Electroporation systems and methods for cargo loading into extracellular vesicles

Abstract

Described herein are electroporation systems and methods for cargo loading into extracellular vesicles. For example, according to one embodiment, there is a flow electroporation system, including: one or more fluid channels configured to receive one or more EV fluid streams; an EV stream inlet for the one or more EV fluid channels; an EV stream flow source fluidly coupled to the sample inlet; a cargo inlet downstream of an electric field region adjacent to the one or more fluid channels; a cargo flow source fluidly coupled to the cargo inlet; one or more cargo flow streams contain a cargo buffer with cargo molecules; at least one pump configured to deliver the one or more EV fluid streams and the one or more cargo flow streams into the one or more fluid channels; and a plurality of electrodes configured to deliver electroporation energy to the one or more EV fluid streams.

Description

ELECTROPORATION SYSTEMS AND METHODS FOR CARGO LOADING INTOEXTRACELLULAR VESICLESCLAIM OF PRIORITY

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 604,810, entitled “ELECTROPORATION SYSTEMS AND METHODS FOR CARGO LOADING INTO EXTRACELLULAR VESICLES,” filed November 30, 2023, and U.S. Provisional Patent Application No. 63 / 695,264, entitled “ELECTROPORATION SYSTEMS AND METHODS FOR CARGO LOADING INTO EXTRACELLULAR VESICLES,” filed September 16, 2024, the contents of both applications are herein incorporated by reference in their entirety.INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.BACKGROUND

[0003] Various methods of loading drugs into extracellular vesicles (EVs) are known including incubation, cell transfection, and physical treatments. Incubation may involve incubation of cargo with an exosome-secreting cell or with an exosome. Transfection may involve overexpression of desired molecules, direct transfection into an exosome, and expression of cargo-loading chaperones. Physical treatments may involve surfactant treatment, sonication, electroporation, extrusion, freeze-thaw treatment, and dialysis.

[0004] Each of these aforementioned methods are problematic. For example, passive incubation has a low loading efficiency and cell transfection provides a challenge to traffic cargo to an exosome and is not compatible with cargo that is toxic to the cell. Similarly, surfactant treatment poses issues of toxicity and biocompatibility of the surfactant with exosomes.

[0005] Another approach is conventional electroporation, or the use of short high-voltage pulses to overcome the barrier of the cell membrane by applying an external electrical field to slightly surpass the capacitance of the cell membrane and create a temporary and reversible breakdown of the cell membrane to introduce cargo. Problematically, issues with conventional electroporation on EVs also exist, particularly involving the limitations ofconventional metal cuvettes. One such issue is low cargo loading efficiency, with a typical maximum loading efficiency around 20-30% due to insufficient and non-uniform electrical fields. Another issue is low exosome recover involving DNA / RNA cargo aggregation due to reaction with metal ions released from metal electrodes and exosome fusion and instability due to non-uniform electoral fields or interaction with by-products from electrolysis reactions. A third issue involves low throughput, with a typical maximum of -ImL batch processing volume. Another important issue is electrolysis, with the formation of bubbles on electrodes which partially or completely disrupt operation as voltage and sample conductivity increase.

[0006] Electroporation has detrimental effects on EVs and cargo molecules. Antibodies, a class of cargo molecules, have been found to exhibit drastic decrease in function such as the ability to bind to cell surface antigens as quantified by Mean Fluorescence Intensity (MFI) under electric fields such as during electroporation and electrical pulsing. The level of decreased antibody function may be correlated with increased electrical pulsing intensity (kV / cm).

[0007] Sheath electroporation has been used for transfecting primary T cells with good transfection efficiency and maintained cell viability. However, there has been no study of feasibility for using sheaths for electroporating small vesicles such as exosomes and EVs.

[0008] Still further, the duration of transient pores on the EVs may only last a fraction of a second, limiting the window for cargo loading into the EVs. Studies using fluorescent labels such as Atto488-biotin (IkDA molecular weight) as a marker have found that EV permeability and cargo loading efficiency decrease sharply within the first few seconds after electroporation (to -40% loading efficiency after 3 seconds post-electroporation) and that EVs become almost non-permeable to small molecules after -30 minutes postelectroporation.

[0009] Thus, what is needed is a high throughput, continuous flow electroporation platform for efficient cargo loading into EVs separated from the detrimental effects of concurrent electrolysis. Furthermore, controlled, efficient, and rapid mixing is needed to achieve the cargo loading into the EVs for delivery through transient pores on the EVs during the very short time that the transient pores are open following electroporation.

[0010] Described herein are electroporation systems and methods for cargo loading into EVs that may address these needs.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] A better understanding of the features and advantages of the methods and apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings.

[0012] FIG. 1 A is a schematic of a two-stage flow electroporation system.

[0013] FIG. IB is a schematic of a three-stage flow electroporation system.

[0014] FIGS. 2A-2B illustrate schematics of specialized channel geometry configured for enhanced flow mixing.

[0015] FIG. 2C-2E illustrate schematics of acoustic wave induced microstreaming for enhanced flow mixing.

[0016] FIGS. 3A-3D illustrate a schematic and views of enhanced flow mixing combining electrolytic gas bubbles and channel wall protrusions.

[0017] FIG. 4 is a graph comparing cargo loading in extracellular vesicles based on the two-stage design considering de-glycosylation and electroporation parameters.

[0018] FIG. 5 depicts a chart illustrating a method for cargo loading into extracellular vesicles via electroporation as described herein.SUMMARY

[0019] Described herein are electroporation systems and methods for cargo loading into extracellular vesicles. For example, according to one embodiment, there is a flow electroporation system for cargo loading into extracellular vesicles, including: one or more fluid channels configured to receive one or more EV fluid streams containing a plurality of de-glycosylated extracellular vesicles (EVs) in an electroporation buffer; an EV stream inlet for the one or more EV fluid channels; an EV stream flow source fluidly coupled to the sample inlet; a cargo inlet downstream of an electric field (electroporation) region adjacent to the one or more fluid channels; a cargo flow source fluidly coupled to the cargo inlet; one or more cargo flow streams contain a cargo buffer with cargo molecules configured to be loaded into the plurality of de-glycosylated EVs during mixing of the cargo flow stream with the one or more EV fluid streams; at least one pump configured to deliver the one or more EV fluid streams and the one or more cargo flow streams into the one or more fluid channels; and a plurality of electrodes disposed along the one or more fluid channels in the electric field (electroporation) region and configured to deliver electroporation energy to the one or more EV fluid streams.

[0020] According to certain embodiments, the EVs are de-glycosylated in one or more separate fluid streams that are merging with the one or more electroporation fluid streams.

[0021] According to certain embodiments, the one or more EV fluid streams are pretreated with the plurality of de-glycosylated EVs.

[0022] According to certain embodiments, the plurality of EVs are de-glycosylated via automation.

[0023] According to certain embodiments, the EVs are liposomes.

[0024] According to certain embodiments, electroporation parameters are optimized to induce maximum opening of the transient pores independent of stability of the cargo molecules, in which the electroporation parameters include pulse voltage, pulse width, repetition rate, and electroporation buffer composition.

[0025] According to certain embodiments, a design of a cargo channel inlet located downstream of the electric field region is configured to adjust a rate of the mixing of the one or more EV fluid streams containing the plurality of de-glycosylated EVs with the one or more cargo flow streams containing the cargo molecules for optimizing loading of the cargo molecules into the de-glycosylated EVs and delivery of the de-glycosylated EVs containing the cargo molecules through the transient pores.

[0026] According to certain embodiments, a geometry of the one or more fluid channels is configured to inducing the mixing of the one or more EV fluid streams containing the plurality of de-glycosylated EVs with the one or more cargo flow streams containing the cargo molecules, in which the geometry includes side-wall channel structures configured as protrusions.

[0027] According to certain embodiments, acoustic wave induced microstreaming is configured to induce the mixing of the one or more EV fluid streams containing the plurality of de-glycosylated EVs with the one or more cargo flow streams containing the cargo molecules.

[0028] According to certain embodiments, there is a plurality of electrolytic gas bubbles produced during delivery of electroporation energy to the one or more electroporation fluid streams and a plurality of channel wall protrusions on the one or more fluid channels configured to induce turbulence configured to mix the one or more EV fluid streams containing the plurality of de-glycosylated EVs with the one or more cargo flow streams containing the cargo molecules.

[0029] According to certain embodiments, the mixed one or more EV fluid streams with the one or more cargo flow streams following the induced turbulence has a flow rate of 16 ml / hr; in which the mixed one or more EV fluid streams with the one or more cargo flow streams following the induced turbulence has an average flow speed of 0.5 m / s.

[0030] According to certain embodiments, the mixing of the one or more EV fluid streams containing the plurality of de-glycosylated EVs with the one or more cargo flow streams containing the cargo molecules occurs within ~20ms.

[0031] According to certain embodiments, one or more electrodes are disposed on, within, or near a region where the first and second fluid channels are merged and configured to deliver electroporation energy to the cargo fluid mixed into the EVs before the transient pores close to enhance cargo loading efficiency.

[0032] According to certain embodiments, the one or more electrodes are patterned into the substrate via a photolithography process, in which the first and second channels are silicon polymers including polydimethylsiloxane (PDMS) microchannels aligned and bonded to the substrate including curing PDMS over photolithographically defined SU-8 polymer old, further in which the substrate is one or more of a silicon water and glass.

[0033] According to certain embodiments, the substrate is formed by one or more of: injection molding and computer numerical control (CNC) micromachining of plastic material, in which the one or more electrodes are embedded within the substrate.

[0034] In yet other aspects, there is a method for cargo loading into extracellular vesicles via electroporation, the method including: optionally de-glycosylating a plurality of extracellular vesicles (EVs); delivering an EV fluid stream having a plurality of EVs into a first fluid channel; applying electroporation energy to the EV fluid stream to induce opening of transient pores on the EVs as the EVs pass through an electroporation region of the first fluid channel; delivering a cargo fluid stream having a cargo into a second fluid channel; mixing, prior to closure of the transient pores, the EV fluid stream from the first fluid channel with the cargo fluid stream from the second fluid channel; and loading cargo from the cargo fluid stream into the transient pores of EVs in the EV fluid stream.

[0035] According to certain examples, the method further includes de-glycosylating the EVs in one or more separate fluid streams before merging the one or more separate fluid streams with the one or more electroporation fluid streams.

[0036] According to certain examples, the method further includes pre-treating one or more EV fluid streams with the plurality of de-glycosylated extracellular vesicles.

[0037] According to certain examples, the method further includes de-glycosylating the plurality of EVs via automation.

[0038] According to certain examples of the method, a time from delivering the electroporation energy to delivering the mixture through the transient pores occurs in <1 sec.

[0039] According to certain examples of the method, the EVs are liposomes.

[0040] According to certain examples of the method, opening of the transient pores is maximized via optimizing electroporation parameters independent of stability of the cargo molecules, in which the electroporation parameters include pulse shape, pulse voltage, pulse width, repetition rate, and electroporation buffer composition.

[0041] According to certain examples, the method further includes applying electroporation energy to the mixed EV fluid stream from the first fluid channel with the cargo fluid stream from the second fluid channel to enhance a loading efficiency of loading the cargo from the cargo fluid stream into the transient pores of EVs in the EV fluid stream.DETAILED DESCRIPTION

[0042] Systems and methods are provided herein for loading cargo into extracellular vesicles (EV), which can be used as engineerable therapeutic delivery vehicles. The systems and methods provided herein allow for loading therapeutic cargo (e.g., proteins) into EVs while retaining cargo activity and EV integrity. The systems and methods discussed herein are configured to provide multi-stage cargo loading into EVs, overcoming many of the deficiencies with other known techniques for EV cargo loading.

[0043] FIG. 1 A is a schematic of a two-stage flow electroporation system, which can include one or more fluid channels 101 and 103 patterned on a substrate. The fluid channels, such as fluid channel 101, are configured to receive a flow of one or more extracellular vesicle (EV) streams 102 having extracellular vesicles (EVs) in an electroporation buffer. In some embodiments, the system includes components configured for generating or providing the EV streams (e.g., reservoirs, mixing chambers, etc.). In certain examples, synthetic lipid vesicles such as liposomes may be used for cargo loading in place of EVs.

[0044] Various methods can be used to produce or manufacture the electroporation systems described herein at scale. In some examples, a photolithography process may be used to pattern metal electrodes on a glass substrate followed by aligning and bonding a silicone polymer such as polydimethylsiloxane (PDMS) microchannels on top. PDMS microchannels may be molded by curing PDMS over photolithographically defined SU-8 polymer mold on a silicon wafer. A similar device can also be made by injection molding or computer numerical control (CNC) micromachining of plastic material with embedded metal electrodes.

[0045] The system of FIG. 1 A is configured to apply a two-stage process for loading cargo into the EVs in the EV stream. The first stage involves applying electroporation to the EVs in the one or more EV streams 102, for example via a plurality of electrodes 104 disposed along, within, or in proximity to fluid channel 101. In some embodiments, the electrodes 104 are configured to deliver electroporation energy to the one or more EVstreams 102 in an electroporation region 107 of the fluid channel 101. The electroporation energy is delivered at energy levels configured to induce transient pores on the EVs at high efficiency without degrading or damaging the EVs.

[0046] Following the first stage, but before the transient pores on the EVs close (e.g., <lsec), the system is configured to provide the EV stream 102 with the EVs with transient pores downstream to mix with cargo stream 106 flowing through fluid channel 103. As shown, the fluid channel 103 is configured to intersect with fluid channel 101 downstream of electrodes 107 and electroporation region 107. The intersection between fluid channels 101 and 103 can optionally be an orthogonal intersection as shown, but other intersection types or angles are also within the scope of this disclosure. In some embodiments, for example, the fluid channels 101 can be parallel, concentric, or join at an angle (e.g., like a Y or V shaped junction). The cargo stream 106 may comprise a fluid stream where cargo (to be loaded into the EVs) is disposed within a cargo buffer.

[0047] During the second stage 108, the system is configured to combine or mix the one or more EV streams 102 from fluid channel 101 (having EVs with transient pores as a result of the first stage) with the one or more cargo streams 106 from fluid channel 103. The mixing of streams can happen in one of fluid channels 101 / 103, or in a separate mixing channel 105, or both. In some embodiments, the mixing channel 105 is an extension or portion of one or both of fluid channels 101 / 103. Since the mixing of the EV stream with the cargo stream must occur before the transient pores in the EVs close (e.g., <lsec), the timing of mixing the streams is very important, and can be controlled by one or more of 1) where the cargo stream 106 intersects with EV stream 102 relative to the electroporation region 107, and 2) the flow rates of the EV and / or cargo streams. Furthermore, the timing of combining the cargo stream 106 to the EV stream 102 may be precisely controlled via the design of a cargo channel inlet 116 downstream of the electroporation region which may control the timing on a scale of microseconds to milliseconds. Control of such timing may be a critical factor for achieving high efficiency cargo loading into the EVs as the transient pores on the EVs may only stay open for a fraction of a second following electroporation.

[0048] Thus, under the two-stage flow electroporation system described herein, the problem of EV and cargo degradation due to electrolysis during electroporation is addressed by de-coupling EV transient pore opening via electroporation from cargo loading into the EVs. Specifically, the cargo (from cargo stream 106) is not present in the EV fluid stream 102 during the electroporation process, but instead is mixed into the EV stream via cargo stream 106 after electroporation but before the transient pores in the EVs close (e.g., less than Isec after electroporation, less than 500ms after electroporation, less than 250ms afterelectroporation, less than 100ms after electroporation, less than 50ms after electroporation, or less than 20ms after electroporation).

[0049] Compared to a single-stage technique where both the EV stream 102 and cargo stream 106 may be electroporated, a two-stage flow electroporation system offers advantages including the ability to optimize electroporation parameters (such as pulse voltage, pulse width, repetition rate, and electroporation buffer composition) to achieve maximum EV pore opening without the requirement of the cargo needing to remain stable under the same electroporation parameters or presence of electroporation energy being applied to the cargo stream 106.

[0050] FIG. IB is a schematic of a three-stage flow electroporation system, which can include one or more fluid channels 101, 103, and 105 patterned on a substrate. As shown here, there may be one or more extracellular vesicle (EV) streams 102 having extracellular vesicles (EVs) in an electroporation buffer flowing within one or more fluid channels, such as fluid channel 101. In certain examples, synthetic lipid vesicles such as liposomes may be used for cargo loading in place of EVs. The system of FIG. IB is similar to that of FIG. 1 A, with the addition of a second plurality of electrodes 112 forming a second electroporation region 114.

[0051] The one or more EV streams 102 is configured to undergo a three-stage process for cargo loading in the EVs. The first stage involves electroporation of the EVs in the one or more EV streams 102, for example via a first plurality of electrodes 104 along fluid channel 101. The first plurality of electrodes 104 are configured to deliver electroporation energy to the one or more EV streams 102 in a first electroporation region 107 of the fluid channel 101. The electroporation energy is delivered at energy levels configured to induce transient pores on the EVs at high efficiency without degrading or damaging the EVs.

[0052] Following the first stage, but before the transient pores on the EVs close (e.g., <lsec), the EV stream 102 with the EVs with transient pores flows downstream to mix with cargo stream 106, which comprises a fluid stream where cargo is loaded into a cargo buffer. This second stage 108 involves combining or mixing the one or more EV streams 102 having EVs with transient pores and the one or more cargo streams 106. The mixing of streams can happen in one of fluid channels 101 / 103, or in a separate mixing channel 105. In some embodiments, the mixing channel 105 is an extension or portion of one or both of fluid channels 101 / 103.

[0053] Following the second stage 108, and before the transient pores on the EVs close (e.g., <1 sec), the EV stream 102 has at least some cargo loaded into the EVs with transient pores. In a third stage 110, electroporation energy may be delivered via a second plurality ofelectrodes 112 within second electroporation region 114 to further increase cargo loading efficiency in mixing channel 105 by enhancing the interaction between the EVs with transient pores and the cargo molecules from the one or more cargo streams 106. Thus, this third stage 110 results in electric field enhanced cargo loading, which in certain examples may induce damage to some cargo molecules but still increase cargo loading efficiency. It should be understood that this third stage can be carried out by the system while the EVs still have open transient pores, to facilitate cargo loading into the EVs. In some aspects, the energy applied to the first plurality of electrodes 104 is different than the energy applied to the second plurality of electrodes 112. For example, more electroporation energy may be required to form transient pores in the EVs during the first stage than is required to enhance mixing in the third stage. In other embodiments, more energy is applied during the third stage than the first stage. In yet other examples, substantially similar energy levels may be applied across the first and third stages.

[0054] Thus, under the three-stage flow electroporation system described herein, cargo loading efficiency 114 may be further enhanced by delivering further electroporation energy to further increase cargo and EV interaction, while not damaging the cargo during the formation of transient pores in the EVs.

[0055] In any of the examples provided herein, the system may increase cargo loading efficiency by providing the functionality for de-glycosylating or enzymatically removing sugar groups on the EVs in EV stream 102 prior to electroporation with electrodes 104 during the first stage. In various examples, the EV stream 102 may be pre-treated with deglycosylated EVs, de-glycosylation of the EVs may be automated, or de-glycosylated EVs may be delivered via one or more separate streams that combine with the EV stream 102.

[0056] As will be discussed further below, cargo stream 106 and EV stream 102 mixing in any of the systems described herein may be induced or further optimized in various ways including formed bubbles or specialized geometry of mixing channel to assist in creating turbulent flows to combine the EVs with transient pores with cargo molecules in the cargo stream.

[0057] FIGS. 2A-2B illustrate schematics of specialized fluid channel geometry configured for enhanced flow mixing are provided. These enhanced geometries may be formed, for example, in fluid channel 105 described above (e.g., where the EV stream and cargo stream are mixed). Enhanced flow mixing may be configured to occur during the second stage as the EV and cargo streams combine or mix. In such examples, various microfluidic mixing strategies may be employed to achieve fast mixing, for example in a timeframe of milliseconds and under high flow rates (ml / hr) which may be above a certainthreshold, as well as under high flow speeds (m / s). Exemplary specialized channel geometry to induce streaming or mixing may include a serpentine channel design shown in FIG. 2A. The serpentine channel design may further include angular channels with abrupt turns (e.g., a zigzag pattern) to further increase mixing or create turbulence within the fluid channel.

[0058] Alternatively, as shown in FIG. 2B, the flow channel may include herringbone style structures within, which may include rough or chevron-like areas, grooves, or gaps in a channel 202 to induce turbulent flow and mixing, for example arranged on a floor and / or sidewall of the channel. Also shown are cross-sectional or slice examples A-A of the turbulent flow or mixing facilitated by the herringbone design structures in the flow channel. In certain examples, the time scale for mixing with such specialized channel geometry may be longer and in the order of seconds or longer.

[0059] FIGS. 2C-2E illustrate schematics of fluid channels that are configured to provide acoustic wave induced microstreaming for enhanced flow mixing. These fluid channels may be formed, for example, in fluid channel 105 described above (e.g., where the EV stream and cargo stream are mixed). Acoustic wave microstreaming may offer the advantage of mixing on a shorter time scale (which may be on the order of milliseconds), to better accommodate the short opening time of the transient pores on the EVs following electroporation but before mixing with the cargo stream.

[0060] As shown in FIG. 2C, a piezoelectric transducer 210 or other wave source generator may be disposed on the substrate adjacent to or in close proximity to the fluid channels 201 / 203 / 205, which may be configured to generate acoustic waves in the fluid channels 201 / 203 / 205 which may contain flow streams such as EV stream 202 and cargo stream 206, or a mix of the fluid streams. In some embodiments, any of the fluid channels, and particularly fluid channel 205 where mixing of the fluid streams occurs, may include one or more channel mixing structures 212 which may include sidewall sharp-edge structures 213 such as protrusions, spikes, or other channel geometry configured to enhance mixing of the combined stream of the EV stream 202 and cargo stream 206 during the second stage 208. While the embodiment of FIG. 2C includes both a piezoelectric transducer 210 and the channel mixing structures 212 to enhance mixing or combination of the EV stream with the cargo stream, it should be understood that embodiments provided herein may include one, both, or none of the piezoelectric transducer and mixing structures depending on the specific application.

[0061] FIG. 2D depicts exemplary microstreaming 214 around one or more channel mixing structures 212 such as sidewall sharp-edge structures 213. Microstreaming 214 maybe induced by the mixing structures themselves or may be further induced by acoustic waves produced by the piezoelectric transducer or other wave source generator of FIG. 2C.

[0062] FIG. 2E depicts exemplary dimensions of channel geometry including the channel structures 212, such as the sidewall sharp-edge structures 213. In one specific example, the sidewall sharp-edge structures 213 may have a height 214 of 250 micrometers, a distance 216 of 600 micrometers between sidewall sharp-edge structures 213 on the same side of the channel, a channel width 218 of 600 microns, and a distance 220 of 300 microns between sidewall sharp-edge structures 213 on opposing sides of the channel. Sidewall sharp-edge structures 213 may feature apexes having various angles 219, such as a, configured to optimize mixing.

[0063] FIGS. 3A-3D illustrate a schematic and views of additional techniques for providing enhanced flow mixing of an EV stream with a cargo stream. In some aspects, any of the systems discussed herein can be configured to produce electrolytic gas bubbles in the EV stream when electroporation is applied to the EV stream (e.g., during the first stage). The systems provided herein may leverage the generation of electrolytic gas bubbles to enhance mixing between the EV stream and the cargo stream. In some aspects, the system and fluid channels can be designed such that electrolytic gas bubbles combined with channel wall protrusions assist in the mixing of cargo streams with EV streams when the transient pores of the EVs are open (e.g., during the second stage discussed above).

[0064] FIG. 3 A shows cargo stream 306 and EV stream 302 as well as channel mixing structures 312 with protrusions 313 as part of a channel wall in a fluid channel. In this example, the EV stream is provided in a vertically arranged fluid channel, and the cargo stream is provided in a horizontally arranged fluid channel that intersects with the EV stream. Providing the EV stream in a vertically arranged fluid channel allows for electrolytic gas bubbles formed during the electroporation process to rise within the channel structures such that they are present in flow channel where the EV stream and the cargo stream intersect. In certain examples, enhanced flow mixing utilizes electrolytic gas bubbles generated in the first stage from the electroporation applied to the EVs in EV stream 302 with the channel geometry including protrusions 313 of channel mixing structures 312 to induce turbulence for active and fast mixing in the second stage. Exemplary flow rates may be 16 ml / hr, with exemplary average flow speeds being 0.5 m / s, and exemplary mixing time scales being within 20 ms.

[0065] FIG. 3B shows an exemplary channel without electroporation, illustrating how the EV stream mixes with the cargo stream without the addition of electrolytic gas bubbles. FIG. 3C shows an exemplary upstream portion of the channel with electroporation in the secondstage showing electrolytic gas bubbles 322 forming on channel mixing structures. The channel mixing structures may cause the electrolytic gas bubbles to burst or collapse, increasing turbulence and mechanical mixing of the EV and cargo streams. FIG. 3D shows an exemplary downstream portion of the channel showing active and fast mixing via the combination of electrolytic gas bubbles 322 with channel mixing structures and protrusions. In certain examples, the downstream portion may be 2cm downstream of the upstream portion previously described.

[0066] As discussed above, de-glycosylation of the EV stream can result in increased cargo loading efficiency. FIG. 4 is a graph comparing cargo loading in extracellular vesicles based on the two-stage design considering de-glycosylation and electroporation parameters. As shown here, percentages of loading efficiency 402 (expressed as number of EVs loaded with cargo / number of EVs recovered after electroporation) and recovery 404 (expressed as number of EVs recovered after electroporation / number of input EVs) for IgG antibody cargo are plotted in two modes: without electrical field (without electroporation) 406 and with an electrical field (with electroporation) 408. Plotted IgG antibody cargo shown here includes a gylcosylated EV such as engineered HEK293 cell line derived EV ExoEV 410 / 420 as well as de-glycosylated EVs P_EV 412 / 422 and N_EV 414 / 424.

[0067] In certain examples, the IgG antibody may be tagged or labeled with fluorescein isothiocyanate (FITC) and the resulting molecular weight may be ~150kDa.

[0068] In certain examples, loading efficiency 402 may be -40% for glycosylated HEK293 derivative Exo EV 420 under electroporation 408, and higher (-70%) for deglycosylated EVs such as P_EV 422 and N_EV 424 under electroporation 408. In certain examples, recovery 404 may be reduced under electroporation conditions 408 to be between 20% and 40%.

[0069] In yet other examples under the two-stage design, loading efficiency may be positively correlated with intensity of an electric field. Compared to a single stage design with concurrent cargo loading and electroporation exposure of EVs and cargo, the two-stage design has been found to exhibit higher cargo / small molecule loading into EVs. Furthermore, under the two-stage design, increasing electric field and loading efficiency correlates with higher gene expression knockdown whereas gene expression knockdown diminishes with increasing electric field in the single stage design, which may be a result of siRNA loaded into EVs being degraded under a high electric field.

[0070] FIG. 5 depicts a chart illustrating a method 500 for cargo loading into extracellular vesicles via electroporation as described herein.

[0071] At block 505, a plurality of extracellular vesicles (EVs) are de-glycosylated (optional step).

[0072] At block 510, one or more EV fluid streams containing the plurality of optionally de-glycosylated extracellular vesicles in an electroporation buffer are delivered into one or more fluid channels.

[0073] At block 515, electroporation energy is delivered to the one or more EV fluid streams via a plurality of electrodes disposed along, near, or within the one or more fluid channels to induce opening of transient pores on the extracellular vesicles as the extracellular vesicles pass through an electric field (electroporation) region.

[0074] At block 520, the one or more EV fluid streams are mixed with one or more cargo flow streams containing cargo molecules suspended in a cargo buffer before closure of the transient pores. In some embodiments, the mixing occurs less than 1 second after the electroporation step.

[0075] Finally, at block 525, the mixture results in the cargo from the one or more cargo flow streams being loaded into the EVs via the open transient pores of the EVs.

[0076] According to certain embodiments of method 500, the EVs are de-glycosylated in one or more separate fluid streams before merging the one or more separate fluid streams with the one or more EV fluid streams.

[0077] According to certain embodiments of method 500, method 500 further includes pre-treating the one or more EV fluid streams with the plurality of de-glycosylated extracellular vesicles.

[0078] According to certain embodiments of method 500, the plurality of EVs are de- glycosylated via automation.

[0079] According to certain embodiments of method 500, a time from delivering the electroporation energy to delivering the mixture through the transient pores occurs in <1 sec.

[0080] According to certain embodiments of method 500, the EVs are liposomes.

[0081] According to certain embodiments of method 500, opening of the transient pores is maximized via optimizing electroporation parameters independent of stability of the cargo molecules, in which the electroporation parameters include pulse voltage, pulse width, repetition rate, and electroporation buffer composition.

[0082] According to certain embodiments of method 500, method 500 further includes adjusting a rate of the mixing of the one or more EV fluid streams containing the plurality of de-glycosylated EVs with the one or more cargo flow streams containing the cargo molecules via a cargo channel inlet located downstream of the electric field region; further in which the cargo channel inlet is configured to optimize maximum loading of the cargo molecules intothe de-glycosylated EVs and delivery of the de-glycosylated EVs loaded with the cargo through the transient pores.

[0083] According to certain embodiments of method 500, method 500 further includes inducing the mixing of the one or more EV fluid streams containing the plurality of de- glycosylated EVs with the one or more cargo flow streams via geometry of the one or more fluid channels.

[0084] According to certain embodiments of method 500, method 500 further includes inducing the mixing of the one or more EV fluid streams containing the plurality of de- glycosylated EVs with the one or more cargo flow streams via acoustic wave induced microstreaming.

[0085] According to certain embodiments of method 500, method 500 further includes inducing the mixing of the one or more EV fluid streams containing the plurality of de- glycosylated EVs with the one or more cargo flow streams containing the cargo molecules via inducing turbulence by combining electrolytic gas bubbles produced during delivery of electroporation energy to the one or more EV fluid streams with a plurality of channel wall protrusions on the one or more fluid channels.

[0086] According to certain embodiments of method 500, the mixed one or more EV fluid streams with the one or more cargo flow streams following the induced turbulence has a flow rate of 16 ml / hr, wherein the mixed one or more EV fluid streams with the one or more cargo flow streams following the induced turbulence has an average flow speed of 0.5 m / s.

[0087] According to certain embodiments of method 500, the mixing of the one or more EV fluid streams containing the plurality of de-glycosylated EVs with the one or more cargo flow streams containing the cargo molecules occurs within ~20ms.

[0088] According to certain embodiments of method 500, method 500 further includes applying electroporation energy to the mixed EV fluid stream from the first fluid channel with the cargo fluid stream from the second fluid channel to enhance a loading efficiency of loading the cargo from the cargo fluid stream into the transient pores of EVs in the EV fluid stream.

[0089] Any of the methods (including user interfaces) described herein may be implemented as software, hardware or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions capable of being executed by a processor (e.g., computer, tablet, smartphone, etc.), that when executed by the processor causes the processor to control perform any of the steps, including but not limited to: displaying, communicating with the user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alerting, or the like. It should be appreciated that allcombinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits described herein.

[0090] When a feature or element is herein referred to as being “on” another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being “connected”, “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being “directly connected”, “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0091] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ”.

[0092] Spatially relative terms, such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. The devicemay be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0093] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present invention.

[0094] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0095] In general, any of the apparatuses and methods described herein should be understood to be inclusive, but all or a sub-set of the components and / or steps may alternatively be exclusive and may be expressed as “consisting of’ or alternatively “consisting essentially of’ the various components, steps, sub-components or sub-steps.

[0096] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “X” is disclosed the “less than or equal to X” as well as “greater than or equal to X” (e.g., where X is a numerical value) is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0097] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0098] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

CLAIMSWhat is claimed is:

1. A flow electroporation system for cargo loading into extracellular vesicles, comprising: a substrate; a first fluid channel patterned in the substrate; one or more electrodes disposed on, within, or near the first fluid channel; a second fluid channel patterned in the substrate, the second fluid channel merging into the first fluid channel; a source of EV fluid containing a plurality of extracellular vesicles (EVs) in an electroporation buffer, the source being fluidly coupled to an inlet of the first fluid channel; and a source of cargo fluid containing a cargo to loaded into the EVs, the source of cargo fluid being fluidly coupled to an inlet of the second fluid channel; wherein the one or more electrodes are controllable to deliver electroporation energy to the EV fluid flowing through the first fluid channel to induce transient pores in the EVs such that cargo fluid flowing in the second fluid channel can be mixed into the EVs before the transient pores close.

2. The system of claim 1, wherein the EVs in the fluid stream are de-glycosylated in one or more separate fluid streams that merge with the first fluid channel.

3. The system of claim 1, wherein the EV fluid is pre-treated with the plurality of deglycosylated EVs.

4. The system of claim 1, wherein the plurality of EVs are de-glycosylated via automation.

5. The system of claim 1, wherein the EVs are liposomes.

6. The system of claim 1, wherein electroporation parameters are optimized to induce maximum opening of the transient pores independent of stability of the cargo molecules, wherein the electroporation parameters include pulse shape, pulse voltage, pulse width, repetition rate, and electroporation buffer composition.

7. The system of claim 1, wherein a geometry of the one or more fluid channels is configured to induce the mixing of the EV fluid with the cargo fluid, wherein the geometry includes side-wall channel structures configured as protrusions.

8. The system of claim 1, further comprising a piezoelectric element disposed on the substrate and configured to induce mixing of the EV fluid with the cargo fluid.

9. The system of claim 7, wherein application of electroporation energy produces gas bubbles within one or more of the EV fluid, the cargo fluid, or a mixture of the EV fluid and the cargo fluid against the side-wall channel structures to assist in mixing the EV fluid and the cargo fluid.

10. The system of claim 1, wherein the cargo fluid is mixed with the EV fluid within ~20ms of applying electroporation energy to the EV fluid.

11. The system of claim 1, further comprising one or more electrodes disposed on, within, or near a region where the first and second fluid channels are merged and configured to deliver electroporation energy to the cargo fluid mixed into the EVs before the transient pores close to enhance cargo loading efficiency.

12. The system of claim 1, wherein the one or more electrodes are patterned into the substrate via a photolithography process; further wherein the first and second channels are silicon polymers including polydimethylsiloxane (PDMS) microchannels aligned and bonded to the substrate via curing PDMS over photolithographically defined SU-8 polymer mold; further wherein the substrate is one or more of a silicon water and glass.

13. The system of claim 1, wherein the substrate is formed by one or more of: injection molding and computer numerical control (CNC) micromachining of plastic material; further wherein the one or more electrodes are embedded within the substrate.

14. A method for cargo loading into extracellular vesicles via electroporation, the method comprising: delivering an EV fluid stream having a plurality of EVs into a first fluid channel;applying electroporation energy to the EV fluid stream to induce opening of transient pores on the EVs as the EVs pass through an electroporation region of the first fluid channel; delivering a cargo fluid stream having a cargo into a second fluid channel; mixing, prior to closure of the transient pores, the EV fluid stream from the first fluid channel with the cargo fluid stream from the second fluid channel; and loading cargo from the cargo fluid stream into the transient pores of EVs in the EV fluid stream.

15. The method of claim 14, further comprising, prior to applying electroporation energy, de-glycosylating the plurality of extracellular vesicles (EVs) in the EV fluid stream.

16. The method of claim 15, further comprising de-glycosylating the EVs in one or more separate fluid streams before merging the one or more separate fluid streams with the one or more electroporation fluid streams.

17. The method of claim 15, further comprising pre-treating the one or more EV fluid streams with the plurality of de-glycosylated extracellular vesicles.

18. The method of claim 15, further comprising de-glycosylating the plurality of EVs via automation.

19. The method of claim 14, wherein a time from delivering the electroporation energy to delivering the mixture through the transient pores occurs in <1 sec.

20. The method of claim 14, wherein the EVs are liposomes.

21. The method of claim 14, wherein opening of the transient pores is maximized via optimizing electroporation parameters independent of stability of the cargo molecules, wherein the electroporation parameters include pulse shape, pulse voltage, pulse width, repetition rate, and electroporation buffer composition.

22. The method of claim 14, further comprising applying electroporation energy to the mixed EV fluid stream from the first fluid channel with the cargo fluid stream from the second fluid channel to enhance a loading efficiency of loading the cargo from the cargo fluid stream into the transient pores of EVs in the EV fluid stream.

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