WO2023130948A1 - Procédé de préparation d'un nano-agent de contraste ultrasonore ciblé biosynthétique, et son application - Google Patents

Procédé de préparation d'un nano-agent de contraste ultrasonore ciblé biosynthétique, et son application Download PDF

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WO2023130948A1
WO2023130948A1 PCT/CN2022/140058 CN2022140058W WO2023130948A1 WO 2023130948 A1 WO2023130948 A1 WO 2023130948A1 CN 2022140058 W CN2022140058 W CN 2022140058W WO 2023130948 A1 WO2023130948 A1 WO 2023130948A1
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nano
contrast agent
ultrasound contrast
biosynthetic
fluorescent probe
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PCT/CN2022/140058
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English (en)
Chinese (zh)
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严飞
郝永胜
李振洲
罗静娜
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中国科学院深圳先进技术研究院
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/08—Detecting organic movements or changes, e.g. tumours, cysts, swellings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
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    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
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    • B82—NANOTECHNOLOGY
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Definitions

  • the invention belongs to the technical field of ultrasonic molecular imaging, and in particular relates to a preparation method and application of a biosynthesis targeting nano-ultrasonic contrast agent.
  • Ultrasound contrast agent is a specific molecule that displays images at the tissue level, cell level, and subcellular level through imaging means, reflecting changes at the molecular level in the living state.
  • Ultrasound molecular imaging technology is a qualitative and Quantitative Research Techniques. Ultrasound molecular imaging has unique advantages, including non-invasive and radiation-free, good safety, strong tissue penetration, high spatial and temporal resolution, and real-time, dynamic, and repeated observation of target tissues.
  • Ultrasonic molecular imaging has achieved good imaging results in disease models such as thrombosis, atherosclerosis, and microvascular inflammation, and has extremely high application value in early diagnosis of diseases.
  • the accuracy rate of ultrasonic molecular imaging in clinical trials of breast cancer was as high as 77%, and the accuracy rate in clinical trials of ovarian cancer was as high as 93%.
  • Ultrasound molecular imaging has been demonstrated in a large number of preclinical studies and in a large number of disease models.
  • the introduction of ultrasound molecular imaging into tumor imaging plays an important role in the early detection stages of cancer.
  • the ultrasound contrast agent currently used clinically is a synthetic ultrasound contrast agent, which consists of lipid, polymer or protein shell-wrapped gas microbubbles, with a diameter of 1-8 ⁇ m and a relatively large volume.
  • the inability to penetrate the vessel wall to image non-vascular sites limits its use in ultrasonographic molecular imaging.
  • Nano-scale ultrasound contrast agents that have been studied more at this stage mainly include acoustic liposomes, fluorocarbon nano-droplets, and nanobubbles, among which acoustic liposomes are mainly prepared by freeze-drying liposomes, and the gas composition depends on Obtained by means of atmospheric pressure infiltration.
  • acoustic liposomes and nanobubbles have different internal gas contents and are easy to leak, which easily leads to poor ultrasound imaging performance; fluorocarbon nanodroplets have better ultrasound imaging performance, but it requires special external stimuli to improve The temperature of the nanodroplet.
  • the above-mentioned nano-scale ultrasound contrast agents are all derived from chemical synthesis, and they all have many problems such as uneven particle size, poor stability, large toxic and side effects, and unfriendly environment.
  • the purpose of the present invention is to provide a preparation method and application of biosynthetic targeted nano-ultrasound contrast agent.
  • the biosynthetic targeted nano-ultrasound contrast agent prepared by the preparation method can recognize the target specifically bound to the target on the cell surface, has the potential to penetrate blood vessels and enter tissue for imaging, and has excellent ultrasonic imaging performance.
  • the biosynthetic targeted nano-ultrasound contrast agent is prepared based on nano-biological airbags produced by microorganisms.
  • the size of the nano-biological airbags is 100-300 nm, and it is completely composed of protein.
  • the particle size of the nano-biological airbags is uniform and stable. Good sex.
  • the present invention provides a method for preparing a biosynthetic targeted nano-ultrasound contrast agent, the method for preparing a biosynthesized targeted nano-ultrasound contrast agent comprises the following steps:
  • the nanobiological airbag is produced by natural vesicle-containing microorganisms and/or genetically engineered vesicle-containing microorganisms.
  • the biosynthetic targeted nano-ultrasound contrast agent is prepared based on nano-biological airbags produced by microorganisms, and the nano-biological airbags are a kind of gas vesicles.
  • GV is an organelle composed of proteins.
  • the shape of GV is a cylindrical or spindle shape with two pointed ends and a round middle.
  • the width is between 45-250 nm, the length can reach 100-300 nm, and the thickness of the capsule wall is 2 nm.
  • the nanobiological airbag is applied to ultrasonic imaging, and the nanobiological airbag is coupled with a targeting substance, so that the nanobiological airbag can recognize the target specifically combined with the targeting substance on the cell surface.
  • the present invention also utilizes the small molecule fluorescent probe to mark the target, occupies the redundant amino groups on the target, and reduces the self-linkage of the target when the nano-biological airbag is coupled to the target.
  • the fluorescent probes include any one or a combination of at least two of coumarin-based probes, fluorescein-based probes, rhodamine-based probes, BODIPY-based probes or Cyanine-based probes.
  • the rhodamine-based probe includes AF488.
  • the preparation method of the nano-bio-airbag comprises: isolating the nano-bio-airbag from natural vesicle-containing microorganisms or isolating the nano-bio-airbag from genetically engineered vesicle-containing microorganisms.
  • the nano-biological airbags can be isolated from natural vesicle-containing microorganisms, and can also be isolated from genetically engineered vesicular microorganisms. Both nano-biological airbags are completely composed of proteins, and their particle diameters are uniform. In the range of 100-300 nm, such as 100 nm, 200 nm or 300 nm, etc., have good stability and have the potential to penetrate blood vessels and enter tissues for imaging.
  • the genetically engineered vesicle-containing microorganisms constructed by genetic engineering are convenient to cultivate, easy to obtain, and can obtain a large number of nanobiological air sacs.
  • the natural vesicle-containing microorganisms include any one or a combination of at least two of halophilic archaea, algae or Bacillus megaterium.
  • the genetically engineered vesicle-containing microorganism contains at least one copy of a plasmid carrying a bio-air sac gene cluster.
  • the chassis cells of the genetically engineered vesicle-containing microorganism include any one or a combination of at least two of Escherichia coli, Streptomyces, yeast, cyanobacteria or Pseudomonas putida.
  • said isolating nanobiological air sacs from natural vesicle-containing microorganisms includes:
  • the lysate is mixed with the natural vesicle-containing microorganism to lyse, and the lysed solution is centrifuged to obtain the nano-biological airbag.
  • the pH of the lysate is 7.3-7.7, such as 7.3, 7.4, 7.5, 7.6 or 7.7.
  • the lysate includes Tris-HCl, MgCl 2 and CaCl 2 .
  • the concentration of Tris-HCl in the lysate is 8-12 mM, such as 8 mM, 9 mM, 10 mM, 11 mM or 12 mM etc.
  • the concentration of MgCl 2 in the lysate is 1.5-2.5 mM, such as 1.5 mM, 1.8 mM, 2 mM, 2.2 mM, 2.4 mM or 2.5 mM.
  • the concentration of CaCl 2 in the lysate is 1.5-2.5 mM, such as 1.5 mM, 1.8 mM, 2 mM, 2.2 mM, 2.4 mM or 2.5 mM.
  • the volume ratio of the lysate mixed with the natural vesicle-containing microorganism is (0.8-3):1, for example, it can be 0.8:1, 1:1, 2:1 or 3:1, etc.
  • the centrifugal force of the centrifuge is 200-400 g, such as 200 g, 250 g g, 300 g, 350 g or 400 g, etc.
  • the centrifugation time is 3-5 h, such as 3 h, 3.5 h, 4 h, 4.5 h or 5 h, etc.
  • the buoyancy of the nanobiological airbags produced by natural bubble-containing microorganisms is greater than the buoyancy of the cell thallus
  • the particle diameter of the nanobiological airbags is 220 ⁇ 20 nm
  • the nanobiological airbags are easy to float on the liquid surface. After low-speed centrifugation at -400 g and 4°C, other cell thalli and organelles sink to the lower layer, and the nanobiological airbags float on the liquid surface, and the nanobiological airbags can be obtained by removing the lower layer of liquid.
  • isolating nanobiological air sacs from genetically engineered vesicle-containing microorganisms comprises:
  • the chassis cells include any one or a combination of at least two of Escherichia coli, Streptomyces, yeast, cyanobacteria or Pseudomonas putida.
  • the mixed volume ratio of the lysate and the induced genetically engineered vesicle-containing microorganism is (5-10):(7-8), for example, it can be 5:7, 6:7, 7:7, 8: 7, 9:7, 10:7, 5:8, 6:8, 7:8, 9:8 or 10:8, etc.
  • the final concentration of the lysozyme is 20-1000 ⁇ g/mL, for example, can be 20 ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL, 200 ⁇ g/mL, 500 ⁇ g/mL or 1000 ⁇ g/mL, etc.
  • the temperature for adding lysozyme to incubate is 25-37°C, for example, it can be 25°C, 27°C, 29°C, 30°C, 31°C, 33°C, 35°C or 37°C, etc.
  • the adding lysozyme The incubation time is 1-3 h, for example, 1 h, 1.5 h, 2 h, 2.5 h or 3 h, etc.
  • the final concentration of the DNase is 20-1000 ⁇ g/mL, such as 20 ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL, 200 ⁇ g/mL, 500 ⁇ g/mL or 1000 ⁇ g/mL wait.
  • the temperature for adding DNase and incubating is 25-37°C, such as 25°C, 27°C, 29°C, 30°C, 31°C, 33°C, 35°C or 37°C, etc.
  • the incubation time It is 1-12 h, for example, it can be 1 h, 2 h, 4 h, 6 h, 8 h, 10 h or 12 h, etc.
  • the centrifugal force of the centrifuge is 300-400 g, such as 300 g, 320 g g, 340 g, 360 g, 380 g or 400 g, etc.
  • the centrifugation time is longer than 1 h, for example, it can be 2 h, 2.5 h, 3 h or 3.5 h, etc.
  • the particle diameter of the nano-biological airbags separated from the genetically engineered vesicle-containing microorganisms is 110 ⁇ 20 nm.
  • the nanobio-air sacs separate.
  • the preparation method of the fluorescent probe modified target comprises:
  • the fluorescent probe is esterified with EDC and NHS, the esterified fluorescent probe, target and medium are mixed for reaction, and the fluorescent probe not combined with the target is removed to obtain the fluorescent probe-modified target.
  • the target itself has an amino group and a carboxyl group.
  • the present invention uses a small molecule fluorescent probe to occupy the amino position of the target. The dots avoid self-ligation, and the amount of target labeling can be calculated from the fluorescence of the fluorescent probe.
  • the target comprises IgG.
  • the targeting substance in the present invention can specifically bind to the target on the cell surface, and through the specific combination of the targeting substance and the target, the biosynthetic targeted nano-ultrasound contrast agent can reach the corresponding tissue and cell surface
  • the target-specific binding realizes targeted imaging.
  • the mass ratio of the esterified fluorescent probe, target and medium mixed (0.5-1):(1-2):(2000-4000), for example, can be 0.5:1:2000, 0.5: 2:2000, 0.5:1:4000, 0.5:2:4000, 1:1:2000, 1:2:2000 or 1:2:4000 etc.
  • the medium includes any one or a combination of at least two of deionized water, sodium borate solution, sodium bicarbonate solution or PBS buffer.
  • the concentration of Na 3 BO 3 in the sodium borate solution is 50-60 mM
  • the pH is 8.0-8.5
  • the concentration of Na 3 BO 3 can be 50 mM, 53 mM, 55 mM, 58 mM or 60 mM, for example. mM, etc.
  • the pH can be, for example, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5, etc.
  • the concentration of NaHCO in the sodium bicarbonate solution is 0.05-0.2 M
  • the pH is 8.3-9.0
  • the concentration of NaHCO can be, for example, 0.05 M, 0.08 M, 0.1 M, 0.13 M, 0.15 M, 0.18 M or 0.2 M, etc.
  • the pH can be, for example, 8.3, 8.5, 8.7, 8.9 or 9.0, etc.
  • the concentration of Na 3 PO 4 in the PBS buffer solution is 0.1-0.2 M
  • the concentration of NaCl is 0.15-0.25 M
  • the pH is 7.2-7.5.
  • the concentration of Na 3 PO 4 can be, for example, 0.1 M, 0.13 M, 0.15 M, 0.18 M or 0.2 M, etc.
  • the concentration of NaCl for example, can be 0.15 M, 0.18 M, 0.2 M, 0.23 M, or 0.25 M, etc.
  • the pH can be, for example, 7.2, 7.3, 7.4 or 7.5, etc.
  • the reaction time is 1-5 h, such as 1 h, 2 h, 3 h, 4 h or 5 h, etc.
  • the removal of the fluorescent probes not bound to the targeting substance is performed through a dialysis bag.
  • the molecular cut-off of the dialysis bag the molecular weight of the fluorescent probe ⁇ the molecular cut-off ⁇ the molecular weight of the target object.
  • the steps of linking the nano-biological airbag and the fluorescent probe-modified target include:
  • step (b) Mixing and reacting the nano-biological airbag solution with the reaction solution in step (a), and centrifuging to obtain the biosynthesis targeting nano-ultrasound contrast agent.
  • the final concentration of the fluorescent probe-modified targeting substance in the mixed solution is 0.05-3 mg/mL.
  • the final concentration of the protective agent in the mixed solution is 3-6 mM, such as 3 mM, 4 mM, 5 mM or 6 mM etc.
  • the protective agent includes NHS or thio-NHS.
  • the final concentration of EDC in the mixed solution is 2-3 mM, for example, can be 2 mM, 2.2 mM, 2.4 mM, 2.6 mM, 2.8 mM or 3 mM, etc.
  • the pH of the MES buffer is 5.5-6.5, for example, it may be 5.5, 5.7, 5.9, 6.0, 6.1, 6.3 or 6.5.
  • the protective agent includes NHS or thio-NHS.
  • the rotational speed of the stirring reaction is 100-400 rpm, such as 100 rpm, 200 rpm rpm, 300 rpm or 400 rpm, etc.
  • the stirring reaction time is 15-30 min, such as 15 min, 18 min, 20 min, 22 min, 25 min, 28 min or 30 min, etc.
  • the nano-bio-air vesicle solution is a buffer containing nano-bio-air vesicles.
  • the concentration of the nano-bio-airbag solution has an OD 500 of 10-120, such as 10, 20, 40, 60, 80, 100 or 120, etc.
  • the pH of the nano-bio-airbag solution is 7.5-8.5, for example, can be 7.5, 7.7, 7.9, 8.0, 8.1, 8.3 or 8.5, etc.
  • the buffer is an amino-free buffer.
  • the volume ratio of the nano-bio-airbag solution mixed with the reaction solution in step (a) is (1.5-4):(0.5-10), for example, it can be 1.5:0.5, 1.5: 2. 1.5:4, 1.5:6, 1.5:8, 1.5:10, 3:10, 3.5:10, 4:0.5, 4:1.5 or 4:3 etc.
  • the mixing reaction time is 2-3 h, for example, it can be 2 h, 2.5 h h or 3 h etc.
  • the centrifugal force of the centrifuge is 200-300 g, such as 200 g, 220 g, 240 g, 260 g, 280 g or 300 g, etc.
  • the centrifugation time is 2-4 h, such as 2 h, 2.5 h, 3 h, 3.5 h or 4 h.
  • the method for calculating the amount of target labeling of the biosynthesized targeted nano-ultrasound contrast agent includes:
  • Labeling amount of target substance (A bubble max ⁇ C target substance ) / (A target substance max ⁇ dilution factor ⁇ C bubble )
  • a bubble max crush the biosynthesized targeted nano-ultrasound contrast agent with an ultrasonic cleaning machine, and measure the absorption value at the absorption peak wavelength of the small molecule fluorescent probe;
  • C target object the molar concentration of the target object
  • a target object max Dilute the target object modified by the small molecule fluorescent probe, and measure the absorption value at the absorption peak wavelength of the small molecule fluorescent probe;
  • Bubble C molar concentration of biosynthesized targeted nano-ultrasound contrast agents.
  • the connected biosynthetic targeted nano-ultrasonic contrast agent is separated from the solution by low-speed centrifugation, and the centrifugal force is The centrifugation time is 2-4 h under the condition of 200-300 g.
  • the biological air bag connected with the target object is suspended in the upper layer of the sample tube, and the unconnected target object is in the solution.
  • Use a syringe to draw the clear solution at the bottom of the sample tube, and then Resuspend in PBS, centrifuge, and repeat this step 2-3 times to obtain biosynthetic targeted nano-ultrasound contrast agent.
  • the preparation method of the biosynthetic targeted nano-ultrasound contrast agent comprises the following steps:
  • the lysate with a pH of 7.3-7.7 and the natural vesicle-containing microorganism are mixed and lysed at a volume ratio of (0.8-3): 1, and the lysate includes 8-12 mM Tris-HCl, 1.5-2.5 mM MgCl 2 and 1.5-2.5 mM CaCl 2 , centrifuge the lysed solution for more than 3-5 h under the condition of a centrifugal force of 200-400 g to obtain the nano-biological airbag.
  • the volume ratio of the lysate to the induced genetically engineered vesicle-containing microorganisms is (5-10): (7-8) mix, add lysozyme, the final concentration of the lysozyme is 20-1000 ⁇ g/mL, incubate at 25-37 °C for 1-3 h, then add DNase, the The final concentration of DNase is 20-1000 ⁇ g/mL, incubate at 25-37°C for 1-12 h, the lysed solution was subjected to a centrifugal force of 300-400 g under the condition of centrifugation for more than 1 h to obtain the nanobiological airbag.
  • step (b) Mixing and reacting the nano-bio-airbag solution with the reaction solution in step (a) in a volume ratio of (1.5-4):(0.5-3) for 2-3 h, and the pH of the nano-bio-airbag solution is 7.5- 8.5.
  • a volume ratio of (1.5-4):(0.5-3) for 2-3 h the pH of the nano-bio-airbag solution is 7.5- 8.5.
  • an OD 500 of 10-120 centrifuge for 2-4 h under the condition of a centrifugal force of 200-300 g to obtain the biosynthetic targeted nano-ultrasound contrast agent.
  • the target marker amount of the biosynthetic targeted nano-ultrasound contrast agent can be calculated by the following formula:
  • Labeling amount of target substance (A bubble max ⁇ C target substance ) / (A target substance max ⁇ dilution factor ⁇ C bubble )
  • a bubble max crush the biosynthesized targeted nano-ultrasound contrast agent with an ultrasonic cleaning machine, and measure the absorption value at the absorption peak wavelength of the small molecule fluorescent probe;
  • C target object the molar concentration of the target object
  • a target object max Dilute the target object modified by the small molecule fluorescent probe, and measure the absorption value at the absorption peak wavelength of the small molecule fluorescent probe;
  • Bubble C molar concentration of biosynthesized targeted nano-ultrasound contrast agents.
  • the present invention provides an application of the preparation method of biosynthetic targeted nano-ultrasound contrast agent in the first aspect in the field of ultrasonic molecular imaging.
  • BODIPY probes boron fluoride dipyrrole probes.
  • Cyanine probes Cyanine probes.
  • EDC 1-ethyl-(3-dimethylaminopropyl)carbodiimide.
  • NHS N-Hydroxy succinimide, N-Hydroxy succinimide.
  • MES Morpholinoethanesulfonic acid, 2-Morpholinoethanesulfonic Acid.
  • DSPC Distearoylphosphatidylcholine.
  • PDI Polymer dispersion index, Polymer dispersion index.
  • the present invention has the following beneficial effects:
  • the present invention has the following beneficial effects:
  • the size of the biosynthetic targeted nano-ultrasound contrast agent of the present invention is 100-300 nm, and the size of the nano-bio-airbag is completely composed of protein.
  • the particle size of the nano-bio-airbag is uniform and stable.
  • Synthetic targeted nano-ultrasound contrast agents can recognize the target that specifically binds to the target on the cell surface, have the potential to penetrate blood vessels into tissue imaging, and have excellent ultrasonic imaging performance.
  • the biosynthetic targeted nano-ultrasound contrast agent prepared by the present invention has no significant difference in the stability test and imaging performance test between the unmodified contrast agent, and also has a targeting effect, and can specifically bind to target cells and target tissues , to achieve targeted imaging.
  • the fluorescent probe is used to occupy the amino site of the target to avoid the self-connection of the target, and the target can be calculated by the fluorescence of the fluorescent probe amount of markers.
  • Fig. 1 is a comparison chart of the fluorescence intensity of the biosynthesized targeted nano-ultrasound contrast agent and the biosynthesized non-targeted nano-ultrasound contrast agent in Test Example 1.
  • Fig. 2 is a comparison chart of in vitro ultrasound imaging of the biosynthesized targeted nano-ultrasound contrast agent and the biosynthesized non-targeted nano-ultrasound contrast agent in Test Example 2.
  • FIG. 3 is a statistical diagram of the signal intensity of the biosynthesized targeted nano-ultrasound contrast agent and the biosynthesized non-targeted nano-ultrasound contrast agent in Test Example 2.
  • Fig. 4 is a comparison chart of the adhesion between biosynthetic targeting nano-ultrasound contrast agent and biosynthetic non-targeting nano-ultrasound contrast agent and human breast cancer MCF-7 cell line with high expression of E-cadherin in Test Example 3.
  • Example 5 is a particle size distribution diagram of the biosynthesized targeted nano-ultrasound contrast agent in Test Example 4 and the phospholipid microbubble contrast agent obtained in Comparative Example 3.
  • FIG. 6 is a statistical graph of the PDI dispersion index of the biosynthesized targeted nano-ultrasound contrast agent in Test Example 5 and the phospholipid microbubble contrast agent obtained in Comparative Example 3.
  • FIG. 6 is a statistical graph of the PDI dispersion index of the biosynthesized targeted nano-ultrasound contrast agent in Test Example 5 and the phospholipid microbubble contrast agent obtained in Comparative Example 3.
  • This embodiment provides a method for preparing a biosynthesized targeted nano-ultrasound contrast agent, wherein the nano-biological airbags in the biosynthesized targeted nano-ultrasound contrast agent come from natural bubble-containing microorganisms.
  • the preparation method of the biosynthetic targeted nano-ultrasound contrast agent comprises the following steps:
  • NRC-1 Isolation of nanobiological air sacs from the halophilic archaea Halobacterium sp. NRC-1 (NRC-1 for short):
  • the lysate with a pH of 7.5 and the NRC-1 are mixed and lysed at a volume ratio of 2:1, and the lysed solution is centrifuged for 4 h under the condition of a centrifugal force of 300 g to obtain the nanobiological airbag; the lysed
  • the solution included 10 mM Tris-HCl, 2.5 mM MgCl 2 and 2 mM CaCl 2 .
  • AF488 was esterified with EDC and NHS to obtain AF488-NHS ester.
  • AF488-NHS ester, IgG and deionized water were mixed and reacted for 3 h at a mass ratio of 0.5:1:2000.
  • AF488 bound by the targeting antibody was obtained to obtain the targeting antibody modified by the fluorescent probe, and the targeting antibody modified by the fluorescent probe was named AF488-IgG-1.
  • step (b) Mix the nano-bio-airbag solution with the reaction solution in step (a) at a volume ratio of 1.5:0.5 and react for 2.5 h.
  • the pH of the nano-bio-airbag solution is 8.0, the OD 500 is 60, and the centrifugal force is 250 g centrifuged under the same conditions for 3 h to obtain the biosynthesized targeted nano-ultrasound contrast agent.
  • the target marker amount of the biosynthesized targeted nano-ultrasound contrast agent can be calculated by the following formula:
  • Labeling amount of target substance (A bubble max ⁇ C target substance ) / (A target substance max ⁇ dilution factor ⁇ C bubble )
  • a bubble max After crushing the biosynthesized targeted nano-ultrasound contrast agent with an ultrasonic cleaning machine, measure the absorption value of the 1 cm optical path at the absorption peak wavelength of the small molecule fluorescent probe;
  • C target object the molar concentration of the target object
  • a target object max the target object modified by the small molecule fluorescent probe measures the absorption value of the 1 cm optical path at the absorption peak wavelength of the small molecule fluorescent probe;
  • Bubble C molar concentration of biosynthesized targeted nano-ultrasound contrast agents.
  • the A bubble max was measured as 10000; the C target object was 1.3 ⁇ 10 -8 M, and the A target object max was 8000; the C bubble was 3.5 ⁇ 10 -13 M; The absorption value of the target substance modified by the small molecule fluorescent probe was measured, and the dilution factor was 1.
  • This embodiment provides a method for preparing a biosynthesis-targeted nano-ultrasound contrast agent, wherein the nano-biological airbags in the biosynthesis-targeted nano-ultrasound contrast agent come from genetically engineered microbes containing bubbles.
  • the preparation method of the biosynthetic targeted nano-ultrasound contrast agent comprises the following steps:
  • AF488 was esterified with EDC and NHS to obtain AF488-NHS ester.
  • AF488-NHS ester, IgG, and deionized water were mixed and reacted for 5 h at a mass ratio of 1:2:4000, and the non-conjugated protein was removed by a dialysis bag with a molecular cutoff of 10 kDa.
  • AF488 bound by the targeting antibody was obtained to obtain the targeting antibody modified by the fluorescent probe, and the targeting antibody modified by the fluorescent probe was named AF488-IgG-2.
  • step (b) Mix the nano-bio-airbag solution with the reaction solution in step (a) at a volume ratio of 4:3 and react for 3 h. centrifuged under the same conditions for 2 h to obtain the biosynthesized targeted nano-ultrasound contrast agent.
  • This comparative example provides a method for preparing a biosynthesized non-targeting nano-ultrasound contrast agent, wherein the nano-biological balloon in the biosynthesized nano-ultrasound contrast agent comes from the halophilic archaea NRC-1.
  • the preparation method of the biosynthetic targeted nano-ultrasound contrast agent comprises the following steps:
  • the lysis solution with a pH of 7.7 and the NRC-1 were mixed and lysed at a volume ratio of 3:1, and the lysis solution included 12 mM Tris-HCl, 2 mM MgCl 2 and 2.5 mM CaCl 2 , and the lysed solution was
  • the biosynthetic nano-ultrasonic contrast agent was obtained by centrifuging for 5 h under the condition of a centrifugal force of 200 g.
  • This comparative example provides a method for preparing a biosynthesized non-targeted nano-ultrasound contrast agent, wherein the nano-biological airbags in the biosynthesized targeted nano-ultrasound contrast agent come from genetically engineered vesicle-containing microorganisms.
  • the preparation method of the biosynthetic nano-ultrasound contrast agent comprises the following steps:
  • This comparative example provides a method for preparing an ultrasound contrast agent, the ultrasound contrast agent is a phospholipid microbubble contrast agent, and the preparation method of the phospholipid microbubble contrast agent is as follows:
  • the buffer solution includes 10 mM Tris-HCl, glycerol, 1,2-propanediol, the volume ratio of Tris-HCl, glycerol and 1,2-propanediol It is 8:1:1.
  • test tube Place the test tube in a 65°C water-bath ultrasonic cleaner, and sonicate for 10 min until a transparent phospholipid solution is obtained.
  • the test method is as follows:
  • the comparison results of the fluorescence intensity of the ultrasound contrast agents in Example 1 and Comparative Example 1 are shown in Figure 1. It can be seen from Figure 1 that after the biosynthesis targeting nano-ultrasound contrast agent is connected with a fluorescent small molecule, it has a very strong Fluorescent signal, indicating that the targeting antibody is successfully linked to the nanobiological airbag.
  • the comparison results of the fluorescence intensities of the ultrasound contrast agents in Example 2 and Comparative Example 2 are similar to the comparison results of the fluorescence intensities of the ultrasound contrast agents in Example 1 and Comparative Example 1.
  • Figure 2 is a comparison of in vitro ultrasound imaging between biosynthesized targeted nano-ultrasound contrast agents and biosynthesized non-targeted nano-ultrasound contrast agents
  • Figure 3 is a comparison of biosynthesized targeted nano-ultrasound contrast agents
  • the signal intensity statistical diagram of the biosynthetic non-targeted nano-ultrasound contrast agent as can be seen from Figure 2 and Figure 3, the biosynthesized targeted nano-ultrasound contrast agent and the biosynthesized non-targeted nano-ultrasound contrast agent ultrasound contrast agent Consistent imaging performance at the same concentration.
  • the biosynthetic targeted nano-ultrasound contrast agent obtained in Example 2 and the biosynthesized non-targeted nano-ultrasound contrast agent obtained in Comparative Example 2 also have similar imaging properties.
  • the biosynthetic targeted nano-ultrasound contrast agent obtained in Example 1 and the biosynthetic non-targeted nano-ultrasound contrast agent obtained in Comparative Example 1 were incubated with the cultured MCF-7 cells for 15 min, and the cells were washed with PBS, and repeated 3 times. 5 min each time, placed under a confocal microscope (A1+ laser confocal system) for observation.
  • the biosynthetic targeted nano-ultrasound contrast agent has a large amount of adhesion on the surface of MCF-7 cells, while the biosynthetic non-targeted nano-ultrasound contrast agent is not seen on the surface of MCF-7 cells
  • the obvious adhesion phenomenon shows that the biosynthetic targeted nano-ultrasound contrast agent obtained in Example 1 has good targeting.
  • the biosynthetic targeted nano-ultrasound contrast agent obtained in Example 2 also has similar targeting properties and can also adhere to the surface of MCF-7 cells.
  • Example 1 The particle diameters of the biosynthetic targeted nano-ultrasound contrast agent obtained in Example 1 and the phospholipid microbubble contrast agent obtained in Comparative Example 3 were measured respectively.
  • the measurement method is as follows:
  • the prepared biosynthetic targeting nano-ultrasound contrast agent and phospholipid microbubble contrast agent were diluted according to a certain ratio, and the particle size distribution of the two contrast agents was measured with a Malvern particle size analyzer (Zetasizer Nano).
  • the average particle size of the biosynthetic targeted nano-ultrasound contrast agent in Example 1 is 200 nm
  • the average particle diameter of the phospholipid microbubble contrast agent described in Comparative Example 3 is 200 nm.
  • the particle size is 1 ⁇ m.
  • the biosynthetic targeted nano-ultrasound contrast agent provided in Example 1 has a smaller particle size and has the potential to penetrate blood vessels and enter tissues for imaging.
  • the PDI dispersion index of the biosynthetic targeted nano-ultrasound contrast agent obtained in Example 1 and the phospholipid microbubble contrast agent obtained in Comparative Example 3 were respectively measured.
  • the measurement method is as follows:
  • the prepared biosynthetic targeting nano-ultrasound contrast agent and phospholipid microbubble contrast agent were diluted according to a certain ratio, and the PDI dispersion index of the two contrast agents was measured with a Malvern particle size analyzer (Zetasizer Nano).
  • the biosynthetic targeted nano-ultrasound contrast agent prepared by the preparation method of the biosynthetic targeted nano-ultrasound contrast agent of the present invention can recognize the target that the cell surface specifically binds to the target substance, has good stability, It has the potential of penetrating blood vessels and entering tissues for imaging.
  • the biosynthetic targeted nano-ultrasound contrast agent in the present invention has excellent ultrasonic imaging performance and has important application prospects in the field of ultrasonic molecular imaging technology.

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Abstract

L'invention concerne un procédé de préparation d'un nano-agent de contraste ultrasonore ciblé biosynthétique, et son application. Le procédé de préparation comprend les étapes suivantes consistant à : préparer un nano-ballonnet biologique et un objet cible modifié par sonde fluorescente, et lier le nano-ballonnet biologique à l'objet cible modifié par sonde fluorescente pour obtenir le nano-agent de contraste ultrasonore ciblé biosynthétique. Le nano-agent de contraste ultrasonore ciblé biosynthétique est préparé sur la base d'un nano-ballonnet biologique généré par un micro-organisme ; le nano-ballonnet biologique présente une taille de 100 à 300 nm, est intégralement composé d'une protéine, et présente une taille de particule uniforme et une bonne stabilité. Le nano-agent de contraste ultrasonore ciblé biosynthétique obtenu par le procédé de préparation permet d'identifier un point cible se liant spécifiquement à un objet cible sur la surface d'une cellule, présente le potentiel de pénétrer dans un vaisseau sanguin pour entrer dans un tissu à des fins d'imagerie, et est excellent en termes de performances d'imagerie ultrasonore.
PCT/CN2022/140058 2022-01-04 2022-12-19 Procédé de préparation d'un nano-agent de contraste ultrasonore ciblé biosynthétique, et son application WO2023130948A1 (fr)

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Citations (3)

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US20140288421A1 (en) * 2013-03-12 2014-09-25 The Regents Of The University Of California Gas vesicle ultrasound contrast agents and methods of using the same
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US20200306564A1 (en) * 2019-03-28 2020-10-01 California Institute Of Technology Compositions, methods and systems for gas vesicle based cavitation
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