WO2019108957A1 - Device and system for loaded cellular vesicles and uses thereof - Google Patents

Abstract

Provided is a device, system, and method for isolating and preparing loaded cellular vesicles, and to their use for diagnosing, imaging, and/or treating certain disorders.

Description

DEVICE AND SYSTEM FOR LOADED CELLULAR

VESICLES AND USES THEREOF

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application 62/593,396, filed on December 1, 2017, the contents of which is hereby incorporated in its entirety.

[0002] The present invention is in the field of medicine. More specifically, the invention relates to a device and system useful for the preparation of cellular vesicles loaded with therapeutic, imaging, and/or diagnostic agents, and to the loaded vesicles and methods of their uses.

BACKGROUND OF THE INVENTION

[0003] Cells must eat, communicate with the world around them, and respond to changes in their environment. To help accomplish these functions, cells continually adjust the composition of their plasma membranes and internal compartments in rapid response to need. They use an elaborate internal membrane system to add and remove cell-surface components.

[0004] Through a process called exocytosis, the secretory pathway delivers newly synthesized proteins, carbohydrates, or lipids to the plasma membrane or the

extracellular space. Using the converse process called endocytosis, cells can uptake extracellular products and can remove plasma membrane components to deliver them to a type of vesicle, called endosomes to be recycled or degraded in lysosomes. To perform its function, each family of vesicles is selective as it takes up only the appropriate molecules and fuses only with the appropriate target membrane.

[0005] Intracellular vesicles, are vesicles released from endoplasmic reticulum,

Golgi Apparatus, and endosome pathway. To date, different intracellular vesicles have been described, including transport vesicles (small vesicles, spherical

vesicles, larger irregular vesicles and tubules), secretory vesicles, early endosomes, multivesicular bodies, and late endosomes. intracellular vesicles are a basic tool used by the cell for organizing cellular substances and in order to release some of them to the extracellular space.

[0006] Extracellular vesicles, are vesicles released from plasma membrane and endosome pathway that can transport cargo between cells representing an endogenous mechanism for intercellular communication. Different extracellular vesicles types, including microvesicles, exosomes, oncosomes, and apoptotic bodies, have been identified. Microvesicles bud directly from the plasma membrane and contain cytoplasmic cargo. Exosomes, although extracellular vesicles, are formed from endosomal multivesicular bodies. Despite this well-established hypothesis, the contribution of other mechanisms to the exosome formation process cannot be ruled out. Exosomes may be involved in distant cell-cell communication because they can enter the circulation when secreted and pass through additional biological barriers. Dying ells release vesicular apoptotic bodies that can be more abundant than exosomes or microvesicles under specific conditions. In addition, membrane protrusions can also give rise to large extracellular vesicles, termed oncosomes, which are produced primarily by malignant cells.

[0007] Despite these differences in origin, no uniform vesicle classification exists, due to the overlap in vesicle sizes, density and the absence of subtype-specific ligands. As a result, it remains difficult, if not impossible, to purify and thereby distinguish between vesicle types and thus to use them e.g., as delivery vehicles for therapeutics.

[0008] Also, the clinical translation of conventional drug delivery platforms has been limited. The efficiency of these platforms to overcome barriers in

macromolecule drug transport, such as reaching the target tissue and engaging intracellular targets, is still limited. Successful development of clinical treatments for certain diseases has stalled due to a lack of proper therapeutic delivery systems. In addition, concerns related to immunogenicity and toxicity of non-natural delivery systems remain.

[0009] Although exosomes may be useful for therapeutic drug delivery, clinical applications depend on the development of scalable vesicle subpopulation isolation techniques and approaches for efficient drug loading. The low recovery of exosomes produced by mammalian cells remains an obstacle for large-scale exosomes production.

[0010] Thus, what is needed is a better in vivo, targeted, biological delivery system which can be prepared in useful, easily scaled up amounts efficacious for therapeutic, diagnostic, and imaging purposes.

SUMMARY OF THE INVENTION

[0011] It has been discovered that both intracellular and extracellular vesicles (or "cellular vesicles") can be isolated from biological tissue samples, and that certain populations of these cellular vesicles can be selected for and loaded with agents useful for therapeutic, diagnostic, and imaging applications.

[0012] These discoveries have been exploited to develop the present invention, which, in part, is directed to a device, system, and method for isolating and preparing such loaded cellular vesicles, and to their use for diagnosing, imaging, and/or treating certain disorders.

[0013] In one aspect, the disclosure provides a device for isolating and cargo loading cellular vesicles. The device comprises: a hollow container with an interior volume having a first end, a second end, and a sidewall spanning between the first end and the second end; a first electrode and first electrical contacts embedded within and extending a length of one side of the sidewall between the first end and the second end; a first electrical connection coupled to the first electrical contacts at the first end and the second end of the hollow container; a second electrode and second electrical contacts embedded within and extending a length of the sidewall on an opposite side of the first electrode and first electrical contacts; and a second electrical connection coupled to the second electrical contacts at the first end and the second end of the hollow container.

[0014] In some embodiments, the device further comprises an affinity

chromatography medium deposed within the interior volume of the hollow container.

In certain embodiments, the affinity chromatography medium comprises magnetic beads or an affinity gel. In particular embodiments, the affinity chromatography medium is coated with a receptor specific for a ligand on a cellular vesicle. In some embodiments, the affinity chromatography medium is a magnetic bead, and wherein an electrical current applied to the first electrical connection and the second electrical connection generates a magnetic field for attracting the magnetic beads to an interior surface of the sidewall.

[0015] In some embodiments, the ligand receptor is an antibody or active fragment thereof, an enzyme which specifically reacts with the ligand, or a receptor.

[0016] In certain embodiments, the first electrode is orientated parallel to the first electrical contacts along the sidewall; and the second electrode is orientated parallel to the second electrical contacts along the sidewall.

[0017] In some embodiments, the hollow container is constructed from

polypropylene or another biocompatible plastic material.

[0018] In some embodiments, the device further comprises: a load valve at the first end of the hollow container; and a remove valve at the second end of the hollow container.

[0019] In other embodiments, the device further comprises: a first opening to the interior volume of the hollow container at a center point of the first end configured to receive a first rubber stopper with electrical contacts; and a second opening to the interior volume of the hollow container at a center point of the second end configured to receive a second rubber stopper with electrical contacts. In certain embodiments, the first electrical connection comprises a first pair of socket connections on opposing sides of the first opening configured to make contact with the electrical contacts of the first rubber stopper; and the second electrical connection comprise second pair of socket connections on opposing sides of the second opening configured to make contact with the electrical contacts of the first rubber stopper. In particular embodiments, insertion of the first rubber stopper in the first opening and the second rubber stopper in the second opening provides an electrical current to the first electrical contacts and the second electrical contacts via the first pair of socket connections and the second pair of socket connections. [0020] In some embodiments, the device further comprises a plurality of cellular vesicles, each of the cellular vesicles have a ligand on its surface. In certain embodiments, the cellular vesicles are selectively bound to the affinity chromatography medium. In particular embodiments, the cellular vesicles are selectively bound to the affinity chromatography medium via a receptor on the affinity chromatography medium specific for the ligand in the vesicle surface. In some embodiments, an electrical current applied to the first electrical connection and the second electrical connection generates an electric field which causes the vesicles to undergo electroporation.

[0021] In certain embodiments, the device further comprises an agent disposed within the hollow space, wherein an electric current applied to the first electrical connection and the second electrical connection generates an electric field which causes the agent to enter the vesicles.

[0022] In another aspect, the disclosure provides a system for isolating and cargo loading cellular vesicles. The system comprises: a cartridge device comprising: a hollow container with an interior volume having a first end, a second end, and a sidewall spanning between the first end and the second end; a first electrode and first electrical contacts embedded within and extending a length of one side of the sidewall between the first end and the second end; a first electrical connection coupled to the first electrical contacts at the first end and the second end of the hollow container; a second electrode and second electrical contacts embedded within and extending a length of the sidewall on an opposite side of the first electrode and first electrical contacts; and a second electrical connection coupled to the second electrical contacts at the first end and the second end of the hollow container. The system also comprises a power supply configured to supply an electrical current; a first wire configured to couple to the power supply and to the first electrical connection of the cartridge device to supply the electrical current to the first electrode; and a second wire configured to couple to the power supply and to the second electrical connection of the cartridge device to supply the electrical current to the second electrode. [0023] In some embodiments, the system further comprises an affinity

chromatography medium disposed within the hollow container. In particular embodiments, the system further comprises a plurality of cellular vesicles disposed within the hollow container.

[0024] In yet another aspect, the disclosure provides a method of preparing a cellular vesicle loaded with a therapeutic, imaging, and/diagnostic agent. The method comprises: obtaining a cellular vesicle fraction from a biological sample; isolating a preselected cellular vesicle from the cellular vesicle by affinity chromatography;

subjecting the isolated vesicle to electrophoresis in the presence of an agent, thereby loading the agent in the vesicle; and eluting the loaded cellular vesicle frirn the medium.

[0025] In some embodiments, the biological sample is a tissue sample, a bodily fluid, or cell culture.

[0026] In certain embodiments, the cellular vesicle fraction is prepared by homogenization and sequential differential ultracentrifugation of the sample. In particular embodiments, the biological sample is homogenized by freezing and thawing, by sonication, by French press, detergent treatment, serine protease treatment, or by saponin-treatment.

[0027] In some embodiments, the ligand on the vesicle is a CD protein, a major histocompatibility complex (MHC) class II, a Rab protein, a histone, ARF6, Tsg 101, Alix, Tetraspanins (CD63, CD37 and CD82), p-selectin, GPib, vSNARE, Secl3/3l, Clathrin, COPI, or COPII.

[0028] In particular embodiments, the isolation step comprises contacting the cellular vesicle in the cellular vesicle fraction with an affinity chromatography medium, the medium comprising a receptor specific for the ligand on the vesicle, thereby immobilizing the cellular vesicle to the affinity chromatography medium.

[0029] In some embodiments, the receptor is an antibody or active fragment thereof or enzyme specific for the ligand. [0030] In certain embodiments, the method further comprises the step of stabilizing the cellular vesicle after electroporation.

[0031] The disclosure also provides a cartridge comprising a cellular vesicle immobilized to an affinity chromatography medium having specificity for a preselected vesicle ligand. In some embodiments, the cellular vesicle is an intracellular or extracellular vesicle, and/or the vesicle ligand is an extracellular or intracellular vesicle ligand.

[0032] In some embodiments, the affinity chromatography medium is a magnetic bead to which a receptor specific for the ligand is attached. In certain embodiments, the receptor on the affinity chromatography medium is an antibody, or active fragment thereof, specific for the microvesicle ligand. In certain embodiments, the ligand is a CD protein, a major histocompatibility complex (MHC) class II, a Rab protein, a histone, ARF6, Tsg 101, Alix, Tetraspanins (CD63, CD37 and CD82), p-selectin, GPib, vSNARE, Sec 13/31, Clathrin, COPI, or COPII.

[0033] In some embodiments, the cartridge further comprises a therapeutic, imaging, and/or diagnostic agent, and in some embodiments, the agent is in the vesicle.

DESCRIPTION OF THF DRAWINGS

[0034] The foregoing and other objects of the present disclosure, the various features thereof, as well as the disclosure itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

[0035] FIG. 1 is a diagrammatic representation of the method of obtaining biological samples useful in the methods of the disclosure;

[0036] FIG. 2 is a diagrammatic representation of one embodiment of the method using homogenization and sequential ultracentrifugation with density adjustments steps;

[0037] FIG. 3 is a diagrammatic representation of the immobilization and purification of cellular vesicles according to one embodiment;

[0038] FIG. 4 is a diagrammatic representation of electroporation and cargo loading of the cellular vesicles;

[0039] FIG. 5 is a diagrammatic representation of the recovery of the loaded cellular vesicles;

[0040] FIG. 6 is a diagrammatic representation of the efficiency of recovery of the agent loaded in the cellular vesicles;

[0041] FIGS. 7A and 7B are diagrammatic representations of a cellular vesicle isolation system;

[0042] FIGS. 8A, 8B, 8C, and 8D are diagrammatic cross-sectional views of a cartridge device for a cellular vesicle isolation system;

[0043] FIGS. 9 A, 9B, 9C, and 9D are diagrammatic representation of a cellular vesicle isolation system during an example operation utilizing magnetic bead affinity chromatography; [0044] FIGS. 10A and 10B are diagrammatic representations of a cellular vesicle isolation system during an example operation utilizing gel affinity chromatography;

[0045] FIG. 11A is a series of representations of transmission electron micrographs of cellular vesicles obtained by sequential ultracentrifugation with density adjustments; and

[0046] FIG. 11B is a representation of a light micrograph of cellular vesicles obtained by sUC.

DESCRIPTION

[0047] The disclosures of these patents, patent applications, and publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. The instant disclosure will govern in the instance that there is any inconsistency between the patents, patent applications, and publications and this disclosure.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated.

[0049] The present disclosure is directed to a device, system, and method useful for isolating cellular vesicles, as opposed to just intracellular or extracellular vesicles, and for loading them with therapeutically efficacious cargo.

[0050] As used herein, the term "cellular vesicles" encompasses the group of all intracellular and extracellular biological vesicles. Intracellular vesicles are those made and found inside cells, such as, but are not limited to, vesicles released from endoplasmic reticulum, Golgi Apparatus, and endosome pathway, including transport vesicles, secretory vesicles, early endosomes, multivesicular bodies, and late endosomes. Extracellular vesicles are those found in the extracellular space and include, but are not limited to, microvesicles, exosomes, oncosomes, and apoptotic bodies.

[0051] The term "biological sample" as used herein refers to samples taken from solid tissue (such as, but not limited to, muscle, bone, organ ,skin, and the like), from bodily fluids (such as, but not limited to, blood, plasma, cerebrospinal fluid, vaginal and lacrimal secretions, mucous, urine, sweat, tears), and cell cultures (including, but not limited to, stem cell, culture, embryonic and adult tissue culture of embryonic and adult tissues). [0052] The term "agent" encompasses diagnostic, imaging, and therapeutic compounds and molecules such as, but not limited to, small molecules, elements, proteins, peptides, nucleic acids, DNA, RNA Genes, antibodies, radionuclides, and fluorescent probes. The term "cargo" also refers to the agent.

[0053] The vesicle fraction isolated by sUC with density adjustments is then subjected to at least the following steps: (1) affinity chromatography either inside or outside of a cartridge device to characterized certain or all cellular vesicles having selected ligands on their surface, (2) electroporation of ligand-bound vesicles inside the device to destabilize vesicle walls, (3) loading an agent into the vesicles; and (4) recovering the loaded cellular vesicles from the cartridge. These steps are described in detail below.

1. Biological Samples

[0054] Cellular vesicles are obtained from autogeneic and allogeneic solid tissue biopsies, mesenchymal stem cell cultures and other type of cell cultures, and from bodily fluids including, but not limited to, whole blood, plasma, urine, saliva, sweat, tears, mucous, vaginal and lacrimal secretions, seen, and cerebrospinal fluid samples. For example, useful biological samples can be excised or biopsied from solid tissue, obtained from a blood draw or bodily fluid aspiration, swab, or sampling, or by lifting of plated cells in culture or from sampling of a liquid culture.

2. Homogenization

[0055] The present method comprises isolation of cellular vesicles from a tissue sample that has been homogenized to rupture cell membranes in the absence of detergents. Homogenization can be done by any method which disrupts at least the cell membrane including, but not limited to, sonication, serial extrusion, pressure (e.g., French Press), freeze/thaw cycles, saponin treatment of intact cells. For example, several freeze-thaw cycles using liquid nitrogen can be used (FIG. 2). The cellular vesicles are then obtained and purified from broken cells as described below. It is useful is homogenization results in at least about a 300-fold increased vesicle yield compared to isolation of exosomes in a supernatant of a cell culture. 3. Isolation of Cellular Vesicles

[0056] Once the sample has been homogenized, it is subjected to any method which results in separation of a cellular vesicle fraction from the rest of the homogenate. One nonlimiting method is sequential ultracentrifugation (sUC) with density adjustments.

[0057] As used herein, the term "density adjustments" refers to the isolation ultracentrifugation procedure based in the property of particles that float in water or density- enhanced solutions, using NaCl or KBr to increase and adjust the density. Cellular vesicles are composed by a mixture of lipids and proteins and they float in a specific range of density.

[0058] Changes in the lipid/protein vesicle ratio cause significant changes in the density of the vesicles. Therefore, cellular vesicles can be isolated based on sedimentation at high g-forces by repeated ultracentrifugation after progressively raising the solvent density. This formed the basis for the sequential flotation of cellular vesicles with density adjustment. Generally, this method comprises low speed spins to remove cell debris. After, density adjustments refers to the sequential centrifugation showed in the Fig 2. Flotation of very small cellular vesicles at <1.006 g/ml for 17 hat 105.000 g, followed by adjustment of the density of the infranadant to 1.060 g/ml with KBr and centrifugation for 24 hat 105.000 g to recover the next cellular vesicle population, followed by adjustment of density of the infranadant to 1.120 g/ml with KBr and centrifugation for 32 hat l05.000g and finally adjustment of the density of the infranadant with KBr to 1.280 g/ml with centrifugation for 40 h to isolate the heaviest cellular vesicle population using a TFT 70.3 fixed angle rotor. The cellular vesicles can then be exhaustively dialyzed against phosphate-buffered saline (PBS), pH 7.4, to remove the salt solution. Sequential ultracentrifugation with density adjustments can be carried out in any commercially available ultracentrifuge.

4. Selection of Cellular Vesicles

[0059] The cellular vesicles are purified form the isolated biological fraction, e.g., from the supernatant of the centrifuged homogenate or from the lighter gel chromatography fractions. The biological fraction may represent only one fraction corresponding to a one specific range of density. Alternatively, the sample may represent different combinations of different fractions, even the combination with all of density ranges.

[0060] Purification is carried out using affinity chromatographic methods. Affinity isolation is based on selective recognition and binding, or selective capture of cellular vesicles that bear specific ligands on their surface. Such ligands include, but are both limited to, proteins, enzymes, and other molecules. These ligands can be utilized to preselect cellular vesicles with the use of affinity chromatography media attached to molecules that specifically recognize and bind to these ligands or markers.

[0061] Useful molecules include receptors or other molecules, such as antibodies and active antibody fragments comprising an antigen binding fragments ( e.g Fv fragments, single chain Fv (scFv) fragments, Fab fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, and multivalent versions of the foregoing: multivalent internalizing moieties including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fab fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, human antibodies and antibody fragments, and multivalent versions of the foregoing: multivalent internalizing moieties including without limitation: monospecific or bispecific antl bodies, such as disulfide stabilized Fv fragmems, scFv tandems ((scfv)₂ fragments), diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized ( i.e ., leucine zipper or helix stabilized) scFv fragments: receptor molecules which naturally interact with a desired target ligand.

[0062] Affinity media attached to antibody-related receptors are

immunoaffinity media. Also useful are media attached to enzymes that specifically react with the vesicle ligand or substrate. Such useful vesicle ligands on the surface of intracellular and/or extracellular vesicles, such as, but not limited to, major histocompatibility complex (MHC) class II, Rab proteins, histones, ARF6, Tsg 101, Alix, Tetraspanins (CD63, CD37 and CD82), p-selectin, GPib, vSNARE,

Seel 3/3 1, Clathrin, COPI, COPII, and the like. For example, different panels of intracellular and extracellular CD (cluster of differentiation) ligands can be used to purify the cellular vesicles by using molecules. Such useful vesicle enzymes on the surface of cellular vesicles (intracellular and/or extracellular vesicles), such as, but not limited to, metalloproteinases, serine proteases, aspartic proteases, and the like.

[0063] Useful affinity chromatography media to which receptors specific for vesicle ligands include, but are not limited to, magnetic beads and gel

filtration/exclusion media such as hydroxy lated methacrylate and TOYOPEARL, TSKgel, and sephacryl.

[0064] In one example, the present method is an isolation method to capture all intracellular and extracellular families derived from antigen-presenting cells (APCs) using methods which immobilize preselected vesicles to a solid support ( e.g receptor-, antibody-, enzyme-coated magnetic beads or affinity media such as gel chromatography media.

[0065] FIG. 3 illustrates one embodiment of the method using antibody-coated magnetic beads. These magnetic beads are small (typically 1 pm - 4 pm diameter) and solid (non- porous) particles that provide the sufficient surface area-to-volume ratio needed for effective ligand immobilization and affinity purification. The coating makes the beads inert (to minimize nonspecific binding) and provides the chemical groups needed for attaching monoclonal antibodies used for isolation of all cellular vesicle populations. Magnetic beads or affinity gel coated with specific ligand receptors are mixed with the homogenized biological samples. During mixing the beads or gel and the isolated homogenized biological samples remain suspended in a binding buffer at physiologic pH and ionic strength, such as, but not limited to, phosphate buffered saline (PBS), pH 7.4 ( + 9 g/L NaCl)" allowing affinity interactions to occur with the ligands on the surface of cellular vesicles. Once the binding interaction occurs (after 2 hours of incubation at 37 C), the support is washed with additional buffer to remove nonbound components of the sample. Nonspecific (e.g., simple ionic) binding interactions can be minimized by adding low levels of detergent or by moderate adjustments to salt concentration in the binding and/or wash buffer. Finally, elution buffer (PBS,+ Glycine) is added to break the binding interaction and release the target molecule, which ls then collected in its purified form. The most widely used elution buffer in the present invention was 1M glycine HCL pH 2.5-3.0. This buffer effectively dissociates most protein: protein and antibody: antigen binding interactions without

permanently affecting protein structure. Mixing can be done in a container to avoid interaction with other biomolecules. Once the cellular vesicles are adhered or bound to the medium, the media can be placed in a container, such as the cartridge device described below according to the disclosure. Alternatively, the unbound medium can be placed in the container with the isolated biological fraction, such that binding occurs therein. The bound affinity medium may then be adhered to the lateral walls of the container.

5. Cartridge and System

[0066] To avoid interaction with biomolecules, affinity purification with receptor-coated medium (magnetic beads or an affinity gel) can be performed in a container, such as a cartridge, according to the disclosure. Generally, the cartridge is a hollow device which can receive different types of affinity chromatography media. The sidewalls of the cartridge have electrodes and electrical connectors embedded therein that can receive an electrical current from a power source ( e.g battery, power supply, etc.). With an affinity chromatography medium deposed within the hollow chamber of the cartridge the electrical current can be applied to the cartridge to cause elements within the affinity chromatography medium to bond with targeted cellular vesicles. With the targeted cellular vesicles bonded within the affinity chromatography medium, those vesicles are isolated from the other objects within the medium and can be utilized thereafter.

[0067] FIGS. 7A and 7B depict a system 700 for isolating and cargo-loading cellular vesicles as discussed throughout. In accordance with an example embodiment, the system 700 includes a cartridge device 702 or other container, a power supply 704, and electrical wires/connectors 706. The cartridge device 702 can be a hollow container with an interior volume having a first end 702a, a second end 702b, and a sidewall 702c spanning between the first end 702a and the second end 702b. The cartridge device 702 includes a first electrode 7lOa and first electrical contacts 712a embedded within and extending a length of one side of the sidewall 702c between the first end 702a and the second end 702b. The cartridge device 702 also includes a first electrical connection 714a coupled to the first electrical contacts 712a at the first end 702a and the second end 702b of the cartridge 702 (or other hollow container). The cartridge device 702 further includes a second electrode 710b and second electrical contacts 712b embedded within and extending a length of the sidewall 702c on an opposite side of the first electrode 7l0a and first electrical contacts 7l2a, as depicted in FIGS. 8A-8D. Similar to the first electrical connection 7l4a, the cartridge device 702 includes a second electrical connection 7l4b coupled to the second electrical contacts 7l2b at the first end 702a and the second end 702b of the cartridge device 702. When the electrodes 7l0a, 7l0b are connected to an electric source (e.g., power supply 704), the electrodes 7l0a, 710b are configured to generate an electric field to induce a voltage of 100V across a vesicle membrane and pores are formed permitting that drugs enter the interior volume of the cartridge device 702, as discussed with respect to FIGS. 9A- 10B.

[0068] FIGS. 8A-8D depict diagrammatic cross-sectional views of the cartridge device for a cellular vesicle isolation system In particular, FIGS. 8A-8D depict the location and orientation of the electrodes 7l0a, 710b, the electrical contacts 712a, 7l2b, and electrical connections 7l4a, 7l4b, as discussed with respect to FIGS.7A and 7B. As would be appreciated by one skilled in the art, the present invention can utilize any combination and number of electrical connections, electrodes, and electrical contacts without departing from the scope of the present invention.

[0069] Continuing with FIGS. 7A and 7B, the power supply 704 is configured to supply an electrical current and the electrical wires/connectors 706 are configured to couple to the power supply and to the first electrical connections 714a, 714b of the cartridge device 702 (e.g., via rubber stoppers with contact points) to supply the electrical current to the first electrode and second electrodes 7l0a, 710b. FIG. 7B depicts how the components of the system 700 are connected together in operation. More specifically, as shown in FIGS. 7A, 7B, and 8A-8D, the cartridge device 702 includes a first opening to the interior volume of the cartridge device 702 at a center point of the first end 702a configured to receive a first rubber stopper with electrical contacts and a second opening to the interior volume of the cartridge device 702 at a center point of the second end 702b configured to receive a second rubber stopper with electrical contacts. The rubber stopper can include separate stopper devices or stoppers coupled to the wires 706. The internal circumference of the first opening and the second opening include a first pair of socket connections and second pair of socket connections, respectively. In an example embodiment, the socket pairs are located on opposing sides of the openings and are configured to make contact with the electrical contacts on the rubber stoppers (when the rubber stoppers are inserted into the openings). Insertion of the first rubber stopper in the first opening and the second rubber stopper in the second opening (with the electrical contacts properly aligned) provides an electrical current to the first electrical contacts 712a and the second electrical contacts 712b via the first pair of socket connections and the second pair of socket connections, respectively.

[0070] As would be appreciated by one skilled in the art, the components of the system 700 can be constructed from any combination of materials known in the art and at any combination of varied dimensions. For example, the cartridge device 702 can be constructed from any biocompatible plastic material such as, but not limited to, polyethylene, PVC, PEEK, polycarbonate, ultem PEI, polysulfone, and polyurethane. The cartridge can be of any useful length, (e.g., 5-50 cm, 10-30 cm, 20-30 cm, 15-25 cm) and width (1-10 cm, 2-9 cm, 3-8 cm, 4-7 cm, or 5-6 cm).

[0071] In operation, an affinity chromatography medium 900 can be deposed within the interior volume of the cartridge device 702. The affinity chromatography medium 900 can include any combination of mediums known in the art. For example, the affinity chromatography medium 900 magnetic beads or an affinity gel. FIGS. 9A- 9D depict an example operation of the cartridge device 702 (discussed with respect to FIGS. 7 A and 7B) utilizing magnetic beads as the affinity chromatography medium 900. As depicted in FIG. 9A, the magnetic beads are coated with a receptor specific for a ligand on a cellular vesicle and are deposited into a suspension with cellular vesicles and loaded into the interior volume of the cartridge device 702. The plurality of cellular vesicles, in the suspension, each of the cellular vesicles have a ligand on its surface.

The ligand receptor is an antibody or active fragment thereof, an enzyme which specifically reacts with the ligand, or a receptor. When the magnetic beads are added to the suspension, the cellular vesicles are selectively bound to the affinity chromatography medium via a receptor on the affinity chromatography medium 900 specific for the ligand in the vesicle surface. As would be appreciated by one skilled in the art, any type of receptor could be implemented in accordance with the present invention.

[0072] FIG. 9B depicts the magnetic immobilization effect that is created when an electrical current is applied to the cartridge device 702 (e.g., via wires 706 providing power view the electrical connections 7l4a, 714). In particular, when an electrical current is applied to the first electrical connection 714a and the second electrical connection 714b a magnetic field is generated for attracting the magnetic beads to an interior surfaces of the sidewall 702c, as discussed with respect to FIG. 7B. Thereafter, a drug can be loaded into the internal volume of the cartridge device 702 via a load valve 716 at the first end of the cartridge device 702.

[0073] Thereafter, when the electrical current is applied to the first electrical connection 7l4a and the second electrical connection 7l4b, the vesicles to undergo electroporation and causes the agent to enter the vesicles, as depicted in FIG. 9C. FIG 9D depicts a wash and elution step in which the inner volume of the cartridge device 702 is flushed and medium is removed via the O valve 718 at the second end 702b of the cartridge device 702.

[0074] FIGS 10A and 10B depict a process in which the affinity chromatography medium 900 is an affinity gel. The cartridge device 702 operates in a similar manner discussed with respect to FIGS. 9A-9D. However, when an electrical current is applied to the cartridge device 702, the affinity gel attracts the cellular vesicles are selectively bound to the affinity chromatography medium 900 via a receptor on the affinity chromatography medium.

6. Cargo-Loading of Cellular Vesicles

[0075] Immobilized cellular vesicles specifically bound the affinity

chromatography medium, e.g., to magnetic coated beads or gel, are then loaded inside the container or the cartridge. Alternatively, the preselected vesicles are already inside the container or cartridge immobilized to affinity chromatographic media there. [0076] The agent to be loaded into the cellular vesicles includes, but is not limited to, therapeutic agents, imaging agents, and diagnostic agents. For example, useful imaging agents include probes or labels that bind e.g., to certain organs or tissues in the body that are effected by a disorder such as cancers, Alzheimer Disease (existence of plaques), coronary artery diseases, atherosclerosis, stroke and cerebral hemorrhages, sepsis, autoimmune disorders, etc. Such probes and labels include fluorescent, colorimetric, and radioactive compounds such as, fluorescent proteins (GFP, YFP, RFR), xanthene derivatives (rhodamine, fluorescein), cyanine derivatives and other non-protein organic fluorophores, technetium-99, fluorine- 18, thallmm 201 chloride. Other useful diagnostic agents include biosensors such as electrochemical biosensors, optical biosensors, electronic biosensors, piezoelectric biosensors, gravimetric biosensors and pyroelectric blOsensors that can measure biochemical and other kind of parameters in bodily fluids and other tissues. Therapeutic agents are any agents useful to treat a disease or disorder. These include, but are not limited to, small molecule drugs, elements, proteins, peptides, antibodies, active antibody fragments, nucleic acids, amino acids genes.

[0077] The agent may be mixed with the vesicle-bound affinity chromatography medium before it is put into the cartridge deice, or it may be placed in the device where it is bound to the vesicle-bound affinity chromatography medium. In the latter case, the chromatography medium may be adhered to the walls of the cartridge or may be free in the cartridge before the agent is loaded into the vesicles.

[0078] The bound vesicles are then subjected to electroporation inside the cartridge (FIG. 7 A) Electroporation causes the spontaneous formation of pores in the vesicle membrane (FIG. 4). These pores are formed to compensate for changes in voltage (about 100 v) after stimulation with an electrical signal. Electroporation may be carried out in the presence of membrane stabilizers such as, but not limited to, glycerol and/or trehalose. (FIG. 7). When the electric pulse is stopped, the cellular vesicles heal spontaneously, thereby retaining the agent inside. The excess agent not captured is removed, e.g., by washing with a washing buffer (e.g., 20 mM sodium phosphate, pH 7.0). 7. Recovery of Loaded Cellular Vesicles

[0079] Loaded vesicles bound to affinity chromatography media, e.g., magnetic beads, are separated from unbound cellular vesicles using an elution buffer (e.g., 0,1 M glycine-HCL pH 2,7) (FIG. 5).

[0080] The efficiency of the procedure can be determined analyzing

concentrations of drugs in elution buffer after lysis of cellular vesicles by Triton X- 100 (lysis buffer 20 mM Sodium Phosphate, pH 7,0 containing 5% Triton X-100) (FIG. 6).

8. Pharmaceutical Formulations and Treatment

[0081] The pharmaceutical formulations useful in the diagnostic, imaging, and therapeutic methods according to the disclosure include the loaded cellular vesicles and a pharmaceutically acceptable carrier for these nanocarriers which enables their administration to a subject via preselected routes such as oral, topical, systemic, intramuscular, or intravenous injection, transdermal, inhalation, transmembrane use, and the like.

[0082] A "therapeutically effective amount" as used herein refers to that amount of agent, and therefore, loaded cellular vesicles which provide a therapeutic and/or prophylactic therapeutic effect to a treated subject. A "therapeutically effective amount” also encompasses that amount of imaging or other agent useful to image and/or diagnose a disorder or lack thereof, in a subject.

[0083] In addition, the pharmaceutical formulations according to the disclosure may also comprise other known therapeutics or diagnostic imaging agents within the pharmaceutically acceptable carrier or within the cellular vesicles also loaded with a first therapeutic or imaging/diagnostic agent.

[0084] Formulations according to the disclosure are prepared with a pharmaceutically acceptable carrier in accordance with known techniques, for example, those described in Remington, The Science And Practice of Pharmacy (9th Ed. 1995). The term "pharmaceutically acceptable carrier" is to be understood herein as referring to any substance that may, medically, be acceptably administered to a patient, together with a compound of this invention, and which does not undesirably affect the pharmacological activity thereof; a

"pharmaceutically acceptable carrier" may thus be, for example, a pharmaceutically acceptable member(s) selected from the group comprising or consisting of diluents, preservatives, solubilizers, emulsifiers, adjuvant, tonicity modifying agents, buffers as well as any other physiologically acceptable vehicle. This pharmaceutical formulation may further contain additional therapeutic, imaging or diagnostic agents.

[0085] The pharmaceutical formulation may be prepared for injectable, topical, oral, inhalation, transdermal, transmembrane, intravenous, intramuscular, or other use and the like.

[0086] Formulations suitable for oral administration may be presented in discrete units or dosage forms, such as capsules, cachets, lozenges, tablets, sublingual tablets, pills, powders, granules, chewing gum, suspensions, solutions, and the like. Each dosage form contains a predetermined amount of agent. If in the form of a solution, the pharmaceutically acceptable carrier may be an aqueous liquid, such as buffered with a pharmaceutically acceptable pH buffer, or in non-aqueous liquid such as DMSO, or be prepared as an oil-in-water or water-in- oil emulsion.

[0087] Injectable dosage forms may be sterilized in a pharmaceutically acceptable fashion, for example by steam sterilization of an aqueous solution sealed in a vial under an inert gas atmosphere at l20°C for about 15 minutes to 20 minutes, or by sterile filtration of a solution through a 0.2 pMor smaller pore-size filter, optionally followed by a lyophilization step, or by irradiation of a composition containing a compound of the present invention by means of emissions from a radionuclide source.

[0088] As a health care professional is aware, an efficacious dose of an agent is dependent on what the agent is. Accordingly, where cellular vesicles loaded with agent are administered, the amount of cellular vesicles is dependent on the amount and type of agent it carries. Once the amount of agent in the vesicles is determined by efficiency determinations (described above), experimental doses can be administered to in vivo mammalian models such as mice or rats and extrapolated to larger animals, such as humans. Dosages of about 10 ng/ml to about lmg/ml are useful. [0089] Reference will now be made to specific examples illustrating the disclosure. It is to be understood that the examples are provided to illustrate exemplary embodiments and that no limitation to the scope of the disclosure is intended thereby.

EXAMPLES

EXAMPLE 1

Biological Sample Collection

A. Solid Tissue

[0090] Allogeneic prostatic biopsies were obtained from patients diagnosed with prostatic cancer, who were on the waiting list for prostate biopsy at the outpatient clinic of the Division of Urology, Hospital Universitario Clinica Otamendi (HUCO) Buenos Aires, Argentina. A total of 7 patients were included. The prostatic biopsies were performed following standard procedures. Prostatic histology was examined by the same experienced urologist pathologist from the Department of Urology of HUCO. The selected samples had a mean size of about 5 mm.

[0091] Allogeneic Adipose tissue samples were obtained by standard liposuction procedures from the Division of Plastic Surgery, Hospital Universitario Clinica Otamendi, (HUCO) Buenos Aires, Argentina. Hundred milliliters of adipose tissue are harvested, filling both the 50 ml FPU syringes, considering this as a lipoaspirate. A total of 7 patients were included.

B. Body Fluid Sample

[0092] For autogeneic cellular vesicle production, the process includes whole blood extraction to obtain a donor cell line and to produce and purified cellular vesicles in a useful amount. For this purpose, cellular vesicles from the same patient, were obtained from peripheral white blood cells including lymphocytes and monocytes. A modified Ficoll- Hypaque's gradient density procedure was used. All experiments were conducted in conformity with institutional guidelines and in compliance with international laws. All volunteers gave written informed consent. Whole blood (100 ml) from healthy volunteers was drawn into heparin-coated vacutainers. Peripheral blood white cells were separated by Ficoll- Hypaque density gradient by centrifugation at 400 g for 30 minutes. Buffy coats (containing cells) were collected using a Pasteur pipehe and erythrocytes were eliminated by treatment with standard lysis buffer procedures. Then, peripheral blood white cells were suspended in complete culture medium RPMI 1640 supplemented with 2 mM L-glutamine, 40 mg/mL gentamicin 100 U/mL penicillin, 100 pg/mL streptomycin) and 10 ng/ml polymyxin-B-sulfate. Finally, cells including medium containing cells were cryopreserved for further procedures.

Viability was > 70% when thawed from cryopreservation.

C. Cell Culture Samples

[0093] For autogeneic cellular vesicle production, the process includes simple whole blood extraction to obtain the donor cell line and to produce and purified cellular vesicles in a useful amount. Peripheral blood white cells suspended in complete culture medium RPMI 1640 supplemented with 10% fetal calf serum, were seeded in 6-well tissue culture plates (5 x 106 cells /mL) and allowed to adhere for 2 h at 37°C in an atmosphere of 5% C02. Monocyte rich cultures were purified by plastic adherence for 2 hat 37°C, 5% C02. Non-adherent cells (lymphocytes) were then removed by washing plates with RPMI 1640 culture medium Monocyte and macrophage differentiation of freshly isolated HBPMCs was carried out under non-stimulated conditions after 10 days of culture at 37°C, 5% C02 with the same media. Finally, cells including medium containing cells were cryopreserved for further procedures. Viability was > 70% when thawed from cryopreservation.

[0094] Cell culture and expand autogeneic peripheral blood monocytes cells to obtain cellular vesicles from immature and mature dendritic cells. For autogeneic cellular vesicle production, the process includes simple whole blood extraction, able to obtain the donor cell line and to produce and purified cellular vesicles in a useful amount.

[0095] Peripheral blood white cells suspended in complete culture medium RPMI 1640 supplemented with 10% fetal calf serum, were seeded in 6-well tissue culture plates (5 x 10^ cells /mL) and allowed to adhere for 2 hat 37°C in an atmosphere of 5% CO2. Monocyte rich cultures were purified by plastic adherence for 2 h at 37°C, 5% C0₂. Non-adherent cells were then removed by washing plates with RPMI 1640 culture medium and monocyte rich cultures were incubated with complete culture medium containing 10% Human Serum, 80 ng/ml hGM- CSF and 80-250 ng/ml rH IL- 4. After 7-day incubation period, loosely adherent cells (considered as immature dendritic cells, iDCs) were collected, washed with RPMI 1640, seeded in to 24-well tissue culture plates ( ri’ cells/mL) and submitted to the different treatments. Some aliquots of iDCs were incubated for 5-days in the presence of 1 pg/ml LPS, 3 pg/ml CD40L, and 50 ng/ml TNF-a (containing hGM-CSF and IL-4) to obtain mature DCs. Cells including medium containing cells, were cryopreserved for further procedures. Viability was > 70% when thawed from cryopreservation.

[0096] Cell culture and expand autogeneic adipose tissue to obtain cellular vesicles from autogeneic and allogeneic adipose-derived mesenchymal stem cells (ASCs).

[0097] Adipose-derived stem cells (ASCs) are multipotent mesenchymal stem cells (MSCs). These cells are precursors to adipocytes and undergo expansion. ASCs have the capacity to undergo osteogenic, chondrogenic, neurogenic, and myogenic differentiation in vitro. ASCs are a source of MSCs as they can be easily harvested in large quantities from adipose tissue fragments with minimal donor site morbidity. For these reasons, for autogeneic cellular vesicle production, ASCs were obtained by lipoaspirate from adipose tissue of volunteers. For allogeneic cellular vesicle production, ASCs were obtained from commercial sources (American Tissue culture collection, ATCC). When enzymatically digested, adipose tissue yields a heterogeneous population of many cell types (pre-adipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells, resident monocytes/macrophages, lymphocytes, and adipose- derived mesenchymal stem cells), which, upon isolation, is termed the stromal vascular fraction (SVF), of which adipose-derived mesenchymal stem cells alone comprise 30%. The freshly harvested lipoaspirate was washed with sterile phosphate buffered saline (PBS), enzymatically digested by collagenase 0.1 g/ml, and subsequently subjected to red blood cell lysis. Adipose-derived stem cells (ASCs) were obtained following the well- known procedure previously reported. At the end of the isolation process, a mean of 21% of the total number of isolated cells, was obtained.

[0098] To characterize the isolated cells and identify ASCs, a flow cytometric assay is required, with a panel of commonly used surface antigens based on current literature. We routinely use the monoclonal antibodies CD34 CD45, CD73, CD31, CD90, and CD105. ASCs were cultured in a low serum (2% FBS) basal medium containing essential and non-essential amino acids, vitamins, other organic

compounds, trace minerals and inorganic salts. To support the proliferation and plating efficiency of ASCs, the basal medium was supplemented with rh FGF basic: 5 ng/mL, rh FGF acidic: 5 ng/mL, rh EGF: 5 ng/mL and L-alanyl-L- Glutamine: 2.4 mM. Antimicrobials and phenol red are not required for proliferation.

EXAMPLE 2

Isolation and Characterization of Cellular Vesicle Populations hv UC

[0099] Cellular vesicles populations present in homogenized samples obtained from prostatic biopsies and cell cultures were isolated by density differences using sequential ultracentrifugation (sUC). To remove cell debris samples were first centrifuged at low speed spins (600 g per 6 min). Density ranges selected were d<

1.006 g/ml; d=l.006-l.060 g/ml; d=l.060-l. l20 g/ml; d=l. l20-l.280 g/ml; and d>l.280 g/ml. Adjustments of the density of each range, were performed with a potassium bromide solution. Moreover, cellular vesicles populations were washed during flotation with a potassium bromide solution of the respective density.

[0100] Sequential ultracentrifugation was carry out with a Centrikon T 2060 ultracentrifuge (Kontron Instruments, Milano, Italy) in a fixed angle rotor TFT 70.38 using polycarbonate thick wall tubes (38.5 ml final volume). The protocol used in each step of ultracentrifugation was the following: (1) to isolate the population with a density < 1.006 g/ml, the samples were ultracentrifuged at 105.000 g for 17 h at 4°C without adjustment of density. After, the layer floating at the top of the tube was recollected and the density of the infranadant was adjusted to d= 1.106 g/ml adding potassium bromide. After, 10 ml of a potassium bromide solution with ad= 1.060 g/ml were layered and the tubes ultracentrifuged at 105.000 g for 24 h at 4°C. Similar procedures for the other density range were done. Cellular vesicles populations were exhaustively dialyzed against phosphate-buffered saline (PBS), pH 7.4, containing 10 mM EDTA. The concentration of cellular vesicles was expressed in terms of protein content, as determined by the Quick Start™ Bradford Protein Method (Bio-Rad), with BSA as the standard. [0101] Flow cytometry in a FACS calibur cytometer was used to determine size (size- scattered light; SSC) of cellular vesicles. To calibrate sizes were used three calibration kits for flow cytometry; Nanobead (50 nm and 100 nm), Submicron (200 nm, 500 nm and 800 nm) and Micron (1 gm, 3 mhi. and 6 mih) Calibration Kits (Bangs Laboratories Inc, Fishers, IN, US). Flow cytometry and immunoblotting were used to determine the expression of intracellular and extracellular vesicle markers (CD63, ARF6, P-Selectin, GPlb, Annexin V, MHC class II, Rab 6 protein, Sec 13, Clathrin, vSNARE). Morphological characterization was performed by transmission electron microscopy (TEM) and light microscopy. Results are shown in Table 1 and in FIGs. 11A and l lB.

Table 1

[0102] Specific density and CD markers expressed on the surface of vesicles of four different populations were identified. A smaller vesicle population (<50 nm) characterized by the presence of intracellular markers on their surfaces. A second population with a size ranged between 50 nm and 200 nm was characterized by presence of endosomal markers, a third population with a size ranged between 500 nm and 800 nm was characterized by membrane plasmatic markers and finally the bigger population with a size> 3000 nm characterized by a mixture of intracellular and extracellular markers.

EXAMPLE 3

Efficiency of Prenarinp Cellular Vesicles Containing

Therapeutic Agents

[0103] To evaluate the device-system efficiency methotrexate was used as the agent to be loaded. This drug is a chemotherapy agent and immune system suppressant used to treat cancer and autoimmune diseases. The Fluorescent Polarization Immunoassay (FPIA, TDx system) was performed to determine the concentration of methotrexate in the vesicles.

[0104] Inside the cartridge, methotrexate at a concentration of 10 mg/ml was incubated with cellular vesicles immobilized to magnetic beads coated with antibodies specific for a panel of surface ligands and electroporation was carried out at 100 v during 2 hat 37 C. The amount of methotrexate that was not loaded inside the cellular vesicles was recovered by washing the cartridge with 10 ml of washing buffer (20 mM sodium phosphate, pH 7.0). The fraction of methotrexate contained in cellular vesicles was recovered with elution buffer (0,1 M glycine- HCL pH 2,7) as shows FIG. 5. Finally, methotrexate concentration was determined by Fluorescent Polarization Immunoassay (FPIA, TDx system) in both fractions. The results are shown in Table 2. Table 2. Efficiency of Preparing Cellular Vesicles Containing Methotrexate 110 mg/mll

[0105] The efficiency of the device-system was of 25 ± 7.

[0106] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

Claims:

1. A device for isolating and cargo-loading cellular vesicles, the device comprising:

a hollow container with an interior volume having a first end, a second end, and a sidewall spanning between the first end and the second end; a first electrode and first electrical contacts embedded within and extending a length of one side of the sidewall between the first end and the second end; a first electrical connection coupled to the first electrical contacts at the first end and the second end of the hollow container; a second electrode and second electrical contacts embedded within and extending a length of the sidewall on an opposite side of the first electrode and first electrical contacts; and a second electrical connection coupled to the second electrical contacts at the first end and the second end of the hollow container.

2. The device of claim 1 , further comprising an affinity chromatography medium deposed within the interior volume of the hollow container.

3. The device of claim 3, wherein the affinity chromatography medium comprises magnetic beads or an affinity gel.

4. The device of claim 2, wherein the affinity chromatography medium is coated with a receptor specific for a ligand on a cellular vesicle.

5. The device of claim 4, wherein the ligand receptor is an antibody or active fragment thereof, an enzyme which specifically reacts with the ligand, or a receptor.

6. The device of claim 4, wherein the affinity chromatography medium is a magnetic bead, and wherein an electrical current applied to the first electrical connection and the second electrical connection generates a magnetic field for attracting the magnetic beads to an interior surface of the sidewall.

7. The device of claim 1, wherein:

the first electrode is orientated parallel to the first electrical contacts along the sidewall; and the second electrode is orientated parallel to the second electrical contacts along the sidewall.

8. The device of claim 1 , wherein the hollow container is constructed from polypropylene or another biocompatible plastic material.

9. The device of claim 1, further comprising:

a load valve at the first end of the hollow container; and

a remove valve at the second end of the hollow container.

10. The device of claim 1, further comprising:

a first opening to the interior volume of the hollow container at a center point of the first end configured to receive a first rubber stopper with electrical contacts; and

a second opening to the interior volume of the hollow container at a center point of the second end configured to receive a second rubber stopper with electrical contacts.

1 1. The device of claim 10, wherein:

the first electrical connection comprises a first pair of socket connections on opposing sides of the first opening configured to make contact with the electrical contacts of the first rubber stopper; and the second electrical connection comprise second pair of socket connections on opposing sides of the second opening configured to make contact with the electrical contacts of the first rubber stopper.

12. The device of claim 11, wherein insertion of the first rubber stopper in the first opening and the second rubber stopper in the second opening provides an electrical current to the first electrical contacts and the second electrical contacts via the first pair of socket connections and the second pair of socket connections.

13. The device of claim 1 , further comprising a plurality of cellular vesicles, each of the cellular vesicles have a ligand on its surface.

14. The device of claim 13, wherein the cellular vesicles are selectively bound to the affinity chromatography medium.

15. The device of claim 13, wherein the cellular vesicles are selectively bound to the affinity chromatography medium via a receptor on the affinity chromatography medium specific for the ligand in the vesicle surface.

16. The device of claim 13, wherein an electrical current applied to the first electrical connection and the second electrical connection generates an electric field which causes the vesicles to undergo electroporation.

17. The device of claim 13, further comprising an agent disposed within the hollow space, wherein an electric current applied to the first electrical connection and the second electrical connection generates an electric field which causes the agent to enter the vesicles.

18. A system for isolating and cargo-loading cellular vesicles, the system comprising: a cartridge device comprising: a hollow container with an interior volume having a first end, a second end, and a sidewall spanning between the first end and the second end;

a first electrode and first electrical contacts embedded within and extending a length of one side of the sidewall between the first end and the second end;

a first electrical connection coupled to the first electrical contacts at the first end and the second end of the hollow container;

a second electrode and second electrical contacts embedded within and extending a length of the sidewall on an opposite side of the first electrode and first electrical contacts; and

a second electrical connection coupled to the second electrical contacts at the first end and the second end of the hollow container;

a power supply configured to supply an electrical current;

a first wire configured to couple to the power supply and to the first electrical connection of the cartridge device to supply the electrical current to the first electrode; and

a second wire configured to couple to the power supply and to the second electrical connection of the cartridge device to supply the electrical current to the second electrode.

19. The system of claim 18, further comprising an affinity chromatography medium disposed within the hollow container.

20. The system of claim 19 further comprising a plurality of cellular vesicles disposed within the hollow container.

21. A method of preparing a cellular vesicle loaded with a therapeutic, imaging, and/diagnostic agent, the method comprising:

obtaining a cellular vesicle fraction from a biological sample; isolating a preselected cellular vesicle from the cellular vesicle by affinity chromatography;

subjecting the isolated vesicle to electrophoresis in the presence of an agent, thereby loading the agent in the vesicle; and

eluting the loaded cellular vesicle frim the medium.

22. The method of claim 21, wherein biological sample is a tissue sample, a bodily fluid, or cell culture.

23. The method of claim 21 , wherein the cellular vesicle fraction is

prepared by homogenization and sequential differential ultracentrifugation of the sample.

24. The method of claim 23, wherein the biological sample is homogenized by freezing and thawing, by soni cation, by French press, detergent treatment, serine protease treatment, or by saponin-treatment.

25. The method of claim 21, wherein the ligand on the vesicle is a CD protein, a major histocompatibility complex (MHC) class II, a Rab protein, a histone, ARF6, Tsg 101, Alix, Tetraspanins (CD63, CD37 and CD82), p-selectin, GPib, vSNARE, Secl3/3l, Clathrin, COPI, orCOPII.

26. The method of claim 21 , wherein the isolation step comprises contacting the cellular vesicle in the cellular vesicle fraction with an affinity chromatography medium, the medium comprising a receptor specific for the ligand on the vesicle, thereby immobilizing the cellular vesicle to the affinity chromatography medium.

27. The method of claim 24, wherein the receptor is an antibody or active fragment thereof or enzyme specific for the ligand.

28. The method of claim 21, further comprising the step of stabilizing the cellular vesicle after electroporation.