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WO2024243514A1 - Exploitation d'un chauffage photothermique pour le traitement de surface d'agents actifs - Google Patents

Exploitation d'un chauffage photothermique pour le traitement de surface d'agents actifs Download PDF

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Publication number
WO2024243514A1
WO2024243514A1 PCT/US2024/031001 US2024031001W WO2024243514A1 WO 2024243514 A1 WO2024243514 A1 WO 2024243514A1 US 2024031001 W US2024031001 W US 2024031001W WO 2024243514 A1 WO2024243514 A1 WO 2024243514A1
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magnetic
active agent
heating
associating
exosomes
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PCT/US2024/031001
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English (en)
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John MOLINSKI
John X.J. ZHANG
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Trustees Of Dartmouth College
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/10—Separation or concentration of fermentation products

Definitions

  • the present disclosure pertain to methods of loading at least one material with at least one active agent.
  • the methods of the present disclosure include: (1) associating the material with a surface; (2) associating the active agent with the material; (3) photothermally heating the surface-associated material to result in the encapsulation of the active agent into the surface-associated material; and (4) dissociating the active agent-loaded material from the surface.
  • the active agent loading methods of the present disclosure include: (1) capturing a material by magnetic beads to form a magnetic bead-material complex; (2) associating the magnetic beads with a magnetic surface; (3) applying a magnetic field to the magnetic surface to result in the immobilization of the magnetic beads on the magnetic surface; (4) associating the active agent with the material; (5) photothermally heating the surface-associated material to result in the encapsulation of the active agent into the surface- associated material; (6) removing the magnetic field from the magnetic surface to result in the dissociation of the active agent-loaded material from the magnetic surface; and (7) dissociating the active-agent loaded material from the magnetic beads.
  • Additional embodiments of the present disclosure pertain to systems operable for loading a material with an active agent.
  • the systems of the present disclosure generally include a surface operable to associate with a material, and a photothermal heating source proximal to the surface and operable to heat the surface.
  • the systems of the present disclosure also include a magnet operable to apply a magnetic field to the surface.
  • FIG. 1A illustrates a method of loading materials with one or more active agents in accordance with various embodiments of the present disclosure.
  • FIG. IB illustrates a method of utilizing a magnetic surface to load materials with one or more active agents in accordance with various embodiments of the present disclosure.
  • FIG. 1C illustrates a method of utilizing a magnetic surface to load exosomes with one or more active agents in accordance with various embodiments of the present disclosure.
  • FIG. ID illustrates the formation of magnetic beads for use in loading exosomes with one or more active agents.
  • FIGS. 2A-2E illustrate the design and validation of a photothermal engineering system.
  • FIG. 2A shows the design of a photothermal engineering platform, which consists of a removable housing for magnetic and magnetic microchip, a light emitting diode (LED), and a thermoelectric cooler (TEC) to provide active cooling.
  • FIG. 2B provides a schematic showing the principle of magnetophoretic capture using flow-invasive micromagnets (bottom) and photothermal heating of patterned micromagnets to enable exosome engineering (top).
  • FIGS. 2C-2E show heatmaps highlighting full system output power (FIG. 2C), binned output levels used for testing (FIG. 2D), and level of loading of doxorubicin at each binned level (FIG. 2E) using fluorescence intensity.
  • FIGS. 4A-4G illustrate a system optimization and proof-of-concept using human colon carcinoma (COLO-1) derived exosome standards.
  • FIG. 4A provides a schematic showing an immunomagnctic capture and engineering process.
  • FIGS. 4B-4C show doxorubicin (DOX) loading amount using fluorescence intensity as proxy at each binned power output, for unpattemed (FIG. 4B) and patterned (FIG. 4C) magnetic microchips.
  • FIG. 4D shows aggregated results comparing DOX loading within unpatterned and patterned microchips (P ⁇ 0.05).
  • FIGS. 4E-4F show system optimization for DOX loading, including number of 50 ms pulses (FIG. 4E) and pulse duration (FIG. 4F).
  • FIG. 4G shows the 3D structure of doxorubicin with a maximum diameter and chemical formula.
  • FIGS. 5A-5I show data related to small molecule exosome loading.
  • FIGS. 5A-5D show 100X confocal microscope images in differential interference contrast (FIG. 5A), DOX channel (Ex. 470/ Em. 595) (FIG. 5B), DiD channel (Ex. 644 nm. Em. 663 nm) (FIG. 5C), and combined DOX and DiD (FIG. 5D). Scale bars are 1 pm.
  • FIG. 5E shows exosome counts loaded with DiD membrane dye as control and DOX as actively loaded compound, showing a -97% permeation efficiency within exosomes.
  • FIG. 5A-5D show 100X confocal microscope images in differential interference contrast (FIG. 5A), DOX channel (Ex. 470/ Em. 595) (FIG. 5B), DiD channel (Ex. 644 nm. Em. 663 nm) (FIG. 5C), and combined DOX and DiD (FIG. 5D). Scale bars are
  • FIGS. 5G-5I show 20X confocal images of magnetic patterning in brightfield (FIG. 5G), DiD channel (FIG. 5H), and DOX channel (FIG. 51), showing successful loading of DOX within exosomes captured, purified, and engineered directly from patient plasma samples. Scale bars are 200 pm.
  • FIGS. 6A-6I show cargo agnostic loading with an EXOtherm platform. Schematics show cargo loaded within exosomes including doxorubicin (FIG. 6A), Cy3-labeled siRNA (FIG. 6B), and Cas9-GFP (FIG. 6C), along with corresponding molecular weights.
  • FIGS. 6D-6I show confocal microscopy images of exosomes captured upon magnetic microchips for doxorubicin (FIGS. 6D and 6G), Cy3-labeled siRNA (FIGS. 6E and 6H), and Cas9-GFP (FIGS. 6F and 61). All scale bares are 200 pm.
  • Exosome cargo loading methods are especially nascent in their development and at present often rely on cell-line engineering or simple incubation steps, with the intention of fusing cargo to the exosome surface upon biogenesis or diffusion into the membrane, respectively. Such processing inefficiencies result in low loading efficiencies for many therapeutically desirable classes.
  • biothcrapcutic manufacturing pipelines are largely static, thereby requiring specific attention and specialization for each cell line or therapeutic cargo class. Such rigidity is especially problematic in the era of personalized medicine.
  • the surface includes a magnetic surface.
  • the active agent loading methods of the present disclosure include: capturing a material by magnetic beads to form a magnetic bead-material complex (step 30); associating the magnetic beads with a magnetic surface (step 32); applying a magnetic field to the magnetic surface (step 34) to result in the immobilization of the magnetic beads on the magnetic surface (step 36); associating the active agent with the material (step 38); photothermally heating the surface-associated material (step 40) to result in the encapsulation of the active agent into the surface-associated material (step 42); and removing the magnetic field from the magnetic surface (step 44) to result in the dissociation of the active agent-loaded material from the magnetic surface (step 46).
  • the methods of the present disclosure also include a step of dissociating the active-agent loaded material from the magnetic beads (step 48).
  • FIGS. 1C-1D A more specific example of an active agent loading method of the present disclosure is illustrated in FIGS. 1C-1D, which pertains to a method of loading active agents into exosomes through the use of a magnetic surface and antibody-magnetic nanoparticle conjugates that are specific for capturing exosomes (FIG. ID).
  • exosomes are first captured by antibody-magnetic nanoparticle conjugates to form a magnetic nanoparticle-exosome complex (steps 1-2). Thereafter, the magnetic nanoparticle-exosome complexes are associated with a magnetic surface by flowing the complexes through the magnetic surface (step 3).
  • photothermal heating source 54 is positioned above surface 52.
  • surface 52 is patterned with a plurality of patterns 53.
  • surface 52 is also in the form of a microfluidic channel 58, which may include an inlet 60 for receiving a material and an active agent, and an outlet 62 for releasing active agent-loaded materials.
  • microfluidic channel 58 is in encapsulated form.
  • system 50 also includes a magnet 56 operable to apply a magnetic field to surface 52. In some embodiments, magnet 56 is positioned below surface 52.
  • system 50 also includes a housing unit 64 operable for housing magnet 56.
  • housing unit 64 is operable to control spacing between magnet 56 and surface 52 to ensure consistent magnetization of surface 52.
  • system 50 also includes a housing unit 66 operable for housing surface 52, a housing unit 70 operable for housing photothermal heating source 54, and a cooling unit 72 operable for cooling photothermal heating source 54.
  • cooling unit 72 is in the form of a thermoelectric cooler.
  • the methods and systems of the present disclosure can have numerous embodiments. As also set forth in more detail herein, the methods and systems of the present disclosure can have numerous advantages and applications.
  • the materials include, without limitation, particles, magnetic particles, nanoparticles, magnetic nanoparticles, polymeric particles, viral particles, virus-like particles, lipid-based particles, nanomaterials, magnetic nanomaterials, materials that include a biomolecular membrane, exosomes, liposomes, cells, or combinations thereof.
  • the materials include materials that include a biomolecular membrane.
  • the materials include exosomes.
  • the materials include cells.
  • the cells include, without limitation, immune cells, T-cells, B-cells, or combinations thereof.
  • the cells include exosomes of varying cellular origins.
  • the exosome origin includes, without limitation, exosomes from T-cells, B- cells, mesenchymal stem cells, plasma-derived exosomes, or combination thereof.
  • the materials are derived from cell culture mediums.
  • the materials are derived from patient biofluids including plasma, whole blood, urine, cerospinal fluid (CSF), or combinations thereof.
  • the active agents include, without limitation, drugs, small molecules, biologies, proteins, peptides, peptoids, nucleic acids, RNA, siRNA, mRNA, miRNA, DNA, genes, gene editing systems, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene editing systems, image contrast agents, quantum dots, or combinations thereof.
  • the active agents include drugs.
  • the active agents include a gene editing system that is operable to insert a gene into a cell.
  • the active agents include a gene for expression in a cell.
  • the methods of the present disclosure may associate active agents with materials in various manners. For instance, in some embodiments, the association of active agents with materials occurs prior to associating the materials with a surface. In some embodiments, the association of active agents with materials occurs during the association of the materials with a surface. In some embodiments, the association of active agents with materials occurs after associating the materials with a surface.
  • association of active agents with materials can occur in various manners. For instance, in some embodiments, the association of the active agents with materials occurs at room temperature. In some embodiments, the association of active agents with materials includes incubating the materials and active agents. In some embodiments, the association of active agents with materials includes mixing the materials and the active agents in a liquid.
  • the methods and systems of the present disclosure may utilize various surfaces for loading active agents into materials.
  • the surface is in the form of a membrane that includes a plurality of photosensitizers.
  • the photosensitizers arc operational to induce localized heating of the membrane upon photothcrmal heating.
  • the photosensitizers include magnetic nanoparticles.
  • the membrane is in the form of an elastomeric matrix.
  • the surfaces of the present disclosure include a patterned surface.
  • the patterned surface is operational for facilitating the association of a material with the surface.
  • the surfaces of the present disclosure may include various patterns.
  • the patterned surface includes one or more shapes.
  • the shapes include, without limitation, squares, herringbones, Y-like shapes (e.g., Y-like shapes 53 of surface 52 in FIG. IE), X-like shapes, or combinations thereof.
  • the patterned surface can be encapsulated within a microfluidic channel.
  • the patterning can be rationally designed to influence a native fluidic environment.
  • the patterning can be rationally designed to influence the microscale magnetic field.
  • the pattern includes one or more shapes.
  • the height of the patterned surface can be changed, from a range of 1 pm to 200 pm.
  • the surfaces of the present disclosure include material binding agents that are operational for associating a material with a surface.
  • the material binding agents include, without limitation, antibodies, aptamers, peptides, peptoids, small molecules, or combinations thereof.
  • the surfaces of the present disclosure are in the form of a microfluidic channel (e.g., microfluidic channel 58 shown in FIG. IE).
  • the microfluidic channel includes an inlet for receiving a material and an active agent (e.g., inlet 60 shown in FIG. IE), and an outlet for releasing an active agent-loaded material (e.g., outlet 62 shown in FIG. IE).
  • the microfluidic channel is in encapsulated form.
  • the surfaces of the present disclosure include a magnetic surface.
  • the magnetic surface includes a plurality of magnetic particles.
  • the magnetic particles include, without limitation, magnetites, iron oxide particles, FeaC -containing particles, y-FeiCh-containing particles, or combinations thereof.
  • the plurality of magnetic particles is encapsulated within a polymer matrix.
  • the plurality of magnetic particles is encapsulated within an elastomer matrix.
  • the weight loading percentage of the magnetic particles within a matrix can range from 1% to 75% weight loading.
  • the magnetic surface is associated with a magnet operational to apply a magnetic field to the magnetic surface (e.g., magnet 56 shown in FIG. IE).
  • the association of materials with a surface includes applying a magnetic field to a magnetic surface.
  • the association of materials with a magnetic surface includes: (1) capturing materials by magnetic beads to form magnetic bead-material complexes; (2) associating the magnetic beads with the magnetic surface; and (3) applying a magnetic field to the magnetic surface to result in the immobilization of the magnetic bead-material complex on the magnetic surface.
  • the association of magnetic beads with a magnetic surface occurs after the capture of materials by magnetic beads.
  • the association of magnetic beads with a magnetic surface occurs before the capture of materials by the magnetic beads.
  • the association of magnetic beads with a magnetic surface occurs during the capture of materials by magnetic beads.
  • the magnetic beads include magnetic particles and material binding agents on the magnetic particles.
  • material capture includes the capture of materials by the material binding agents.
  • the material binding agents include, without limitation, antibodies, aptamers, peptides, peptoids, small molecules, or combinations thereof.
  • Photothermal heating generally refers to the utilization of photons to heat surface-associated materials.
  • the methods of the present disclosure may photothermally heat surface-associated materials in various manners.
  • photothermal heating includes photothermally heating a surface.
  • the photothermal heating of the surface results in the heating of the material by the surface.
  • the photothermal heating results in the photoporation of the material.
  • the photothermal heating of a surface-associated material includes light irradiation from a light source.
  • the light source includes a laser.
  • the light source includes a light emitting diode (LED).
  • the light source is positioned above a surface during photothermal heating.
  • the photothermal heating of a surface-associated material occurs at sequential intervals. In some embodiments, each interval lasts from about 5 ms to about 250 ms. In some embodiments, each interval is separated by at least 5 ms. In some embodiments, the time of each interval is separated by an equal time between. In some embodiments, the interval time is longer than the time between. In some embodiments, the interval time is shorter than the time between. In some embodiments, the photothermal heating occurs during at least 3 sequential intervals. In some embodiments, the photothermal heating occurs with more sequential intervals, such as up to 10 sequential intervals. In some embodiments, only a single photothermal heating interval is used. In some embodiments, each interval within a sequence has a different time duration. In some embodiments, each interval has a different power density. In some embodiments, each interval has the same power density.
  • the photothermal heating source includes a light source operable to emit light irradiation.
  • the light source includes a laser.
  • the light source includes a light emitting diode (LED).
  • the light source includes a Chip-On-Board (COB) LED.
  • the light source includes a Surface-Mounted Device (SMD) LED.
  • the light source includes a Multiple- Chips-On-Board (MCOB) LED.
  • the wavelength of the light source corresponds to the absorbance of the patterned materials.
  • the wavelength of the light source corresponds to the absorbance of the surface-associated materials. In some embodiments, the wavelength is within the visible regime (380-700 nm). In some embodiments, the wavelength in is the near-IR regime (800-2500 nm).
  • the methods of the present disclosure also include a step of washing a surface-associated material.
  • the washing step occurs prior to dissociating active agent-loaded materials from a surface.
  • the washing step separates unloaded active agents from surface-associated and active agent-loaded materials.
  • the washing step includes flowing a wash buffer through a surface.
  • the wash buffer includes phosphatc-buffcrcd saline (PBS).
  • the methods of the present disclosure also include a step of dissociating active-agent loaded materials from a surface.
  • the dissociation step includes a step of flowing an elution buffer through the surface.
  • the elution buffer includes a Tris-Cl buffer.
  • the dissociation step includes a step of removing a magnetic field from the magnetic surface to result in the dissociation of active agent-loaded materials from the magnetic surface.
  • the active agent-loaded materials may be immobilized on the magnetic surface through magnetic beads that form magnetic bead-material complexes on a magnetic surface.
  • the methods and systems of the present disclosure can have various advantages. For instance, in some embodiments, the methods of the present disclosure may be utilized to load various active agents into various materials in a consistent, facile, cost effective and adaptable manner.
  • the materials include T-cells and the active agents include one or more genes encoding a T-cell receptor.
  • the methods of the present disclosure may be used to engineer chimeric antigen receptor T-cells for various therapeutic applications.
  • Example 1 Rapid and scalable screening and drug loading within patient-derived exosomes for autologous therapies
  • EXOtherm a microtechnology-based system for the capture and engineering of autologous exosomes using magnetic microchips and photothermal heating, respectively.
  • DOE small molecule chemotherapeutic drug doxorubicin
  • system parameters i.c., doses and dose rate
  • EXOtherm makes significant steps towards enabling integrated immunomagnetic exosome isolation and cargo agnostic photothermal loading within a modular benchtop system.
  • Example 1,1, EXOtherm Platform for Integrated Exosome Isolation and Cargo Loading [0071] As illustrated in FIGS. 2A-2B, the EXOtherm platform can be broken down into two main components, namely (1) magnetic microchips, and (2) a photothermal engineering system. Magnetic microchips are fabricated using a scalable laser-based fabrication developed by Applicant’s group that is suitable for rapid and scalable microchip fabrication and enables efficient immunomagnetic exosome sorting via flow-invasive micromagnets which generate high gradient fields throughout the biofluid medium. Surrounding this main component, Applicant has designed and developed a photothermal engineering system, EXOtherm, that enables integrated on-chip cargo loading within captured exosomes and fits within a minimal form factor enabling benchtop use (FIG. 2A).
  • EXOtherm photothermal engineering system
  • EXOtherm consists of a 3D-printed housing included of three interconnected components: the base, microchip holder, and thermoelectric cooler (TEC) holder, plus a separate LED module and programmable power supply (FIG. 2A).
  • the base harbors a permanent neodymium magnet which magnetizes the patterned micromagnets whereas the microchip holder controls spacing between the permanent magnet and magnetic microchip ensuring consistent magnetization.
  • Magnetic microchips are aligned and affixed in place within the holder by magnetic clips which can easily be removed following processing to release the microchip.
  • the TEC holder controls the spacing between magnetic microchips and the LED module, ensuring consistent and repeatable sample irradiation.
  • each component including the microchip and TEC holder, are connected to one another with magnetic fixtures that allow for modular assembly and rapid sample processing.
  • Applicant sought to enable efficient large-area engineering and as such rather than a laser source chose to utilize a high- powcr chip-on-board LED (Luminus, CLM-22) for irradiation.
  • a programmable power supply (Keysight 36155A) provides fine-tuned control over optical dose, and tunability in pulse width, duration, and number of pulses.
  • Optical power density is controlled via interchangeable chassis mount resistors and preset voltages on the programmable power supply.
  • EXOtherm showed precise control dose control from 0.1 W/cm 2 - 100 W/cm 2 (FIGS. 2C-2E), pulse durations as short as 20 ms, and reliable and repeatable dose control, enabling high levels of tunability.
  • the microchip holder and LED module were positioned such that irradiation occurred at a fixed and known distance (5.13 mm), and all optical recordings were measured at this distance to ensure consistency (FIG. 2B).
  • nanoparticle formulations including gold, carbon, and poly dopamine-based nanoparticles have been explored, due to their favorable optical properties and biocompatibility.
  • sizes of nanoparticle, nanoparticle material, and optical configurations can all play significant roles in transfection efficiency and biomolcculc viability, and more importantly that often nanoparticles sensitizers can be difficult from solution following incorporation.
  • efforts have been made to develop methods towards transient photoporation by imbedding photosensitizers within matrices with promising results.
  • Micromagnets arc included of a dense (50% wt/wt) and homogeneous packing of magnetic nanoparticles (-150 nm) within an elastomer matrix. Optical characterization has shown an absorption maximum at -640 nm, suggesting that micromagnets could act as matrix-based photosensitizers (FIG. 3C).
  • this microchip architecture was designed to enable robust patterning of magnetically tagged exosomes, which promotes localization of exosomes near photosensitive micromagnets. Taken together, this microchip provides an ideal substrate upon which photothermal engineering of exosomes can occur.
  • Doxorubicin hydrochloride a first line cancer chemotherapy drug for multiple cancers.
  • Doxorubicin hydrochloride (Sigma) was kept at a stock concentration of 1 mM and within loading tests diluted to a concentration of 150 pM. Characterization of loading magnitude was completed by measuring doxorubicin fluorescence (Ex/Em 470nm / 595 nm) upon a plate reader (Molecular Devices SpectraMax Paradigm). To remove potential heterogeneity resulting from in-house exosome isolation, exosome standards (COLO-1, Novus Biologies) reconstituted in molecular grade water according to manufactures specifications were utilized for initial optimization testing.
  • exosomes Prior to introduction within magnetic microchips, exosomes were magnetically tagged with streptavidin-coated magnetic nanoparticles (MGB magnetic beads, 500 nm, Luna Nanotech) conjugated with biotinylated antibodies (BioLegend) targeting pan-exosome markers CD9, CD63, and CD81. Previous efforts from Applicant’s group have optimized the antibody to magnetic bead ratio to maximize conjugation efficiency, which was adopted within this Example. [0085] As illustrated in FIG. 4A, doxorubicin loading optimization tests within EXOtherm were performed as follows: A solution (200 pL working volume) containing magnetically tagged exosomes (1 pg exosomes) is combined with DOX and this solution is immediately transferred onto the magnetic microchip.
  • exosomes are rapidly patterned upon micromagnets.
  • the TEC and LED module are positioned and secured above the microchip, where LED irradiation commences to initiate nanoscale photothermal heating.
  • still-captured exosomes are rigorously washed to remove residual unloaded DOX.
  • the exosomes are then removed from the microchip holder and washed thoroughly to release exosomes. Loading optimization was completed across the full operational range of the EXOtherm system, by binning the available power range into nine binned and distributed power outputs, with a 50 ms irradiation pulse.
  • Results from the power density testing are shown in FIGS. 4B-4G, for unpatterned and patterned microchips, with an accumulated comparison comparing each shown in FIG. 4D.
  • the results highlight the benefit of matrix-based photosensitizers.
  • Unpatterned microchips represent traditional photoporation and reflect the contribution of LED irradiation alone, or minor heating resulting from LED irradiation to capture magnetic particles.
  • Applicant investigated alterations to pulse duration and number of pulses. Within traditional photoporation using laser-based sources, increased pulse duration leads to higher accumulated doses, eventually leading to irreversible membrane damage and reduced cellular viability. To explore this relationship for exosomes, Applicant selected pulse durations of 20, 50, 100, 150, 200, and 250 ms, and performed loading experiments as completed previously, data from which can be seen in FIGS. 4E-4F.
  • Example 1 Loading of Labile Molecular Cargo
  • EXOtherm was modularly constructed using a combination of custom 3D printed parts which include the housing and control spacing, and off-the-shelf components.
  • 3D printed parts include the base, microchip holder, and thermoelectric cooler (TEC) holder.
  • the base provides overall structure for the system and provides a housing for the permanent magnet (1” x 1” x 0.5”, N52, KJMagnetics).
  • the microchip holder sits atop the base, is locked in place with magnetic fixturing, and has a space that allows for the alignment of a 3” x 2” glass slide, which was the substrate used for magnetic microchips. Once aligned, the magnetic microchip is also locked in place with magnetic fixturing.
  • a holder for the TEC (Laird Thermal Systems, DA-044-12-02-00-00) is posited, which finely controls the distance between the LED (Luminus, CLM-22) and magnetic patterning to enable consistent irradiation.
  • the LED optical output is controlled by a programmable power supply (Keysight 36155A), connected with interchangeable chassis mount resistors to protect against current spikes and enable high power operation.
  • micromagnets were mechanically stabilized using a solution-based dip-coating treatment with AI2O3, optimized to form a nanoscale thick coating (-100 nm), and a microwell placed above forming a fluidic cell.
  • 3D printed rings designed to hold -200 pL, were printed (Bambu PIP) followed by dipping the bottom of the well within PDMS (Sylgard 184, 10:1 base, curing agent) and aligning over pattern and bringing into contact, whereby the PDMS was cured on a hot plate (60°C for 2 hours).
  • PDMS Sylgard 184, 10:1 base, curing agent
  • an oxygen plasma treatment 29.8W, 3 minutes, Herrick Plasma PDC-001 was performed to increase wettability.
  • Biotinylated anti-human antibodies (CD9, CD81, CD63) were purchased from BioLegend and used without further modification.
  • Streptavidin-coated iron oxide nanoparticles (500 nm diameter) were purchased from Luna Nanotech, and prior to use, beads were washed twice within a magnetic stand with IX PBS. Magnetic bead conjugates were prepared as described previously. Briefly, 20 pL stock antibody solution was combined with 175 pL magnetic beads and diluted to 400 pL with PBST (PBS + 0.1% Tween-20), resulting in a concentration of 0.025 pg antibody/pL.
  • PBST PBS + 0.1% Tween-20
  • Exosome standards (COLO-1, Novus Biologies) were purchased and reconstituted according to the manufacturer’ s recommendations with molecular grade DI water at a concentration of 1 pg/pL. Magnetic labeling of exosomes was completed in batch-based preparations, such that a single batch could be used for each experiment and was scaled down and up as necessary. In a typical experiment, 5 pL of reconstituted exosome standard was added to 20 pL conjugate (6.66 pL of CD9, CD63, and CD81), and diluted to 500 pL with PBST + 0.1% BSA.
  • Labeling was completed with mixing (Argos Technologies RotoFlex Plus) for 3 hours at room temperature, followed by washing twice within a magnetic rank with PBST + 0.1% BSA and resuspending in 1000 pL, yielding a final concentration of 0.5 pg/100 pL solution.
  • plasma obtained from Dartmouth Health was thawed at 37 °C for 15 minutes.
  • 200 pL of clinical plasma was incubated with 0.2 pg of antibody (included of an equal ratio of CD9, CD63, and CD81) for 2 hours at room temperature and used directly after for on-chip testing.
  • Doxorubicin was purchased from Sigma Aldrich as a power and kept as a stock concentration within IX PBS at 1 mM, hidden from light, and stored at 4 °C. A standard curve was created to quantify doxorubicin concentration in solutions. Due to the ability to thoroughly wash exosomes upon magnetic capture chips, Applicant elected to use high concentrations of doxorubicin in solution for loading studies. Following magnetic tagging of exosomes, washing away excess antibody, and resuspending, a 200 pL working volume was aliquoted and doxorubicin added to a final concentration of 150 pM and briefly mixed by pipetting the solution up and down.
  • the sample was introduced into the magnetic microchip placed within its holder to enable magnetic patterning and subject to LED irradiation.
  • samples were washed twice using PBST with 0.1 % BSA, then removed from their holder enabling the release of captured exosomes, which was completed by resuspending them in 200 pL PBST with 0.1% BSA.
  • samples were used directly after conjugation. Once on-chip loading was performed, the exosomes were again thoroughly washed on-chip using PBST with 0.1% BSA before the microchip was removed and exosomes resuspended for subsequent processing.
  • Labile cargo including Cy3-labeled siRNA (Thermo, SilencerTM CyTM3-labeled Negative Control No. 1 siRNA) and Cas9-GFP (Sigma, CAS9GFPPRO), were purchased and reconstituted as necessary per manufacturer recommendations. Due to the significantly lower quantity of both reagents compared to doxorubicin, stock solutions of 25 pM were made. Loading testing followed an identical protocol to initial small molecule testing where COLO-1 exosome standards (1 pg) within a 200 pL working volume were aliquoted from the prepared stock.
  • Cy3-labclcd siRNA or Cas9-GFP were added to the solution to a concentration of 1.25 pM, mixed briefly with a pipette to ensure homogenous incorporation, and added to the microchip. Following loading, an identical washing procedure used for doxorubicin was used prior to on-chip confocal imaging.
  • Exosome visualization was completed using a DiD lipid membrane stain (Invitrogen, Ex./Em. 644/665 nm), prepared by mixing 25 mg of DiD in 4 mL of dimethyl sulfoxide. Staining of exosomes was completed with a 15-minute incubation at 37°C, using 2.5 pL/1 pg of exosomes, followed by thoroughly washing of the sample five times using PBST with 0.1% BSA within a magnetic rack. DiD staining was completed following cargo loading for exosome standards and plasma samples alike, with exosomes that were removed from capture microchips.
  • DiD lipid membrane stain Invitrogen, Ex./Em. 644/665 nm
  • Imaging was completed with an Andor W 1 Spinning Disc confocal microscope under brightfield and fluorescence using a 488 nm laser for excitation for doxorubicin and Cas9-GFP, 532 nm excitation for Cy3-labed siRNA, and 637 nm excitation for DiD membrane dye. Images were processed within the NIS Elements Workstation or Fiji ImageJ. Processed plasma samples were finally reconstituted to a final volume of 200 pL with PBST with 0.1% BSA.

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Abstract

Des modes de réalisation de la présente divulgation concernent des procédés de chargement d'un matériau avec un agent actif par (1) association du matériau à une surface ; (2) association de l'agent actif au matériau ; (3) chauffage photothermique du matériau associé à la surface pour conduire à l'encapsulation de l'agent actif dans le matériau associé à la surface ; et (4) dissociation du matériau chargé d'agent actif de la surface. Des modes de réalisation supplémentaires de la présente divulgation concernent des systèmes utilisables pour charger un matériau avec un agent actif. Les systèmes de la présente divulgation peuvent comprendre (1) une surface utilisable pour s'associer à un matériau, (2) une source de chauffage photothermique proximale à la surface et utilisable pour chauffer la surface, et (3) un aimant utilisable pour appliquer un champ magnétique à la surface.
PCT/US2024/031001 2023-05-25 2024-05-24 Exploitation d'un chauffage photothermique pour le traitement de surface d'agents actifs WO2024243514A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030022370A1 (en) * 2001-07-27 2003-01-30 Rocco Casagrande Magnetic immobilization of cells
US20130335703A1 (en) * 2011-01-13 2013-12-19 Optos Plc Ophthalmology
US20150197720A1 (en) * 2011-05-13 2015-07-16 The Regents Of The University Of California Photothermal substrates for selective transfection of cells
US20180258391A1 (en) * 2010-12-09 2018-09-13 The Trustees Of The University Of Pennsylvania Compositions and Methods for Treatment of Cancer
US20200062813A1 (en) * 2017-02-22 2020-02-27 Evox Therapeutics Ltd Improved Loading of EVs with Therapeutic Proteins
US20200071727A1 (en) * 2018-08-31 2020-03-05 The Charles Stark Draper Laboratory, Inc. Method and Apparatus for High Throughput High Efficiency Transfection of Cells

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030022370A1 (en) * 2001-07-27 2003-01-30 Rocco Casagrande Magnetic immobilization of cells
US20180258391A1 (en) * 2010-12-09 2018-09-13 The Trustees Of The University Of Pennsylvania Compositions and Methods for Treatment of Cancer
US20130335703A1 (en) * 2011-01-13 2013-12-19 Optos Plc Ophthalmology
US20150197720A1 (en) * 2011-05-13 2015-07-16 The Regents Of The University Of California Photothermal substrates for selective transfection of cells
US20200062813A1 (en) * 2017-02-22 2020-02-27 Evox Therapeutics Ltd Improved Loading of EVs with Therapeutic Proteins
US20200071727A1 (en) * 2018-08-31 2020-03-05 The Charles Stark Draper Laboratory, Inc. Method and Apparatus for High Throughput High Efficiency Transfection of Cells

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