comprises a polymer hydrogel of chitosan, different grades of chitosan, or a combination of different molecular weights of chitosan.

[0065] In an embodiment of the invention, the therapeutic effect of the compositionincluding, without limitation, prevention, inhibition or reduction of NLRP3 activation, IL- 18 production, IL- 1 production, or inflammasome activation is achieved independent of the polymer selected to prepare the hydrogel.

[0066] In an embodiment of the invention, the therapeutic effect of the compositionincluding, without limitation, prevention, treatment of attenuation of atrial arrhythmia, atrial fibrosis, or inflammation in a patient is achieved independent of the polymer selected to prepare the hydrogel.

[0067] In an embodiment of the invention, the therapeutic effect of the compositionincluding, without limitation, prevention, inhibition or reduction of NLRP3 activation, IL- 18 production, IL- 10 production, or inflammasome activation; or prevention, treatment of attenuation of atrial arrhythmia, atrial fibrosis, or inflammation in a patient; is achieved independent of the polymer selected to prepare the hydrogel, wherein the hydrogel composition is configured to meet the injectability, thermoresponsive handling, and in situ gelation requirements described herein.

[0068] In certain embodiments, the therapeutic effect is obtained irrespective of whether the polymer selected for the hydrogel is natural, synthetic, semi-synthetic, or a combination thereof, including but not limited to polymers selected fromalginate, starch, cellulose, carboxymethylcellulose, chitosan fibrin, gelatin, collagen, hyaluronic acid, poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA), poly(glycolic acid) (PGA), poly(3-hydroxybutyrate)-co-3-hydroxyvalerate (PHBV), poly(hydroxybutyrate) (PHB), poly(cyanoacrylates), poly(p-dioxanone) (PPDO), poly(acrylic acid) (PAA), poly(amides) (PA), poly(anhydrides), polyethylene glycol) (PEG), poly(ortho esters) (PES), poly(vinyl alcohol) (PVA), PLGA-PEG-PLGA blocks, or combinations thereof.Gelation Agent

[0069] With respect to the above composition, in an exemplary embodiment 0-glycerophosphate acts as a thermosensitive / thermoresponsive gelation agent. 0-glycerophosphate is a crosslinker which converts the chitosan polymer solution into hydrogel under physiological pH and temperature (Figure 1). In addition to 0-glycerophosphate, several other thermosensitive / thermoresponsive gelation agents can be used for hydrogel systems that transition from solution to gel under physiological pH and temperature. Poloxamers (e.g., Poloxamer 407) are widely used triblock copolymers that gel at body temperature. Chitosan-based systems, including chitosan with glycerophosphate salts or alginate blends, are also effective. Methylcellulose (MC) forms thermoreversible gels upon heating, and Poly(N-isopropylacrylamide) (PNIPAAm) exhibits a lower critical solution temperature (LCST) near body temperature. Gelatin can be used as a thermoreversible gelation agent, often stabilized by cross-linking with genipin. Modified alginate systems, combined with calcium ions or other agents, can exhibit thermos responsiveness / thermosensitivity. Carboxymethylcellulose (CMC) and hyaluronic acid derivatives, such as hyaluronic acid combined with PNIPAAm or methylcellulose, offer additional options. Pluronic F127 (Poloxamer 407) is another common choice due to its gelation at temperatures above 30°C. Additionally, polyethylene glycol-poly(lactic acid) (PEG-PLA) copolymers and their derivatives exhibit temperature-responsive properties, making them suitable for various applications. Additional anionic cross-linkers that could be used include triphosphate groups (e.g. sodium tri -phosphate) sodium hydroxide, sodium hydrogen carbonate with phosphate buffer. Other chemical cross-linkers that could be used are genipen, glutaraldehyde, EDC-NHS (N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), citric acid, vanillin, d-fructose, and transglutaminase (T). However, change in crosslinker can change the physical and chemical properties of the hydrogel.

[0070] In addition to chitosan derivatives, other thermoresponsive polymers that may be used for hydrogel compositions are Poly (N-isopropylacrylamide) (PNIPAM), pluronics, Elastin-like polypeptides, Poly(N-vinylcaprolactam) (PNVCL), Poly(organophosphazene) (PPZN), PF 127, poly (e-caprolactone / polyethylene glycol (PCL / PEG), PCL-PEG-PCL, PEG-PCL-PEG), Polyphosphazene, Pluronic, cellulose, Gelatin, Collagen, Agarose, POEGMA (poly (oligo ethylene glycol methacrylate)), N-Isopropylacrylamide (NiPAAm),PLGA-PEG-PLGA, Poloxamer 407 (P407), Poloxamer 188, N-(2-hydroxypropyl)methacrylamide (HPMA), methylcellulose (MC) and their derivatives. These are natural, synthetic and semi-synthetic polymers which can gel or be modified to gel around physiological temperature (32-27 °C). Changing the hydrogel polymer could change the properties of hydrogel significantly (e.g. it will change the release profile of EVs, the mechanical strength of the hydrogel, and gelation time / temperature).

[0071] Hydrogel Characteristics and Release Profile

[0072] In a further embodiment, the chitosan hydrogel in the above composition comprises 1-1.5% w / v chitosan. However, in certain embodiments chitosan concentrations can range from 0.5-3.3% w / v, depending on the applications and formulations. Lower concentrations (0.5-1.0% w / v) are suitable for applications requiring softer hydrogels, improved diffusion of small molecules, or reduced viscosity during application. Moderate concentrations (1.0-1.5% w / v) provide a balance between mechanical strength, biocompatibility, and ease of handling, making them ideal for many biomedical applications. Higher concentrations (1.5-3.0% w / v) are beneficial for applications requiring stiffer hydrogels or enhanced structural integrity, such as tissue engineering scaffolds or prolonged drug release systems. However, changing concentration of polymer may have an effect on gelation temperature.

[0073] In certain embodiments, lower polymer concentrations are selected to achieve faster extracellular vesicle (EV) release, for example chitosan hydrogels at about 0.5-1.0% w / v, which provide softer gels and increased diffusion, thereby facilitating an earlier and more rapid liberation of EVs following administration; such selections can also reduce formulation viscosity during handling.

[0074] In certain embodiments, intermediate polymer concentrations, for example about 1.0-1.5% w / v chitosan, are employed to balance injectability and structural integrity in order to provide a controlled, staged release profile timed to anticipated inflammatory peaks (e.g., within the first 2-3 days post-procedure), with gelation and stability parameters adjusted via gelation agent concentration to fine-tune release kinetics without compromising biocompatibility.

[0075] In certain embodiments, higher polymer concentrations are used to delay EV release, for example chitosan hydrogels at about 1.5-3.0% w / v which form denser, stiffer networks that prolong diffusion-limited transport and retention of EVs within the gel matrix; in some cases, increasing polymer molecular weight and / or density further slows EV release without exceeding the mechanical constraints of atrial tissue.

[0076] In certain embodiments, the gelation agent level is co-varied with polymer concentration to modulate release timing, for instance 2%-5.5% [3-glycerophosphate, wherein lower gelation agent levels favor relatively quicker EV release and higher levels such as 8.5%-10% [3-glycerophosphate progressively slow release over at least 72 hours, thereby enabling independent or combined tuning of release via polymer and crosslinker densities to meet clinical timing needs.

[0077] In certain embodiments, the foregoing compositions are selected to achieve a viscosity window sufficient to maintain depot integrity while permitting target diffusion, recognizing that excessive viscosity may impede EV transport whereas very low viscosity may accelerate degradation; thus, polymer percentage and gelation agent are adjusted to remain within an operational range suitable for the desired release timeframe.

[0078] In a further embodiment, [3-glycerophosphate is present at a concentration of 2.0-10% w / v depending on the desired gelation properties and applications. In someembodiments, P-glycerophosphate is present at 5.5-8.5 % w / v, preferably 6.5-8.5 % w / v, in the above composition. Lower concentrations (2.0-5.5% w / v) may work for formulations requiring slower gelation or softer gels, while higher concentrations (8.5-10.0% w / v) can enhance gel stiffness and accelerate gelation under physiological conditions. However, concentrations above 8.5% may affect cell viability. The range of 6.5-8.5% w / v remains optimal for most applications due to its balance of gelation speed, stability, and mechanical properties.

[0079] In certain embodiments, the chitosan hydrogel comprises medium molecular weight (MMW) chitosan or high molecular weight (HMW) chitosan. “Medium molecular weight” chitosan is between 50 and 190 kDa, while “high molecular weight” chitosan is between 190 and 310 kDa (thus making “low molecular weight” chitosan less than 50 kDa). The practical difference between MMW and HMW is that the HMW chitosan hydrogel will have a higher mechanical strength and density. Increasing the density of the hydrogel can be advantageous to delay the release of the encapsulated EVs. This density can be fine-tuned to obtain the desired release profile for the given use. In some embodiments, it is envisioned that a mix of medium and high molecular weight chitosan could be used, depending on the desired properties of the hydrogel (e.g. EV release rate).

[0080] In any of the afore-mentioned embodiments, the composition provides a release profile configured to sustain extracellular vesicle (EV) delivery across to yield a prevention, inhibition, or reduction inNLRP3 activation, IL-18 production, IL-ip production, or inflammasome activation that is greater than that achieved by an equivalent number of suspended EVs administered directly.

[0081] In any of the afore-mentioned embodiments, the composition provides a release profile configured to sustain extracellular vesicle (EV) delivery across to yield a prevention, treatment or attenuation of atrial arrhythmia, atrial fibrosis or inflammation, that is greater than that achieved by an equivalent number of suspended EVs administered directly.

[0082] In an embodiment of the invention, a greater or better therapeutic effect (i.e. prevention, inhibition or reduction of NLRP3 activation, IL- 18 production, IL-ip production, or inflammasome activation; or prevention, treatment of attenuation ofatrial arrhythmia, atrial fibrosis, or inflammation in a patient) is achieved using fewer EVs than an equivalent dosage of suspended EVs administered without the hydrogel.

[0083] In a further embodiment, the method prevents or treats an atrial arrhythmia, atrial fibrosis, or inflammation using a reduced EV dose relative to suspended EV administration lacking the hydrogel, while achieving a comparable or superior therapeutic endpoint.

[0084] In certain embodiments, the composition exhibits dose-sparing or requires a reduced number of EVs to achieve the therapeutic effect of prevention, inhibition or reduction of NLRP3 activation, IL-18 production, IL-1 production, or inflammasome activation; or prevention, treatment of attenuation of atrial arrhythmia, atrial fibrosis, or inflammation in a patient; as compared to an equivalent administration of suspended EVs.

[0085] In certain embodiments, the composition achieves the therapeutic effect of prevention, inhibition or reduction of NLRP3 activation, IL- 18 production, IL- 10 production, or inflammasome activation; or prevention, treatment of attenuation of atrial arrhythmia, atrial fibrosis, or inflammation in a patient; at an EV dosage range of 105- 107particles as compared to an administration of suspended EVs in the range of 109- 1012particles to achieve a similar therapeutic effect.

[0086] In an embodiment of the invention, the hydrogel composition is configured to provide a faster release profile for clinical scenarios requiring early EV availability and a delayed or sustained release profile for clinical scenarios requiring prolonged EV availability, with release kinetics adjusted by polymer concentration, viscosity and molecular weight, and gelation agent level, whereas variable release kinetics are difficult to achieve via suspended EV administration lacking the hydrogel.

[0087] In an embodiment of the invention, the composition’s release kinetics are configured to follow first-order delivery / behavior, wherein from about 60% to about 90% of the encapsulated EVs are released over a period of about 24 to about 28 hours, thereby achieving an early therapeutic effect on inflammatory mediators while maintaining residual release profile to continue the therapeutic effect release thereafter.

[0088] In certain embodiments, the composition’s release kinetics are configured to provide a limited burst followed by sustained delivery, wherein no more than about 20% of the encapsulated EVs are released within the first about 6-12 hours and at least about 50% are released by about 48-72 hours, thereby aligning the release profile with anticipated inflammatory peaks / trajectories.

[0089] In a further embodiment, the release kinetics are configured to provide near zero-order delivery / behavior over at least about 48 to about 72 hours, maintaining a substantially constant EV release sufficient to inhibit or reduce NLRP3 activation, IL-18 production, IL-1 production, or inflammasome activation across the peak inflammatory window and into the early resolution phase.

[0090] In an embodiment of the invention, the release profile comprises an initial release phase (e.g., about 10% to about 30% release within the first about 6-12 hours) followed by a sustained phase (e.g., about 40% to about 70% cumulative release by about 48-72 hours), thereby initiating early paracrine signaling while supporting prolonged modulation of inflammatory pathways.

[0091] In certain embodiments, the release kinetics are selected to achieve a predefined therapeutic endpoint, including prevention or reduction of postoperative atrial fibrillation, atrial fibrosis, or inflammation, wherein the hydrogel maintains EV availability through approximately 48 to 72 hours after the procedure and for an additional period sufficient to exert a therapeutic effect.

[0092] Extracellular Vesicles

[0093] Extracellular vesicles are secreted into the extracellular space and can transfer their constituents to other cells by binding and fusing to their plasma membrane (Murphy, de Jong et al. 2019; "Extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking." Exp Mol Med 51(3): 1-12). Constituents of both the extracellular vesicle lipid bi-layer and the extracellular vesicle cargo can be incorporated into the receiving cell. In one embodiment, the transfer of protein from the extracellular vesicle to the receiving cell may determine absorption of extracellular vesicle constituents. In another embodiment, the transfer of lipids from the extracellular vesicle to the receiving cell may determine absorption of extracellular vesicle constituents. In certain embodiments, the human heart cellsabsorbing the extracellular vesicles may be immune cells, endothelial cells, myocytes and / or fibroblasts. In certain embodiments, the human heart cells absorbing the extracellular vesicles may be in vitro. In certain embodiments, the human heart cells absorbing the extracellular vesicles may be in vivo.

[0094] Extracellular vesicles may be obtained from bodily fluids (examples, including but not limited to, blood, milk, and urine), primary cell lines or conditioned cell culture medium. As bodily fluids may contain extracellular vesicles from many bodily sources, a person of skill will recognize that derivation of extracellular vesicles from cell culture medium minimizes heterogeneity of the vesicle source compared to bodily fluids. In some embodiments, said human heart cells may have been grown in vitro. In certain embodiments, said human heart cells may have been expanded in vitro. As will be understood, the presently described extracellular vesicles compositions, uses and methods thereof, may be amenable to Good Manufacturing Practices (GMP) standards and / or other such pharmaceutical industry standards. In some embodiments, GMP standards may be used, for example, culturing heart explant derived cells as described in US Pat. 11,083,756. Standard operating procedures (SOPs) may be developed for producing and using the extracellular vesicle compositions described herein, examples of which are provided in US Pat. 11,083,756. In certain embodiments, said human heart cells may have been grown and expanded in vitro using GMP conditions. In certain embodiments, GMP conditions may comprise controlled physiological cell culture conditions, said conditions comprising continuous atmosphere around 1% to around 10% oxygen, and around 1% to around 10% carbon dioxide, relative humidity around 50% to around 90% RH, and temperature around 32 °C to around 42 °C, serum-free, xenogen-free growth media, and extracellular vesicle depleted fetal bovine serum. In certain embodiments, said human heart cells may have been expanded in vitro using a GMP compliant enzyme to disassociate cells from culture plates or culture dishes, said GMP compliant enzyme comprising one or more of TrypLETM Select, collagenase I and collagenase II, or combination thereof. In certain embodiments, said extracellular vesicles are isolated after about 1 hours to about 196 hours or more incubation, any values defining a range therein, including, for example, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, 24 hrs, 25 hrs, 26hrs, 27 hrs, 28 hrs, 29 hrs, 30 hrs, 31 hrs, 32 hrs, 33 hrs, 34 hrs, 35 hrs, 36 hrs, 37 hrs, 38 hrs, 39 hrs, 40 hrs, 41 hrs, 42 hrs, 43 hrs, 44 hrs, 45 hrs, 46 hrs, 47 hrs, 48 hrs, 49 hrs, 50 hrs, 51 hrs, 52 hrs, 53 hrs, 54 hrs, 55 hrs, 56 hrs, 57 hrs, 58 hrs, 59 hrs, 60 hrs, 61 hrs, 62 hrs, 63 hrs, 64 hrs, 65 hrs, 66 hrs, 67 hrs, 68 hrs, 69 hrs, 70 hrs, 71 hrs, 72 hrs, 73 hrs, 74 hrs, 75 hrs, 76 hrs, 77 hrs, 78 hrs, 79 hrs, 80 hrs, 81 hrs, 82 hrs, 83 hrs, 84 hrs, 85 hrs, 86 hrs, 87 hrs, 88 hrs, 89 hrs, 90 hrs, 91 hrs, 92 hrs, 93 hrs, 94 hrs, 95 hrs, 96 hrs, 97 hrs, 98 hrs, 99 hrs, 100 hrs, 101 hrs, 102 hrs, 103 hrs, 104 hrs, 105 hrs, 106 hrs, 107 hrs, 108 hrs, 109 hrs, 110 hrs, 111 hrs, 112 hrs, 113 hrs, 114 hrs, 115 hrs, 116 hrs, 117 hrs, 118 hrs, 119 hrs, 120 hrs, 121 hrs, 122 hrs, 123 hrs, 124 hrs, 125 hrs, 126 hrs, 127 hrs, 128 hrs, 129 hrs, 130 hrs, 131 hrs, 132 hrs, 133 hrs, 134 hrs, 135 hrs, 136 hrs, 137 hrs, 138 hrs, 139 hrs, 140 hrs, 141 hrs, 142 hrs, 143 hrs, 144 hrs, 145 hrs, 146 hrs, 147 hrs, 148 hrs, 149 hrs, 150 hrs, 151 hrs, 152 hrs, 153 hrs, 154 hrs, 155 hrs, 156 hrs, 157 hrs, 158 hrs, 159 hrs, 160 hrs, 161 hrs, 162 hrs, 163 hrs, 164 hrs, 165 hrs, 166 hrs, 167 hrs, 168 hrs, 169 hrs, 170 hrs, 171 hrs, 172 hrs, 173 hrs, 174 hrs, 175 hrs, 176 hrs, 177 hrs, 178 hrs, 179 hrs, 180 hrs, 181 hrs, 182 hrs, 183 hrs, 184 hrs, 185 hrs, 186 hrs, 187 hrs, 188 hrs, 189 hrs, 190 hrs, 191 hrs, 192 hrs, 193 hrs, 194 hrs, 195 hrs, 196 hrs or more.

[0095] The interrogation of extracellular vesicle constituents has uncovered several proteins and nucleic acids related to heart conditions and diseases. By way of example, over-expression of miR-24 improved heart function and attenuated fibrosis in a rodent model of myocardial infarction (Barile, Lionetti et al. 2014; "Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction." Cardiovasc Res 103(4): 530-541). The person of skill in the art will further recognize that extracellular vesicle constituents may positively impact heart conditions or function (examples including, but not limited to, (Quattrocelli, Crippaetal. 2013; "Long-term miR-669a therapy alleviates chronic dilated cardiomyopathy in dystrophic mice." J Am Heart Assoc 2(4): e000284), (Yang, Qin et al. 2019; "An in Vivo miRNA Delivery System for Restoring Infarcted Myocardium." ACS Nano 13(9): 9880-9894), US 9,828,603) or negatively impact heart conditions and heart function (examples including, but not limited to, (Adam, Lohfelm et al. 2012; "Role of miR-21 in the pathogenesis of atrial fibrosis." Basic Res Cardiol 107(5): 278), (Cardin, Guasch et al. 2012; "Role for MicroRNA-21 in atrial profibrillatory fibroticremodeling associated with experimental postinfarction heart failure." Circ Arrhythm Electrophysiol 5(5): 1027-1035), (Cao, Shi etal. 2017; "miR-21 enhances cardiac fibrotic remodeling and fibroblast proliferation via CADM1 / STAT3 pathway." BMC Cardiovasc Disord 17(1): 88), (Zhi, Xu et al. 2019; "Effective Delivery of Hypertrophic miRNA Inhibitor by Cholesterol-Containing Nanocarriers for Preventing Pressure Overload Induced Cardiac Hypertrophy." Adv Sci (Weinh) 6(11): 1900023)).

[0096] The person of skill in the art having regard to the teachings herein will further recognize that it is contemplated that compositions of extracellular vesicles as described here may be further prepared by performing genetic reprogramming / genetic modification on the human heart cells including, but not limited to, to increase specific constituents (discussed in, for example, (Hall, Prabhakar et al. 2016; "Delivery of Therapeutic Proteins via Extracellular Vesicles: Review and Potential Treatments for Parkinson's Disease, Glioma, and Schwannoma." Cell Mol Neurobiol 36(3): 417-427)), decrease specific constituents (discussed in, for example, (Pfeifer, Werner et al. 2015; "Role and Function of MicroRNAs in Extracellular Vesicles in Cardiovascular Biology." Biomed Res Int 2015: 161393)), target extracellular vesicles to specific locations (discussed in, for example, (Kooijmans, Schiffelers et al. 2016; "Modulation of tissue tropism and biological activity of exosomes and other extracellular vesicles: New nanotools for cancer treatment." Pharmacol Res 111: 487-500)), target extracellular vesicles to specific cells (discussed in, for example, (Kooijmans, Schiffelers et al. 2016)), increase the production of extracellular vesicles (discussed in, for example, (Park, Bandeira et al. 2019; "Enhancement of therapeutic potential of mesenchymal stem cell-derived extracellular vesicles." Stem Cell Res Ther 10(1): 288)), thus boosting extracellular vesicle function, as described in the aforementioned references. By way of example, in certain embodiments it is contemplated that human heart cells as described herein may be subjected to genetic reprogramming to over-express the KCNN4 gene, which may promote extracellular vesicle production. Such approaches may involve lentivirus reprogramming, CRISPR / Cas9 editing, or other methods known to the person of skill such as mini circle DNA, for example.

[0097] Extracellular vesicles are naturally secreted, replication incompetent particles, enclosed by a lipid-bilay er, which can transfer its constituents to other cells (Murphy, de Jong et al. 2019). Herein, the term ‘constituents’ refers to the molecular contents of the extracellular vesicle, including but not limited to, proteins, lipids, nucleic acids, metabolites, and organelles. Extracellular vesicles may be referred to by the term poly disperse, as they are a population of particles with non-uniform size, shape, and constituents. Extracellular vesicle constituents can vary substantially with factors including but not limited to tissue type, cell type, and physiological conditions. As extracellular vesicle constituents and function can vary between similar cell types, even with modest modifications to their derivation, it is important to identify specific cells and conditions for these compositions. An exemplary nonlimiting example of extracellular vesicle compositional diversity including, but not limited to, extracellular vesicles derived from heart-explant derived cells prepared under physiological conditions were reported to have an average size of 120 nm, however, when prepared under standard conditions the average extracellular vesicle size was 148 nm (Mount, Kanda et al. 2019; "Physiologic expansion of human heart-derived cells enhances therapeutic repair of injured myocardium." Stem Cell Res Ther 10(1): 316). In addition, the same study reported changes to 51 miRNA constituents under physiological conditions compared to standard conditions. Therefore, determining an ideal composition of extracellular vesicles may be critical in developing more effective therapies for atrial arrhythmias, as well as other heart and non-heart conditions.

[0098] Extracellular vesicles may treat various conditions. In a preferred embodiment of the present invention, it is envisioned that the EVs are isolated from heart cells and used to treat atrial arrhythmias such as atrial fibrillation. However, it is also envisioned that this hydrogel platform may be used to treat other conditions with either heart-derived EVs or EVs derived from other cell types.

[0099] The chosen encapsulation technique for encapsulating extracellular vesicles (EVs) or other payloads in polymer hydrogels, should focus on preserving bioactivity and achieving the desired release profile.

[0100] Encapsulation by in situ gelation (mix-then-gel): In a common workflow, EVs can gently be mixed into a liquid polymer precursor under cold or ambientconditions, and the mixture can then be triggered to gel in situ at the target site. Thermoresponsive systems (for example, chitosan with a suitable gelation agent) remain flowable at room temperature for syringe or catheter delivery and undergo sol-gel transition at body temperature, physically entrapping EVs within the forming network. This approach minimizes shear and avoids organic solvents, helping to preserve EV integrity. Tuning polymer concentration, temperature, and gelation agent level adjusts mesh size and gelation time, which together control initial burst and sustained release.

[0101] Ionic or physical gelation (solvent-free, mild conditions): For ionic or physically gelling matrices, EVs are blended with the polymer solution and gelation is induced by adding a counterion or changing pH / ionic strength. Examples include alginate crosslinking with divalent cations (e.g., Ca2+) or chitosan systems triggered by basic / ionic additives. Because these methods run in aqueous media at mild temperatures, they are well-suited for EVs. The rate of ion diffusion and polymer concentration govern gel uniformity and pore size, enabling control over encapsulation efficiency and release kinetics.

[0102] Covalent and affinity-assisted encapsulation (for tighter retention): When slower release or higher retention is desired, gentle covalent or affinity strategies can be employed. Covalent options use biocompatible click-type reactions (e.g., thiolene, catechol-mediated, or carbodiimide couplings) executed under aqueous, nearneutral conditions to form or densify the network around EVs. Affinity approaches incorporate moi eties (e.g., heparin, charged groups, or specific peptide ligands) into the gel to interact with EV surface components, enhancing loading and retarding diffusion without harsh chemistry. These methods can be helpful in reducing burst release and extend delivery windows, provided reaction conditions that avoid damaging vesicle membranes.

[0103] Microgel, particle-in-gel, and layered depots (spatiotemporal control): EVs can be loaded into microgels or nanoparticles first, then dispersed within a secondary bulk hydrogel (particle-in-gel). Alternatively, layered architectures are formed by casting or co-injecting EV -rich and EV-poor zones, creating gradients that shape early versus late release. These constructs let you decouple injectabilityfrom release behavior, increase loading capacity, and spatially contain EVs, which is useful in dynamic environments like the pericardial space.

[0104] These approaches can be matched to clinical goals: use softer, larger-mesh networks or weaker affinity for faster release, and denser networks, stronger affinity, or particle-in-gel designs for delayed or sustained delivery.

[0105] In the present disclosure, the inventors leverage the electrostatic attraction between the negatively charged EVs and the positively charged amides present in the polymer (such as chitosan) to enhance EV retention. Furthermore, the inventors explored the effects of enhancing the molecular weight of chitosan, a modification that leads to a concurrent increase in both the number of amide groups and the density of the hydrogel. The inventors expected this tactic would not only improve the electrostatic encapsulation efficacy of EVs but also physically secure EVs within the hydrogel, to further sustain release. The inventors then assessed the persistence of the hydrogel at the injection site within the atria and explored the capacity of sustained EV delivery to reduce the EV dosage required for inhibiting atrial fibrosis, inflammation, and fibrillation.

[0106] In an embodiment, the EV s within the composition comprise one or more miRNA selected from miR-23a-3p, miR-199a-3p+miR-199b-3p, miR-4454+miR-7975, let-7a-5p, let-7b-5p, miR-125b-5p, miR-100-5p, miR-29b-3p, miR-21-5p, miR-191-5p, miR-199b-5p, miR-29a-3p, miR-22-3p, let-7i-5p, miR-181a-5p, miR-25-3p, miR-127-3p, let-7g-5p, miR-15b-5p, miR-320e, miR-221-3p, let-7d-5p, miR-16-5p, miR-424-5p, miR-3180, miR-374a-5p, miR-15a-5p, miR-130a-3p, miR-376a-3p, miR-199a-5p, miR-222-3p, miR-4286, miR-4516, miR-34a-5p, miR-1255a, miR-365a-3p+miR-365b-3p, miR-323a-3p, let-7c-5p, miR-27b-3p, miR-134-3p, miR-451a, miR-23b-3p, miR-423-5p, miR-28-5p, miR-125a-5p, miR-24-3p, miR-382-5p, miR-1228-3p, miR-20a-5p+miR-20b-5p, let-7e-5p, miR-18a-5p, miR-337-5p, miR-320e, miR-106a-5p+miR-17-5p, miR-19b-3p, miR-140-5p, let-7f-5p, miR-323a-5p, miR-148a-3p, miR-132-3p, miR-136-5p, miR-376c-3p, miR-379-5p, miR-26a-5p, miR-202-3p, miR-1290, miR-154-5p, miR-214-3p, miR-377-3p, miR-381-3p, miR-188-5p, miR-26b-5p, miR-363-3p, miR-337-3p, miR-137, miR-1973, miR-193a-5p+miR-193b-5p, miR-411-5p, a functional equivalent or a combination thereof.

[0107] In certain embodiments, the EV s in the composition may comprise one or more of the miRNAs identified in any one or more of the tables herein. In a particular embodiment, the EVs in the composition may comprise miR-199, miR22-3p, and miR-374a-5p.

[0108] In certain embodiments, the EV s in the composition may be substantially lacking or devoid of one or more of miR-1, miR-133, miR-328, miR-590 or any combination thereof. In certain embodiments, EVs may be substantially lacking or devoid of one or more of miR-210, miR-146a or a combination thereof. The aforementioned miRNA are known pathological transcripts (miR-1 (Girmatsion, Biliczki et al. 2009)), miR-133 (Tsoporis, Fazio et al. 2018), miR-328 (Small, Frost et al. 2010) and miR-590 (Shan, Zhang et al. 2009)). Furthermore, some miRNA known to be in heart derived EVs include those associated with reduced fibrotic atrial remodeling (miR-26 (Luo, Pan et al. 2013) and miR-29 (vanRooij, Sutherland et al. 2008)).In certain embodiments, the EVs in the composition may comprise one or more of the proteins from one or more of the tables herein.

[0109] In some embodiments, EV s in the composition may comprise one or more cytosolic markers defined by ALIX, any ANXA polypeptide, any ACT polypeptide, TSG101, any CHMP polypeptide, PDCD6IP, VPS4A, VPS4B, ARRDC1, any CAV polypeptide, any EHD polypeptide, RHOA, HSPA8, HSPA1A, any actin polypeptide, any tubulin polypeptide, GAPDH, HSP90AB1, ARF6, SDCBP, MAPT, HSC70, HSP70, HSP84, and TSG101, or a combination thereof.

[0110] In a further embodiment, EVs in the composition may comprise one or more transmembrane markers defined by CD3, CD9, CD14, CD37, CD41, CD45, CD47, CD53, CD55, CD59, CD61, CD63, CD73, CD81, CD82, CD90, any GNA polypeptide, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, any H2-A polypeptide H2-K, H2-D, H2-Q, any ITGA polypeptide, any ITGB polypeptide, TFR2, LAMP1, LAMP2, any SDC polypeptide, BSG, ADAM10, SHH, TSPAN8, PECAM1, ERBB2, EPCAM, CDC42a, GYP A, AChE-S, AChE-E, A A4, APP, ABCC1, FLOT1, FLOT2, ICAM1, EpCam, or a combination thereof.

[0111] In a further embodiment, the compositions described herein comprise one or more transmembrane markers defined by CD9, CD61, CD63, CD81, FLOT1, ICAM1, EpCam, or any combination thereof.

[0112] In a further embodiment, the compositions described herein may comprise one or more cytosolic markers defined by ALIX, ANXA5, and TSG101, or a combination thereof.

[0113] In certain embodiments, the EVs in the composition may comprise acetylcholinesterase activity.

[0114] In certain embodiments, the EV s in the composition may be substantially lacking or devoid of one or more of the group comprising GM130, APOA1, APOA2, APOB, APOB 100, ALB, ribosomal proteins, UMOD and protein / nucleic acid aggregates. In a preferred embodiment, the EVs in the composition are substantially lacking or devoid of GM130.

[0115] The dosage of the EV s in the compositions described herein may be from about 102particles to about IO20particles, any values defining a range therein, including, for example, IxlO2particles to 5xl02particles, 5xl02particles to IxlO3particles, IxlO3particles to 5xl03particles, 5xl03particles to IxlO4particles, IxlO4particles to 5xl04particles, 5xl04particles to IxlO5particles, IxlO5particles to 5xl05particles, 5xl05particles to IxlO6particles, IxlO6particles to 5xl06particles, 5xl06particles to IxlO7particles, IxlO7particles to 5xl07particles, 5xl07particles to IxlO8particles, IxlO8particles to 5xl08particles, 5xl08particles to IxlO9particles, IxlO9particles to 5xl09particles, 5xl09particles to IxlO10particles, IxlO10particles to 5xlO10particles, 5xlO10particles to IxlO11particles, IxlO11particles to 5xlOnparticles, 5xlOnparticles to IxlO12particles, IxlO12particles to 5xl012particles, 5xl012particles to IxlO13particles, IxlO13particles to 5xl013particles, 5xl013particles to IxlO14particles, IxlO14particles to 5xl014particles, 5xl014particles to IxlO15particles, IxlO15particles to 5xl015particles, 5xl015particles to IxlO16particles, IxlO16particles to 5xl016particles, 5xl016particles to IxlO17particles, IxlO17particles to 5xl017particles, 5xl017particles to IxlO18particles, IxlO18particles to 5xl018particles, 5xl018particles to IxlO19particles, IxlO19particles to 5xl019particles and 5xl019particles to IxlO20particles. In certainembodiments, the dosage of the extracellular vesicle composition may be related to the total number of particles received. In other embodiments, the dosage of the extracellular vesicle composition may be related to the number of particles received in each individual injection. In certain other embodiments, the dosage of the extracellular vesicle composition may be related to the sum of extracellular vesicles received over multiple inj ections, said inj ections may be received at the same time or different times.

[0116] In a preferred embodiment, the number of EVs in the composition is 106-109particles. The range of EVs in the composition could vary depending on the condition being treated and the target tissue. For conditions requiring localized or less intensive modulation, such as wound healing or certain inflammatory diseases, a lower range (10s— 106particles) may be sufficient. Conversely, for systemic conditions or those requiring higher therapeutic impact, such as cancer or fibrosis in larger organs, a higher range ( 109- 1012particles) may be more appropriate to ensure adequate biodistribution and therapeutic efficacy.

[0117] In certain embodiments, a unit dosage of EVs ranges from 1O2-1O20particles per milliliter, any values defining a range therein, including, for example, IxlO2particles / mL to 5xl02particles / mL, 5x102parti cl es / mL to IxlO3particles / mL, IxlO3particles / mL to 5xl03particles / mL, 5xl03particles / mL to lxl04particles / mL, IxlO4particles / mL to 5xl04particles / mL, 5x104parti cl es / mL to IxlO5particles / mL, IxlO5parti cles / mL to 5x105parti cles / mL, 5x105parti cl es / mL to IxlO6particles / mL, IxlO6parti cles / mL to 5x106parti cles / mL, 5x106parti cl es / mL to lxl07particles / mL, IxlO7parti cles / mL to 5x107parti cles / mL, 5x107parti cl es / mL to lxl08particles / mL, IxlO8parti cles / mL to 5x108parti cles / mL, 5x108parti cl es / mL to lxl09particles / mL, IxlO9particles / mL to 5xl09particles / mL, 5xl09particles / mL to IxlO10particles / mL, IxlO10particles / mL to 5xlO10particles / mL, 5xlO10particles / mL to IxlO11particles / mL, IxlO11particles / mL to 5xlOnparticles / mL, 5x10" particles / mL to IxlO12particles / mL, IxlO12particles / mL to 5xl012particles / mL, 5xl012particles / mL to IxlO13particles / mL, IxlO13particles / mL to 5xl013particles / mL, 5xl013particles / mL to IxlO14particles / mL, IxlO14particles / mL to 5xl014particles / mL, 5xl014particles / mL to IxlO15particles / mL, IxlO15particles / mL to 5xl015particles / mL, 5xl015particles / mL to IxlO16particles / mL,IxlO16particles / mL to 5xl016particles / mL, 5xl016particles / mL to IxlO17particles / mL, IxlO17particles / mL to 5xl017particles / mL, 5xl017particles / mL to IxlO18particles / mL, IxlO18particles / mL to 5xlO18particles / mL, 5xlO18particles / mL to IxlO19particles / mL, IxlO19particles / mL to 5xl019particles / mL and 5xl019particles / mL to IxlO20particles / mL.

[0118] In a further embodiment, the EVs are isolated from heart cells. EVs are isolated from the heart cells by the standard method known in the art, with several key modifications to enhance purity, eliminate contaminants, and improve EV yield. The standard procedure typically involves differential centrifugation, ultracentrifugation, or precipitation-based methods to isolate EVs. However, the inventors introduce a chromatography step following tangential flow filtration (TFF) to refine the isolation process, which provides superior separation of EVs from contaminants such as proteins, nucleic acids, and non-vesicular particles. Additionally, the inventors modified the tangential flow filtration parameters, including flow rate, pressure, and membrane pore size, to optimize EV yield and purity. These changes allow us to eliminate the ultracentrifugation steps traditionally used in EV isolation, thereby reducing potential contaminants, minimizing vesicle damage, and ensuring a more streamlined and scalable procedure.

[0119] The optimal viscosity range for a hydrogel to facilitate controlled EV release typically balances the need for sustained release with adequate diffusion. A hydrogel that is too viscous (>300 Pa-s) may excessively impede EV diffusion, reducing therapeutic efficacy, while a hydrogel with very tow viscosity (<50 Pa- s) may degrade too quickly, failing to sustain the release over the desired timeframe. Based on this, an ideal viscosity range would likely fall between 100-300 Pa-s, where the hydrogel provides sufficient structural integrity to maintain its presence in the target tissue while allowing gradual diffusion of EVs.

[0120] In a further embodiment, the composition may comprise EVs which comprise acetylcholinesterase activity. Acetylcholinesterase activity may be measured using FluoroCet Exosome Quantitation kit or equivalent methodology. In certain embodiments, acetylcholinesterase activity may be used to quantify extracellular vesicle particle number.

[0121] Desirable polymer and hydrogel characteristics

[0122] The selected polymer should be biocompatible and suitable for injection in human beings, exhibiting low cytotoxicity, low immunogenicity, and degradation byproducts compatible with tissue homeostasis. In some embodiments, material properties of the polymer can be carefully chosen to remain below relevant cardiac tissue mechanical limits while maintaining sufficient integrity for the intended therapeutic duration.

[0123] Thermoresponsiveness is also an important feature of the polymer system. The selected polymer should be capable of being in a liquid state at room temperature (about 18-25 °C) to facilitate preparation, loading, and precise delivery through a catheter or syringe, and capable of gelling or solidifying at approximately 37 °C to create an in situ gel depot at the target atrial site. Such temperature-triggered behavior enables surface deposition or intramural placement without intraprocedural curing steps and promotes localized retention of the payload following administration.

[0124] The injectability / viscosity window should also be suitable for clinical delivery. The selected polymer should be capable of passing smoothly through clinically used catheters or syringes without clogging or premature gelation, while forming a cohesive depot that resists washout and remains localized on or within atrial tissue. Gelation kinetics are tuned to avoid gelling within the delivery device yet to set sufficiently quickly at the target site to limit unintended dispersion.

[0125] The polymer matrix should be capable of encapsulating a therapeutically meaningful payload of extracellular vesicles (EVs) while preserving EV integrity and bioactivity. The gel network is configured to retain EVs within the depot and to provide a controlled release profile aligned with the intended therapeutic window. In some embodiments, adjusting polymer concentration, molecular weight, or crosslinking density modulates EV diffusion and release timing, enabling selection of softer, faster-releasing depots for early -phase delivery or stiffer, slower-releasing depots for prolonged therapeutic effect.

[0126] In an embodiment of the invention, the selected polymer is capable of being in a liquid state at approximately 18°C, at approximately 19°C, atapproximately 20°C, at approximately 21 °C, at approximately 22°C, at approximately 23°C, at approximately 24°C, or at approximately 25°C, and capable of solidifying of forming a gel at approximately 35°C, at approximately 36°C, at approximately 37°C, at approximately 38°C, or at approximately 37°C.

[0127] Gelation timing

[0128] In certain embodiments, the time required for gelation may be selected to balance procedural practicality with reliable gel formation and retention at the target site. A hydrogel composition that solidifies immediately upon exposure to physiologic conditions can be unsuitable because premature gelation may occur within the syringe, or at the needle tip, impeding delivery and risking incomplete or imperfect deposition. Conversely, a composition that requires an extended period to gel (an hour or more) can be impractical in the clinical setting, as it prolongs the procedure, increases patient and operator burden, and may allow undesirable dispersion or washout before the gel depot is established. Accordingly, the gelation interval is chosen to allow controlled placement and shaping of the depot while ensuring that solidification proceeds promptly enough to localize the material at the intended site.

[0129] In certain embodiments, the preferred gelation time will depend on what the clinician considers a clinically reasonable window that would permit smooth administration and reliable localization, preferably between 5-25 minutes. For instance, for an epicardial surface deposition via pericardial catheter, gelation may be desired to be rapid enough to resist displacement by cardiac motion and pericardial fluid circulation once delivered, yet not so rapid as to preclude even spreading and conformal contact with the atrial surface. In another instance, for delivery into myocardial tissue, gelation is desirably fast enough to minimize backflow along the inj ection tract and to confine spread within the myocardial plane, while allowing sufficient working time for precise needle placement and controlled injection.

[0130] In certain embodiments, the clinically reasonable gelation time may be tailored to the patient’s condition, the anatomical target, and the procedural workflow. For example, patients with hemodynamic instability, atrial dilation, orhigh pericardial fluid turnover may benefit from a shorter gelation interval to secure the depot promptly, whereas stable patients undergoing more extensive mapping or multi-site treatment may accommodate a modestly longer working time to facilitate accurate placement. The gelation profile can be tuned by adjusting polymer concentration, molecular weight, gelation agent concentration, and temperature handling conditions to achieve the desired balance between injectability and rapid, reliable setting at approximately 37 °C.

[0131] In certain embodiments, the gelation time is coordinated with viscosity and handling temperature to maintain a flowable state during preparation and delivery (e.g., at room temperature) and to trigger gelation upon reaching the target tissue. This coordination mitigates the risks of premature setting in the device and excessive delay at the target. By selecting a gelation interval aligned with procedural steps-catheter positioning, target confirmation, and controlled dispensing-the composition forms a cohesive depot that preserves extracellular vesicle integrity, achieves the intended spatial localization, and delivers the therapeutic payload over the desired timeframe.

[0132] In certain embodiments, the selected polymer hydrogel is capable of solidifying, forming a gel, or forming an in situ gel, at approximately 36°C - 38°C, or preferably 37°C, within 1 minute, within 2 minutes, within 3 minutes, within 4 minutes, within 5 minutes, within 6 minutes, within 7 minutes, within 8 minutes, within 9 minutes, within 10 minutes, within 11 minutes, within 12 minutes, within 13 minutes, within 14 minutes, within 15 minutes, within 16 minutes, within 17 minutes, within 18 minutes, within 19 minutes, within 20 minutes, within 21 minutes, within 22 minutes, within 23 minutes, within 24 minutes, within 25 minutes, within 26 minutes, within 27 minutes, within 28 minutes, within 29 minutes, within 30 minutes, within 31 minutes, within 32 minutes, within 33 minutes, within 34 minutes, within 35 minutes, within 36 minutes, within 37 minutes, within 38 minutes, within 39 minutes, within 40 minutes, wilthin 4 minutes, within 42 minutes, within 43 minutes, within 44 minutes, within 45 minutes, within 46 minutes, within 47 minutes, within 48 minutes, within 49 minutes, within 50 minutes, within 51 minutes, within 52 minutes, within 53 minutes, within 54 minutes, within 55 minutes, within 56 minutes, within 57minutes, within 58 minutes, within 59 minutes, within 60 minutes, or more, after being injected into or released into the target atrial space (myocardial, epicardial or pericardial space).

[0133] Release profile

[0134] In certain embodiments, the preferred extracellular vesicle (EV) release profde varies according to the clinical objective and timing of the biological process to be modulated. A relatively quicker release may be preferred when immediate paracrine signaling is advantageous -for example, to influence early wound-healing events shortly after the procedure-where a lower molecular weight hydrogel can facilitate earlier diffusion of EVs from the depot. Conversely, a more delayed or sustained release may be preferred when prolonged exposure is needed to maintain effects over days to weeks, in which case a denser or more highly crosslinked network, higher polymer concentration, or stronger polymer-EV interactions can slow transport and extend residence time within the gel.

[0135] In certain embodiments, the release profde may be aligned with anticipated inflammatory kinetics. For instance, if inflammation is expected to peak at approximately 48-72 hours following the procedure, the hydrogel is configured to retain and deliver EVs through that window and for a period thereafter to allow the EVs to exert their anti-inflammatory effects. In such cases, formulation parameters (e.g., polymer percentage, molecular weight, crosslinking density, charge balance, and gelation agent level) are selected to reduce early release while sustaining a therapeutically meaningful level across the peak inflammatory period and the subsequent resolution phase.

[0136] In certain embodiments, the release profde of EVs is adjusted to be within 5 minutes, within 6 minutes, within 7 minutes, within 8 minutes, within 9 minutes, within 10 minutes, within 11 minutes, within 12 minutes, within 13 minutes, within 14 minutes, within 15 minutes, within 16 minutes, within 17 minutes, within 18 minutes, within 19 minutes, within 20 minutes, within 21 minutes, within 22 minutes, within 23 minutes, within 24 minutes, within 25 minutes, within 26 minutes, within 27 minutes, within 28 minutes, within 29 minutes, within 30 minutes, within 31 minutes, within 32 minutes, within 33 minutes, within 34minutes, within 35 minutes, within 36 minutes, within 37 minutes, within 38 minutes, within 39 minutes, within 40 minutes, wilthin 4 minutes, within 42 minutes, within 43 minutes, within 44 minutes, within 45 minutes, within 46 minutes, within 47 minutes, within 48 minutes, within 49 minutes, within 50 minutes, within 51 minutes, within 52 minutes, within 53 minutes, within 54 minutes, within 55 minutes, within 56 minutes, within 57 minutes, within 58 minutes, within 59 minutes, within 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, 24 hrs, 25 hrs, 26 hrs, 27 hrs, 28 hrs, 29 hrs, 30 hrs, 31 hrs, 32 hrs, 33 hrs, 34 hrs, 35 hrs, 36 hrs, 37 hrs, 38 hrs, 39 hrs, 40 hrs, 41 hrs, 42 hrs, 43 hrs, 44 hrs, 45 hrs, 46 hrs, 47 hrs, 48 hrs, 49 hrs, 50 hrs, 51 hrs, 52 hrs, 53 hrs, 54 hrs, 55 hrs, 56 hrs, 57 hrs, 58 hrs, 59 hrs, 60 hrs, 61 hrs, 62 hrs, 63 hrs, 64 hrs, 65 hrs, 66 hrs, 67 hrs, 68 hrs, 69 hrs, 70 hrs, 71 hrs, 72 hrs, 73 hrs, 74 hrs, 75 hrs, 76 hrs, 77 hrs, 78 hrs, 79 hrs, 80 hrs, 81 hrs, 82 hrs, 83 hrs, 84 hrs, 85 hrs, 86 hrs, 87 hrs, 88 hrs, 89 hrs, 90 hrs, 91 hrs, 92 hrs, 93 hrs, 94 hrs, 95 hrs, 96 hrs, 97 hrs, 98 hrs, 99 hrs, 100 hrs, 101 hrs, 102 hrs, 103 hrs, 104 hrs, 105 hrs, 106 hrs, 107 hrs, 108 hrs, 109 hrs, 110 hrs, 111 hrs, 112 hrs, 113 hrs, 114 hrs, 115 hrs, 116 hrs, 117 hrs, 118 hrs, 119 hrs, 120 hrs, 121 hrs, 122 hrs, 123 hrs, 124 hrs, 125 hrs, 126 hrs, 127 hrs, 128 hrs, 129 hrs, 130 hrs, 131 hrs, 132 hrs, 133 hrs, 134 hrs, 135 hrs, 136 hrs, 137 hrs, 138 hrs, 139 hrs, 140 hrs, 141 hrs, 142 hrs, 143 hrs, 144 hrs, 145 hrs, 146 hrs, 147 hrs, 148 hrs, 149 hrs, 150 hrs, 151 hrs, 152 hrs, 153 hrs, 154 hrs, 155 hrs, 156 hrs, 157 hrs, 158 hrs, 159 hrs, 160 hrs, 161 hrs, 162 hrs, 163 hrs, 164 hrs, 165 hrs, 166 hrs, 167 hrs, 168 hrs, 169 hrs, 170 hrs, 171 hrs, 172 hrs, 173 hrs, 174 hrs, 175 hrs, 176 hrs, 177 hrs, 178 hrs, 179 hrs, 180 hrs, 181 hrs, 182 hrs, 183 hrs, 184 hrs, 185 hrs, 186 hrs, 187 hrs, 188 hrs, 189 hrs, 190 hrs, 191 hrs, 192 hrs, 193 hrs, 194 hrs, 195 hrs, 196 hrs or more.

[0137] Method of treating and Administration Strategy

[0138] In an embodiment of the present invention, there is provided a method of treating or preventing atrial fibrillation, wherein the method comprises administering the above composition to a subject in need thereof.

[0139] In another embodiment, there is provided a method of treating or preventing inflammation or fibrosis, wherein the method comprises administeringthe above composition to a subject in need thereof. For instance, a non-limiting example is the treatment of liver, kidney, cardiac, or pulmonary fibrosis. With respect to inflammation, treatment could include administration of the composition to treat post-injury or surgery -induced inflammation. Alternatively, the composition may be used to treat or prevent types of chronic inflammation associated with conditions or diseases such as arthritis, cardiovascular disease, cancer, Crohn’s disease, inflammatory bowel disease, lung diseases, and neurodegenerative diseases, without being limiting.

[0140] In an embodiment of the present invention, there is provided a method of preventing postoperative atrial fibrillation, wherein said method comprises administering the above composition at the time of surgery to a subject in need thereof. However, it is also envisioned that the above composition may be administered before surgery, for instance, several days or several weeks prior to surgery, without being limiting. This may be beneficial so that the tissue is well conditioned at the time of surgery.

[0141] In a further embodiment of the above methods, administering comprises intramyocardial injection of the composition into one or both atria of the heart. As mentioned previously, the composition will be in a liquid state under room temperature conditions (approximately 25 °C), which allows for the composition to be loaded into a syringe for inj ection. Once inj ected, the composition will form a gel structure at normal body temperature (37 °C), which allows for prolonged release of encapsulated EVs and efficient disease treatment. It is important to consider that the exact formulation of the composition is critical in order to obtain a composition with the desired properties. For instance, it is desirable to obtain a composition that is of the right viscosity for easy injection, but that forms a gel of sufficient density relatively quickly to ensure retention of the gel in the area of interest. However, gelation cannot be too rapid or it is conceivable that gelation would occur in the needle or quickly upon exiting the needle. This could negatively impact gel dispersion and therefore treatment of the disease or condition in question.

[0142] Administering may comprise injection of the composition onto one atrial surface, both atrial surfaces, one ventricle surface, both ventricle surfaces, or any combination thereof. In this embodiment, the composition is injected onto thesurface of the tissue, where it forms into a gel once exposed to body temperature. Again, the properties of the composition will be critical for effective treatment. Placing the hydrogel on the surface of the tissue has the added advantages of being less invasive to the patient, and the external positioning on the heart prevents exposure to perfusion, which may prolong the hydrogel’s life in the body. It is also envisioned that the hydrogel solution could be injected on surfaces around / near the heart, which may allow for more disperse release of EVs in the area, and may prevent any negative impacts of the hydrogel being placed directly on the tissue surfaces. Of course, since the composition may be used to treat inflammation or fibrosis associated with a number of different tissues, it is conceivable that the hydrogel solution be injected onto / into various other tissues (or in the vicinity of these tissues) where the EVs are supposed to take their effect.

[0143] In a further embodiment, the composition may be administered by injection into one atrial wall, both atrial walls, one ventricle wall, both ventricle walls, the septum, or any combination thereof. The advantage of injecting the hydrogel composition into the walls of the heart tissue is potentially better retention of the gel for extended release of EVs at the location of interest. Injection into the walls of the heart tissue would also reduce the exposure of the hydrogel composition to perfusion, again supporting the retention of the gel and prolonged release of EVs.

[0144] In a further embodiment, the composition may be formulated for inj ection onto the inner surface of one atrial wall, both atrial walls, one ventricle wall, both ventricle walls, or a combination thereof. This method of administration may be more advantageous forthose applications where prolonged EV release is not desired and a shortened treatment time is preferred, as the perfusion of the heart may degrade the hydrogel more quickly.

[0145] In certain embodiments, the composition is administered via a catheterbased approach in which a catheter is advanced within the pericardial space and positioned outside the heart over one or both atria, followed by injection of the composition onto the atrial epicardial surface to form a localized gel at physiological temperature for sustained release application. This route leverages the composition’s liquid state prior to delivery and thermogelling behavior at 37 °C. Procedurally, the catheter is placed outside the heart, and the composition is injected onto one or bothatrial surfaces where it solidifies in situ. Inventors have also contemplated related surface- and vicinity -based administration options which may be selected based on clinical considerations.

[0146] In a further embodiment, the hydrogel composition may be administered in the vicinity of the heart tissue. “In the vicinity” refers to a location in the body that is close enough to the heart to have a treatment effect when EVs are released from the hydrogel. This may allow for more disperse release of the EVs in the area of interest and potentially treat a wider area with less overall hydrogel being injected into the subject.

[0147] It is also contemplated that the hydrogel composition may be injected using a combination of the above methods. For instance, the hydrogel composition may be injected on the inner surface of one or more heart chambers, on the surface of one or more heart chambers, in the wall(s) of one or more heart chambers, and in the vicinity of one or more heart chambers, or any combination thereof.

[0148] In an embodiment of the invention, administering comprises endocardial delivery to an atrial endocardial surface, optionally at a single site or at multiple sites, under imaging guidance and / or mapping guidance.

[0149] In certain embodiments, administering comprises epicardial delivery to an atrial epicardial surface, optionally at a single site or at multiple sites, under imaging guidance and / or mapping guidance.

[0150] In a further embodiment, administering comprises transmural delivery spanning endocardial to epicardial layers, optionally at a single site or at multiple sites, under imaging guidance and / or mapping guidance.

[0151] It is envisioned that injection of the hydrogel composition in the above scenarios could be one injection or a series of injections, over a broad or small surface area, without being limiting.

[0152] Furthermore, these administration strategies could be applied to other tissues, if hydrogel compositions were formed with EVs to treat a disease associated with another tissue. For instance, if EVs were used to treat a liver condition, it isenvisioned that the hydrogel composition could be injected on the surface, in the vicinity, into the liver tissue itself, or any combination thereof.

[0153] In certain embodiments, the composition can be applied during open or minimally invasive surgical procedures. For example, during open cardiac surgery or a limited thoracoscopic / minithoracotomy approach, the hydrogel can be dispensed directly onto the atrial surface as a conformal layer, brushed into place, or spread within a defined region using a delivery tip configured for broad coverage.

[0154] In certain embodiments, transthoracic percutaneous delivery may be employed under imaging guidance without intravascular access. A fine needle may be advanced through the chest wall under ultrasound, fluoroscopy, or CT guidance to deliver the composition into or onto cardiac structures. For intramural placement, the needle may be advanced to a predetermined myocardial depth and the composition injected to form a confined depot. For surface placement, the tip may be positioned immediately superficial to the epicardium to deposit a cohesive layer that gels in situ.

[0155] In certain embodiments, an endocardial route may be used to reach atrial tissue from within the heart chambers. A transseptal or transvenous catheter can position a needle-equipped tip against the endocardial surface to inject the composition intramurally, forming a depot within the atrial wall. Alternatively, a controlled surface application to the endocardium may be performed using a low-profile delivery head designed to minimize washout by circulating blood.

[0156] In certain embodiments, the composition may be dispensed using specialized applicators that tailor distribution and adhesion. Spray or atomized delivery heads can create a thin, uniform coating over a defined area, while multilumen or coaxial tips can co-deliver gelation agent and polymer at the point of contact to accelerate set time and reduce run-off. Microneedle or microjet arrays can deliver controlled microdoses across a grid to generate a mosaic of micro-depots with predictable aggregate coverage and release kinetics.

[0157] In certain embodiments, retention strategies may be combined with any of the foregoing routes to enhance localization and durability. Examples include using a lightly adhesive primer, a biodegradable overlayer or barrier film to shield the gelfrom shear and fluid washout, or a mechanical scaffold (e.g., a sutureless frame or ring) that maintains the depot’s footprint during early cardiac cycles.

[0158] In certain embodiments, the compositions and methods disclosed herein are implemented using minimally invasive, catheter-based epicardial administration over one or both atria, with the hydrogel maintained in a liquid state prior to delivery and undergoing in situ gelation at physiological temperature upon deposition, as already described for surface injection and catheter placement outside the heart over the atria. Consistent with these teachings, large-animal feasibility studies demonstrate pericardial access, epicardial catheter positioning, and targeted atrial-surface delivery, thereby supporting clinical translation of mapping-guided epicardial localization and hydrogel deposition to the atrial epicardium (see Example 2). In some embodiments, intra-procedural confirmation and post-deposition assessment may utilize detectable labels within the hydrogel to verify localization and retention at the target epicardial site, analogous to fluorescence-based confirmation of hydrogel presence and localization demonstrated in vivo.

[0159] In an embodiment of the present invention, there is provided a method of treating atrial fibrillation, wherein the method comprises:a) catheter ablation;b) inserting a catheter outside the heart, over one or both atria; andc) injecting the composition defined above on the one or both atrial surfaces, wherein the composition will solidify on the atrial surface and gradually release encapsulated EVs for the treatment of atrial fibrillation.

[0160] Catheter ablation and the insertion of a catheter outside the heart may be performed as would be known in the art.

[0161] In certain embodiments, the extracellular vesicle composition may be administered in a treatment regimen simultaneously, sequentially, or in combination with other drugs, pharmaceutical compositions or treatments, either separately or as a combined formulation or combination, to treat or prevent arrhythmia. In certain embodiments, the extracellular vesicle composition may be administered in atreatment regimen simultaneously, sequentially, or in combination with rhythm control drugs (including but not limited to, for example, flecainide, propafenone, quinidine, sotalol, amiodarone and dronedarone), rate control drugs (including but not limited to, for example, beta blockers, calcium channel blocks and cardiac glycosides) or surgical treatment (including but not limited to, for example, electrical cardioversion, catheter ablation, pacemaker insertion, defibrillator implantation, and the Maze procedure), either separately or as a combined formulation or combination, to treat or prevent arrhythmia.

[0162] In a further embodiment, the compositions defined herein may be used to treat, prevent, or reduce inflammation, such as, but not limited to, atrial inflammation, ventricular inflammation, or both.

[0163] In another embodiment, the compositions as described herein may be used to inhibit or treat conditions in other tissues and / or systems, unrelated to the heart, for example, but not wishing to be limiting, preventing, inhibiting or treating inflammation in non-cardiac cells, tissues, organs or systems or a combination thereof.

[0164] Delivery System

[0165] The inventors have also envisioned a target atrial surface delivery system. In an embodiment of the invention, a target atrial surface (or a pericardial surface) delivery system is provided that is configured to dispense a flowable, thermoresponsive hydrogel composition onto a target atrial surface and to form an in situ gel depot at physiological temperature.

[0166] The system comprises a catheter, preferably having a distal outlet configured for delivery within the pericardial space. Optionally, the catheter may further comprise a handle configured for controlled infusion, and lumen geometry and materials selected to maintain injectability of the composition at handling temperatures while resisting premature gelation within the device.

[0167] In a further embodiment, the system includes a guidance system configured to localize the catheter relative to the target atrial surface / predefined atrial landmarks. The guidance system can provide imaging guidance, mappingguidance, or both, and is configured to furnish real-time spatial registration sufficient to confirm catheter location during targeting and delivery. Imaging guidance can include fluoroscopy, intracardiac echocardiography, transthoracic or transesophageal ultrasound, magnetic resonance imaging, or any combination thereof. Mapping guidance can include electroanatomic mapping configured to generate three-dimensional spatial information to guide the catheter to a predefined region and to verify localization prior to dispensing the composition.

[0168] In certain embodiments, the system further comprises a mechanical landmark configured to provide a visible positional reference on the epicardial surface. The accessory can include a pericardial wire with a mechanical marker that is visible under imaging or direct visualization and that serves as a spatial reference for confirming the target site before delivery. The mechanical landmark can be used alone or in combination with the guidance system, and is configured to improve targeting accuracy and reproducibility across multiple placements.

[0169] In a further embodiment, the system is configured to verify delivery and depot localization using a detectable label associated with the hydrogel composition. The composition can include a fluorescent label or other detectable marker, and the system can be used with an imaging modality suitable to detect the label intra-procedurally or post-procedurally to confirm surface-adherent deposition and retention at the target atrial site. The system can be employed to deliver the composition at one or more sites on the atrial surface, and in certain embodiments is configured to facilitate multi-site delivery to promote even distribution of the encapsulated extracellular vesicles across a predefined treatment region.

[0170] In certain embodiments, the system is adapted to support a range of procedural workflows and anatomical targets within the pericardial space. The catheter can be positioned over one or both atria, including contralateral surfaces relative to an access site, and the guidance system is configured to maintain localization despite cardiac motion.

[0171] In certain embodiments, the system further includes temperaturemanagement features, such temperature-controlled syringes or insulated lines, tomaintain the composition in a liquid state prior to delivery and to enable a reliable sol-gel transition upon contact with tissue at approximately 37 °C.

[0172] The present invention will be further illustrated in the following examples.Example 1: Density-Modulated Hydrogel Platforms for Controlled Extracellular Vesicle Release in Atrial Fibrosis and Inflammation Therapy

[0173] Atrial fibrillation (AF) — the most prevalent cardiac arrhythmia — continues to challenge clinical management due to its impact on quality of life and stroke risk. Current treatments, including antiarrhythmic drugs and ablation, are constrained by limited efficacy and side effects. The inventors present a strategy employing chitosan-based hydrogels for sustained, controlled release of extracellular vesicles (EVs) as a targeted AF intervention.MethodsFabrication of chitosan hydrogel

[0174] To fabricate the chitosan hydrogel, a chitosan stock solution (2-2.2% w / v) was prepared in 0.1 N HC1 and autoclaved at 121°C for 15 minutes to ensure sterility. A [3-glycerophosphate (P-GP) stock solution (50% w / v) was prepared in sterile water and filtered using a 0.2 pm filter for purification. Chitosan hydrogels (100 pL) were subsequently fabricated using 1% and 1.5% w / v chitosan, sourced as low and medium molecular weight (LMW and MMW; Sigma), combined with 8.5% w / v P-GP. Following the addition of -GP to the chitosan solution, the mixture was gently hand-tapped to ensure homogeneity. The solutions were incubated at 37°C to facilitate gelation.

[0175] To fabricate the HMW chitosan hydrogel, a chitosan stock solution of 2.2 % w / v was prepared. The stock solution was prepared in 0. IN HC1 with heating at around 40-50°C until it is dissolved. This solution was then combined with 8.5% w / v P-GP. Following the addition of P-GP to the chitosan solution, the mixture was gently hand-tapped to ensure homogeneity. The solutions were incubated at 37°C to facilitate gelation.Thermoresponsiveness and injectability of chitosan hydrogel:

[0176] To assess thermoresponsiveness, hydrogels were fabricated using different concentrations of P-glycerophosphate as described above. After mixing chitosan (1% w / v) with P-glycerophosphate (5.5-8.5% w / v), the solutions were kept at room temperature (25 °C) for 30-60 minutes to observe gelation. These solutions did not form a hydrogel at 25 °C, so they were then transferred to a water bath at 37 °C to evaluate gelation potential.

[0177] For injectability testing, the chitosan / p-glycerophosphate solution was allowed to gel at 37 °C, then subjected to manual shear stress to liquefy it. The liquefied solution was drawn into a syringe and then returned to a vial to re-gel at 37 °C.Atrial fibroblast isolation

[0178] Primary cultures of rat atrial fibroblasts were isolated from the hearts of 6- month-old Sprague-Dawley rats (Charles River) using enzymatic digestion with Collagenase Type II (Worthington Biochemical) at 37 °C. The cells were cultured in Dulbecco’s Modified Eagle High Glucose Medium, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 1% L-glutamine (Thermo Fisher Scientific), and 1% penicillin-streptomycin (Thermo Fisher Scientific). Fibroblasts at the second or third passage were used in all subsequent experiments. Manual cell counting was performed after staining with trypan blue dye (Sigma).Assessment of metabolic activity

[0179] The metabolic activity of rat atrial fibroblasts was analyzed using a fluorescent commercial assay (CCK-8, Dojindo) according to the manufacturer’s instructions. Briefly, cells were seeded in a 96-well plate and incubated overnight at 37 °C in a humidified atmosphere of 5% CO2. Chitosan hydrogels with varying concentrations of P-GP (5.5-8.5% w / v) were then prepared, and Dulbecco’s Modified Eagle Medium (Sigma) was added to the hydrogels, followed by incubation for 24 and 48 hours. Hydrogel extract medium, which is the culture medium exposed to the hydrogel, was collected, added to the cells and incubated for an additional 24 or 48 hours. This is done to determine the cytotoxicity of those components leaching from the hydrogel. Absorbance was measured at 450 nm using a microplate reader.Live-dead cell imaging

[0180] Cell viability studies were conducted using live-dead cell imaging. Hydrogel extract was collected at 24 and 48 hours as described above and applied to atrial fibroblasts for a 48-hour incubation. Following incubation, cells were washed and stained with Calcein AM (live cells) and ethidium homodimer (EthD-1, dead cells). Images were captured using a fluorescence microscope at 1 OX magnification. The average number of live and dead cells per field of view was quantified using ImageJ after 48 hours of cell culture and expressed as the average cell count per field of view.Viscosity analysis of chitosan hydrogel

[0181] Viscosity analysis of chitosan hydrogels with varying concentrations of f>- GP (5.5-8.5% w / v) was conducted using a Brookfield R / S Plus rheometer (Brookfield) at 37 °C. A C25-2 / 30 conical spindle was used to compress the material. The material was pre-heated at 37 °C at a shear rate of 6 sec1for 5 minutes. The viscosity of the chitosan hydrogels was then measured using a ramp rotational block, starting with a shear rate of 0-2 sec1over 30 seconds, followed by a ramp from 2 to 100 sec1over a period of 4 minutes.Swelling and degradation analysis

[0182] Chitosan hydrogels were fabricated using varying concentrations of -GP (5.5, 6.5, 7.5, and 8.5% w / v) and pre-heated at 37 °C for gelation. To each vial, phosphate-buffered saline was added, and samples were incubated at 37 °C with shaking at 120 rpm. At specified intervals, the supernatant was removed, gels were dried, and weight changes were analyzed. Weight gain was considered as swelling, while weight loss was considered as degradation.Human heart explant-derived cell culture, EV isolation and EV encapsulation

[0183] Human heart explant-derived cells were obtained from left atrial appendages donated by patients undergoing clinically indicated heart surgery, with informed consent under a protocol approved by the University of Ottawa Heart Institute Research Ethics Board. Cells were cultured using serum-free, xenogen-freemethods in the Ottawa Hospital Clinical Cell Manufacturing Facility as previously described.[9]Briefly, cardiac biopsies were minced, digested (Roche), and plated in Nutristem media (Biological Industries) under physiologic (5%) oxygen conditions in a GMP cell manufacturing facility. Once a week for 4 weeks, cells were harvested from the plated tissue using TrypLE Select (Thermo Fisher Scientific) for direct experimentation. Conditioned media was collected after 48 hours of culture in 1% EV-depleted serum (System Biosciences) and 1% oxygen. This conditioned media was then centrifuged at 10,000g for 30 minutes, followed by 100,000g for 3 hours, to pellet EVs.

[0010]

[0184] EVs (108particles) were encapsulated within a chitosan hydrogel, maintained on ice throughout the process. Gentle manual tapping was performed to facilitate the mixing of components. The solution was subsequently allowed to gel at 37°C. After a 10-minute incubation period, the hydrogels were immersed in phosphate-buffered saline within vials maintained at 37°C. An aliquot of the release medium, which contain the EVs released from the hydrogel, was collected from the vials at time points of 2, 6, 24, 48, and 72 hours. Following each collection, an equivalent volume of fresh dissolution medium was replenished to maintain the conditions. The quantification of released EVs was conducted using the micro-BCA protein assay.NLRP3 activation

[0185] THP-1 macrophages were treated with 108EVs or EVs encapsulated in chitosan hydrogel (1.5% w / v chitosan, 8.5% w / v 0-GP) in a transwell system, followed by exposure to 100 ng / mL LPS (Sigma) and 10 pM nigericin (Sigma) to activate the NLRP3 inflammasome. IL- 18 and IL - 1 P in the culture supernatant were measured using ELISA (Abeam). To assess long-term EV release from the hydrogel, encapsulated EVs were incubated in a transwell insert for 48 hours and then transferred to a new well with THP- 1 macrophages for up to 96 hours in the presence of LPS and nigericin. Suspended EVs and hydrogel alone were used as controls. Supernatants were collected and analyzed for IL-18 and IL- 1 using ELISA. Human caspase-1 activation was also assessed under similar conditions for up to 96 hours using a Caspase-1 ELISA (R&D Systems).- Ill -Surgical procedures

[0186] Rats were fed rat chow and housed under a 12: 12-hour light / dark cycle at 21°C and 50% humidity. All animals had free access to tap water and food. After preoperative buprenorphine (0.03 mg / kg subcutaneous), rats were anesthetized with 3% isoflurane, intubated, and ventilated. The thorax was shaved and sterilized with 2% w / v chlorhexidine gluconate in 70% v / v isopropyl alcohol. Animals were then randomized to sham operation (n=24; 12 female, 12 male), induction of sterile pericarditis with intramyocardial injection of 108atrial EVs (n=35; 18 female, 17 male), or induction of sterile pericarditis with intramyocardial injection of vehicle (n=34; 17 female, 17 male) using a sealed envelope approach. Animals randomized to sterile pericarditis underwent a thoracotomy and the atrial surfaces were dusted with sterile talcum powder (Thermo Fischer Scientific). Animals randomized to a sham procedure underwent a superficial incision that was closed in a manner indistinguishable from thoracotomy animals. Intramyocardial injections were performed using a total volume of 100-pL injected using a Hamilton microsyringe (27-gauge needle) into the left atrial wall at 5 separate injection points

[0011] . Injections into the atrial wall ensured that the hydrogels solidified within the wall, creating a localized depot for the delayed release of EVs. Moreover, the use of five separate injection sites allowed for even distribution of the hydrogel throughout the targeted region, maximizing tissue exposure to the therapeutic EVs and ensuring consistent delivery across the atrial wall. While fewer injection sites might be feasible, reducing the number could potentially limit the distribution and efficacy of the treatment. After surgery, animals were placed in a 30 degrees Celsius incubator with supplemental oxygen and moistened food until they returned to a physiological state. Additional doses of buprenorphine (0.03 mg / kg subcutaneous) were administered 6 and 12 hours postoperatively. A University of Ottawa Animal Care Technician monitored animals twice daily for 2 days after surgery. Investigative staff were blinded to the treatment received and analysis was conducted by individuals blinded to group allocation. Group allocations were kept in a separate password-protected list for unblinding after analysis of the primary study outcome was completed. Ninety-six rats underwent surgery, and all completed the study with no adverse events or protocol deviations.

[0187] Female Sprague Dawley rats (6 months old, Charles River) underwent induction of sterile pericarditis or sham operation under a protocol approved by the University of Ottawa Animal Care Committee. The inventor selected to focus on female recipients as previous work has shown recipient sex did not alter the ability of EVs to prevent inducible AF.

[0188] Middle-aged Male Sprague-Dawley rats (6-8 months old, Charles River) underwent induction of sterile pericarditis or sham operation under a protocol approved by the University of Ottawa Animal Care Committee. Rats were housed in an accredited animal facility for acclimatization. Prior to surgery, rats were given buprenorphine (0.03 mg / kg subcutaneous) prior to anesthesia and then intubated and ventilated. For the animal studies, the inventors elected to focus on male recipient as our previous studies has shown that receipt sex didn’t alter the ability of EVs to induce AF. For all the animal studies, lab staff were blinded to the treatment and analysis was done in a blinded group allocation manner.

[0189] A study was designed to test whether intramyocardial injection of hydrogel alone into the left atria would be able to stay in the atria. Chitosan-rhodamine conjugate was procured from PEGWorks (CS-323), 100 pl of intramyocardial injection of chitosan-rhodamine was administered using a 20G needle. Rats were sacrificed 1 hour post-surgery and left and right atria were extracted for histology. Histological studies were carried out by staining the rhodamine with antirhodamine antibody (green) (Rhodamine monoclonal antibody, Thermofisher (11H10), dilution 1 : 750) and DAPI (Blue). Images were taken using a fluorescent microscope.

[0190] Quantification of fibrosis: Rats were injected with EVs (IxlO9particles), hydrogel encapsulated EVs (100 ul of hydrogel containing 1 dose (i.e. 106, 107, or 109EV particles) of EVs, and hydrogel alone into the left atria followed by induction of sterile pericarditis. After 3 days, rats were euthanized and left atrial collagen was quantified based on hydroxyproline content measurement (K555-100, BioVision). Intramyocardial injection of different doses of suspended EVs and hydrogel encapsulated EVs (106, 107and 109) were given using a Hamilton micro syringe (27-gauge needle) into the left atrial wall at 6 separate injection points at the time of talc application.[la]A sham control group was included to demonstrate the backgroundinducibility . Animals were allowed to recover in a 30-degree Celsius incubator with supplied oxygen and moistened food. Rats were given buprenorphine (0.03 mg / kg subcutaneous) at 6 and 12 h post-operative surgery. Animals were scarified after 3 days to extract left atria for quantification of fibrosis using hydroxy proline assay.

[0191] In a similar experiment, rats were given different concentrations of suspended EVs and hydrogel encapsulated EVs without talc application to analyze NT4 expression where animals were scarified at 24 h and 3 days post-treatment.

[0192] RESULTS

[0193] The chitosan hydrogel was initially prepared with a 1% weight per volume (w / v) medium molecular weight chitosan solution (50-190 kDa). The inventors then added varying concentrations of [3-glycerophosphate (P-GP), a thermosensitive gelation agent, to initiate and control the gelation process at elevated temperatures. At room temperature, the mixture remained liquid (Figure 1). The vials were subsequently transferred to a 37°C non-circulatory water bath. Here, the 5.5% w / v P-GP mixture began to gel but did not achieve full gelation within 10 minutes. In contrast, concentrations between 6.5% and 8.5% w / v fully gelled within 10 minutes.

[0194] The inventors then analyzed the effect of -GP concentration on gel viscosity. As shown in Figure 2A, the hydrogel with the highest P-GP concentration (8.5% w / v) displayed the greatest viscosity at 260 Pascal-second (Pa.s). Lower concentrations corresponded to reduced viscosity, with the 5.5% w / v hydrogel showing negligible viscosity, likely due to its extended gelation time. These trends also influenced the swelling and degradation properties of the hydrogels (Figure 2B). Gels containing 6.5-8.5% w / v P-GP swelled up to 30% before beginning to degrade. Conversely, the 5.5% P-GP hydrogels started degrading after only two days while those with greater P-GP concentrations began degrading only after seven days.

[0195] In vivo, moderate swelling of the hydrogel is often beneficial as it allows the hydrogel to absorb surrounding fluids, creating a hydrated environment conducive to controlled release of encapsulated therapeutic agents, such as EVs, and facilitating integration with surrounding tissue. However, excessive swelling can be undesirable, as it may cause mechanical stress on the surrounding tissue or lead topremature degradation, compromising the hydrogel’s structural integrity and therapeutic efficacy.

[0196] In certain embodiments, pre-degradation moderate swelling of the hydrogel is advantageous because the gradual increase in water content enables the depot to conform more closely to irregular cardiac surfaces and fill small tissue gaps or microcrevices, thereby improving contact, coverage, and localization at the target site. Swelling can also increase interfacial adhesion and reduce backflow or washout immediately after placement, which is particularly beneficial in dynamic environments such as the pericardial space or within myocardial tissue planes. In addition, controlled swelling can serve to modulate release of payload by increasing the mesh size and diffusivity over time, allowing an initially protective, low-diffusion state that transitions into a sustained-release regime aligned with certain therapeutic requirements (for example, bridging the period before inflammatory peaks).

[0197] Where fluorescent or radiopaque labeling is employed, a modest increase in depot size due to swelling may further aid visualization and confirmation of placement. While beneficial, the extent and rate of swelling are preferably tuned (e.g., via polymer percentage, molecular weight, and gelation agent levels) to avoid excessive expansion that could impinge on adjacent structures or alter local mechanics.

[0198] In certain embodiments, minimal swelling of the hydrogel and quicker degradation of the hydrogel may be preferred in clinical setting, especially for patients with a severe heart condition, or where excessive strain on the atrial tissue may cause adverse effects.

[0199] To probe for a toxic effect attributable to unreacted crosslinkers or polymers, the inventors evaluated the effect of supernatants collected during the first 48 hours after complete gelation on primary rat atrial fibroblasts. As shown in Figure 2C, fibroblast proliferation was preserved when gels were formed using higher concentrations of -GP. In contrast, a colorimetric assay of metabolic activity indicated that the activity of all treated cells remained relatively unaffected by hydrogel supernatants. This observation was further supported by live-dead cellimaging, which demonstrated that any leaching components from the hydrogels had no effect on the proportion of live (green-colored) cells (Figure 6). The quantification data also revealed no differences in live / dead cells in all the treatments as compared to positive / negative control (Figure 2D).

[0200] The influence of varying -GP concentrations on EV release was then evaluated by quantifying protein release into the gel supernatant after EV encapsulation. As shown in Figure 2E, chitosan with 5.5% -GP showed a comparatively fast release of EVs while increasing -GP concentrations progressively slowed EV release out to 72 hours. Based on these findings, the inventors selected the chitosan hydrogel with 8.5% -GP for further characterization, as it demonstrated optimal cell viability, no evidence of cell toxicity, enhanced viscosity / stability and prolonged EV release compared to hydrogels with lower -GP concentrations.

[0201] Having established that increasing -GP concentrations slows EV release, the inventors explored the effects of polymer density and concentration on EV release using medium (MMW, 50-190 kDa) and high (HMW, 190-310 kDa) molecular weight chitosan at varying concentrations (1% and 1.5% w / v). Given that postoperative AF typically occurs 2-3 days after surgery, the ideal biomaterial for this condition would release EVs both before and during the peak of inflammation (i.e., 2-3 days after implant). The inventors based the rationale for increasing hydrogel density on the observation that high molecular weight chitosan forms denser gels, potentially delaying EV release. This densification results from its increased chain length, enhanced potential for hydrogen bonding, greater physical entanglement, and extensive cross-linking, all facilitated by its longer polymer chains and abundant active sites. As expected, rheological analysis showed that the mechanical strength of HMW chitosan hydrogel was twice that of MMW chitosan hydrogel (491±25 vs. 245±10 Pa-s, p<0.05; Figure 3A). Reassuringly, this improved mechanical strength remained below the mechanical tensile strength of the atrial tissue, suggesting it would not impact diastolic relaxation or impair atrial filling.[5]It is important to note that if the hydrogel's mechanical properties, such as viscosity or stiffness, exceed the mechanical tensile strength of the atrial tissue, it could impair the natural compliance of the atrium. This may hinder diastolic relaxation and reduceatrial filling, compromising overall cardiac function. Ensuring that the hydrogel's mechanical strength remains below that of the atrial tissue is crucial to avoid these adverse effects while still providing sufficient stability and controlled release of therapeutic agents like EVs. As predicted, encapsulating EVs within HMW chitosan hydrogel slowed EV release over the first 72 hours by 10±2% compared to MMW chitosan hydrogel (p<0.05, Figure 3B). Akin to findings with MMW hydrogels, the swelling and degradation profile of HMW hydrogels indicated that the gel initially swelled and then began to degrade after 8 days (p<0.05 vs. MMW hydrogels; Figure 3C). Moreover, applying HMW hydrogel supernatants collected during the first 48 hours after complete gelation had no effect on the proliferation, metabolic activity, or viability of rat atrial fibroblasts (Figure 3D). Taken as a whole, this data support the notion that altering the molecular weight of hydrogels effectively prolongs EV retention within chitosan, without compromising the mechanical strength or safety of the hydrogels.

[0202] Fibroblasts comprise approximately 25% of the cell population in the atria,[6]where they proliferate and differentiate into myofibroblasts upon exposure to pro-fibrotic stimuli. These changes have adverse effects on atrial physiology, ultimately impairing atrial function. In our previous study, the inventors demonstrated that EVs attenuate fibroblast proliferation and reduce fibrosis. In the current study, the inventors evaluated the effects of hydrogel-encapsulated EVs on atrial fibroblast proliferation using a transwell system. Rat atrial fibroblasts were exposed to TGF 1 to stimulate proliferation, as evidenced by an increase in manual cell counts and cell metabolic activity (Figure 4A). Cells exposed to suspended EVs and hydrogel-encapsulated EVs showed a reduction in atrial fibroblast proliferation by 51±9% and45±10%, respectively, in manual cell counts (p<0.05 vs. TGF-treated cells), and by l.l±0.1-fold and 1.2±0.1-fold, respectively, in cell metabolic activity (p<0.05 vs. TGF-treated cells).

[0203] Previous studies have shown that inactivation of the NLRP3 (NACHT, LRR, and PYD domains -containing protein-3) inflammasome plays a key role in EV -based prevention of atrial fibrillation (AF) by reducing inflammation in the postoperative period.[la]Based on these findings, the inventors evaluated the functionality of hydrogel-encapsulated EVs on the NLRP3 inflammasome usingTHP-1 macrophages activated by lipopolysaccharide (LPS) and nigericin in a transwell system. The application of either suspended EVs or EVs encapsulated in high molecular weight (HMW) chitosan inhibited NLRP3 activation and the production of downstream mediators, such as interleukin- 18 (IL-18), to a similar extent over the first 24 and 48 hours (Figure 4B).

[0204] To explore the potential for extended EV release, the HMW EV hydrogel was first incubated in a transwell for 48 hours and then transferred to a new transwell insert containing THP-1 macrophages to assess its sustained activity over 96 hours. As shown in Figure 4C, sufficient EVs remained in the hydrogel to suppress IL- 18 production for up to 96 hours, achieving a 96±2% reduction with hydrogel-encapsulated EV s compared to a 46±21 % reduction with suspended EV s at 24 hours (£><0.05 vs. untreated cells).

[0205] To assess the intrinsic effects of the hydrogel on inflammation, hydrogel alone was incubated in a transwell insert for 48 hours and then transferred to a new well containing THP-1 macrophages. Surprisingly, hydrogel alone also reduced IL-18 production by 39±2% at 96 hours, though this reduction was less pronounced than with hydrogel-encapsulated EVs (Figure 4C). The inventors speculate that this effect could be attributed to the inherent anti-inflammatory properties of the chitosan polysaccharide, which may suppress LPS-induced inflammation and oxidative stress by inhibiting the NF-KB pathway.[7]

[0206] Similarly, the inventors analyzed another downstream mediator of NLRP3, IL-10, and observed a 73±10% reduction in IL-1 expression at 96 hours with hydrogel-encapsulated EVs, compared to a 50±8% reduction with suspended EVs (£><0.05 vs. untreated cells). Hydrogel alone also inhibited IL-10 expression, showing a 27±12% reduction at 96 hours ( ><0.05 vs. untreated cells) (Figure 4D). These results were further corroborated by analyzing caspase- 1 expression in hydrogel-encapsulated EVs versus EVs alone. Sufficient EVs remained in the depleted hydrogel, resulting in a 51±5% reduction in caspase-1 expression at 72 hours and a 32±9% reduction at 96 hours (£><0.05 vs. untreated cells) (Figure 4E).

[0207] Inspired by these promising results, the inventors examined the retention of fluorescent-tagged chitosan hydrogels in the thin atrial wall (~2-3 mm).Histological analysis of rat left atrial sections revealed rhodamine-conjugated hydrogel presence (Figure 5 A), confirmed by anti -rhodamine staining (Figure 5B). The network pattern of the hydrogel within the atrial tissue suggests potential for enhancing and sustaining EV retention.

[0208] Previously, the inventors demonstrated that a single dose of suspended EVs provides a dose-dependent reduction in atrial fibrosis, inflammation, and fibrillation in a rat model of sterile pericarditis, with maximal effects observed after injecting 109EVs.[la’8]Given that the atrial muscle in rats is well-perfused, the inventors hypothesized that encapsulating EVs within hydrogels could enhance their retention, potentially preventing the significant loss of EVs that typically occurs due to normal blood flow and lymphatic clearance in the atria. To test this hypothesis, the inventors administered both suspended and hydrogel-encapsulated EVs into the rat atria. Three days later, the animals were euthanized, and atrial tissue was collected. Analysis revealed that rats receiving either hydrogel-encapsulated EVs or suspended EVs exhibited a significant reduction in pericarditis-induced atrial fibrosis, with reductions of 64±11% and 59±14%, respectively (hydroxyproline content; / V0.05 vs. vehicle control, Figure 5C). These results indicate that hydrogel-encapsulated EVs provide similar efficacy in reducing fibrosis compared to EVs alone after 3 days post-treatment.

[0209] The inventors further speculated that the hydrogel might enable effective delivery at lower doses of EVs. To test this, the inventors investigated whether a lower dose of hydrogel-encapsulated EVs could effectively reduce atrial fibrosis in the rat model of sterile pericarditis. For this purpose, different doses of EVs (106, 107, and 109EVs) were inj ected into the rat atria following encapsulation in hydrogel and compared with suspended EVs and vehicle alone. After 3 days, the animals were sacrificed, and atrial tissue samples were collected and analyzed by estimating hydroxy proline content. The results showed that an EV dose of 107led to a reduction in fibrosis with and without hydrogel encapsulation (40±20% and 49±9%, respectively). Surprisingly, the lowest dose of hydrogel-encapsulated EVs (106particles) significantly reduced fibrosis by 60±17% (hydroxyproline content; / V0.05 vs. vehicle control) compared to suspended EVs at the same dose, which showed no reduction (Figure 5C). These findings suggest that hydrogel encapsulation not onlyimproves EV retention within the atria but also enables lower EV doses to achieve the efficacy of a higher dose (109particles), which could be clinically relevant in treating patients with persistent AF. Therefore, encapsulating EVs within a biomaterial could not only improve efficacy at lower doses but also offer a cost-effective approach.

[0210] Example 2: Catheter-Based Epicardial Delivery of Thermoresponsive Chitosan / p-Glycerophosphate Hydrogels

[0211] This example demonstrates feasibility of minimally invasive, catheterbased access to the pericardial space (FIGURE 7), mapping-guided epicardial localization over atrial surfaces (FIGURE 8), and targeted delivery of a thermoresponsive chitosan / p-gly cerophosphate hydrogel that remains liquid at low temperature and undergoes in situ gelation at physiological temperature upon deposition on atrial epicardium. These studies complement the previously described administration methods in which the composition is injected onto one or both atrial surfaces and solidifies at body temperature. The hydrogel system employs chitosan and P-gly cerophosphate in concentrations previously shown to be thermoresponsive and biocompatible and to control viscosity and release, consistent with the formulations disclosed herein (e.g., 1% w / v chitosan with 5.5-8.5% w / v P-gly cerophosphate).

[0212] Materials and Methods

[0213] Hydrogel materials and preparation

[0214] A thermoresponsive chi tosan / p-gly cerophosphate hydrogel was used. A chitosan stock solution (2% w / v) was prepared in 0.1 N HC1 and sterilized by autoclaving at 121 °C, consistent with prior preparations described herein. A P-gly cerophosphate stock solution (50% w / v) was prepared in sterile water and sterile-filtered using a 0.2 pm filter. Immediately prior to use, the formulation was prepared by combining chitosan to a final concentration of 1% w / v with P-gly cerophosphate at concentrations ranging from approximately 5.5-8.5% w / v, followed by gentle mixing to ensure homogeneity, consistent with the thermoresponsive systems described herein. The formulation was maintained at low temperature (liquid phase)to prevent premature gelation during handling and catheter delivery, and it formed a gel upon warming to physiological temperature.

[0215] Large-animal feasibility studies (porcine model)

[0216] Large-animal studies evaluated catheter-based pericardial access, epicardial catheter positioning, and targeted hydrogel delivery to atrial surfaces. Delivery sites were localized using electroanatomic mapping and fluoroscopic guidance, with direct visualization where applicable, to provide three-dimensional spatial registration relative to atrial chambers and appendages. This mapping-guided epicardial localization aligns with catheter-based surface administration disclosed herein.

[0217] Pericardial access via atrial appendage

[0218] In one non-limiting embodiment, pericardial access was obtained via controlled perforation of the right atrial appendage using standard electrophysiology tools under imaging and mapping guidance. From this access point, a catheter was advanced within the pericardial space and positioned on the epicardial surface of the left atrium, including regions adjacent to the left atrial appendage, as shown in FIGURE 7. This approach demonstrates that atrial-appendage-based access can reproducibly reach contralateral atrial epicardial surfaces without open surgical exposure, consistent with surface-directed delivery contemplated herein. Other pericardial access routes may also be used without departing from the scope of the invention.

[0219] Electroanatomic mapping and catheter localization

[0220] Electroanatomic mapping catheters were positioned within both the left atrial appendage and the right atrial appendage to generate three-dimensional registration of intracardiac structures and to confirm the trajectory of the epicardial delivery catheter within the pericardial space, as shown in FIGURE 7. These data demonstrate that intracardiac mapping systems can guide and confirm epicardial delivery site localization relative to atrial anatomy, and that alternative imaging and localization modalities (including fluoroscopy, ultrasound / ICE, and MRI) may beemployed, consistent with the guidance and administration strategies disclosed herein.

[0221] Mechanical landmarking for target confirmation

[0222] To further confirm the epicardial target location prior to delivery, a mechanical marker coupled to a pericardial wire positioned within the pericardial space served as a spatial reference, as shown in FIGURE 8. The marker was readily visualized and enabled confirmation of the precise atrial surface region selected for delivery, providing a simple and reproducible method for correlating pericardial tool position with mapping / imaging data. This approach can be used alone or in combination with other localization techniques to effect surface administration contemplated herein.

[0223] Epicardial hydrogel delivery and in situ gelation

[0224] F oilowing localization, the hydrogel formulation was infused directly onto the epicardial surface of the atria via the pericardial catheter. The material underwent in situ gelation upon warming to physiological temperature, forming a localized deposit adherent to the epicardial surface that remained stably associated with the target region despite cardiac motion, consistent with the liquid-at-low-temperature / gel-at-37 °C behavior described herein. To confirm localization and retention, a version of the hydrogel incorporating a detectable fluorescent label was delivered, as shown in FIGURE 9. Fluorescent signal was detected at the intended atrial epicardial surface following delivery and remained localized for a sustained period, without visible dispersion throughout the pericardial space. Control conditions lacking a gelling formulation did not exhibit comparable surface retention. These observations are consistent with prior in vivo fluorescence-histology confirmation of hydrogel localization described herein.

[0225] Results

[0226] These studies establish the feasibility of: (i) safe and reproducible pericardial access via an atrial appendage (FIGURE 7); (ii) accurate catheter positioning on contralateral atrial epicardial surfaces (FIGURE 7); (iii) guidance and confirmation of epicardial localization using electroanatomic mapping and imaging(FIGURE 7); and (iv) targeted epicardial delivery of a thermoresponsive hydrogel that undergoes in situ gelation and sustained surface retention on atrial epicardium (FIGURE 8, 9). These capabilities align with and substantiate the catheter-based surface administration methods claimed and described herein.

[0227] Embodiments

[0228] In an embodiment of the invention, a composition is provided comprising a hydrogel and a therapeutically effective amount of extracellular vesicles (EVs).

[0229] In certain embodiments, the hydrogel or the composition comprising the hydrogel is thermoresponsive. In a further embodiment, the hydrogel is in a liquid state, or remains flowable, at a temperature ranging from about 18 °C to about 25 °C and solidifies or forms a gel at approximately 37 °C, or at a temperature ranging from 36 °C to 38 °C.

[0230] In an embodiment of the invention, the hydrogel swells, or is capable or swelling at approximately 37 °C to form the gel and continues to swell prior to degradation, preferably for a period ranging from about 1 hour to about 96 hours. In certain embodiments, the hydrogel swells upon administration at an atrial target tissue and continues to swell prior to degradation, preferably at the atrial target tissue for a period ranging from about 1 hour to about 96 hours.

[0231] In a further embodiment, the hydrogel has a viscosity ranging from about 50 Pa- s to about 550 Pa- s.

[0232] In an embodiment of the invention, the hydrogel comprises a low molecular weight polymer (less than about 50 kDa), a medium molecular weight polymer (ranging from about 50 kDa to about 200 kDa) or a high molecular weight polymer (greater than about 200 kDa but less than about 500 kDa), or a combination thereof. In certain embodiments, the polymer has a mechanical strength or tensile strength lower than a mechanical strength or tensile strength of an atrial target, preferably wherein the mechanical or tensile strength of the polymer is lower than about 550 Pa-s, lower than about 450 Pa-s, lower than about 350 Pa-s, lower than about 250 Pa- s, lower than about 150 Pa- s, or lower than about 50 Pa-s.

[0233] In a further embodiment, the low, medium or high molecular weight polymer is selected from alginate, starch, cellulose, carboxymethylcellulose, fibrin, gelatin, collagen, hyaluronic acid, poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA), poly(glycolic acid) (PGA), poly(3-hydroxybutyrate)-co-3-hydroxyvalerate (PHBV), poly(hydroxybutyrate) (PHB), poly(cyanoacrylates), poly(p-dioxanone) (PPDO), poly(acrylic acid) (PAA), poly(amides) (PA), poly(anhydrides), poly(ethylene glycol) (PEG), poly(ortho esters) (PES), poly(vinyl alcohol) (PVA), PLGA-PEG-PLGA blocks, or a combination thereof. In certain embodiments, the polymer is chitosan.

[0234] In an embodiment of the invention, the hydrogel additionally comprises a thermoresponsive gelation agent. In a further embodiment, the thermoresponsive gelation agent is selected from [3-glycerophosphate, poloxamers, methylcellulose, poly(N-isopropylacrylamide), sodium triphosphate, sodium hydroxide, or sodium hydrogen carbonate with phosphate buffer. In certain embodiments, the thermoresponsive gelation agent is [3-glycerophosphate. In certain embodiments, f>-glycerophosphate is present at a concentration of about 5.5-10% w / v, preferably at a concentration of about 6.5-8.5% w / v.

[0235] In an embodiment of the invention, the hydrogel comprises about 0.5-3.3% w / v chitosan. In certain embodiments, the hydrogel comprises about 1-1.5% w / v chitosan. In a further embodiment, the hydrogel comprises a low molecular weight chitosan, a medium molecular weight chitosan, a high molecular weight chitosan, or a combination thereof.

[0236] In an embodiment of the invention, the EVs are encapsulated within the hydrogel. In certain embodiments, the hydrogel is configured to release EVs with a predefined release profile around the atrial target tissue. In a further embodiment, the predefined release profile ranges from about 1 hour to about 240 hours. In certain embodiments, the composition has a slow-release profile, or a sustained release profile or a rapid release profile depending on polymer viscosity, polymer mass concentration, and concentration of gelation agent.

[0237] In an embodiment of the invention, the number of EV s encapsulated in the hydrogel ranges from about 10Al to about 10A9 particles. In certain embodiments,the number of EVs encapsulated in the hydrogel ranges from about 10A6 to about 10A9 particles. In a further embodiment, the EVs are isolated from heart cells.

[0238] In an embodiment of the invention, the EVs comprise one or more microRNA (miRNA). In certain embodiments, the one or more miRNA is selected from miR-23a-3p, miR-199a-3p+miR-199b-3p, miR-4454+miR-7975, let-7a-5p, let-7b-5p, miR-125b-5p, miR-100-5p, miR-29b-3p, miR-21-5p, miR-191-5p, miR-199b-5p, miR-29a-3p, miR-22-3p, let-7i-5p, miR-181a-5p, miR-25-3p, miR-127-3p, let-7g-5p, miR-15b-5p, miR-320e, miR-221-3p, let-7d-5p, miR-16-5p, miR-424-5p, miR-3180, miR-374a-5p, miR-15a-5p, miR-130a-3p, miR-376a-3p, miR-199a-5p, miR-222-3p, miR-4286, miR-4516, miR-34a-5p, miR-1255a, miR-365a-3p+miR-365b-3p, miR-323a-3p, let-7c-5p, miR-27b-3p, miR-134-3p, miR-451a, miR-23b-3p, miR-423-5p, miR-28-5p, miR-125a-5p, miR-24-3p, miR-382-5p, miR-1228-3p, miR-20a-5p+miR-20b-5p, let-7e-5p, miR-18a-5p, miR-337-5p, miR-320e, miR-106a-5p+miR-17-5p, miR-19b-3p, miR-140-5p, let-7f-5p, miR-323a-5p, miR-148a-3p, miR-132-3p, miR-136-5p, miR-376c-3p, miR-379-5p, miR-26a-5p, miR-202-3p, miR-1290, miR-154-5p, miR-214-3p, miR-377-3p, miR-381-3p, miR-188-5p, miR-26b-5p, miR-363-3p, miR-337-3p, miR-137, miR-1973, miR-193a-5p+miR-193b-5p, miR-411-5p, a functional equivalent, or a combination thereof. In a further embodiment, the one or more miRNA is miR-125b-5p, miR-21-5p, miR-22-3p, miR-199, miR-22-3p, miR-374a-5p, or a combination thereof.

[0239] In an embodiment of the invention, the composition comprises about 1-1.5% w / v chitosan with a viscosity of about 240-280 Pa- s, EVs in the range of about 10A6-10A7 particles, and [3-glycerophosphate in the range of about 6.5% to about 8.5%.

[0240] In certain embodiments, the hydrogel is labeled with a fluorescent label.

[0241] In an embodiment of the invention, the composition forms a gel in situ upon administration into the atrial target tissue of a patient.

[0242] According to the present invention there is provided a composition comprising chitosan hydrogel, extracellular vesicles (EVs), and [3-glycerophosphate.

[0243] In a further embodiment of the present invention, there is provided the above composition, wherein the chitosan hydrogel comprises 0.5%-3.3% w / v chitosan, or 1-1.5% w / v chitosan, or a combination of varying mass concentrations of chitosan.

[0244] In a In a further embodiment of the present invention, there is provided the above composition, wherein the chitosan hydrogel comprises 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 23%, 3.1%, 3.2%, 3.3% w / v chitosan, or a combination thereof.

[0245] In a further embodiment of the present invention, there is provided the above composition wherein [3-glycerophosphate is present at a concentration of 5.5-10 % w / v, preferably 6.5-8.5 % w / v, or a combination of varying concentrations of P-glycerophosphate.

[0246] In a further embodiment of the present invention, there is provided the above composition wherein the chitosan hydrogel comprises medium molecular weight chitosan or high molecular weight chitosan.

[0247] In a further embodiment of the present invention, there is provided the above composition, wherein the number of EVs is 106-l 09particles.

[0248] In a further embodiment of the present invention, there is provided the above composition wherein the EVs are isolated from heart cells.

[0249] In a further embodiment of the present invention, there is provided the above composition wherein the EVs comprise one or more miRNA.

[0250] In a further embodiment of the present invention, there is provided the above composition wherein the EVs comprise miR-199a-5p, miR-125b-5p, miR-21-5p, miR-22-3p, or combinations thereof. In certain embodiments, the EVs in the composition may comprise miR-199, miR22-3p, and miR-374a-5p.

[0251] In an embodiment of the invention, a method is provided for inhibiting NLRP3 activation comprising administering the composition described herein to a subject in need thereof. In certain embodiments, a method is provided for inhibitingor reducing the production of IL- 18 or IL- 1 for a period of about 1 hour to about 96 hours comprising administering the composition described herein to a subject in need thereof. In a further embodiment, a method is provided for preventing or reducing inflammasome activation comprising administering the composition described herein to a subject in need thereof.

[0252] In an embodiment of the invention, a method is provided for treating or reducing inflammation in a patient either pre-surgery, at the time of surgery, or postsurgery comprising administering the composition described herein to a subject in need thereof. In certain embodiments, a method is provided for treating or preventing atrial fibrillation, atrial fibrosis, or inflammation comprising administering the composition described herein to a subject in need thereof. In a further embodiment, a method is provided for treating or preventing atrial fibrillation, atrial fibrosis, or inflammation by preventing, inhibiting, or reducing NLRP3 activation comprising administering the composition described herein to a subj ect in need thereof. In certain embodiments, a method is provided for treating or preventing atrial fibrillation, atrial fibrosis, or inflammation by preventing, inhibiting, or reducing the production of IL-18 or IL-10 comprising administering the composition described herein to a subject in need thereof. In a further embodiment, a method is provided for preventing postoperative atrial fibrillation, atrial fibrosis, or inflammation, wherein the method comprises administering the composition described herein at the time of surgery to a subject in need thereof.

[0253] In an embodiment of the invention, administering comprises an intramy ocardial inj ection or an epicardial inj ection of the composition into the atrial target tissue, preferably into one or both atria of the patient’s heart. In certain embodiments, administering comprises inserting a catheter outside the heart, over one or both atria, preferably in the pericardial space, optionally using imaging guidance and / or mapping guidance, and administering the intramy ocardial injection or the epicardial injection of the composition into the atrial target tissue, preferably into one or both atria of the patient’s heart.

[0254] In an embodiment of the invention, a method of treating atrial fibrillation comprises: a) catheter ablation; b) inserting a catheter outside the heart, over one or both atria; and c) injecting the composition described herein on one or both atrialsurfaces, wherein the composition solidifies on the atrial surface, thereby gradually releasing encapsulated EVs for the treatment of atrial fibrillation.

[0255] In a further embodiment, a method of treating atrial fibrillation comprises : a) inserting a catheter outside the heart, preferably in the pericardial space, over one or both atria; and b) delivering the composition described herein by injecting the composition on the target atrial surface, preferably on the myocardial surface or the epicardial surface, wherein the composition is maintained in a liquid state prior to delivery and solidifies at physiological temperature on the atrial surface, thereby gradually releasing encapsulated EVs for the treatment of atrial fibrillation. In certain embodiments, the catheter is inserted using imaging guidance and / or mapping guidance. In a further embodiment, imaging guidance is provided by fluoroscopy, intracardiac echocardiography, transthoracic ultrasound, transesophageal ultrasound, magnetic resonance imaging, or any combination thereof. In certain embodiments, mapping guidance is provided by electroanatomic mapping, preferably configured to provide three-dimensional spatial registration sufficient to confirm catheter location. In a further embodiment, the method comprises mechanical landmarking of the myocardial surface or epicardial surface using a wire with a visible mechanical marker to assist with catheter insertion. In certain embodiments, the method further comprises confirming delivery of the composition using fluorescent imaging. In a further embodiment, delivering the composition involves injecting the composition at two or more sites at the target atrial surface, preferably at two, three, four, five, six or more sites, and in certain embodiments, more than one injection promotes even distribution of encapsulated EVs at the target atrial surface.

[0256] In an embodiment of the invention, a method of inhibiting, preventing, or reducing NLRP3 activation, IL-18 production, IL-1 production, or inflammasome activation in a subject comprises: maintaining a flowable composition as described herein at a temperature sufficient to keep the composition in a liquid phase prior to delivery; positioning a catheter relative to a target atrial surface, preferably epicardial or myocardial, under imaging guidance and / or mapping guidance; and dispensing the composition in an amount effective to form, upon exposure to physiological temperature, an in situ gel configured to release an effective amount ofEVs. In certain embodiments, imaging guidance is provided by fluoroscopy, intracardiac echocardiography, transthoracic ultrasound, transesophageal ultrasound, magnetic resonance imaging, or any combination thereof. In a further embodiment, mapping guidance is provided by electroanatomic mapping, preferably configured to provide three-dimensional spatial registration sufficient to confirm catheter location. In certain embodiments, the method further comprises mechanical landmarking of the myocardial surface or epicardial surface using a wire with a visible mechanical marker to assist with catheter insertion. In a further embodiment, the method further comprises confirming delivery of the composition by detecting the fluorescent label of the composition using fluorescent imaging. In certain embodiments, dispensing comprises injecting the composition (i) onto an atrial epicardial surface to form a surface-adherent depot or (ii) into atrial myocardial tissue to form an intramural depot. In a further embodiment, dispensing at the target atrial surface causes release of the EVs which provides the therapeutic effect of inhibiting, preventing, or reducing NLRP3 activation, IL-18 production, IL-1 production, or inflammasome activation in the subject. In certain embodiments, the method prevents or treats an atrial arrhythmia, atrial fibrosis, and / or inflammation in the subject.

[0257] In an embodiment of the invention, an atrial target tissue delivery system is provided comprising: a catheter configured to dispense a composition onto a target atrial surface; a guidance system configured to provide imaging and / or mapping guidance to localize the catheter relative to a predefined mechanical landmark on the target atrial surface; and a composition adapted to remain flowable prior to delivery and to undergo in situ gelation upon exposure to physiological temperature to form a depot at the target atrial surface. In certain embodiments, the composition is configured for release of an active agent. In a further embodiment, the guidance system provides imaging guidance by fluoroscopy, intracardiac echocardiography, transthoracic ultrasound, transesophageal ultrasound, magnetic resonance imaging, or any combination thereof. In certain embodiments, the guidance system provides mapping guidance by electroanatomic mapping, preferably configured to provide three-dimensional spatial registration sufficient to confirm catheter location. In a further embodiment, the mechanical landmark on the target atrial surface is a wire with a visible mechanical marker.

[0258] One or more illustrative embodiments have been described by way of example. It will be understood to persons skilled in the art that a number of variations and modifications may be made without departing from the scope of the invention as defined in the claims.Therefore, the present disclosure is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular embodiments disclosed above are illustrative only, as the present disclosure may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Although individual embodiments are discussed, the disclosure covers all combinations of all those embodiments. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. Also, the terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. It is therefore evident that the particular illustrative embodiments disclosed above may be altered or modified and all such variations are considered within the scope and spirit of the present disclosure. If there is any conflict in the usages of a word or term in this specification and one or more patent(s) or other documents that may be incorporated herein by reference, the definitions that are consistent with this specification should be adopted.

[0259] All citations and references are hereby incorporated by reference.REFERENCES[1] a)S. Parent, R. Vaka, J. S. Amant, S. Kahn, S. VanRemortel, C. Bi, D. Courtman, D. J. Stewart, D. R. Davis, Theranostics 2024, 14, 608; b)S. Parent, J. St Amant, S. Van Remortel, S. Kahn, R. Vaka, D. Courtman, D. J. Stewart, D. R. Davis, J ACC Clin Electrophysiol 2024, In press.[2] R. Vaka, S. Parent, Y. Risha, S. Khan, D. Courtman, D. J. Stewart, D. R. Davis, Mol Ther Nucleic Acids 2023, 32, 80.[3] B. Pena, M. Laughter, S. Jett, T. J. Rowland, M. R. G. Taylor, L. Mestroni, D. Park, Macromol Biosci 2018, 18, e!800079.[4] K. Malhotra, S. Van Remortel, V. Ly, D. R. Davis, Adv Healthc Mater 2023, 12, e2301980.[5] a)S. McLaughlin, B. McNeill, J. Podrebarac, K. Hosoyama, V. Sedlakova, G. Cron, D. Smyth, R. Seymour, K. Goel, W. Liang, K. J. Rayner, M. Ruel, E. J. Suuronen, E. I. Alarcon, Nat Commun 2019, 10, 4866; b)G. Musotto, A. Monteleone, D. Vella, S. Di Leonardo, A. Viola, G. Pitarresi, B. Zuccarello, A. Pantano, A. Cook, G. M. Bosi, G. Burriesci, Frontiers in Cardiovascular Medicine 2022, 9.[6] M. Litvinukova, C. Talavera-Lopez, H. Maatz, D. Reichart, C. L. Worth, E. L. Lindberg, M. Kanda, K. Polanski, M. Heinig, M. Lee, E. R. Nadelmann, K. Roberts, L. Tuck, E. S. Fasouli, D. M. DeLaughter, B. McDonough, H. Wakimoto, J. M. Gorham, S. Samari, K. T. Mahbubani, K. Saeb-Parsy, G. Patone, J. J. Boyle, H. Zhang, H. Zhang, A. Viveiros, G. Y. Oudit, O. A. Bayraktar, J. G. Seidman, C. E. Seidman, M. Noseda, N. Hubner, S. A. Teichmann, Nature 2020, 588, 466.[7] a)J. Tu, Y. Xu, J. Xu, Y. Ling, Y. Cai, International Journal of Biological Macromolecules 2016, 86, 848; b)W. Tao, G. Wang, X. Pei, W. Sun, M. Wang, Antioxidants (Basel) 2022, 11; c)H.-t. Liu, P. Huang, P. Ma, Q.-s. Liu, C. Yu, Y.-g. Du, Acta Pharmacologica Sinica 2011, 32, 478.[8] S. Parent, R. Vaka, Y. Risha, C. Ngo, P. Kanda, S. Nattel, S. Khan, D. Courtman, D. J. Stewart, D. R. Davis, JCI Insight 2023, 8, el 63297.[9] a)D. R. Davis, E. Kizana, J. Terrovitis, A. S. Barth, Y. Zhang, R. R. Smith, J. Miake, E. Marban, JMCC 2010, 49, 312; b)N. Latham, B. Ye, R. Jackson, B. Lam, D. Kuraitis, M. Ruel, E. J. Suuronen, D. J. Stewart, D. R. Davis, Circulation 2013, 128, SI; c)S. Mount, P. Kanda, S. Parent, S. Khan, C. Michie, L. Davila, V. Chan, R. A. Davies, H. Haddad, D. Courtman, D. J. Stewart, D. R. Davis, Stem Cell Research & Therapy 2019, 10, 316.

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Claims

WHAT IS CLAIMED IS:

1. A composition comprising a hydrogel and a therapeutically effective amount of extracellular vesicles (EVs).

2. The composition of claim 1, wherein the hydrogel is thermoresponsive.

3. The composition of claim 1 or claim 2, wherein the hydrogel is in a liquid state at a temperature ranging from 18°C - 25°C and solidifies / forms a gel at approximately 37°C.

4. The composition of claim 3, wherein the hydrogel swells at approximately 37°C to form the gel and continues to swell prior to degradation, preferably wherein the hydrogel swells for a period ranging from 1 hour to 96 hours.

5. The composition of claim 4, wherein the hydrogel swells upon administration at an atrial target tissue and continues to swell prior to degradation, preferably wherein the hydrogel swells at the atrial target tissue for a period ranging from 1 hour to 96 hours6. The composition of any one of claims 1-5, wherein the hydrogel has a viscosity ranging from 50 Pa- s to 550 Pa- s.

7. The composition of any one of claims 1-6, wherein the hydrogel comprises alow molecular weight polymer (less than 50 kDa), a medium molecular weight polymer (ranging from 50kDa - 200 kDa) or a high molecular weight polymer (greater than 200 kDa but less than 500 kDa), or a combination thereof.

8. The composition of claim 7, wherein the polymer has a mechanical strength / tensile strength lower than a mechanical strength / tensile strength of an atrial target, preferably wherein the mechanical / tensile strength of the polymer is lower than 550 Pa- s, lower than 450 Pa-s, lower than 350 Pa-s, lower than 250 Pa-s, lower than 150 Pa-s or lower than 50 Pa-s.

9. The composition of claim 7 or 8, wherein the polymer is alginate, starch, cellulose, carboxymethylcellulose, fibrin, gelatin, collagen, hyaluronic acid, poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA), poly(glycolic acid) (PGA), poly(3-hydroxybutyrate)-co-3-hydroxyvalerate (PHBV),poly(hydroxybutyrate) (PHB), poly(cyanoacrylates), poly(p-dioxanone) (PPDO), poly(acrylic acid) (PAA), poly(amides) (PA), poly(anhydrides), polyethylene glycol) (PEG), poly(ortho esters) (PES), poly(vinyl alcohol) (PVA), PLGA-PEG-PLGA blocks, or a combination thereof.

10. The composition of claim 7 or 8, wherein the polymer is chitosan.

11. The composition of any one of claims 1-10, wherein the hydrogel additionally comprises a thermoresponsive gelation agent.

12. The composition of claim 11 , wherein the thermoresponsive gelation agent is f>-glycerophosphate, Poloxamers, Methylcellulose, Poly(N-isopropylacrylamide), sodium triphosphate, sodium hydroxide, or sodium hydrogen carbonate with phosphate buffer.

13. The composition of claim 11, wherein the thermoresponsive gelation is 0-glycerophosphate.

14. The composition of any one of claims 10-13, wherein the hydrogel comprises 0.5-3.3% w / v chitosan.

15. The composition of any one of claims 10-13, wherein the hydrogel comprises 1-1.5% w / v chitosan.

16. The composition of any one of claims 10-15, wherein the hydrogel comprises a low molecular weight chitosan, a medium molecular weight chitosan, a high molecular weight chitosan, or a combination thereof.

17. The composition of any one of claims 13-16, wherein 0-glycerophosphate is present at a concentration of 5.5-10 % w / v, preferably at a concentration of 6.5-8.5 % w / v.

18. The composition of any one of claims 1-17, wherein the EVs are encapsulated within the hydrogel.

19. The composition of any one of claims 1-18, wherein the hydrogel is configured to release EVs with a predefined release profile around the atrial target tissue.

20. The composition of any one of claims 1-19, wherein the predefined release profile of the hydrogel ranges from 1 hour to 240 hours.

21. The composition of any one of claims 1-20, wherein the number of EVs encapsulated in the hydrogel ranges from 10x-109particles.

22. The composition of any one of claims 1-20, wherein the number of EVs encapsulated in the hydrogel ranges from 106-109particles.

23. The composition of any one of claims 1-22, wherein the EVs are isolated from heart cells.

24. The composition of any one of claims 1-23, wherein the EVs comprise one or more miRNA.

25. The composition of claim 24, wherein the one or more miRNA is miR-23a-3p, miR-199a-3p+miR-199b-3p, miR-4454+miR-7975, let-7a-5p, let-7b-5p, miR-125b-5p, miR-100-5p, miR-29b-3p, miR-21-5p, miR-191-5p, miR-199b-5p, miR-29a-3p, miR-22-3p, let-7i-5p, miR-181a-5p, miR-25-3p, miR-127-3p, let-7g-5p, miR-15b-5p, miR-320e, miR-221-3p, let-7d-5p, miR-16-5p, miR-424-5p, miR-3180, miR-374a-5p, miR-15a-5p, miR-130a-3p, miR-376a-3p, miR-199a-5p, miR-222-3p, miR-4286, miR-4516, miR-34a-5p, miR-1255a, miR-365a-3p+miR-365b-3p, miR-323a-3p, let-7c-5p, miR-27b-3p, miR-134-3p, miR-451a, miR-23b-3p, miR-423-5p, miR-28-5p, miR-125a-5p, miR-24-3p, miR-382-5p, miR-1228-3p, miR-20a-5p+miR-20b-5p, let-7e-5p, miR-18a-5p, miR-337-5p, miR-320e, miR-106a-5p+miR-17-5p, miR-19b-3p, miR-140-5p, let-7f-5p, miR-323a-5p, miR-148a-3p, miR-132-3p, miR-136-5p, miR-376c-3p, miR-379-5p, miR-26a-5p, miR-202-3p, miR-1290, miR-154-5p, miR-214-3p, miR-377-3p, miR-381-3p, miR-188-5p, miR-26b-5p, miR-363-3p, miR-337-3p, miR-137, miR-1973, miR-193a-5p+miR-193b-5p, miR-411-5p, a functional equivalent or a combination thereof.

26. The composition of claim 24, wherein the one or more miRNA is miR-125b-5p, miR-21-5p, miR-22-3p, miR-199, miR22-3p, miR-374a-5p, or a combination thereof.

27. The composition of any one of claims 10-26, wherein the composition comprises:- 1-1.5% w / v chitosan with a viscosity of 240 - 280 Pa-s- EV’s in the range of 106- 107; and- P-glycerophosphate in the range of 6.5% to 8.5%.

28. The composition of any one of claims 1-27, wherein the hydrogel is labelled with a fluorescent label.

29. A composition comprising:- 1-1.5% w / v chitosan with a viscosity of 240 - 280 Pa-s- EV’s in the range of 106- 107; and- P-glycerophosphate in the range of 6.5% to 8.5%.

30. The composition of any one of claims 1 -29, wherein the composition forms a gel in situ upon administration into the atrial target tissue of a patient.

31. A method of inhibiting or reducing the production of 1 L- 18 or IL- 1 P for a period of 1 hour - 96 hours comprising the step of administering the composition as defined in any one of claims 1-30 to a subject in need thereof.

32. A method of inhibiting, preventing or reducing NLRP3 activation or inflammasome activation comprising the step of administering the composition as defined in any one of claims 1-30 to a subject in need thereof.

33. A method of treating or reducing inflammation in a patient either pre-surgery , at the time of surgery or post-surgery comprising the step of administering the composition as defined in any one of claims 1-30 to a subject in need thereof.

34. A method of treating or preventing atrial fibrillation, atrial fibrosis or inflammation, comprising the step of administering the composition as defined in any one of claims 1-30 to a subject in need thereof.

35. A method of treating or preventing atrial fibrillation, atrial fibrosis or inflammation by preventing, inhibiting or reducing NLRP3 activation comprising the step of administering the composition as defined in any one of claims 1-30 to a subject in need thereof.

36. A method of treating or preventing atrial fibrillation, atrial fibrosis or inflammation by preventing, inhibiting or reducing the production of IL- 18 or IL- 1 , comprising the step of administering the composition as defined in any one of claims 1-30 to a subject in need thereof.

37. A method of preventing postoperative atrial fibrillation, atrial fibrosis or inflammation, wherein said method comprises administering the composition as defined in any one of claims 1-30 at the time of surgery to a subject in need thereof.

38. The method of any one of claims 31-36, wherein the step of administering the composition comprises administering an intramyocardial injection or an epicardial injection of the composition into the atrial target tissue, preferably into one or both atria of the patient’s heart.

39. The method of any one of claims 31-38, wherein administering comprises inserting a catheter outside the heart, over one or both atria, preferably in the pericardial space, preferably using imaging guidance and / or mapping guidance, and administering the intramyocardial inj ection or the epicardial inj ection of the composition into the atrial target tissue, preferably into one or both atria of the patient’s heart.

40. A method of treating atrial fibrillation, wherein the method comprises:a) catheter ablation;b) inserting a catheter outside the heart, over one or both atria; and c) injecting the composition as defined in any one of claims 1-30 on the one or both atrial surfaces, wherein the composition solidifies on the atrial surface, thereby gradually releasing encapsulated EVs for the treatment of atrial fibrillation.

41. A method of treating atrial fibrillation, wherein the method comprises:a) inserting a catheter outside the heart, preferably in the pericardial space, over one or both atria,b) delivering the composition as defined in any one of claims 1-30 by injecting the composition on the target atrial surface, preferably on the myocardial surface or the epicardial surface, wherein the composition is maintained in a liquid state prior to delivery and solidifies at physiological temperature on the atrial surface, thereby gradually releasing encapsulated extracellular vesicles for the treatment of atrial fibrillation.

42. The method of claim 41 , wherein the catheter is inserted using imaging guidance and / or mapping guidance.

43. The method of claim 41 or claim 42, wherein imaging guidance is provided by fluoroscopy, intracardiac echocardiography, transthoracic ultrasound, transesophageal ultrasound, magnetic resonance imaging, or any combination thereof.

44. The method of claim 42 or 43, wherein mapping guidance is provided by electroanatomic mapping, preferably wherein the electroanatomic mapping is configured to provide three-dimensional spatial registration sufficient to confirm catheter location.

45. The method of any one of claims 41-44, wherein the method further comprises mechanical landmarking of the myocardial surface or epicardial surface using a wire with a visible mechanical marker to assist with catheter insertion.

46. The method of any one of claims 41-45, wherein the method further comprises confirming delivery of the composition using fluorescent imaging.

47. The method of any one of claims 41-46, wherein the step of delivering the composition involves injecting the composition at two or more sites at the target atrial surface, preferably wherein the composition is inj ected at two sites, three sites, four sites, five sites, six sites or more, at the target atrial surface.

48. The method of claim 47, wherein more than one injection of the composition promotes even distribution of encapsulated EVs at the target atrial surface.

49. A method of reducing atrial fibroblast proliferation in a patient either pre-surgery, at the time of surgery or post-surgery comprising the step of administering the composition as defined in any one of claims 1-30 to a subject in need thereof.

50. A method of inhibiting, preventing or reducing NLRP3 activation, IL- 18 production, IL- 1 production or inflammasome activation in a subject, comprising the steps of:maintaining a flowable composition as defined in any one of claims 1-30 at a temperature sufficient to keep the composition in a liquid phase prior to delivery;positioning a catheter relative to a target atrial surface, preferably epicardial / myocardial under imaging guidance and / or mapping guidance;and dispensing the composition in an amount effective to form, upon exposure to physiological temperature, an in situ gel configured to release an effective amount of EVs.

51. The method of claim 50, wherein imaging guidance is provided by fluoroscopy, intracardiac echocardiography, transthoracic ultrasound, transesophageal ultrasound, magnetic resonance imaging, or any combination thereof.

52. The method of claim 50 or 51, wherein mapping guidance is provided by electroanatomic mapping, preferably wherein the electroanatomic mapping is configured to provide three-dimensional spatial registration sufficient to confirm catheter location.

53. The method of any one of claims 50-52, wherein the method further comprises mechanical landmarking of the myocardial surface or epicardial surface using a wire with a visible mechanical marker to assist with catheter insertion.

54. The method of any one of claims 50-53, wherein the method further comprises confirming delivery of the composition by detecting the fluorescent label of the composition using fluorescent imaging.

55. The method of any one of claims 50-54, wherein the dispensing comprises injecting the composition (i) onto an atrial epicardial surface to form a surface-adherent depot or (ii) into atrial myocardial tissue to form an intramural depot.

56. The method of any one of claims 50-55, wherein the dispensing of the composition at the target atrial surface causes release of the EVs which provides the therapeutic effect of inhibiting, preventing or reducing NLRP3 activation, IL- 18 production, IL- 1 production or inflammasome activation in the subject.

57. The method of any one of claims 50-56, wherein the method prevents or treats an atrial arrhythmia, atrial fibrosis, and / or inflammation in the subject.

58. An atrial target tissue delivery system comprising:• a catheter configured to dispense a composition onto a target atrial surface;• a guidance system configured to provide imaging and / or mapping guidance to localize the catheter relative to predefined mechanical landmark on the target atrial surface; and• a composition adapted to remain flowable prior to delivery and to undergo in situ gelation upon exposure to physiological temperature to form a depot at the target atrial surface59. The delivery system of claim 58, wherein the composition is configured to release of an active agent.

60. The delivery system of claim 58 or 59, wherein the guidance system provides imaging guidance by fluoroscopy, intracardiac echocardiography, transthoracic ultrasound, transesophageal ultrasound, magnetic resonance imaging, or any combination thereof.

61. The delivery system of any one of claims 58-60, wherein the guidance system provides mapping guidance by electroanatomic mapping, preferably wherein the electroanatomic mapping is configured to provide three-dimensional spatial registration sufficient to confirm catheter location.

62. The delivery system of any one of claims 58-61, wherein the mechanical landmark on the target atrial surface is a wire with a visible mechanical marker.

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