WO2024059715A2 - Microbulles et leurs procédés de fabrication - Google Patents

Microbulles et leurs procédés de fabrication Download PDF

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WO2024059715A2
WO2024059715A2 PCT/US2023/074198 US2023074198W WO2024059715A2 WO 2024059715 A2 WO2024059715 A2 WO 2024059715A2 US 2023074198 W US2023074198 W US 2023074198W WO 2024059715 A2 WO2024059715 A2 WO 2024059715A2
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microbubbles
surfactant
water
certain embodiments
mbs
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PCT/US2023/074198
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WO2024059715A3 (fr
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Brian Oeffinger
Margaret Wheatley
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Drexel University
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Publication of WO2024059715A3 publication Critical patent/WO2024059715A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/20—Mixing gases with liquids
    • B01F23/23—Mixing gases with liquids by introducing gases into liquid media, e.g. for producing aerated liquids
    • B01F23/237—Mixing gases with liquids by introducing gases into liquid media, e.g. for producing aerated liquids characterised by the physical or chemical properties of gases or vapours introduced in the liquid media
    • B01F23/2373—Mixing gases with liquids by introducing gases into liquid media, e.g. for producing aerated liquids characterised by the physical or chemical properties of gases or vapours introduced in the liquid media for obtaining fine bubbles, i.e. bubbles with a size below 100 µm
    • B01F23/2375—Mixing gases with liquids by introducing gases into liquid media, e.g. for producing aerated liquids characterised by the physical or chemical properties of gases or vapours introduced in the liquid media for obtaining fine bubbles, i.e. bubbles with a size below 100 µm for obtaining bubbles with a size below 1 µm

Definitions

  • the invention provides a method for preparing microbubbles.
  • the method for preparing microbubbles comprises the steps of i. providing a first system comprising at least one water-soluble surfactant; ii. providing a second system comprising at least one water-insoluble surfactant, wherein providing the second system optionally comprises the step of autoclaving the second system and optionally cooling the resulting autoclaved system; and iii. contacting the first system with the second system at the temperature below the melting point of the second surfactant, so as to produce a mixture comprising microbubbles.
  • the first system is aqueous.
  • the first system comprises a micelle.
  • the second system comprises a cream.
  • the method further comprises purging the surfactant mixture of step (iii) with a purging gas.
  • the purging gas is a perfluorocarbon (PFC).
  • the method further comprises washing the mixture comprising microbubbles with DI water and separating the microbubbles from the mixture by gravity filtration.
  • the method further comprises freeze-drying and capping the separated microbubbles under vacuum.
  • the method further comprises filling the freeze-dried microbubbles with a filling gas.
  • the filling gas is oxygen, octafluoropropane, and sulfur hexafluoride.
  • the at least one water soluble surfactant is selected from the group consisting of tocopheryl polyethylene glycol succinate (TPGS), cetyltrimethylammonium Bromide (CTAB), and mixtures thereof.
  • the at least one water-insoluble surfactant is selected from the group consisting of sorbitan monostearate, sorbitan fatty acid esters, sorbitan monopalmitate, (+) A tolopherol acid succinate (vitamin E) and mixtures thereof.
  • the contacting is performed at room temperature.
  • At least one of the first system and the second system independently comprises an additive.
  • the at least one additive is selected from the group consisting of a drug, a dye, and a charged molecule for surface modification of the microbubbles.
  • the charged molecule alters the charge (zeta potential) of the microbubbles.
  • the microbubbles with altered zeta potential are further loaded with oppositely charged molecules.
  • the microbubbles are acoustically-sensitive.
  • FIG. 1 depicts the chemical structures of sorbitan monostearate and tocopheryl polyethylene glycol succinate (TPGS).
  • FIG. 2 depicts a schematic showing different methods, i.e., original method (OM), micelle method (MM), and cream method (CM), for preparing the microbubbles (MBs).
  • OM original method
  • MM micelle method
  • CM cream method
  • FIG. 3 depicts that the presently developed method (cream method) allows TPGS and sorbitan monostearate to be combined at lower temperatures, which is better for heat sensitive drug loading.
  • the cream method also allows chemical addition to sorbitan monostearate phase at room temperature (such as drug or cationic species).
  • sorbitan monostearate formed large chunks when cooled (left picture) in previously disclosed (micelles) method.
  • FIG. 4 is a schematic depicting certain steps for microbubble production after combining a first system comprising water-soluble surfactant and a second system comprising a water insoluble-surfactant.
  • FIG. 5 demonstrates that SE61 ’s (formed by combining Span 60 and TPGS) the zeta potential , potential) is likely negative.
  • FIG. 6 show tables depicting size and C, potential of SE61’s MB population prefreeze dry step for SE61’s MBs prepared vial OM, MM, and CM.
  • FIG. 7 show tables depicting size and potential of SE61 ’s MB population postfreeze dry step SE61’s MBs prepared vial OM, MM, and CM.
  • FIG. 8 shows a comparison of dose response curve post freeze drying for SE61 MBs prepared vial OM, MM, and CM.
  • FIG. 9 shows examples of possible additions for positively charged bubbles, the examples include cationic surfactants, cationic lipids, cationic cholesterols.
  • FIG. 10. shows N 4 -Cholesteryl-Spermine HC1 Salt (GL67) structure, zeta potential, size distribution, and dose response for MBs prepared from GL67.
  • FIG. 11 A shows the chemical structure of Rose Bengal (RB) sodium salt.
  • FIG. 1 IB shows a cartoon of proposed/putative SE61 microbubble structure prepared from Span 60 and TPGS.
  • FIG. 12 shows a schematic of sonodynamic therapy, wherein the MB are destroyed using low frequency ultrasound to release a therapeutic agent.
  • FIG. 13 shows a cartoon of putative CTAB-RB microbubble.
  • FIG. 14 shows a schematic of a process for making CTAB-RB microbubbles, according to the embodiments of the present invention.
  • FIG. 15 shows a set-up of in vitro acoustical testing of microbubbles to assess MB dose and time response. Bottom two images are taken under fluorescence. In the SE61-RB batch, there is no glowing indicating no RB attachment. In the CTAB-RB batch there is a glowing shell indicating successful RB attachment.
  • FIG. 16 is a table showing examples of cationic additives as well as their respective addition methods.
  • FIGS. 17A-17C size distributions of MBs with 5% (FIG. 17A), 15%(FIG. 17B), and 25% (FIG. 17C) cationic additive (post-sonication).
  • Increasing the concentration of DDAB and DOTOP reduces the number of MBs created, with 15% and 25% DOTAP not producing sufficient MBs for testing.
  • FIGS. 18A-18C show that all cationic additives (5% (FIG. 18A), 15%(FIG. 18B), and 25% (FIG. 18C)) increased the zeta potential of SE61 (-40 mV pre-freeze dried, -30 mV post-freeze dried). Approximately 15% additive is needed to reach the target of 30 mV.
  • FIG. 19A-19C all successful cationic modified SE61 MBs comprising 5% (FIG. 19 A), 15%(FIG. 19B), and 25% (FIG. 19C) additives) produced an enhancement greater than 15 dB.
  • FIGS. 20A-20D are images of Unmodified SE61-RB brightfield (FIG.20A) and fluorescence (FIG.20B); 15% CT AB SE61-RB brightfield (FIG. 0C), and fluorescence (FIG. 20D).
  • FIG. 21A-21B are images of in vivo ultrasound of a human head and neck tumor (arrows) (CAL27) in a nude mouse model, pre-injection (FIG. 21A) and post-injection (FIG. 2 IB).
  • FIG. 22 is a cartoon/putative structure of SE61MB with examples of gases that can be filled within the MBS.
  • FIG. 23 is a schematic showing fabrication of SE61 (cream method).
  • FIG. 24 shows a dose response curves and sizes for MBs surface-modified using DMTAP at different concentrations.
  • FIG. 25 shows zeta potential for MBs surface-modified using l,2-dimyristoyl-3- trimethylammonium-propane chloride (DMTAP) at different concentrations.
  • DMTAP l,2-dimyristoyl-3- trimethylammonium-propane chloride
  • FIG. 26 is a graph showing DNA loading on MBs surface-modified using DMTAP at different concentrations
  • the present invention is related to a process of making MBs comprised of at least two surfactant phases, one of which is water soluble and the other that is insoluble.
  • the method allows for the creation of the two surfactant phases independently with the ability to incorporate additional species, such as drugs or charged molecules, in either phase at temperatures near room temperature to preserve the integrity of the additive.
  • This method produces MBs with similar sizes distributions, surface charges, and acoustical properties as previous methods.
  • the present invention is related to incorporating a charged species into the MB shell. This charged species can be added to either the soluble or the insoluble phase of the preparation. This charged species can change the surface charge of the resulting MB and be used to facilitate the attachment of oppositely charged species. Previous methods had a surface charge of about -30 mV, while adding cationic molecules changed the charged surface to around +30 mV.
  • the present invention is related to attaching charged species to a surface modified MB, wherein the charged species is ,for examples, Rose Bengal (RB) or nucleic acid to facilitate delivery using MBs and ultrasound to release drug at a target site.
  • the charged species is ,for examples, Rose Bengal (RB) or nucleic acid
  • RB and nucleic acids are water soluble, typical encapsulation in the hydrophobic MB shell results in minimal loading.
  • the MB becomes positively charged, allowing the negatively charged RB or nucleic acid to be attached.
  • Attaching RB to a MB allows for drug and encapsulated gas e.g., O2 delivery through Sonodynamic Therapy (SDT), which, uses ultrasound to initiate inertial cavitation.
  • MB collapse releases drug and gas (O2) from the core.
  • SDT Sonodynamic Therapy
  • Collapse triggers emission of photo-luminescence light which excites RB; and a further reaction with the released oxygen provokes biological damage.
  • SDT is an improved alternative to chemotherapy. Chemotherapy is systemic, whereas SDT is a localized treatment and can target a specific tumor site.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the invention provides a method for preparing microbubbles (MBs), wherein the method comprises the steps of providing a first system comprising at least one water-soluble surfactant (first surfactant); providing a second system comprising at least one water-insoluble surfactant (second surfactant), wherein providing the second system optionally comprises the step of autoclaving the second system and optionally cooling the resulting autoclaved system; and contacting the first system with the second system at the temperature below the melting point of the second surfactant to form a surfactant mixture comprising microbubbles.
  • first surfactant water-soluble surfactant
  • second surfactant water-insoluble surfactant
  • the contacting is performed at about 100 °C, 95 °C, 90 °C, 85 °C, 80 °C, 75 °C, 70 °C, 65 °C, 60 °C, 55 °C, 50 °C, 45 °C, 40 °C, 35 °C, 30 °C, 25 °C, 20 °C, 15 °C, 10 °C, 5 °C, or 0 °C.
  • the contacting is performed at the room temperature.
  • the first system is aqueous.
  • the first system comprises a micelle.
  • the concentration of the water- soluble/micelle-forming surfactant is above the critical micelle concentration (CMC).
  • the micelle-forming surfactant may include one or more micelle-forming surfactants such as diblock copolymers.
  • the hydrophobic portions of such diblock copolymers may include one or more hydrophobic polymers, such as polyesters, polyanhydrides, polyglycolic acids, polybutrylactones, polyhydroxybutyrates, polylactic acids and polylacaprolactones.
  • the hydrophobic portion of the copolymer may include one or more different hydrophobic polymers in random or block orientation.
  • Preferred hydrophilic portions of micelle forming copolymers that may be used in this invention have a molecular weight of about 750 or greater up to about 8000.
  • Non-limiting examples of the water-soluble surfactants include TPGS and/or suitable polyoxyethylene fatty acid esters such as TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 65 and TWEEN 80, each of which are commercially available from ICI Americas, Inc. of Wilmington, Del.
  • the TWEEN surfactants are each mixtures of various polyoxyethylene fatty acid esters in liquid form.
  • TWEEN 20 comprises polyoxyethylene (POE) esters of about 60 weight percent lauric acid (dodecanoic acid); about 18% myristic acid (tetradecanoic acid); about 7% caprylic acid (octanoic acid) and about 6% capric acid (decanoic acid).
  • POE polyoxyethylene
  • TWEEN 40 generally comprises POE esters of about 90% palmitic acid (hexadecanoic acid).
  • TWEEN 60 generally comprises POE esters of about 49% stearic acid (octadecanoic acid) and about 44% palmitic acid.
  • TWEEN 80 generally comprises POE esters of about 69% oleic acid (cis-9-octadecanoic acid); about 3% linoleic acid (linolic acid); about 3% linolenic acid (9,12,15- octadecatrienoic acid); about 1% stearic acid and about 1% palmitic acid.
  • Additional examples include other water-soluble species that from micelles such as water-soluble diblock copolymers. Examples include those based on hydrophilic poly(ethylene glycol)(PEG).
  • the water-insoluble surfactant is a dispersible surfactant, although one of ordinary skill in the art would understand that the second surfactant may be partially or fully soluble in a solvent. In certain embodiments, it is preferred that the second surfactant is non-ionic.
  • Non-limiting examples of the water-insoluble surfactants include sorbitan fatty acid esters, such as sorbitan monostearate, sorbitan monopalmitate and mixtures thereof.
  • Preferred mixtures of sorbitan fatty esters include SPAN 40 and SPAN 60, each of which are dry powders commercially available from ICI Americas, Inc.
  • SPAN 40 comprises sorbitan esters of about 93% palmitic acid; about 2.5% myristic acid and less than about 1% pentadecanoic acid.
  • SPAN 60 comprises sorbitan esters of about 50% stearic acid, about 45% palmitic acid and about 2% myristic acid.
  • Additional second components could be compounds such as (+) A tolopherol acid succinate (vitamin E), Brij 52, or Polyethylene-block-poly(ethylene glycol) MW 575.
  • the second system comprises a cream. In certain embodiments, the second system comprises an emulsion.
  • the method further comprises the step of agitating the surfactant mixture to form microbubbles. In certain embodiments, the method comprises sonicating the surfactant mixture to form the microbubbles. In certain embodiments, a homogenizer/homogenization is used to form the microbubbles.
  • the method further comprises purging the surfactant mixture with a purging gas.
  • the purging gas is a perfluorocarbon (PFC). In certain embodiments, the purging is performed with octafluropropane. In certain embodiments the purging gas is oxygen, sulfur hexafluoride (SF6), Perfluorobutane (C4F10) or other non toxic gas.
  • PFC perfluorocarbon
  • the purging is performed with octafluropropane. In certain embodiments the purging gas is oxygen, sulfur hexafluoride (SF6), Perfluorobutane (C4F10) or other non toxic gas.
  • the method further comprises washing the mixture comprising microbubbles with PBS or DI water and separating the microbubbles from the mixture by gravity filtration. In certain embodiments, the method further comprises freeze-drying and capping the separated microbubbles under vacuum.
  • the frozen samples were lyophilized in vials with stoppers placed on the vials to the first groove, and placed on a previously chilled (-20 °C) shelf in a Virtis Benchtop freeze-dryer (Gardiner, NY). At the end of the cycle, prior to venting, a piston was lowered to seal the stoppers on the vials under vacuum.
  • the method further comprises filling the freeze-dried microbubbles with a filling gas.
  • the method comprises reconstituting the freeze-dried bubbles with a water or PBS.
  • the filling gas is for example, oxygen, octafluoropropane xenon, nitrous oxide, nitrogen, C4F10 or sulfur hexafluoride, which is encapsulated by the MBs.
  • the encapsulated gas is a therapeutic such as oxygen to facilitate treatment of hypoxia and to generate reactive oxygen species.
  • the encapsulated gas is released upon insonation.
  • At least one of the first system and the second system independently comprises an additive.
  • At least one additive is selected from the group consisting of a drug, a dye, a polymer comprised of a nucleic acid and a charged molecules for surface modification.
  • the method allows for the separate manipulation of the two surfactants prior to the final mixture to allow for the addition of additives in either the micelle (first system comprising water-soluble surfactant) or the cream (second system comprising water-insoluble surfactant), thus avoiding deleterious effects of heat, drug interaction and/or surfactant precipitation.
  • Additives can include drugs, dyes, and/or charged molecules for surface modification.
  • Charged molecules can be cationic, anionic, or zwitterionic: surfactants, lipids, sterols such as cholesterols e.g., negatively charged cholesterol sulfate (cholesterol-SCh) and positively charged 3 -[N-( dimethylaminoethane )carbamoyl ]- cholesterol (DC-cholesterol), zwitterionic surfactants such as CHAPS (3-[(3- Cholamidopropyl)dimethylammonio]-1 -propanesulfonate ).
  • surfactants e.g., negatively charged cholesterol sulfate (cholesterol-SCh) and positively charged 3 -[N-( dimethylaminoethane )carbamoyl ]- cholesterol (DC-cholesterol), zwitterionic surfactants such as CHAPS (3-[(3- Cholamidopropyl)dimethylam
  • addition of additives can change the surface charge of the microbubble. In certain embodiments, additives create MBs having positive charge.
  • the examples of additives that modify the surface of the MBs include CTAB, DSTAP, DMTAP, DDAB, TAP 18:0, GL67, and DOTOP.
  • the charged MB can be further loaded with oppositely charged molecules, such as but not limited to DNA, RNA, and/or Rose Bangel.
  • the charged MB is subjected to a wash step in which species of the opposite charge are attracted loaded on to the shell by ionic interaction.
  • the MBs with positive zeta potential can further be loaded with negatively charged molecules such as, for example, therapeutic agents including Rose Bengal and DNA.
  • MBs can be loaded with a charged species such as genetic material in cases such but not limited to DNA, RNA, siRNA, shRNA, and/or CRISPR.
  • the charged species is a sonosensitizer.
  • the charged species is a positively charged protein or peptide, for example (but not limited to) to allow for delivery across the high negative fixed charge density (FDC) of the anionic proteoglycans in various tissues (including but not limited to cartilage, meniscus and ligaments of the knee, ankle, shoulder and hip joints etc., intervertebral discs. (IVDs) in the spine, mucosal membrane of the gastrointestinal (GI) tract and the vitreous humor of the eye, and skin).
  • FDC negative fixed charge density
  • opsonization upon intravenous injection is minimized by a positive surface charge.
  • the charged species is effective against multi drug resistance.
  • the MBs are acoustically-sensitive.
  • the size of the separated microbubbles is less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 pm.
  • the invention provides a kit comprising surfactants and reagents for making the MBs as described elsewhere herein and instruction material for making the MBs..
  • Bubble Counting and Sizing' AccuSizer (A7000 Single Particle Optical Sizing, Entegris). Three samples taken from each vial and bubble size and diameter were averaged.
  • ZetaSizer Zetasizer Nano ZS, Malvern Instruments
  • Time Response Reflected signal from one dose (180 pl/L) over 10 min. Normalize to determine half-life
  • Microscope Imaging Fluorescent filter TRITC, images taken at an exposure of 39 ms. Photos were taken in brightfield before transferring to the fluorescent filter to confirm bubble presence.
  • the transducer was focused through an acoustic window of a custom-made sample vessel submerged in a deionized water bath (37 °C), with the contents continuously stirred during testing.
  • Cumulative dose response curves that is signal returned to the transducer as a function of microbubble dose, were constructed by pipetting increments of MBs into the sample chamber containing 50 mL of phosphate buffered saline at 37 °C while measuring the acoustic response.
  • Example 1 Synthesis of Surfactant-stabilized Microbubbles (SE61) Using the Cream Method
  • Span 60 mixture was added to the TPGS micelle solution, and the final mixture allowed to cool to room temperature.
  • CM Cream Method
  • TPGS and NaCl was added to 25 mL of 2x PBS to form micelles, and separately, Span 60 was added to 25 mL of DI water and autoclaved.
  • the Span 60 mixture was allowed to cool to room temperature while stirring. Once cooled to room temperature and a cream had formed (FIG 3), the TPGS solution was added to the Span 60 cream for the final mixture.
  • CM Cosmetical Testing'.
  • MM Micelle Method
  • OM original Method
  • CM produces SE61 MBs with the same size population, surface charge, and acoustical properties as the MM, while allowing both the TPGS and the SPAN components to cool to room temperature before mixing.
  • the CM also allows for additions to the Span post autoclaving.
  • Example 2 Modifying SE61 by Adding Cationic Species
  • SE61 MBs were modified by adding cationic species to either the water soluble or water insoluble portion of the cream method (CM). While maintaining an overall 85mM surfactant mixture and relative 4:1 ratio of SPAN 60 to TPGS, three molar ratios were tested: 5% Cationic Additive, 76% SPAN 60, 19% TPGS; 15% Cationic Additive, 68% SPAN 60, 17% TPGS; 25% Cationic Additive, 60% SPAN 60, 15% TPGS. Cationic additive and their respective addition methods are shown in the table depicted in FIG. 16.
  • Microbubble Testing analyses post sonication, pre-freeze drying, and post-freeze drying were performed.
  • the testing included testing bubble populations (using AccuSizer A7000, Entegris Inc.) zeta analysis (using Zetasizer Nano ZS, Malvern Inst.), and testing dose response(Freeze dried only), which included measuring reflected signal (dB) as function of increasing dose. Minimum 15 dB needed for in vivo contrast imaging.
  • size distributions show MBs created with all 5% cationic additives.
  • Increasing the concentration of DDAB and DOTOP reduces the number of MBs created, with 15% and 25% DOTAP not producing sufficient MBs for testing.
  • Example 3 Using 15% CTAB SE61 for loading negatively charged therapeutics
  • SE61 MBs were modified by adding CTAB to the water soluble portion of the cream method (CM) using a molar ratio of 15% CTAB, 68% SPAN 60, 17% TPGS.
  • Double stranded DNA used to model siRNA had a sequence of (5’- AATGAGCCCTTGCATCTAAGAA-3’) (SEQ ID No: 1).
  • the process of DNA loading onto MB after wash and collection included 1 part 250 mM dsDNA, 1 part 20% w/v glucose (cryoprotectant), and 2 parts collected microbubbles.
  • the MBs were washed with deionized water allowing dsDNA-loaded MB to separate due to gravity.
  • Example 4 Characterization of MBs with various amounts of DMTAP (TAP 14)
  • SE61 MBs were modified by adding DMTAP to the water insoluble soluble portion of the cream method (CM). While maintaining an overall 85mM surfactant mixture and relative 4: 1 ratio of SPAN 60 to TPGS, MBs with increasing amount of DMTAP (TAP 14) (0, 10, 15, 20, 25%) were prepared. MBs were loaded with double stranded DNA (dsDNA), wherein 320pL collected microbubbles were loaded with 160pL dSDNA (250 pM) in the presence of 160pL 20% glucose cryoprotectant.
  • dsDNA double stranded DNA
  • 320pL collected microbubbles were loaded with 160pL dSDNA (250 pM) in the presence of 160pL 20% glucose cryoprotectant.
  • MBs were analyzed and the results related to dose response curves, size, Zeta Potential, and DNA loading are shown in FIGS. 24-26. These results indicate that a ratio of 20% DMTAP is optimal for loading nucleic acid.
  • Embodiment 1 comprises a method for preparing microbubbles, wherein the method comprises: i. providing a first system comprising at least one water-soluble surfactant; ii. providing a second system comprising at least one water-insoluble surfactant, wherein providing the second system optionally comprises the step of autoclaving the second system and optionally cooling the resulting autoclaved system; and iii. contacting the first system with the second system at the temperature below the melting point of the second surfactant, so as to produce a mixture comprising microbubbles.
  • Embodiment 2 provides the method of embodiment 1, wherein the first system is aqueous.
  • Embodiment 3 provides the method of embodiments 1-2, wherein the first system comprises a micelle.
  • Embodiment 4 provides the method of embodiments 1-3, wherein the second system comprises a cream.
  • Embodiment 5 provides the method of embodiments 1-4, wherein the method further comprises purging the surfactant mixture of step (iii) with a purging gas.
  • Embodiment 6 provides the method of embodiments 1-5, wherein the purging gas is a perfluorocarbon (PFC).
  • Embodiment 7 provides the method of embodiments 1 -6, wherein the method further comprises washing the mixture comprising microbubbles with DI water and separating the microbubbles from the mixture by gravity filtration.
  • Embodiment 8 provides the method of embodiments 1-7, wherein the method further comprises freeze-drying and capping the separated microbubbles under vacuum.
  • Embodiment 9 provides the method of embodiments 1-8, the method further comprises filling the freeze-dried microbubbles with a filling gas.
  • Embodiment 10 provides the method of embodiments 1-9, wherein the filling gas is oxygen, octafluoropropane, and sulfur hexafluoride.
  • Embodiment 11 provides the method of embodiments 1-10, wherein the at least one water soluble surfactant is selected from the group consisting of tocopheryl polyethylene glycol succinate (TPGS), cetyltrimethylammonium Bromide (CTAB), and mixtures thereof.
  • TPGS tocopheryl polyethylene glycol succinate
  • CTAB cetyltrimethylammonium Bromide
  • Embodiment 12 provides the method of embodiments 1-11, wherein the at least one water-insoluble surfactant is selected from the group consisting of sorbitan monostearate, sorbitan fatty acid esters, sorbitan monopalmitate, (+) A tolopherol acid succinate (vitamin E) and mixtures thereof.
  • the at least one water-insoluble surfactant is selected from the group consisting of sorbitan monostearate, sorbitan fatty acid esters, sorbitan monopalmitate, (+) A tolopherol acid succinate (vitamin E) and mixtures thereof.
  • Embodiment 13 provides the method of embodiments 1-12, wherein the contacting is performed at room temperature.
  • Embodiment 14 provides the method of embodiments 1-13, wherein at least one of the first system and the second system independently comprises an additive.
  • Embodiment 15 provides the method of embodiments 1-14, wherein the at least one additive is selected from the group consisting of a drug, a dye, and a charged molecule for surface modification of the microbubbles.
  • Embodiment 16 provides the method of embodiments 1-15, wherein the charged molecule alters the charge (zeta potential) of the microbubbles.
  • Embodiment 17 provides the method of embodiments 1-16, wherein the microbubbles with altered zeta potential are further loaded with oppositely charged molecules.
  • Embodiment 18 provides the method of embodiments 1-17, wherein the microbubbles are acoustically-sensitive.
  • Embodiment 19 provides the method of embodiments 1-18, wherein the size of the separated microbubbles is below 10 pm in diameter.

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  • Colloid Chemistry (AREA)

Abstract

La présente invention concerne des procédés de préparation de microbulles acoustiquement sensibles. Le procédé comprend les étapes consistant à fournir un premier système comprenant au moins un tensioactif soluble dans l'eau ; fournir un second système comprenant au moins un tensioactif insoluble dans l'eau, la fourniture du second système comprenant éventuellement l'étape d'autoclavage du second système et éventuellement le refroidissement du système autoclavé obtenu ; et mettre en contact le premier système et le second système à la température inférieure au point de fusion du second tensioactif, de façon à produire un mélange comprenant des microbulles.
PCT/US2023/074198 2022-09-14 2023-09-14 Microbulles et leurs procédés de fabrication WO2024059715A2 (fr)

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AU2004308757B2 (en) * 2003-12-22 2010-06-17 Bracco Suisse S.A. Assembly of gas-filled microvesicle with active component for contrast imaging
WO2014103475A1 (fr) * 2012-12-27 2014-07-03 株式会社林原 Composition antivieillissement pour l'extérieur de la peau et son procédé de production
US11305013B2 (en) * 2014-08-26 2022-04-19 Drexel University Surfactant microbubbles and process for preparing and methods of using the same

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