[go: up one dir, main page]

WO2025117977A1 - Systèmes d'électroporation et procédés pour le chargement de cargaisons dans des vésicules extracellulaires - Google Patents

Systèmes d'électroporation et procédés pour le chargement de cargaisons dans des vésicules extracellulaires Download PDF

Info

Publication number
WO2025117977A1
WO2025117977A1 PCT/US2024/058141 US2024058141W WO2025117977A1 WO 2025117977 A1 WO2025117977 A1 WO 2025117977A1 US 2024058141 W US2024058141 W US 2024058141W WO 2025117977 A1 WO2025117977 A1 WO 2025117977A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluid
cargo
evs
electroporation
channel
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/058141
Other languages
English (en)
Inventor
Ting-Hsiang Wu
Tung-Yu Wu
Jason Hong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Met Biotechnology Inc
Original Assignee
Met Biotechnology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Met Biotechnology Inc filed Critical Met Biotechnology Inc
Publication of WO2025117977A1 publication Critical patent/WO2025117977A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • EVs extracellular vesicles
  • 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.
  • 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.
  • problems with conventional electroporation on EVs also exist, particularly involving the limitations of conventional 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.
  • 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.
  • MFI Mean Fluorescence Intensity
  • the level of decreased antibody function may be correlated with increased electrical pulsing intensity (kV/cm).
  • Sheath electroporation has been used for transfecting primary T cells with good transfection efficiency and maintained cell viability.
  • sheaths for electroporating small vesicles such as exosomes and EVs.
  • 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.
  • Atto488-biotin IkDA molecular weight
  • FIG. 1 A is a schematic of a two-stage flow electroporation system.
  • FIG. IB is a schematic of a three-stage flow electroporation system.
  • FIGS. 2A-2B illustrate schematics of specialized channel geometry configured for enhanced flow mixing.
  • FIG. 2C-2E illustrate schematics of acoustic wave induced microstreaming for enhanced flow mixing.
  • FIGS. 3A-3D illustrate a schematic and views of enhanced flow mixing combining electrolytic gas bubbles and channel wall protrusions.
  • FIG. 4 is a graph comparing cargo loading in extracellular vesicles based on the two-stage design considering de-glycosylation and electroporation parameters.
  • FIG. 5 depicts a chart illustrating a method for cargo loading into extracellular vesicles via electroporation as described herein.
  • 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 EVs are de-glycosylated in one or more separate fluid streams that are merging with the one or more electroporation fluid streams.
  • the one or more EV fluid streams are pretreated with the plurality of de-glycosylated EVs.
  • the plurality of EVs are de-glycosylated via automation.
  • the EVs are liposomes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PDMS polydimethylsiloxane
  • 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.
  • CNC computer numerical control
  • a method for cargo loading into extracellular vesicles via electroporation 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.
  • EVs extracellular vesicles
  • 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.
  • the method further includes pre-treating one or more EV fluid streams with the plurality of de-glycosylated extracellular vesicles.
  • the method further includes de-glycosylating the plurality of EVs via automation.
  • a time from delivering the electroporation energy to delivering the mixture through the transient pores occurs in ⁇ 1 sec.
  • the EVs are liposomes.
  • 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.
  • 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.
  • Systems and methods are provided herein for loading cargo into extracellular vesicles (EV), which can be used as engineerable therapeutic delivery vehicles.
  • EV extracellular vesicles
  • the systems and methods provided herein allow for loading therapeutic cargo (e.g., proteins) into EVs while retaining cargo activity and EV integrity.
  • therapeutic cargo e.g., proteins
  • 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.
  • 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.
  • the system includes components configured for generating or providing the EV streams (e.g., reservoirs, mixing chambers, etc.).
  • synthetic lipid vesicles such as liposomes may be used for cargo loading in place of EVs.
  • 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.
  • CNC computer numerical control
  • 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.
  • the electrodes 104 are configured to deliver electroporation energy to the one or more EV streams 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the mixing channel 105 is an extension or portion of one or both of fluid channels 101/103.
  • 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.
  • 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 after electroporation, less than 100ms after electroporation, less than 50ms after electroporation, or less than 20ms after electroporation).
  • Isec after electroporation less than 500ms after electroporation, less than 250ms after electroporation, less than 100ms after electroporation, less than 50ms after electroporation, or less than 20ms after electroporation.
  • 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.
  • electroporation parameters such as pulse voltage, pulse width, repetition rate, and electroporation buffer composition
  • 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.
  • 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.
  • 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.
  • 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.
  • the mixing channel 105 is an extension or portion of one or both of fluid channels 101/103.
  • the EV stream 102 has at least some cargo loaded into the EVs with transient pores.
  • electroporation energy may be delivered via a second plurality of electrodes 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 certain threshold, 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.
  • 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.
  • cross-sectional or slice examples A-A of the turbulent flow or mixing facilitated by the herringbone design structures in the flow channel may be longer and in the order of seconds or longer.
  • 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.
  • 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.
  • 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.
  • FIG. 2D depicts exemplary microstreaming 214 around one or more channel mixing structures 212 such as sidewall sharp-edge structures 213.
  • Microstreaming 214 may be 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.
  • FIG. 2E depicts exemplary dimensions of channel geometry including the channel structures 212, such as the sidewall sharp-edge structures 213.
  • 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.
  • FIGS. 3A-3D illustrate a schematic and views of additional techniques for providing enhanced flow mixing of an EV stream with a cargo stream.
  • 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.
  • 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).
  • 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.
  • the EV stream is provided in a vertically arranged fluid channel
  • 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.
  • 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.
  • 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 second stage 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.
  • 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.
  • 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.
  • the IgG antibody may be tagged or labeled with fluorescein isothiocyanate (FITC) and the resulting molecular weight may be ⁇ 150kDa.
  • FITC fluorescein isothiocyanate
  • 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.
  • recovery 404 may be reduced under electroporation conditions 408 to be between 20% and 40%.
  • loading efficiency may be positively correlated with intensity of an electric field.
  • the two-stage design has been found to exhibit higher cargo/small molecule loading into EVs.
  • 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.
  • FIG. 5 depicts a chart illustrating a method 500 for cargo loading into extracellular vesicles via electroporation as described herein.
  • a plurality of extracellular vesicles (EVs) are de-glycosylated (optional step).
  • 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.
  • 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.
  • 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.
  • the mixing occurs less than 1 second after the electroporation step.
  • 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.
  • 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.
  • method 500 further includes pre-treating the one or more EV fluid streams with the plurality of de-glycosylated extracellular vesicles.
  • the plurality of EVs are de- glycosylated via automation.
  • a time from delivering the electroporation energy to delivering the mixture through the transient pores occurs in ⁇ 1 sec.
  • the EVs are liposomes.
  • 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.
  • 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 into the de-glycosylated EVs and delivery of the de-glycosylated EVs loaded with the cargo through the transient pores.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a processor e.g., computer, tablet, smartphone, etc.
  • a feature or element 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.
  • 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 device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
  • the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.
  • 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.
  • 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.
  • all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear.
  • 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 appropriately understood 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.

Landscapes

  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Mechanical Engineering (AREA)
  • Physics & Mathematics (AREA)
  • Electromagnetism (AREA)
  • Physical Or Chemical Processes And Apparatus (AREA)

Abstract

L'invention concerne des systèmes d'électroporation et des procédés de chargement de cargaison dans des vésicules extracellulaires (VE). Par exemple, selon un mode de réalisation, il existe un système d'électroporation en flux, comprenant : un ou plusieurs canaux fluidiques conçus pour accueillir un ou plusieurs flux fluidiques de VE ; une entrée de flux de VE pour le ou les canaux fluidiques de VE ; une source de flux de VE couplée fluidiquement à l'entrée de l'échantillon ; une entrée de cargaison en aval d'une région de champ électrique adjacente au ou aux canaux fluidiques ; une source de flux de cargaison couplée fluidiquement à l'entrée de la cargaison ; un ou plusieurs flux de cargaison contiennent un tampon de cargaison avec des molécules de cargaison ; au moins une pompe conçue pour délivrer le ou les flux de fluide de VE et le ou les flux de cargaison dans le ou les canaux fluidiques ; et une pluralité d'électrodes conçues pour délivrer de l'énergie d'électroporation au ou aux flux de fluide de VE.
PCT/US2024/058141 2023-11-30 2024-12-02 Systèmes d'électroporation et procédés pour le chargement de cargaisons dans des vésicules extracellulaires Pending WO2025117977A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363604810P 2023-11-30 2023-11-30
US63/604,810 2023-11-30
US202463695264P 2024-09-16 2024-09-16
US63/695,264 2024-09-16

Publications (1)

Publication Number Publication Date
WO2025117977A1 true WO2025117977A1 (fr) 2025-06-05

Family

ID=95898256

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/058141 Pending WO2025117977A1 (fr) 2023-11-30 2024-12-02 Systèmes d'électroporation et procédés pour le chargement de cargaisons dans des vésicules extracellulaires

Country Status (1)

Country Link
WO (1) WO2025117977A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050019921A1 (en) * 2001-11-27 2005-01-27 Orwar Owe E. Method for combined sequential agent delivery and electroporation for cell structures and use thereof
US20190247852A1 (en) * 2018-02-15 2019-08-15 Ohio State Innovation Foundation Microfluidic devices and methods for high throughput electroporation
US20200199628A1 (en) * 2014-03-28 2020-06-25 The Regents Of The University Of California Efficient delivery of large cargos into cells on a porous substrate
US20210207150A1 (en) * 2013-03-15 2021-07-08 The Regents Of The University Of California High-throughput cargo delivery into live cells using photothermal platforms
WO2023039389A1 (fr) * 2021-09-07 2023-03-16 University Of Florida Research Foundation, Incorporated Électroporation efficace à haut débit pour le chargement de vésicules extracellulaires (ev) et d'exosomes
EP4163380A1 (fr) * 2021-10-08 2023-04-12 ETH Zurich Dispositif et procédé de manipulation de vésicules extracellulaires

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050019921A1 (en) * 2001-11-27 2005-01-27 Orwar Owe E. Method for combined sequential agent delivery and electroporation for cell structures and use thereof
US20210207150A1 (en) * 2013-03-15 2021-07-08 The Regents Of The University Of California High-throughput cargo delivery into live cells using photothermal platforms
US20200199628A1 (en) * 2014-03-28 2020-06-25 The Regents Of The University Of California Efficient delivery of large cargos into cells on a porous substrate
US20190247852A1 (en) * 2018-02-15 2019-08-15 Ohio State Innovation Foundation Microfluidic devices and methods for high throughput electroporation
WO2023039389A1 (fr) * 2021-09-07 2023-03-16 University Of Florida Research Foundation, Incorporated Électroporation efficace à haut débit pour le chargement de vésicules extracellulaires (ev) et d'exosomes
EP4163380A1 (fr) * 2021-10-08 2023-04-12 ETH Zurich Dispositif et procédé de manipulation de vésicules extracellulaires

Similar Documents

Publication Publication Date Title
US7811603B2 (en) Microfluidic device for forming monodisperse lipoplexes
JP7334127B2 (ja) 多孔質支持体上の細胞への大きなカーゴの効率的な送達
US11377652B2 (en) Micro flow-through electroporation devices and methods of cell transfection
Rasouli et al. An ultra-rapid acoustic micromixer for synthesis of organic nanoparticles
Rad et al. A comprehensive review on intracellular delivery
Morshedi Rad et al. A comprehensive review on intracellular delivery
US20240261782A1 (en) Microfluidic devices and methods for high throughput electroporation
Sun et al. Recent advances in micro/nanoscale intracellular delivery
US20240084236A1 (en) Method and Apparatus for High Throughput High Efficiency Transfection of Cells
Aghaamoo et al. High‐Throughput and Dosage‐Controlled Intracellular Delivery of Large Cargos by an Acoustic‐Electric Micro‐Vortices Platform
US20240367172A1 (en) Microfluidic Delivery Method Utilizing An Electric Field
US11559809B2 (en) Enhanced cell/bead encapsulation methods and apparatuses
Hsieh et al. Nonviral gene vector formation in monodispersed picolitre incubator for consistent gene delivery
WO2025117977A1 (fr) Systèmes d'électroporation et procédés pour le chargement de cargaisons dans des vésicules extracellulaires
Tong et al. The evolution of microfluidic‐based drug‐loading techniques for cells and their derivatives
KR20200109229A (ko) 관성을 기반으로 한 세포 내 전달 미세 유체 플랫폼 및 이를 이용한 세포 내 물질 전달방법
CN113842960B (zh) 一种利用新型微流控装置制备核酸脂质纳米粒的方法
WO2007053802A2 (fr) Apport de medicament et de genes par timbre cellulaire a nanobuses et nanopointes polymeres
KR101472160B1 (ko) 나노입자, 나노입자의 제조 방법 및 제조 장치
Lee et al. Highly efficient mRNA transfection with droplet cell squeezing for cellular engineering
KR20200127546A (ko) 세포 처리 장치 및 세포 처리 방법
EP4600342A1 (fr) Cartouche de transformation, dispositif de transformation, système de transformation la comprenant, et procédé de transformation l'utilisant
Chen Dynamic Control of Chimeric Antigen Receptor Expression via External Shear Induced
Hsieh et al. Monodisperse Lipoplex Generation by Integrated Picoliter Micro Reactor and Incubator
Huang et al. Exosome-Based Drug Delivery

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24898916

Country of ref document: EP

Kind code of ref document: A1