[0001] This application claims priority to U.S. Provisional Application Ser. No. 62/428,407, entitled "Nanovesicles for Compound Delivery" to Kwon et al., filed November 30, 2016, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Governmental support under Grant No. DGE- 1321846 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention is directed to extracellular vesicles, including methods of synthesis and applications thereof, more particularly, extracellular vesicles created from living cells, which are created with more homogeneity in size and increased production levels. The present invention is also directed to medicaments delivered via an extracellular vesicle as well as method to deliver an extracellular vesicle to a patient.

BACKGROUND OF THE INVENTION

[0004] The fields of drug delivery and gene therapy rely on nano-sized carriers for effective delivery of precious cargo to the designated target site. (See, e.g., Thomas, C. E., et al., Nat. Rev. Genet. 4, 346-58 (2003); Lv, H., Zhang, et al., J. Control. Release 1 14, 100-9 (2006); and Silva, A. K. A. et al. Nanoscale 5, 1 1374-84 (2013), the disclosures of which are incorporated herein by reference.) Therapeutic delivery agents have two key objectives: protect cargo from the harsh environment of the body and release cargo at the appropriate site without inducing immunogenic response. In order to achieve these goals, a variety of viral and non-viral nanocarriers with highly specific properties, dependent upon the type of cargo and desired site of delivery, have been designed. Common viral nanocarriers include retroviruses and adenoviruses. Common nonviral nanocarriers include liposomes, polyplexes, and dendrimers. In addition, l compounds can be delivered to cells by passive membrane transport or forceful entry that disrupts the membrane (e.g. particle bombardment, sonication). Nevertheless, issues with non-specific cytotoxicity, poor biocompatibility, and low efficacy of compound delivery still remain major challenges in the field.

[0005] Cells emit extracellular vesicles (EVs) to transport vital biomacromolecules such as mRNA and microRNA between cells and exogenous RNA-loaded exosomes have been used to achieve targeted, tissue-specific delivery. One common type of EV is an exosome, which have been studies as a possible method for therapeutic delivery. Cells in culture naturally produce EVs but at a rate significantly below the requirements for therapeutic administration, which has been attempted to be mitigated by exposing cells to endosomal trafficking regulators, modified proteins, and external stressors. However, these time- and labor-intensive processes directly affect cellular activities and make it difficult to preserve the composition and biological functions of EVs at a desired cellular stage. Thus, it is essential to explore alternative methods for rapid and large-scale production of EVs.

SUMMARY OF THE INVENTION

[0006] In one embodiment of this disclosure is directed to a method to generate extracellular vesicles. In various embodiments, these vesicles are created by exposing at least one mammalian cell to a solution comprising sulfhydryl blocking reagents.

[0007] In many such embodiments the methods may consist of collecting extracellular vesicles that are generated by exposing the at least one mammalian cell to the solution comprising sulfhydryl blocking reagents.

[0008] In some embodiments, the solution may also consist of a buffer. In some such embodiments, the buffer concentration used in this solution may be altered to modify the size and distribution of extracellular vesicles produced by the method of this embodiment.

[0009] In more embodiments, numerous sulfhydryl blocking reagents may be used. In some such embodiments, the method may use sulfhydryl blocking reagents comprising a cross-linking reagent and a reducing agent. In some such embodiments, the cross- linking agent may be selected from formaldehyde or paraformaldehyde, while the reducing agent may be selected from dithiothreitol, cysteine, and glutathione. [0010] In yet other embodiments, paraformaldehyde may be used as the cross-linking reagent, dithiothreitol as the reducing agent, and phosphate buffered saline as the buffer.

[0011] In still yet other embodiments, the specific concentration of paraformaldehyde may range from about 2.5 mM to about 2.5 M, while the specific concentration of dithiothreitol may range from about 0.2 mM to about 200 mM. In some such embodiments, the specific concentration of paraformaldehyde may be 25 mM, while the specific concentration of dithiothreitol may be 2 mM. In still other such embodiments, the phosphate buffered saline may have a concentration of at least 5X, the phosphate buffered saline may have a concentration of less than 0.5X, or the phosphate buffered saline may have a concentration of less than 0.5X.

[0012] In still yet other embodiments, the extracellular vesicles produced by the method of this embodiment may range in size from 25-100 nm, 100-700 nm, 700-2000 nm, or 1000-10000 nm. In these size ranges, the extracellular vesicles may have a polydispersity index of less than about 0.6, such as less than about any of 0.5, 0.4, 0.3, 0.2, or 0.1 .

[0013] Another embodiment of this disclosure is drawn to methods to provide extracellular vesicles to a treatment subject.

[0014] In many such embodiments, the method may include the steps of obtaining at least one extracellular vesicle generated from at least one mammal cell.

[0015] In other embodiments, the method includes purifying the at least one extracellular vesicle in a saline buffer.

[0016] In still other embodiments, the method may include the step of loading the at least one extracellular vesicle with a medicament. In some such embodiments, the loading step may be performed by incubating the at least one extracellular vesicle with a medicament at 37°C for at least one hour.

[0017] In yet other embodiments, the loaded extracellular vesicles may have a concentration of at least 100 pg/mL and may be selected from the doxorubicin and a vaccine.

[0018] In still yet other embodiments, the at least one mammal cell may come from the treatment subject and may be an antigen-presenting cell. In some such embodiments, these cells may be selected from a dendritic cell and a tumor cell. [0019] In still yet other embodiments, the administration of the at least one extracellular vesicle may be performed in proximity to the tumor tissue.

[0020] In still yet other embodiments, the administering step may effect a response in the treatment subject, wherein the response may consist of T-cell activation or immune response stimulation.

[0021] Still another embodiment of this disclosure is directed to a composition for the delivery of a compound.

[0022] In many such embodiments, the compound may comprise at least one extracellular vesicle derived from a mammalian cell and is substantially free of a nuclear component.

[0023] In other embodiments, the extracellular vesicle has a diameter of between about 10 nm and 10000 nm.

[0024] In still other embodiments, the mammalian cell may come from a stem cell, a cancer cell, a dendritic cell presenting an antigen, and a red blood cell.

[0025] In yet other embodiments, the mammalian cell may come from a primary cell or a cell derived from a cell line.

[0026] In still yet other embodiments, at least one extracellular vesicle may be a plurality of extracellular vesicles having an average diameter of between about 10 nm and about 200 nm.

[0027] In still yet other embodiments, the at least one extracellular vesicle may also be generated through sulfhydryl blocking, may be stable for at least six hours in serum at 37°C, and may be loaded with a medicament.

[0028] In still yet other embodiments, this medicament may be selected from a therapeutic agent, an imaging agent, an anticancer agent, doxorubicin, an antigenic peptide, and a polynucleotide encoding an antigenic peptide.

[0029] In still other embodiments, the compound of this embodiment may comprise a saline buffer.

[0030] Additional embodiments and features are set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the specification or may be learned by the practice of the disclosure. A further understanding of the nature and advantages of the present disclosure may be realized by reference to the remaining portions of the specification and the drawings, which forms a part of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings where:

[0032] Figure 1 illustrates various extracellular bodies that may be emitted from a cell along with general size ranges that these bodies may possess.

[0033] Figure 2 illustrates a process for treating a host with extracellular vesicles produced from isolated cells of the host in accordance with an embodiment of the invention.

[0034] Figure 3 illustrates the alteration of a biological process associated with extracellular vesicle formation in accordance with various embodiments of the invention.

[0035] Figure 4 illustrates the alteration of a biological process associated with extracellular vesicle formation in accordance with various embodiments of the invention.

[0036] Figure 5A is a bar chart depicting concentrations of paraformaldehyde and effects on extracellular vesicle associated protein content generated in accordance with various embodiments of the invention.

[0037] Figure 5B is a data graph depicting concentrations of sulfhydryl blockers and effects on extracellular vesicle associated protein content generated in accordance with various embodiments of the invention.

[0038] Figure 6 depicts the removal of formaldehyde from various embodiments of the invention after purification.

[0039] Figure 7 is an electron microscope image of extracellular vesicles in accordance with various embodiments of the invention.

[0040] Figure 8 provides electronic microscope images comparing supernatants of EL4 cells treated without or with sulfhydryl blocking reagents generated in accordance with various embodiments of the invention.

[0041] Figure 9 is a data graph depicting extracellular vesicles production in the presence and absence of sulfhydryl blocking reagents. [0042] Figure 10 describes the stability of the extracellular vesicles produced by various embodiments of this invention as compared to naturally occurring exosomes.

[0043] Figure 1 1 is a composite of three electron microscope images of supernatants derived from EL4 cells with various treatments: untreated and unloaded (Figure 1 1 A), treated with sulfhydryl blocking agents (Figure 1 1 B), treated with sulfhydryl blocking agents and loaded with DOX (Figure 1 1 C), generated in accordance with various embodiments of the invention.

[0044] Figure 12A provides the results of a dynamic light scattering assay showing the size range of nano-scale extracellular vesicles produced by different concentrations of phosphate buffered saline of various embodiments of this invention.

[0045] Figure 12B is a data graph depicting the correlation between extracellular vesicle size and osmolarity in accordance with various embodiments of the invention.

[0046] Figure 12C provides the results of a dynamic light scattering assay showing the size range of micro-scale extracellular vesicles produced by different concentrations of phosphate buffered saline of various embodiments of this invention.

[0047] Figure 12D is a bar graph showing the average size and standard distribution of extracellular vesicles produced by different concentrations of phosphate buffered saline in accordance with various embodiments of the present invention.

[0048] Figure 12E shows images of extracellular vesicles produced by varying the concentration of phosphate buffer in accordance with various embodiments of the invention.

[0049] Figure 13A provides the results of a dynamic light scattering assay showing the size range of extracellular vesicles produced by different buffers of various embodiments of this invention.

[0050] Figure 13B is a bar graph showing the amount of extracellular vesicles produced by various buffers under the same conditions of various embodiments of the invention.

[0051] Figure 13C is a bar graph depicting the polydispersity index of extracellular vesicles produced by different buffers of various embodiments of the invention.

[0052] Figure 13D shows the size and distribution of nano-scale extracellular vesicles produced in the presence of HEPES and saline buffers in accordance with various embodiments of the invention. [0053] Figure 13E shows the size and distribution of micro-scale extracellular vesicles produced in the presence of HEPES and saline buffers in accordance with various embodiments of the invention.

[0054] Figure 14A and Figure 14B depicts microscope images of extracellular vesicles being produced by various embodiments of the invention along with the presence of actin in the cell and vesicle.

[0055] Figure 15 shows electron microscope images of cells and extracellular vesicles, indicating the possible size range of extracellular vesicles that may be produced by various embodiments of the invention.

[0056] Figure 16 illustrates a process for treating a tumor with DOX-loaded extracellular vesicles in accordance with an embodiment of the invention.

[0057] Figure 17A illustrates the process of loading DOX into extracellular vesicles in accordance with an embodiment of the invention.

[0058] Figure 17B is a data graph depicting the effects of concentration of compound, temperature, and time on compound loading of extracellular vesicles generated in accordance with various embodiments of the invention.

[0059] Figure 17C depicts the effects of extracellular vesicle to DOX concentration of loading extracellular vesicles in accordance with various embodiments of the invention.

[0060] Figure 18A is a data graph depicting the protein concentration in the recovered and filtrate portions after centrifugal filtration generated in accordance with various embodiments of the invention.

[0061] Figure 18B is a data graph comparing size of unloaded extracellular vesicles with DOX-loaded extracellular vesicles generated in accordance with various embodiments of the invention.

[0062] Figure 19A is a pair of data graphs depicting DOX release in PBS with or without serum, comparing DOXIL and DOX-loaded extracellular vesicles in support of various embodiments of the invention.

[0063] Figure 19B is a composite of confocal microscope images depicting EL4 cells treated with DOX, DOXIL or DOX-loaded extracellular vesicles, and fluorescently labelled for endosomes or lysosomes generated to support various embodiments of the invention. [0064] Figure 20A is a pair of data graphs depicting the viability of EL4 cells treated with DOX, DOXIL, DOX-loaded extracellular vesicles, and unleaded extracellular vesicles in support of various embodiments of the invention.

[0065] Figure 20B is a quartet of data graphs depicting the viability of EL4 and HeLa cells treated with DOX-loaded extracellular vesicles derived from both EL4 and HeLa cells in support of various embodiments of the invention.

[0066] Figure 21 A is a data graph depicting tumor volume treated with PBS, unloaded extracellular vesicles, DOX-loaded extracellular vesicles, DOX, and DOXIL, in support of various embodiments of the invention.

[0067] Figure 21 B is a data graph depicting a survival curve of mice having tumors treated with PBS, unloaded extracellular vesicles, DOX-loaded extracellular vesicles, DOX, and DOXIL, PBS, unloaded extracellular vesicles, DOX-loaded extracellular vesicles, DOX, and DOXIL, in support of various embodiments of the invention.

[0068] Figure 21 C is a pair of data graphs depicting the level of DOX, DOXIL, and DOX- loaded extracellular vesicles in the serum, lung, liver, heart, spleen, and tumor of treated mice PBS, unloaded extracellular vesicles, DOX-loaded extracellular vesicles, DOX, and DOXIL, in support of various embodiments of the invention.

[0069] Figure 22 illustrates a process for immunizing a treatment subject against tumor- causing cells using SIINFEKL-presenting extracellular vesicles in accordance with an embodiment of the invention.

[0070] Figure 23A is a data graph comparing size of unloaded extracellular vesicles with SIINFEKL-presenting extracellular vesicles generated in accordance with various embodiments of the invention.

[0071] Figure 23B is a data graph depicting extracellular vesicles derived from dendritic cells can present the antigen SIINFEKL in accordance with various embodiments of the invention.

[0072] Figure 24 is a data graph depicting the activation of T-cells by extracellular vesicles presenting the antigen SIINFEKL in accordance with various embodiments of the invention.

[0073] Figure 25A is a data graph depicting the activation of T-cells by extracellular vesicles derived from immature dendritic cells, immature dendritic cells presenting the SIINFEKL antigen, mature dendritic cells, and mature dendritic cells presenting the

SIINFEKL antigen in accordance with various embodiments of the invention.

[0074] Figure 25B is a data graph depicting the level of SIINFEKL presentation on extracellular vesicles derived from immature dendritic cells, immature dendritic cells presenting the SIINFEKL antigen, mature dendritic cells, and mature dendritic cells presenting the SIINFEKL antigen in accordance with various embodiments of the invention.

DETAILED DISCLOSURE OF THE INVENTION

[0075] Turning now to the diagrams and figures, embodiments of the invention are generally directed to EVs, methods of their manufacture, and applications thereof (Figure 1 ). Several embodiments are directed to the EVs themselves, which are chemically modified vesicles that resemble naturally occurring EVs and exosomes. In many of these embodiments, the EVs have altered actin-myosin function that lead to an altered structure within the vesicles.

[0076]Various embodiments are also directed to EVs that are compound loaded. In numerous embodiments, the EVs are treated to retain a compound for a particular treatment. Accordingly, the compounds loaded onto the vesicles are chosen for the proper application. In several embodiments, antineoplastic compounds are loaded onto EVs for treatment of neoplasms, tumors, or cancer. In more particular embodiments, the antineoplastic compound is doxorubicin. In many other embodiments, EVs present an antigenic compound to induce a particular immune response, such as, for example, elicit T-cell activation. In more particular embodiments, the EVs present the cancer antigen SIINFEKL.

[0077] Methods of EV manufacture in accordance with many embodiments are also described. In many of these embodiments, EVs can be manufactured from any animal cell, dependent on the application. For example, to treat a patient, the patient's own cells can be extracted and used to manufacture the EVs having several benefits, which may include a lack of an allogenic response. Accordingly, multiple embodiments are directed to personalized EVs derived from an animal host source. The cell-type for EV manufacture may also vary, dependent on application. In some embodiments, the cell- type chosen are blood cells, fibroblasts, or tumor cells. In various embodiments, the cell type is chosen for ease of extraction and culture. In other embodiments, the cell type is chosen based on characteristics of the cell. For example, dendritic cells may be chosen for their antigen presenting capability, which can yield EVs presenting an antigen. Accordingly, many embodiments are directed to EVs derived from antigen-presenting cells.

[0078] In numerous embodiments, EVs are produced by incubating cells with sulfhydryl blocking reagents. These reagents alter the function of a natural cellular phenomenon known as blebbing. The alteration of blebbing by sulfhydryl blocking reagents results in vesicles of altered structure. In many embodiments, the sulfhydryl blocking reagents may be comprised of a non-specific cross-linker and a reducing agents. In some embodiments, the non-specific cross-linker is selected from N-ethyl maleamide, formaldehyde, and paraformaldehyde. In some embodiments, the reducing agent is selected from dithiothreitol, cysteine, and glutathione. In several embodiments, the nonspecific cross-linker may be paraformaldehyde, and the reducing agent may be dithiothreitol. In more particular embodiments, the concentrations of the reagents are 25 mM paraformaldehyde with 2 mM dithiothreitol. It should be noted that paraformaldehyde and its monomeric subunit, formaldehyde, are non-specific cross-linking reagents, while dithiothreitol is a reducing agent. Additionally, certain embodiments are directed to scalable manufacture of EVs. Accordingly, embodiments of the processes described within yield large amounts personalized EVs.

[0079] In some embodiments, EVs may be produced in the presence of a buffer along with sulfhydryl blocking reagents. In some embodiments, the buffer concentration is any of 0.1X, 0.2X, 0.3X, 0.4X, 0.5X, 0.6X, 0.7X, 0.8X, 0.9X, 1X, 2X, 3X, 4X, 5X, 6X, 7X, 9X, and 10X. In some embodiments, a buffer may be selected from the group consisting of PBS, saline, DMEM, GPMV, HEPES, DPBS, or any other buffer known in the art. In particular embodiments, the buffer is 1X PBS, while in some embodiments the buffer is 0.1X, and in some embodiments the buffer is 10X.

[0080] Various embodiments are also directed to methods of treatment. In many embodiments, the method of treatment is utilizing compound-loaded EVs on an animal subject, such as, for example, a human patient. In various other embodiments, the method of treatment is utilizing antigen-presenting EVs on an animal subject. The disorder to be treated depends on the application. Accordingly, in several embodiments, the EVs can be used treat any disorder that utilizes a compound for treatment. In more particular embodiments, EVs loaded with antineoplastic compounds are used to treat neoplasms, tumors, or cancer. In other particular embodiments, antigen-presenting EVs are utilized to elicit an immune response to treat a disorder. In more particular embodiments, the disorder to be treated by the antigen-presenting EVs is cancer or a pathogenic infection.

[0081] In some embodiments, EVs may be generated to carry a therapeutic agent. In some embodiments, the therapeutic agent may be Doxorubicin (DOX). In order to generate EVs carrying DOX, EVs may be produced by inducing vesiculation in harvested cells via sulfhydryl blocking in the presence of a buffer, specifically, vesiculation induced by paraformaldehyde and dithiothreitol in the presence of PBS. The EVs generated by this means may be isolated by centrifugation and incubated in the presence of DOX to load DOX into the EVs. Once loaded with DOX, the EVs may be filtered again to remove excess DOX from the solution. At this point, the DOX-loaded EVs (DOX-EVs) may be administered to an individual.

[0082] In some embodiments, EVs may be generated to display certain surface moieties. In some embodiments, the surface moiety may be the SIINFEKL antigen. In order to generate SIINFEKL-loaded EVs (SIINFEKL-EVs), cells may be harvested from an individual. Harvested cells may be pulsed in the presence of the SIINFEKL antigen to cause the harvested cells to display SIINFEKL. Upon loading SIINFEKL onto the harvested cells, vesiculation may be induced via sulfhydryl blocking in the presence of a buffer, specifically, vesiculation induced by paraformaldehyde and dithiothreitol in the presence of PBS. The EVs generated by this vesiculation may be collected via centrifugation to remove any cells or excess chemicals. The resultant SIINFEKL-EVs may be administered to an individual to induce a response in the individual to the presence of SIINFEKL.

[0083] Turning now to Figure 2, an embodiment of a process for personalized treatment with EVs is presented. It should be noted that Figure 2 is only exemplary, and embodiments of the invention are not limited to personalized treatment. In a personal treatment regime as described in Process 200, the host to be treated is the same host that provided the animal cells to manufacture the EV. For example, a patient having a cancer can get personalized treatment by isolated cells from the patient (202), production of EVs from the patient's isolated cells (204), loading neoplastic compounds onto the derived EVs (206), and treating the patient with the personalized, compound-loaded EVs (208). Although humans are suggested, it should be understood, however, that veterinary or experimental treatments on respective nonhuman animals could also be performed.

[0084] Process 200 can begin with extracting and isolating animal cells from a host source (202). In several embodiments, the host is any animal to be treated, such as, for example a human patient. The cell type to be extracted is dependent on the application. In many embodiments, the cell type is one that are of easy access and culture, such as blood cells or fibroblasts. In a non-personalized context, cells may be harvested from another source that is not directly from a patient or host. Harvesting cells from other sources may include cells harvested from cell lines established in cell culture or harvesting cells from another host source, such as another animal or human.

[0085] Process 200 can continue with production of EVs from the isolated animal cells (204). Application of sulfhydryl blocking reagents on the isolated the isolated animal cells can yield mass quantity of EVs that is scalable. In particular embodiments, the sulfhydryl blocking reagents are 25 mM paraformaldehyde with 2 mM dithiothreitol. It should be understood, however, the sulfhydryl blocking reagents and their concentration to be used can vary, dependent on the application and optimization. Once the EVs have been produced, the vesicles can be concentrated and purified by acceptable protocols, which may include centrifugation, filtration, chromatography, or other applicable methods.

[0086] Isolated EVs can be loaded with a compound of interest (206). The compound to be loaded is determined by the application. For example, if the personalized treatment is directed at cancer, the compound could be an antineoplastic drug. Accordingly, the EVs are incubated with the compound of interest in order to load the vesicles with the drug. The procedure for compound loading will vary, dependent on the properties of the EVs and the compounds. For example, loading EVs with antineoplastic drug doxorubicin is dependent on compound concentration, temperature, and incubation time. [0087] Once the EVs are loaded with the compound of interest, the compound-loaded vesicles can be used for treatment of the host (208). The site of treatment will depend on the application. For example, with EVs can be administered locally at the tumor site or systematically by any appropriate mechanism (e.g., oral, intravenous).

[0088] It should be noted that the steps present in Process 200 do not necessarily have to be completed in the order as described in Figure 2. Such that in some embodiments, loading vesicles with a compound of interest step may occur prior to producing vesicles by sulfhydryl blocking. Such an instance may occur in situations where the vesicles are being loaded with a compound (e.g., mRNA, protein, or peptide) is produced by the cell, from which the vesicles will be produced.

[0089] EVs have the potential to be an exciting option for nanoscale delivery. EVs present a unique chance to harness near natural biological carriers for treatments of a host. As diagrammed in Figure 3, extracellular vesicles offer a compelling opportunity to develop into personalized therapeutic delivery carriers. In one embodiment, cells are harvested from a patient and used to produce vesicles in vitro. In more embodiments, these vesicles are loaded with compounds for delivery to the patient's diseased tissue. In some embodiments, the EVs contain surface modification to improve targeting. Personalized treatment, as depicted by example in Figure 3, would mitigate an immunogenic response associated with non-self compound carriers. Furthermore, EVs could also improve targeting, as determined by surface modifications on the vesicles and interaction of the modifications with the targeted host cells.

[0090] Despite their high potential in therapeutic delivery, vesicle-based therapeutics have been slow to progress to clinical trials due to problems associated with mass production. The low yield associated with ex vivo production of vesicles is a major challenge that leads to a bottleneck in the production process. In addition, traditional use of sulfhydryl blocking reagents, such as formaldehyde and N-ethyl maleamide, to produce EVs typically renders giant plasma membrane vesicles (e.g., as shown in membrane raft studies; E. Sezgin et al. Nat. Protoc. 7, 1042-51 (2012), the disclosure of which is incorporated herein by reference). Due to their large size and polydispersivity, giant plasma membrane vesicles are not suitable therapeutic delivery carriers. Compositions for Delivery of a Compound

[0091] In some embodiments, EVs may be used to deliver a compound derived from a mammalian cell. EVs may also be free of a nuclear component. A nuclear component may be characterized by proteins and nucleic acid which are understood to be localized within the nucleus of a cell. A nuclear component does not include cellular components, which are naturally or artificially designed to be exported from the nucleus. As such, a nuclear component may not consist of carbohydrates, proteins, nucleic acids, which may be exported. Such nucleic acids may consist of plasm id DNA, plastid DNA, mitochondrial DNA, nuclear DNA, RNA, or RNA-DNA hybrid molecules, which naturally or artificially are exported from the nucleus.

[0092] EVs of some embodiments may come from nucleated or non-nucleated cells, which may come from the group consisting of stem cells, dendritic cells, red blood cells, or cancer cells. In certain embodiments, the EV may come from a dendritic cell. In some embodiments, the EV may come from a dendritic cell displaying an antigen. In some embodiments, EVs may come from a primary cell harvested from an individual, or an EV may come from a cell line established in cell culture.

[0093] In embodiments of a composition for the delivery of a compound, where the EV comes from nucleated or non-nucleated cells, including any of stem cells, dendritic cells, red blood cells, or cancer cells, where the cells display or do not display an antigen, these embodiments may comprise a plurality of EVs, where the average diameter of EVs is between about 10 nm and about 200 nm. In some of these embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the EVs may be between about 10 nm and about 200 nm.

[0094] In any of the above embodiments for a composition for delivery of a compound, the composition may include a sulfhydryl blocking reagent. Additionally, in any of the embodiments for a composition for delivery of a compound, the EVs may comprise cross- linked actin, cross-linked myosin, or both cross-linked actin and cross-linked myosin. In embodiments containing cross-linked actin, cross-linked myosin, or both cross-linked actin and cross-linked myosin, an average of at least 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the actin, myosin, or actin and myosin are cross-linked. [0095] In any of the above embodiments for a composition for delivery of a compound, the EVs may be stable for at least 2, 4, 6, 8, 10, 12, 16, 24, or 48 hours in serum at 37°C.

[0096] Additionally, in any of the above embodiments for a composition for delivery of a compound, the EVs may be loaded with compound. In some of these embodiments, the compound is therapeutic or an imaging agent. In embodiments, where the compound is therapeutic, the compound may be an anti-cancer agent. In embodiments, where the compound is an anti-cancer agent, the compound may be doxorubicin. Additionally, in embodiments, where the compound is therapeutic, the compound may be selected from an antigenic peptide and a polynucleotide that encodes an antigenic peptide.

[0097] In any of the above embodiments for a composition for delivery of a compound, the composition may include an agent to maintain osmotic pressure of the EV. In embodiments including an agent to maintain osmotic pressure of the EV, the agent may be a saline buffer.

Methods of producing EVs derived from a mammalian cell

[0098] In some embodiments, EVs may be produced by incubating a mammalian cell with a cross-linking agent and a reducing agent. In these embodiments to produce an EV, the cross-linking reagent may be selected from formaldehyde or paraformaldehyde. In either of the above embodiments of methods to produce EVs, the reducing agent may be selected from dithiothreitol, cysteine and glutathione. In any of the above embodiments of methods to produce EVs, the cross-linking agent may be paraformaldehyde, and the reducing agent may be dithiothreitol. In the above embodiments of methods to produce EVs, where the cross-linking agent is paraformaldehyde, the paraformaldehyde may be at a concentration of about 2.5 mM to about 2.5 M. In the above embodiments of methods to produce EVs, where the reducing agent is dithiothreitol, the dithiothreitol may be at a concentration of about 0.2 mM to about 200 mM. In the above embodiments of methods to produce EVs, where the cross-linking agent is paraformaldehyde and the reducing agent is dithiothreitol, the concentration of paraformaldehyde may be at a concentration of 25 mM, and the dithiothreitol may be at a concentration of 2 mM.

[0099] In any of the above embodiments of methods to produce EVs, the EVs may be incubated with a compound. In embodiments of methods to produce EVs, where the EVs are incubated with a compound, the EVs may be incubated with a compound at about 37°C. In embodiments where the EVs may be incubated with a compound, the concentration may be at concentration of at least 100 pg/mL. Further, in embodiments where the EVs may be incubated with a compound, the EVs may be incubated with the compound for at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours. In any of the embodiments where the EVs may be incubated with a compound, the compound may be doxorubicin.

[0100] In any of the above embodiments of methods to produce EVs, the method may further include treating the composition with an agent to maintain the osmotic pressure of the EVs. In embodiments where the method to produce EVs includes treating the composition with an agent to maintain the osmotic pressure of the EVs, the agent to maintain the osmotic pressure may be a saline buffer.

Methods to treat a disease or disorder

[0101] Some embodiments of the present invention may be a method of treating a disease or disorder comprising administering a composition for the delivery of a compound as described above— in which the composition includes a therapeutic agent, for example an anticancer agent, doxorubicin, or an antigenic peptide and a polynucleotide that encodes an antigenic peptide— to a subject suffering from a disease or disorder. In the above embodiment of a method of treating a disease or disorder, the mammalian cell used in the compound may be harvested from the subject. In either of the above embodiments, the cell may be a tumor cell. In embodiments, where the cell is harvested from a tumor cell, the EVs may be administered to or in proximity to tumor tissue.

[0102] In embodiments of a method of treating a disease or disorder, where the cell is a mammalian cell from the subject, the mammalian cell may be an antigen-displaying cell from the subject. In embodiments of a method of treating a disease or disorder, where the mammalian cell is an antigen-displaying cell, the cell may be a dendritic cell. In embodiments of a method of treating a disease or disorder, where the EVs are loaded with a compound— where the compound is a therapeutic agent, an imaging agent, an anti-cancer agent, or doxorubicin— the compound may be a vaccine for the disease or disorder. Methods to activate T Cells

[0103] Some embodiments of the invention may consist of a method of activating T cells comprising administering a composition for the delivery of a compound as described above— in which the composition is therapeutic, an anti-cancer agent, doxorubicin, or an antigenic peptide and a polynucleotide that encodes an antigenic peptide— into a subject. In the above embodiment of a method of activating T cells, the mammalian cell may be a dendritic cell.

Methods to stimulate an immune response

[0104] Some embodiments of the present invention may consist of a method to stimulate an immune response comprising administering a composition for the delivery of a compound as described above— in which the composition is therapeutic, an anti-cancer agent, doxorubicin, or an antigenic peptide and a polynucleotide that encodes an antigenic peptide— into a subject. In the above embodiment of a method to stimulate an immune response, the mammalian cell may be a dendritic cell.

Embodiments of EV Production and Characterization

[0105] Formation of EVs requires the biological process known as blebbing, which is the protrusion and retraction of portions of the plasma membrane. Blebbing is a result of changes in hydrostatic pressure, which are counteracted by cytoskeletal mechanisms (G. T. Charras, et al., J. Cell Biol. 175,477-90 (2006), the disclosure of which is incorporated herein by reference). The opposing forces between the hydrostatic pressure of the cytoplasm and the retraction of the actin filaments determines whether a vesicle is released or the bleb retracts (J. Hagmann, M. M. Burger, & J. A. Theriot, J. Cell. Biochem. 73, 488-99 (1999), the disclosure of which is incorporated herein by reference). Accordingly, blebbing relies heavily on actin and myosin function. Figure 3 depicts crosslinking-derived EVs prior to and following isolation and purification.

[0106] As shown in Figure 4, exposure of cells to sulfhydryl blocking reagents induces a state of irreversible blebbing. Concentration of sulfhydryl blocking reagents affects actin- myosin function and also correlates with media osmolarity. This implies significance of hydrostatic pressure and actin-myosin function on blebbing.

[0107] EV formation by sulfhydryl blocking reagents, in accordance with various embodiments of the invention, is highly advantageous because of its extremely rapid rate of EV production. Other methods to produce EVs, such as calcium- and stress-induced EV production, requires between 12 hours and several days to achieve significantly improved yields. Methods in accordance with embodiments described here can form similar amount of EVs in just one hour. Additionally, EVs, generated as described, are relatively simple to characterize and modify because the vesicles have similar membrane composition and intravesicular cytosol components to the cells they are derived from.

[0108] In Figure 5 describes embodiments of the production of EVs, where the concentrations of sulfhydryl blocking reagents have been altered. Specifically, Figure 5A demonstrates embodiments where the concentration of paraformaldehyde has been altered to show maximum production levels at 25 mM paraformaldehyde with 2mM dithiothreitol. Figure 5B shows embodiments where HeLa cells were exposed to various concentrations of sulfhydryl blocking reagents, such that 1X = 25 mM paraformaldehyde with 2mM dithiothreitol. EVs were isolated and quantified using a BCA protein assay. These data show that the production of EVs with HeLa cells is highest at 25mM paraformaldehyde with 2mM dithiothreitol, as compared to other concentrations tested.

[0109] In various embodiments, EVs may be collected by any suitable means to separate EVs from cells or cell debris. In some embodiments, to isolate EVs, cells were removed by centrifugation at 1 ,200 rpm for 5.5 minutes followed by removal of cell debris and micro-sized vesicles at 10,000 rpm for 10 minutes. EVs were concentrated with a 30 kDa centricon (4,500 rpm for 15 minutes); the EV-containing supernatant was concentrated and washed with an equivalent volume of PBS twice. Upon production of EVs in embodiments of this disclosure, formaldehyde residue may be removed using centrifugation, as shown in Figure 6. The presence of formaldehyde in the resulting EV collection was measured by ¹ H NMR. It should be noted that an embodiment removing formaldehyde by centrifugation is only exemplary, and residue from any added reagents may be removed by any suitable means, including centrifugation, absorption, neutralization, or other means known in the art. [0110] To analyze the lower size limit for EV production by sulfhydryl blocking reagents, EVs may be analyzed by means such as transmission electron microscopy (TEM). Figure 7 demonstrates an embodiment where the supernatant of vesiculating HeLa cells was analyzed by TEM. HeLa cells treated with paraformaldehyde (PFA) and dithiothreitol (DTT), in accordance with various embodiments of the invention, generate a large amount of EVs as secondarily confirmed by TEM measurements. Since the control (inset) does not show EVs, these nano-sized EVs are unlikely to be exosomes generated by normal cellular processes. Instead, sulfhydryl blocking reagent-induced blebbing results in cells shedding nano-sized EVs. Additionally, Figure 8 shows a TEM image of EL4 cells treated with and without PFA/DTT. TEM images in Figure 8A demonstrate that the supernatant from EL4 cells incubated in serum-free/reagent-free media have little or no vesicles, while the EL4 cells incubated with serum-free media containing 25 mM paraformaldehyde and 2 mM dithiothreitol for 4 hours, however, had many nano-sized EVs as indicated by the circles. In order to more clearly examine the EVs, the cells, debris and giant plasma membrane vesicles were removed by centrifugation at 16, 100xg. The EVs were then isolated and concentrated by centrifugation at 100,000xg.

[0111] EVs are a promising potential therapeutic carrier. Low yield of intrinsic vesicle production is a problem. However, sulfhydryl blocking reagents can be used to produce EVs appropriate in size for carrying therapeutic cargo. Figure 9 demonstrates embodiments of EV production in the presence and absence of sulfhydryl blocking reagents. EL4 cells were incubated at 100,000 cells/mL in 5 mL of either DMEM (without FBS) for 24 hours or PBS with 90 μί 4%PFA solution and 10 μί 1 M DTT for 2 hours at 37°C. After cells and cell debris were removed by centrifugation at 1200 rpm for 5.5 min, micro-sized vesicles were pelleted at 13,200 rpm for 10 minutes, leaving the EVs in the supernatant. Then, EVs were isolated by 30 kDa centricons at 4,500 rpm for 10 minutes. The EVs were washed three times with 5 mL of DPBS to remove free protein and sulfhydryl blocking reagents. Protein content was assessed using a BCA protein assay (Figure 9). Vesicles produced from cells that were not exposed to sulfhydryl blocking reagents are called exosomes. Sulfhydryl blocking reagents produce a significantly greater (10-fold) amount of EVs compared to naturally occurring exosomes, based on protein quantification over a significantly shorter time-frame of production. [0112] Additionally, the stability of EVs produced by embodiments of the invention may be measured by various means. Figure 10 describes the stability of exosomes versus EVs produced according to certain embodiments. In this figure, the size range of exosomes and EVs of some embodiments are shown as measured by dynamic light scattering (DLS) as measured at 0, 24, and 48 hours. Additionally, the polydispersity index (PDI) shows that as time passes, exosomes increase in polydispersity, while EVs of certain embodiments remain stable. Also, the anionic surface charge of exosomes decreased over time, while EVs of some embodiments remain relatively stable after 24 and 48 hours, when compared to the initial measurement.

[0113] Turning now to Figure 1 1 , describing the ability to generate and use EVs to carry a therapeutic drug. Specifically, Figure 1 1 demonstrates the size of EVs produced in accordance with various embodiments of the invention. In particular, Figure 1 1A is a TEM image of supernatant collected from control EL4 cells, which have not been exposed to sulfhydryl blocking reagents. Figure 1 1 B shows a representative TEM image of EL4 EVs of some embodiments produced via sulfhydryl blocking. Figure 1 1 C shows EL4 EVs, which have been loaded with doxorubicin (DOX) after being produced by sulfhydryl blocking. The scale bars in Figure 1 1 equal 200 nm.

[0114] Additionally, in embodiments of the invention, the size of EVs being produced can be altered by adjusting the osmolarity of the buffer (Figure 12). In some embodiments, phosphate-buffered saline (PBS) may be used along with the sulfhydryl blocking reagents to generate EVs. In this figure, EVs were generated using 25 mM PFA, 2 mM DTT along with PBS at 0.1X, 1X, and 10X concentrations. The size distribution of EVs produced in these embodiments was measured via a DLS assay. The increased concentration of PBS led to smaller EVs produced in some embodiments. These results indicate that the size of EVs produced in some embodiments may be customized to suit specific needs.

[0115] Specifically, Figure 12A demonstrates the effect of increasing the buffer concentration to generate EVs in the 10 nm to 1 ,000 nm size-range (nano-scale EVs) of some embodiments. Embodiments of nano-scale EVs may be generated by inducing vesiculation in cells, followed by a 30 kDa centrifugal filtration as described in this disclosure. In some embodiments, cells may be removed from the solution by an initial 1 ,200 rpm centrifugation prior to the 30 kDa centrifugal filtration. Additionally, Figure 12B demonstrates a similar result showing a linear relationship between increased osmolarity and smaller EVs.

[0116] Similarly, Figures 12C and 12D demonstrate the effect of increasing the buffer concentration to generate EVs in the 500 nm to greater than 15,000 nm size-range (micro- scale EVs) in other embodiments. Embodiments of micro-scale EVs may be generated by inducing vesiculation in cells followed by centrifugation at 1 ,200 rpm to remove cells. The remaining supernatant may be further centrifuged at 9,300 xg to isolate micro-scale EVs. It should be noted that the supernatant remaining after isolation of micro-scale EVs may also be submitted to a 30 kDa centrifugal filtration to further isolate nano-scale EVs. Images of micro-scale EVs of various embodiments are shown in Figure 12E. In these images, the size and distribution of micro-scale EVs generated using sulfhydryl blocking reagents along with varying concentrations of PBS are shown.

[0117] The size of EVs produced in certain embodiments may also be adjusted by using alternative buffers. Figure 13A demonstrates the effect of several buffers on the size of EVs produced by some embodiments as measured by DLS. In this figure, the buffers DPBS, DMEM, and GPMV were shown to produce EVs with sulfhydryl blocking in various size ranges, including into the larger, pm-sized EVs.

[0118] Additionally, not all buffers produce EVs at the same rate. Figure 13B demonstrates the production of EVs by various buffers as determined by a BCA assay to assess the protein content. As shown, PBS and DPBS buffers are more efficient than GPMV and DMEM buffers. Figure 13C demonstrates PDI of EVs produced by PBS and GPMV buffers. As indicated in Figure 13C, PBS buffer produces EVs with a lower PDI, indicating that PBS creates more uniformly sized EVs over GPMV, which has a PDI of approximately 1 , which indicates nearly complete polydispersity of EVs produced with GPMV. Further, Figure 13D demonstrates nano-sized EVs produced by vesiculation with 0.1 M HEPES buffer and 0.9% saline, which show a very broad size distribution and high polydispersity among these buffers. Similarly, Figure 13E shows production of micro- sized EVs using 0.1 M HEPES buffer and 0.9% saline, which show a very broad size distribution and high polydispersity among these buffers.

[0119] Each of these buffers shown in Figure 13 may contain various components to balance osmotic pressure as well as supplement cellular growth. The results shown in Figure 13 indicate that changing osmolarity of the solution is not the only factor in adjusting the average size, size distribution, or production rate of EVs produced by various embodiments.

[0120] Turning now to Figure 14, which demonstrates the ability to assess the presence of actin within EVs produced in accordance of various embodiments of the invention. In Figure 14A, left panel shows a light microscope image of a micro-scale EV being formed from a host cell, while Figure 14A, right panel shows the presence of actin as stained with a fluorescent dye. Similarly, Figure 14B overlays a fluorescent image onto a light image to show that EVs produced by some embodiments may be substantially free of actin.

[0121] Biological data supports the generation and characteristic description of the EVs. Furthermore, the data supports the notion that the EVs can be used to a compound delivery system capable as a therapeutic treatment. The following data also details the scalability and enhanced production of EVs from a host source. Accordingly, these data support the various embodiments of the invention as described. Turning now to Figure 15, certain embodiments may utilize various sizes of EVs produced by other embodiments. Specifically, smaller, nano-scale EVs may be more suitable for drug delivery, while larger micro-scale EVs may be more suitable for applications in immunotherapy.

Embodiments of EV Drug Delivery Vehicles

[0122] Some embodiments of the present disclosure may be used to deliver drugs or other therapeutic agent to an individual. Such a delivery mechanism could also be used to transport peptides, proteins, nucleic acids, or imaging agents. Examples of therapeutic agents could be synthetic or natural compounds, such as small molecules, nucleic acids, or peptides. Examples of such agents include drugs, hormones, enzymes, proteins, lipids, carbohydrates, glycoproteins, transcription factors, DNA, RNA, mRNA, modified mRNA, small RNAs, siRNA, miRNA, genes, transgenes, and dyes. Using many natural compounds may elicit a desired effect directly, such as an mRNA encoding a peptide for immediate transcription. Alternatively, some compounds may cause the desired tissue to generate the response, such that delivery of a transcription factor may activate an innate gene. Additionally, delivery of specific imaging agents may allow accumulation of dyes to a specific cell-type or tissue for imaging without background imaging signal being produced by neighboring tissue.

[0123] In some embodiments, agents of interest, including therapeutic, diagnostic, or a combination of therapeutic and diagnostic, may be loaded into cells prior to, concurrently with, after, or any combination thereof of the production of EVs. In such embodiments, harvested cells may be loaded with therapeutic agents or harvested cells may be induced to produce the agents of interest. In embodiments where the cells are loaded with agents of interest, the cells may be incubated with agents of interest in similar conditions as described within this disclosure to allow the cell to uptake the therapeutic agent. In embodiments where the cells are loaded by inducing a cell to produce the agents of interest, the cell may produce specific peptides, nucleic acids, or both peptides and nucleic acids with therapeutic properties. Therapeutic peptides may include small peptides, protein subunits, entire proteins, or any combination of the above. Therapeutic nucleic acids may include DNA or RNA, including genie sequences, plasmid DNA, tRNA, rRNA, mRNA, small RNAs, miRNA, siRNA, shRNA, crRNA, or any combination of nucleic acids produced within the cell. Additionally, ribonucleoproteins or any other form of protein-nucleic acid complex may be produced within a cell. In some embodiments, once the cells are loaded with the agents of interest, vesiculation may be induced. In other embodiments, vesiculation may be induced during the loading of the therapeutic agents into the cells. Situations were vesiculation may occur during loading may include where the cell is producing the therapeutic agents. Upon inducing vesiculation, EVs produced from the loaded cells may contain the agents of interest. EVs produced in accordance with embodiments of the invention may also be loaded with agents of interest via direct membrane penetration, chemical labeling and conjugation, electrostatic coating, adsorption, absorption, electroporation, or any combination thereof. Further, EVs produced in accordance with certain embodiments may undergo multiple loading steps, such that some agents of interest may be loaded prior to vesiculation, while additional agents of interest may be loaded during or after vesiculation. Additionally, EVs may be loaded with an agent of interest during vesiculation, and further loaded with another agent of interest after vesiculation. [0124] Figure 16 demonstrates an example of one strategy to deliver a therapeutic drug to an individual in accordance with various embodiments. It should be noted that Figure 16 is only exemplary and does not describe all possible ways to deliver a therapeutic drug to an individual in accordance with embodiments of the present invention. Specifically, Figure 16 demonstrates the delivery of the drug, DOX to a mouse with an established tumor. In this figure, EL4 cells from a mouse have been isolated and placed into culture (1602). Additionally, a tumor is established in a mouse using EL4 cells (1604). Further, EVs have been produced from these cells by the use of sulfhydryl blocking reagents (1606). In this figure, formaldehyde is demonstrated as a possible reagent. EVs produced by sulfhydryl blocking are loaded with DOX (1608). Finally, the DOX-loaded EVs are provided to the mouse to treat the tumor established by the EL4 cells.

[0125] Figure 17 further demonstrates examples of various conditions for loading EVs with a therapeutic agent in accordance with embodiments of the present invention. Specifically, Figure 17A demonstrates the production of EVs by incubating EL4 cells with PFA and DTT. EVs produced may be collected by centrifugation. DOX or another therapeutic may be loaded into to the EVs, then collected through centrifugation. EVs have great potential as therapeutic carriers due to their small size and high biocompatibility. The EL4-derived EVs were loaded with a common chemotherapeutic drug, doxorubicin (DOX). DOX is known for high instances of cardiotoxicity, and therefore is an ideal candidate for targeted therapeutic delivery. Anticancer agent, DOX, was selected for studies due to its relatively low solubility and bioavailability and subsequent potential for improved biodistribution when delivered via drug-loaded EVs. DOX's intrinsic fluorescence also aids in confirmation of drug entrapment within EVs. DOX, a chemotherapeutic anthracycline antibiotic, exhibits red fluorescence (excitation: 480 nm, emission: 580 nm). Passive DOX loading of EVs was tested at different drug concentrations, temperatures and incubation periods (Figure 17B). As expected, all three factors factor in DOX loading. Additionally, when the ratio of DOX to EVs was increased, the amount of DOX loaded into EVs also increased (Figure 17C).

[0126] In embodiments of EV drug delivery vehicles, the EVs may be loaded with a compound by incubating cells or empty EVs with 25 pg/mL, 50 pg/mL, 100 pg/mL, 200 pg/mL, 300 pg/mL, 400 pg/mL, or 500 pg/mL of the compound. Additionally, the incubation may occur for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, or 48 hours. Alternatively, the loading conditions may occur at a ratio of EVs to a compound of 1 :0.1 , 1 :0.2, 1 :0.3, 1 :0.4, 1 :0.5, 1 :0.6, 1 :0.7, 1 :0.8, 1 :0.9, 1 : 1 , 1 :2, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.

[0127]Additionally, the polydispersity of compound-loaded EVs may have a similar polydispersity index (PDI) of unloaded EVs. As such, compound-loaded EVs may have a PDI of about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 .0.

[0128] Turning now to figure 18, which shows non-limiting examples to assess the drug- loading of EVs in accordance with embodiments. Specifically, to assess the DOX-loading of EL4-derived EVs, the EVs were loaded with DOX via incubation with 1 mg/mL of DOX for 12 hours prior to purification with 30 kDa centrifugal filters. The filter size was selected to be more than sufficient to isolate EVs while still removing free proteins and DOX. Initial studies with 30 kDa centrifugal filtration showed that a single filtration step led to removal of more than 85% of protein from the original sample (Figure 18A). This high protein loss indicates that the method should be sufficient for removal of free protein and free drug from samples of EVs loaded with DOX (herein called DOX-EVs). The size of the DOX- EVs was further assessed via DLS (Figure 18B). DLS identified that the DOX-EVs range in size from 50 nm to several hundreds of nanometers in diameter (Figure 18B). Representative TEM images of DOX-EVs can be seen in Figure 1 1 , where Figure 1 1A shows the supernatant of EL4 cells, which have not been subjected to sulfhydryl blocking reagents; Figure 1 1 B shows EL4-derived unloaded EVs, and Figure 1 1 C shows DOX- EVs.

[0129] Turning now to Figure 19, EVs produced in accordance with various embodiments may show different release and uptake characteristics from other forms of drug delivery. Specifically, Figure 19 shows DOX release from and uptake. Specifically, Figure 19A demonstrates the release of DOX from DOX-EVs with and without serum added to PBS buffer as compared to the release of DOX from liposomal DOX (DOXIL). DOX-EVs release drug at a much more rapid rate than DOXIL. In fact, DOX-EVs show a more rapid release profile than DOXIL in both serum-free media (Figure 19A, left panel) and in serum-containing media (Figure 19B, right panel). Drug release reached 50% of the maximum at 51 , 13, 23, and 10 minutes for DOXIL and DOX-EVs in serum-free media and DOXIL and DOX-EVs in serum-containing media, respectively.

[0130] Similarly, DOX-EVs were taken up by cells more rapidly than DOXIL (Figure 19B). Figure 19B shows EL4 cells incubated with 100 mg/mL of DOX, DOXIL, and DOX-EVs after 3, 6, and 12 hours prior to confocal imaging. In Figure 19B, endosomes were stained with CellLight Early Endosomes-GFP and lysosomes were stained with LysoTracker Green DND-26. A representative image is shown for each time point. While DOXIL shows minimal uptake over the timeframe shown, DOX-EVs are taken up in the endosome within 6 hours and drug appears in the nucleus by 12 hours. EL4 cells treated with DOX-EVs for up to 12 hours exhibit DOX fluorescence in the cytoplasm while DOXIL treated cells do not.

[0131] Turning now to Figure 20. Figure 20 describes the effect of therapeutic agents delivered by EVs of various embodiments as compared to other forms of drug delivery. Specifically, Figure 20 shows the viability of EL4 cells was assessed using an MTT assay in triplicate (n = 3), and error bars show standard deviation. At the highest concentrations tested, DOX and DOX-EVs were statistically significant (p<0.01 ) from the controls as analyzed by a one-way ANOVA and the Tukey HSD post-hoc test. Although the mechanism behind the superior uptake of DOX-EVs compared to DOXIL has not been studied in this work, it is mores likely related to the PEGylatio of DOXIL. PEGylation has been shown to sterically stabilize liposomes allowing for slower release and uptake. (See, Immordino, et al., Int. J. Nanomedicine 1 , 297-315 (2016), the disclosures being incorporated herein by reference.)

[0132] Figure 20A shows the results of an in vitro MTT assay to test the viability of EL4 cells treated with DOX-loaded EVs. EL4 cells were treated over 24 or 48 hours with DOX, DOXIL, DOX-EVs, or unloaded EVs. The results show that there are some significant differences between cells treated over 24 hours and those treated over 48 hours. After a 24-hour treatment, DOX-loaded EVs do not perform as well as free DOX, but they seem to "catch up" over the longer incubation time. It is important to note that DOX-loaded EVs would not be expected to outperform free DOX in in in vitro study since their primary benefits (improved biocompatibility and extended release) are critical factors in an in vivo delivery setting. [0133] Figure 20B shows the effect of DOX-loaded HeLa-derived EVs and EL4-derived EVs at varying concentrations of DOX on both HeLa and EL4 cells. Interestingly, the cell lines did not show a preference for the EVs derived from other cell lines.

[0134]Turning now to Figure 21. Figure 21 describes the effect of therapeutic agents delivered by EVs of various embodiments as compared to other forms of drug delivery. Specifically, data from in vivo studies showing the efficacy of DOX are shown in Figure 21 . In these studies, EL4 tumors were established in C57BL/6 mice eight days before treatment. Treatment began on Day 0, where an equivalent DOX concentration of 8 mg/kg was given to the mice via intravenous injection into the tail vein. Figure 21A shows that DOX-EVs provided the slowest tumor growth, indicating that DOX-EVs were more effective than free DOX or DOXIL in decreasing tumor size. Figure 21 B shows that DOX- EVs also provided the highest survival rate among the mice.

[0135]Turning to Figure 21 C, serum was collected from C57BL/6 mice treated with DOX, DOXIL or DOX-EVs and analyzed for drug content. Concentration of DOX in the serum (n=3) was measured over 12 hours by a series of blood collections and fluorescence quantifications. Both DOXIL and DOX-EVs remain in the serum longer than free DOX (Figure 21 C, left panel). Most likely due to the protective effect of PEGylation, DOXIL has a longer circulation time than DOX-EVs. Twenty-four hours after treatment, the mice were sacrificed and their organs were assessed for drug content. Figure 21 C, right panel, shows that free DOX tends to accumulate in the lung while DOXIL has a tendency to be cleared by the liver. DOX-EVs, on the contrary, show minimal accumulation in lung, liver, and all other tissue examined.

[0136] In the past decade, the goal of developing biocompatible, targeting nano-carriers in the form of EVs has become the goal of many researchers in the field therapeutic delivery. The theoretical process would involve isolating EVs from primary cells derived from a patient and then using those EVs as a therapeutic carrier for delivery of cargo to a specific site in the original patient's body. A key challenge in achieving this goal is the successful production of therapeutically effective levels of EVs. (See, Smith, J. A. et al. Bioprocess Int. 13, 1-13 (2015), the disclosure of which is incorporated herein by reference.) These procedures provide a relatively simple and highly scalable protocol for producing large quantities of nano-sized EVs. [0137] Cell-derived EVs are expected to have lower immunogenicity than polymeric, viral, or lipid-based carriers. Additionally, the DOX-EVs accumulate less in the liver and lungs than DOX and DOXIL. This may be in part related to their small size; their average diameter is half that of liposomal DOX. It could also be due to vesicles' ability to specifically associate with cells from the line that they were derived from. These biodistribution characteristics result in highly improved survival outcomes for tumor- bearing mice treated with DOX-EVs compared to the controls (50% survival versus 0% survival over 40 days, Figure 21 B).

[0138] Although the exemplary data shown here use DOX-delivery vehicles for cancer therapy, EVs have a broad range of potential health applications. A wide variety of therapeutics have poor biodistribution which could be improved by delivery via EVs. Additionally, EVs could also be utilized to improve delivery of RNAs for gene therapy applications. EVs are highly promising to the field of therapeutic delivery, and the described method for scalable mass production could allow them to reach their potential in the field.

Embodiments of EVs for Therapy, including Immunotherapy

[0139] Some embodiments of the present disclosure may be used to elicit an immune response, signal a reaction, or produce any other form of response in an individual. These responses may be produced by the EV itself or elicited by the display of surface moieties on EVs. These surface moieties may include antigens, receptors, antibodies, chemically labeled, or conjugated molecules of interest, or any other form of moiety which may be displayed on a cell membrane. Conjugated molecules of interest may include small molecules, polymers, inorganic materials or any combination thereof. These surface moieties may be made of proteins, carbohydrates, lipids, nucleic acids, small molecules, inorganic materials, or any type of molecule that may be produced by a cell or labeled or conjugated prior to, concurrently with, after, or any combination thereof of EV production. These surface moieties may be artificially placed on the surface of a cell prior to vesiculation, during vesiculation, after vesiculation, or any combination of prior to, during, or after vesiculation. In some embodiments, the displaying of surface moieties may occur artificially through an act such as pulsing, heat shock, electroporation, covalent conjugation, and noncovalent coating, or any other method or combination of methods to place surface moieties on a plasma membrane. In other embodiments, the displaying of surface moieties may occur through a natural process, such as inducing a cell to produce the surface moieties and locating them on the plasma membrane. In some embodiments, some surface moieties may be loaded in combination with other agents of interest as described within this disclosure. In such situations, it may be beneficial to conduct multiple loading steps, where surface moieties may be loaded prior to vesiculation and other agents of interest, including additional surface moieties, may be loaded during or after vesiculation. Additionally, the surface moieties may be loaded after the loading of an agent of interest, such that an agent of interest, including surface moieties, may be loaded prior to vesiculation, while additional surface moieties may be loaded during or after vesiculation. Using EVs alone or an EV displaying a surface moiety, may be important for applications such as immune transplant rejection or multiple sclerosis.

[0140] Figure 22 demonstrates an example of one strategy to elicit an immune response in an individual in accordance with various embodiments. It should be noted that Figure 22 is only exemplary and does not describe all possible ways to elicit an immune response in an individual in accordance with embodiments of the present invention. Specifically, Figure 22 demonstrates an example of the delivery of an EV displaying specific antigens, to create an immune response. In Figure 22, cells may be isolated from an individual (2202). These cells may be manipulated in a way to display an antigen of choice (2204) prior to inducing vesiculation (2206) to produce EVs displaying the antigen. These antigen-displaying EVs may be administered to an individual (2208) to generate an immune response, when the individual is immune-challenged by something displaying the same antigen (2210).

[0141] Specifically, Figure 22 describes a method of several embodiments where bone marrow dendritic cells (BMDC) were used for immunotherapy, especially in the field of cancer. EVs derived via exposing BMDCs to sulfhydryl blocking reagents can be used for cancer immunotherapy in the form of a cell-free vaccine. Dendritic cells (DCs) activate T cells against antigens, and therefore can be used for developing an immunization against antigens, including cancer-specific antigens. A common laboratory model for cancer immunotherapy relies on E.G7-OVA cells, a lymphoma cell line which expresses the antigen SIINFEKL. EVs from SIINFEKL-presenting BMDCs were produced to use as a model vaccine against E.G7-OVA. DC-derived exosomes have been shown to be an alternative to DC adoptive therapy. Vesiculation, in accordance with embodiments of processes described herein, would enable a more efficiently produced cell-free vaccine than is currently available using exosomes.

[0142] In the method described by Figure 22, BMDC were isolated from C57BL/6 mice (2202). These cells were pulsed in the presence of the SIINFEKL antigen to cause the cells to display the antigen (2204). The BMDC cells were treated with sulfhydryl blocking reagents (2206) to produce EVs displaying the SIINFEKL antigen. The SIINFEKL- displaying EVs were administered to a mouse (2208) through an intraperitoneal injection (2208). The mouse was further challenged to with EG7-OVA cells (2210) to assess efficacy of using antigen-displaying EVs to produce an immune response.

[0143] In embodiments of EV for use for immunotherapy, the EVs may be loaded with a surface moiety by incubating cells or empty EVs with 25 pg/mL, 50 pg/mL, 100 pg/mL, 200 Mg/mL, 300 pg/mL, 400 pg/mL, or 500 pg/mL concentration of the surface moiety. Additionally, the incubation may occur for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, or 48 hours. Alternatively, the loading conditions may occur at a ratio of EVs to a surface moiety of 1 :0.1 , 1 :0.2, 1 :0.3, 1 :0.4, 1 :0.5, 1 :0.6, 1 :0.7, 1 :0.8, 1 :0.9, 1 : 1 , 1 :2, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.

[0144]Turning now to Figure 23. Figure 23 demonstrates how a person may determine the efficacy of loading a surface moiety onto an EV. Specifically, Figure 23 shows the results of SIINFEKL loading onto EVs are shown. In Figure 23A, the size distribution of EVs produced from BMDC and BMDC displaying SIINFEKL (BMDC-SIINFEKL) as measured by DLS.

[0145] In Figure 23B, the presentation of the antigen SIINFEKL by EVs was examined. BMDC cells (either pulsed with SIINFEKL at 1 mg/ml for 1 hour at 37°C and washed twice or control cells) were vesiculated at 10 mil cells/ml in 10 mL PBS with 180 μΙ 4%PFA solution and 20 μΙ 1 M DTT for 4 hours at 37°C. After cell and cell debris removal by centrifugation at 1200 rpm for 5.5 min, micro-sized EVs were concentrated at 13,200 rpm for 10 minutes and redispersed in 100 μΙ PBS. Vesicles were labeled with fluorescently labeled antibody specific for SIINFEKL for 1 hour at 4°C and washed twice and then characterized by flow cytometry. Based on forward and side scattering data, micro-sized vesicle gating was set and the percentage of fluorescent micro-sized vesicles was found. Figure 23B shows the percentage of fluorescent events in the micro-sized vesicle region. Over 70% of SIINFEKL-pulsed BM DC-derived EVs show SIINFEKL presentation.

[0146] Additionally, the polydispersity of surface moiety displaying EVs may have a similar polydispersity index (PDI) of unloaded EVs. As such, surface moiety displaying EVs may have a PDI of about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

[0147] Turning now to Figure 24, showing how the efficacy of EVs loaded with surface moieties may be at eliciting an immune response. Specifically, Figure 24 demonstrates the ability of these EVs to activate T-cells after determining EVs presented the antigen SIINFEKL. In embodiments, ten thousand cells of the T-cell line B3Z were plated in 95 μΙ_ of media (RPMI) in a 96 well plate. 5 μΙ_ of EVs were added and the cells were incubated overnight. (Note that BMDC and BMDC-SIINFEKL EVs were produced from equivalent numbers of BMDC cells.) After removing the media from the cells, a beta-galactosidase assay (CPRG) was performed and absorbance was measured at 570 nm. High activity of beta-galactosidase indicate activation of the T-Cells. Thus, Figure 24 is an indication that BMDC-SIINFEKL EVs can activate B3Z cells.

[0148]Turning now to Figure 25, which demonstrates examples where some cells may be more suitable for surface moiety loading than others. Specifically, immature BMDC- SIINFEKL EVs were found to be more effective than other BMDC EVs at eliciting an immune response. Figure 25A shows data from a CPRG assay describing how mature BMDC-SIINFEKL produced a similar T-cell response to mature BMDC and immature BMDC, while immature BMDC-SIINFEKL cells produced a higher level of response. Following an assay using fluorescently labeled antibodies specific for SIINFEKL, only immature BMDC-SIINFEKL EVs presented the SIINFEKL antigen.

Methods and Materials of Exemplary Embodiments

[0149] Cell culture. A mouse lymphoma cell line (EL4) was obtained from the American Type Culture Collection (ATCC) and grown in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 1 % penicillin-streptomycin. Cell were kept at 37°C with [0150] E\/ production and isolation. 107 cells/mL were incubated with 25 mM paraformaldehyde and 2 mM dithiothreitol in PBS for 6 hours at 37°C with 5% CO2. To isolate EVs, cells were removed by centrifugation at 1 ,200 rpm for 5.5 minutes followed by removal of cell debris and micro-sized EVs at 9,300 xg for 10 minutes. EVs were concentrated with a 30 kDa centricon (4,500 rpm for 15 minutes); the EV-containing supernatant was concentrated and washed with an equivalent volume of PBS twice.

[0151] E\/ characterization. EV size distribution was characterized by dynamic light scattering analysis using a Malvern Zetasizer. Size was confirmed and structured was analyzed via transmission electron microscopy (TEM). Formvar carbon coated copper grids coated with 10 μί of EVs stained with 1 % uranyl acetate were imaged with a Philips/FEI CM-20 Transmission Electron Microscope operated at 200 kV. EVs were quantified using a BCA Protein Assay Kit (Pierce Biotechnology).

^52] Doxorubicin-loading of EVs. EVs (25 ug/mL by protein content) were incubated with DOX (1 mg/mL) in PBS mixing for 12 hours at 37°C. Free drug was removed and EVs were concentrated with a 30 kDa centricon (4,500 rpm for 15 minutes); the collected EVs were washed with an equivalent volume of PBS three times. A sample of vesicles was lysed via sonication for 15 minutes and the drug content was quantified by fluorescence spectroscopy (ex. 485 nm, em. 595 nm).

[0 ^* 53] Doxorubicin release. DOXIL and DOX-loaded EVs were diluted to 10ug/ml_ in PBS and kept incubating at 37°C on a shaking plate. At each time point, a sample was removed and centrifuged with a 30 kDa centricon at 4,500 rpm for 15 minutes. The EVs remaining concentrated on the filter were resuspended in an equivalent volume of PBS and analyzed by florescence spectroscopy (ex. 485 nm, em. 595 nm) to determine the concentration of DOX remaining in the liposomes and EVs.

[0154] /n vitro viability studies. EL4 cells were plated at 20,000 cells/well in 100 μΙ_ of DMEM with 10% FBS. Cells were incubated for 24 hours or 48 hours with the concentrations of DOX, DOXIL or DOX-EVs described in Figure 20. An MTT assay was performed and cells were incubated with 1 mg/mL of MTT for 1 hour. The crystals were dissolved in 100 μί of DMSO and the absorbance of each well was characterized at 560 nm. [0155] /n vivo therapeutic experiments. EL4 tumors were established in the right thighs of 12-week old female C57BL/6 mice by subcutaneous injection of 106 EL4 cells in 100 μΙ_ DPBS. After eight days, tumors were clearly visible, and the mice were intravenously injected with a single dose of the treatment of control DPBS in a total volume of 100 μΙ_. Tumor size was measured over 40 days post treatment.

[0156] For pharmacokinetics and biodistribution experiments, tumors were established and mice were treated as described above. Tail vein blood collection was completed at the time points indicated and suspended in acidified alcohol. The blood was analyzed for DOX concentration by fluorescence spectroscopy (ex. 485 nm, em. 595 nm). 24 hours post treatment, the mice were sacrificed and the organs were extracted. The organs were cryopulverized and resuspended in acidified alcohol for fluorescence spectroscopy (ex. 485 nm, em. 595 nm) analysis.

EXEMPLARY EMBODIMENTS

[0157] Biological data supports the generation and characteristic description of the nanovesicles. Furthermore, the data supports the notion that the nanovesicles can be used to a compound delivery system capable as a therapeutic treatment. The following data also details the scalability and enhanced production of nanovesicles from a host source. Accordingly, these data support the various embodiments of the invention as described.

Optimizing Sulfhydryl Blocking to Produce EVs

[0158] Figure 5 describes embodiments of the production of EVs, where the concentrations of sulfhydryl blocking reagents have been altered. Specifically, Figure 5A demonstrates embodiments where the concentration of paraformaldehyde has been altered to show maximum production levels at 25 mM paraformaldehyde. Figure 5B shows embodiments where HeLa cells were exposed to various concentrations of sulfhydryl blocking reagents, such that 1X = 25 mM paraformaldehyde with 2mM dithiothreitol. EVs were isolated and quantified using a BCA protein assay. These data show that the production of EVs with HeLa cells is highest at 25mM paraformaldehyde with 2mM dithiothreitol, as compared to other concentrations tested. Collecting and Purifying EVs

[0159] In various embodiments, EVs may be collected by any suitable means to separate EVs from cells or cell debris. In some embodiments, to isolate EVs, cells were removed by centrifugation at 1 ,200 rpm for 5.5 minutes followed by removal of cell debris and micro-sized vesicles at 10,000 rpm for 10 minutes. EVs were concentrated with a 30 kDa centricon (4,500 rpm for 15 minutes); the EV-containing supernatant was concentrated and washed with an equivalent volume of PBS twice. Upon production of EVs in embodiments of this disclosure, formaldehyde residue may be removed using centrifugation, as shown in Figure 6. The presence of formaldehyde in the resulting EV collection was measured by ¹ H NMR. It should be noted that an embodiment removing formaldehyde by centrifugation is only exemplary, and residue from any added reagents may be removed by any suitable means, including centrifugation, absorption, neutralization, or other means known in the art.

Producing EVs by Sulfhvdryl Blocking

[0160] Figure 7 demonstrates an embodiment where the supernatant of vesiculating HeLa cells was analyzed by TEM. HeLa cells treated with paraformaldehyde (PFA) and dithiothreitol (DTT), in accordance with various embodiments of the invention, generate a large amount of EVs as secondarily confirmed by TEM measurements. Since the control (inset) does not show EVs, these nano-sized EVs are unlikely to be exosomes generated by normal cellular processes. Instead, sulfhydryl blocking reagent-induced blebbing results in cells shedding nano-sized EVs. Additionally, Figure 8 shows a TEM image of EL4 cells treated with and without PFA/DTT. TEM images in Figure 8A demonstrate that the supernatant from EL4 cells incubated in serum-free/reagent-free media have little or no vesicles, while the EL4 cells incubated with serum-free media containing 25 mM paraformaldehyde and 2 mM dithiothreitol for 4 hours, however, had many nano-sized EVs as indicated by the circles. In order to more clearly examine the EVs, the cells, debris and giant plasma membrane vesicles were removed by centrifugation at 16, 100xg. The EVs were then isolated and concentrated by centrifugation at 100,000xg. [0161] EVs are a promising potential therapeutic carrier. Low yield of intrinsic vesicle production is a problem. However, sulfhydryl blocking reagents can be used to produce EVs appropriate in size for carrying therapeutic cargo. Figure 9 demonstrates embodiments of EV production in the presence and absence of sulfhydryl blocking reagents. EL4 cells were incubated at 100,000 cells/mL in 5 ml_ of either DMEM (without FBS) for 24 hours or PBS with 90 μΙ_ 4%PFA solution and 10 μΙ_ 1 M DTT for 2 hours at 37°C. After cells and cell debris were removed by centrifugation at 1200 rpm for 5.5 min, micro-sized vesicles were pelleted at 13,200 rpm for 10 minutes, leaving the EVs in the supernatant. Then, EVs were isolated by 30 kDa centricons at 4,500 rpm for 10 minutes. The EVs were washed three times with 5 ml_ of DPBS to remove free protein and sulfhydryl blocking reagents. Protein content was assessed using a BCA protein assay (Figure 9). Vesicles produced from cells that were not exposed to sulfhydryl blocking reagents are called exosomes. Sulfhydryl blocking reagents produce a significantly greater (10-fold) amount of EVs compared to naturally occurring exosomes, based on protein quantification over a significantly shorter time-frame of production.

Determining Stability of EVs

[0162] Figure 10 describes the stability of exosomes versus EVs produced according to certain embodiments. In this figure, the size range of exosomes and EVs of some embodiments are shown as measured by dynamic light scattering (DLS) as measured at 0, 24, and 48 hours. Additionally, the polydispersity index (PDI) shows that as time passes, exosomes increase in polydispersity, while EVs of certain embodiments remain stable. Also, the anionic surface charge of exosomes decreased over time, while EVs of some embodiments remain relatively stable after 24 and 48 hours, when compared to the initial measurement.

Adjusting EV Size for Specific Uses

[0163] Figure 12 shows some embodiments, where phosphate-buffered saline (PBS) may be used along with the sulfhydryl blocking reagents to generate EVs. In this figure, EVs were generated using 25 mM PFA, 2 mM DTT along with PBS at 0.1X, 1X, and 10X concentrations. The size distribution of EVs produced in these embodiments was measured via a DLS assay. The increased concentration of PBS led to smaller EVs produced in some embodiments. These results indicate that the size of EVs produced in some embodiments may be customized to suit specific needs.

[0164] Specifically, Figure 12A demonstrates the effect of increasing the buffer concentration to generate EVs in the 10 nm to 1 ,000 nm size-range (nano-scale EVs) of some embodiments. Embodiments of nano-scale EVs may be generated by inducing vesiculation in cells, followed by a 30 kDa centrifugal filtration as described in this disclosure. In some embodiments, cells may be removed from the solution by an initial 1 ,200 rpm centrifugation prior to the 30 kDa centrifugal filtration. Additionally, Figure 12B demonstrates a similar result showing a linear relationship between increased osmolarity and smaller EVs.

[0165] Similarly, Figures 12C and 12D demonstrate the effect of increasing the buffer concentration to generate EVs in the 500 nm to greater than 15,000 nm size-range (micro- scale EVs) in other embodiments. Embodiments of micro-scale EVs may be generated by inducing vesiculation in cells followed by centrifugation at 1 ,200 rpm to remove cells. The remaining supernatant may be further centrifuged at 9,300 xg to isolate micro-scale EVs. It should be noted that the supernatant remaining after isolation of micro-scale EVs may also be submitted to a 30 kDa centrifugal filtration to further isolate nano-scale EVs. Images of micro-scale EVs of various embodiments are shown in Figure 12E. In these images, the size and distribution of micro-scale EVs generated using sulfhydryl blocking reagents along with varying concentrations of PBS are shown.

[0166] The size of EVs produced in certain embodiments may also be adjusted by using alternative buffers. Figure 13A demonstrates the effect of several buffers on the size of EVs produced by some embodiments as measured by DLS. In this figure, the buffers DPBS, DMEM, and GPMV were shown to produce EVs with sulfhydryl blocking in various size ranges, including into the larger, pm-sized EVs.

[0167] Additionally, not all buffers produce EVs at the same rate. Figure 13B demonstrates the production of EVs by various buffers as determined by a BCA assay to assess the protein content. As shown, PBS and DPBS buffers are more efficient than GPMV and DMEM buffers. Figure 13C demonstrates PDI of EVs produced by PBS and GPMV buffers. As indicated in Figure 13C, PBS buffer produces EVs with a lower PDI, indicating that PBS creates more uniformly sized EVs over GPMV, which has a PDI of approximately 1 , which indicates nearly complete polydispersity of EVs produced with GPMV. Further, Figure 13D demonstrates nano-sized EVs produced by vesiculation with 0.1 M HEPES buffer and 0.9% saline, which show a very broad size distribution and high polydispersity among these buffers. Similarly, Figure 13E shows production of micro- sized EVs using 0.1 M HEPES buffer and 0.9% saline, which show a very broad size distribution and high polydispersity among these buffers.

[0168] Each of these buffers shown in Figure 13 may contain various components to balance osmotic pressure as well as supplement cellular growth. The results shown in Figure 13 indicate that changing osmolarity of the solution is not the only factor in adjusting the average size, size distribution, or production rate of EVs produced by various embodiments.

Assessing Actin Content of EVs

[0169] In Figure 14A, left panel shows a light microscope image of a micro-scale EV being formed from a host cell, while Figure 14A, right panel shows the presence of actin as stained with a fluorescent dye. Similarly, Figure 14B overlays a fluorescent image onto a light image to show that EVs produced by some embodiments may be substantially free of actin.

Loading EVs with Doxorubicin

[0170] Figure 17 further demonstrates examples of various conditions for loading EVs with a therapeutic agent in accordance with embodiments of the present invention. Specifically, Figure 17A demonstrates the production of EVs by incubating EL4 cells with PFA and DTT. EVs produced may be collected by centrifugation. DOX or another therapeutic may be loaded into to the EVs, then collected through centrifugation. EVs have great potential as therapeutic carriers due to their small size and high biocompatibility. The EL4-derived EVs were loaded with a common chemotherapeutic drug, doxorubicin (DOX). DOX is known for high instances of cardiotoxicity, and therefore is an ideal candidate for targeted therapeutic delivery. Anticancer agent, DOX, was selected for studies due to its relatively low solubility and bioavailability and subsequent potential for improved biodistribution when delivered via drug-loaded EVs. DOX's intrinsic fluorescence also aids in confirmation of drug entrapment within EVs. DOX, a chemotherapeutic anthracycline antibiotic, exhibits red fluorescence (excitation: 480 nm, emission: 580 nm). Passive DOX loading of EVs was tested at different drug concentrations, temperatures and incubation periods (Figure 17B). As expected, all three factors factor in DOX loading. Additionally, when the ratio of DOX to EVs was increased, the amount of DOX loaded into EVs also increased (Figure 17C).

[0171] To assess the DOX-loading of EL4-derived EVs, the EVs were loaded with DOX via incubation with 1 mg/ml_ of DOX for 12 hours prior to purification with 30 kDa centrifugal filters. The filter size was selected to be more than sufficient to isolate EVs while still removing free proteins and DOX. Initial studies with 30 kDa centrifugal filtration showed that a single filtration step led to removal of more than 85% of protein from the original sample (Figure 18A). This high protein loss indicates that the method should be sufficient for removal of free protein and free drug from samples of EVs loaded with DOX (herein called DOX-EVs). The size of the DOX-EVs was further assessed via DLS (Figure 18B). DLS identified that the DOX-EVs range in size from 50 nm to several hundreds of nanometers in diameter (Figure 18B). Representative TEM images of DOX-EVs can be seen in Figure 1 1 , where Figure 1 1 A shows the supernatant of EL4 cells, which have not been subjected to sulfhydryl blocking reagents; Figure 1 1 B shows EL4-derived unloaded EVs, and Figure 1 1 C shows DOX-EVs.

Assessing EV-delivered Doxorubicin Release, Uptake, and Efficacy

[0172] Figure 19 shows DOX release from and uptake. Specifically, Figure 19A demonstrates the release of DOX from DOX-EVs with and without serum added to PBS buffer as compared to the release of DOX from liposomal DOX (DOXIL). DOX-EVs release drug at a much more rapid rate than DOXIL. In fact, DOX-EVs show a more rapid release profile than DOXIL in both serum-free media (Figure 19A, left panel) and in serum-containing media (Figure 19B, right panel). Drug release reached 50% of the maximum at 51 , 13, 23, and 10 minutes for DOXIL and DOX-EVs in serum-free media and DOXIL and DOX-EVs in serum-containing media, respectively. [0173] Similarly, DOX-EVs were taken up by cells more rapidly than DOXIL (Figure 19B). Figure 19B shows EL4 cells incubated with 100 mg/mL of DOX, DOXIL, and DOX-EVs after 3, 6, and 12 hours prior to confocal imaging. In Figure 19B, endosomes were stained with CellLight Early Endosomes-GFP and lysosomes were stained with LysoTracker Green DND-26. A representative image is shown for each time point. While DOXIL shows minimal uptake over the timeframe shown, DOX-EVs are taken up in the endosome within 6 hours and drug appears in the nucleus by 12 hours. EL4 cells treated with DOX-EVs for up to 12 hours exhibit DOX fluorescence in the cytoplasm while DOXIL treated cells do not.

[0174] Turning now to Figure 20. Figure 20 describes the effect of therapeutic agents delivered by EVs of as compared to other forms of drug delivery. Specifically, Figure 20 shows the viability of EL4 cells was assessed using an MTT assay in triplicate (n = 3), and error bars show standard deviation. At the highest concentrations tested, DOX and DOX-EVs were statistically significant (p<0.01 ) from the controls as analyzed by a oneway ANOVA and the Tukey HSD post-hoc test. Although the mechanism behind the superior uptake of DOX-EVs compared to DOXIL has not been studied in this work, it is mores likely related to the PEGylatio of DOXIL. PEGylation has been shown to sterically stabilize liposomes allowing for slower release and uptake. (See, Immordino, et al., Int. J. Nanomedicine 1 , 297-315 (2016), the disclosures being incorporated herein by reference.)

[0175] Figure 20A shows the results of an in vitro MTT assay to test the viability of EL4 cells treated with DOX-loaded EVs. EL4 cells were treated over 24 or 48 hours with DOX, DOXIL, DOX-EVs, or unloaded EVs. The results show that there are some significant differences between cells treated over 24 hours and those treated over 48 hours. After a 24-hour treatment, DOX-loaded EVs do not perform as well as free DOX, but they seem to "catch up" over the longer incubation time. It is important to note that DOX-loaded EVs would not be expected do outperform free DOX in in in vitro study since their primary benefits (improved biocompatibility and extended release) are critical factors in an in vivo delivery setting. [0176] Figure 20B shows the effect of DOX-loaded HeLa-derived EVs and EL4-derived EVs at varying concentrations of DOX on both HeLa and EL4 cells. Interestingly, the cell lines did not show a preference for the EVs derived from other cell lines.

[0177] Turning now to Figure 21. Figure 21 describes the effect of therapeutic agents delivered by EVs as compared to other forms of drug delivery. Specifically, data from in vivo studies showing the efficacy of DOX are shown in Figure 21 . In these studies, EL4 tumors were established in C57BL/6 mice eight days before treatment. Treatment began on Day 0, where an equivalent DOX concentration of 8 mg/kg was given to the mice via intravenous injection into the tail vein. Figure 21 A shows that DOX-EVs provided the slowest tumor growth, indicating that DOX-EVs were more effective than free DOX or DOXIL in decreasing tumor size. Figure 21 B shows that DOX-EVs also provided the highest survival rate among the mice.

[0178]Turning to Figure 21 C, serum was collected from C57BL/6 mice treated with DOX, DOXIL or DOX-EVs and analyzed for drug content. Concentration of DOX in the serum (n=3) was measured over 12 hours by a series of blood collections and fluorescence quantifications. Both DOXIL and DOX-EVs remain in the serum longer than free DOX (Figure 21 C, left panel). Most likely due to the protective effect of PEGylation, DOXIL has a longer circulation time than DOX-EVs. Twenty-four hours after treatment, the mice were sacrificed and their organs were assessed for drug content. Figure 21 C, right panel, shows that free DOX tends to accumulate in the lung while DOXIL has a tendency to be cleared by the liver. DOX-EVs, on the contrary, show minimal accumulation in lung, liver, and all other tissue examined.

[0179] In the past decade, the goal of developing biocompatible, targeting nano-carriers in the form of EVs has become the goal of many researchers in the field therapeutic delivery. The theoretical process would involve isolating EVs from primary cells derived from a patient and then using those EVs as a therapeutic carrier for delivery of cargo to a specific site in the original patient's body. A key challenge in achieving this goal is the successful production of therapeutically effective levels of EVs. (See, Smith, J. A. et al. Bioprocess Int. 13, 1-13 (2015), the disclosure of which is incorporated herein by reference.) These procedures provide a relatively simple and highly scalable protocol for producing large quantities of nano-sized EVs. [0180] Cell-derived EVs are expected to have lower immunogenicity than polymeric, viral, or lipid-based carriers. Additionally, the DOX-EVs accumulate less in the liver and lungs than DOX and DOXIL. This may be in part related to their small size; their average diameter is half that of liposomal DOX. It could also be due to vesicles' ability to specifically associate with cells from the line that they were derived from. These biodistribution characteristics result in highly improved survival outcomes for tumor- bearing mice treated with DOX-EVs compared to the controls (50% survival versus 0% survival over 40 days, Figure 21 B).

Loading of Surface Moieties on EVs

[0181] Figure 23 shows the results of SIINFEKL loading onto EVs are shown. In Figure 23A, the size distribution of EVs produced from BMDC and BMDC displaying SIINFEKL (BMDC-SIINFEKL) as measured by DLS.

[0182] In Figure 23B, the presentation of the antigen SIINFEKL by EVs was examined. BMDC cells (either pulsed with SIINFEKL at 1 mg/ml for 1 hour at 37°C and washed twice or control cells) were vesiculated at 10 mil cells/ml in 10 mL PBS with 180 μΙ 4%PFA solution and 20 μΙ 1 M DTT for 4 hours at 37°C. After cell and cell debris removal by centrifugation at 1200 rpm for 5.5 min, micro-sized EVs were concentrated at 13,200 rpm for 10 minutes and redispersed in 100 μΙ PBS. Vesicles were labeled with fluorescently labeled antibody specific for SIINFEKL for 1 hour at 4°C and washed twice and then characterized by flow cytometry. Based on forward and side scattering data, micro-sized vesicle gating was set and the percentage of fluorescent micro-sized vesicles was found. Figure 23B shows the percentage of fluorescent events in the micro-sized vesicle region. Over 70% of SIINFEKL-pulsed BMDC-derived EVs show SIINFEKL presentation.

[0183] Figure 24 demonstrates the ability of these EVs to activate T-cells after determining EVs presented the antigen SIINFEKL. In embodiments, ten thousand cells of the T-cell line B3Z were plated in 95 μί of media (RPMI) in a 96 well plate. 5 μί of EVs were added and the cells were incubated overnight. (Note that BMDC and BMDC- SIINFEKL EVs were produced from equivalent numbers of BMDC cells.) After removing the media from the cells, a beta-galactosidase assay (CPRG) was performed and absorbance was measured at 570 nm. High activity of beta-galactosidase indicate activation of the T-Cells. Thus, Figure 24 is an indication that BMDC-SIINFEKL EVs can activate B3Z cells.

[0184] Figure 25 demonstrates that immature BMDC-SIINFEKL EVs were found to be more effective than other BMDC EVs at eliciting an immune response. Figure 25A shows data from a CPRG assay describing how mature BMDC-SIINFEKL produced a similar T- cell response to mature BMDC and immature BMDC, while immature BMDC-SIINFEKL cells produced a higher level of response. Following an assay using fluorescently labeled antibodies specific for SIINFEKL, only immature BMDC-SIINFEKL EVs presented the SIINFEKL antigen.

DOCTRINE OF EQUIVALENTS

[0185] While the above description contains many specific embodiments of the invention, these should not be construed as limitations on the scope of the invention, but rather as an example of one embodiment thereof. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their equivalents.