WO2020239058A1 - 表面修饰气囊及其制备和应用 - Google Patents

表面修饰气囊及其制备和应用 Download PDF

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WO2020239058A1
WO2020239058A1 PCT/CN2020/093171 CN2020093171W WO2020239058A1 WO 2020239058 A1 WO2020239058 A1 WO 2020239058A1 CN 2020093171 W CN2020093171 W CN 2020093171W WO 2020239058 A1 WO2020239058 A1 WO 2020239058A1
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lipid
targeted
gas
filled
cancer
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French (fr)
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孙雷
宋林
王国浩
侯璇迪
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Hong Kong Polytechnic University HKPU
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Hong Kong Polytechnic University HKPU
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Priority to CN202410224293.5A priority Critical patent/CN118105491A/zh
Priority to CN202080009821.5A priority patent/CN113301904B/zh
Priority to US17/595,855 priority patent/US20220257503A1/en
Publication of WO2020239058A1 publication Critical patent/WO2020239058A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/08—Clinical applications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A61K41/0033—Sonodynamic cancer therapy with sonochemically active agents or sonosensitizers, having their cytotoxic effects enhanced through application of ultrasounds
    • 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
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • 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; Ultrasonic imaging preparations
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present disclosure relates to surface-modified targeted GVs (such as PH-GV), lipid GVs, lipid targeted GVs, preparation methods thereof, and applications in tumor diagnosis, imaging, and treatment.
  • surface-modified targeted GVs such as PH-GV
  • lipid GVs such as lipid GVs
  • lipid targeted GVs preparation methods thereof, and applications in tumor diagnosis, imaging, and treatment.
  • Contrast-enhanced ultrasound (CEUS) with gas-filled microbubbles as a contrast agent plays an important role in the diagnosis and management of many diseases.
  • a molecular targeted contrast agent that is surface-modified by attaching a binding ligand to the microbubble shell helps to visualize over-expressed biomarkers at the molecular level and significantly improves imaging sensitivity and specificity. Since the microbubbles are a few microns in diameter, they only remain in the vascular cavity. This characteristic makes it particularly suitable for visualizing molecular markers expressed on tumor neovascular systems.
  • microbubbles in imaging and therapy have shown encouraging results, their potential applications in biomedicine are limited by their larger hydrodynamic size (1-8 ⁇ m) because of the larger fluid size.
  • the kinetic size hinders its penetration into surrounding non-vascular tissues after intravenous injection.
  • the gas content of these microbubbles will quickly diffuse into the surrounding medium, resulting in a very short half-life ( ⁇ 20 minutes).
  • nano-scale ultrasound contrast agents with a particle size of less than 1000 nm exhibit the following advantages: small molecular weight, strong penetration and high stability.
  • This type of contrast agent includes fluorocarbon emulsion nanodrops, nanobubbles, and nanoparticles. Due to their nano-scale size, they can overcome the above-mentioned limitations, being able to penetrate the leaky and defective vascular system of the tumor, and at the same time, they can also stay due to the enhanced permeability and retention (Enhanced Permeability and Retention, EPR) effects. In the vascular system of healthy tissues.
  • nano-scale ultrasound contrast agents As a supplement to diagnostic imaging, these nano-scale ultrasound contrast agents have the potential to serve as active drug carriers for local drug delivery, and due to the instantaneous increase in permeability to the vascular system and cell membranes, nano-scale ultrasound contrast agents can also be used in space and time. Actively trigger drug release in a specific way. Although nano-scale ultrasound contrast agents have attractive benefits, their clinical transformation for diagnostic purposes has not achieved great success. The low gas density and low contrast-to-noise ratio of nano-scale ultrasound contrast agents do not allow high-contrast imaging at diagnostic frequencies under ultrasound.
  • GV gas vesicle isolated from planktonic photosynthetic microorganisms
  • the newly reported gas vesicle (GV) isolated from planktonic photosynthetic microorganisms has great potential as a new type of ultrasound imaging nano-level contrast agent.
  • GV produces strong ultrasound contrast in the picomolar frequency range and exhibits harmonic scattering, so it can enhance the detection in vivo relative to the background.
  • the nonlinear oscillation of the GV generates harmonics of incident ultrasound, which can be specifically detected to further enhance the contrast-to-noise ratio, thereby improving sensitivity and specificity.
  • GV has a width of 45-250nm and a length of 200-600nm, so it can enter the interstitial space through the EPR effect.
  • GV 2nm-thick protein shell repels water, but allows gas to diffuse in and out of the surrounding medium freely, making it difficult to It is physically stable and not affected by its nanometer size.
  • genetic engineering of GV in the existing technology can design GV-based contrast agents with new mechanical, acoustic, surface and functional properties, so as to achieve harmonic, multiplexed and multimodal ultrasound. Imaging and cell-specific molecular targeting. Due to the above advantages, GV is a very promising nanoparticle for ultrasound (US) molecular imaging and further cancer treatment.
  • US ultrasound
  • RES reticuloendothelial system
  • tumor hypoxia has been regarded as a key problem in oncology.
  • Pathological hypoxia can cause changes in the proteome and genome in tumor cells and changes in the tumor microenvironment. These changes may alter the local metabolism of tumor cells and activate adaptive cellular responses that contribute to tumor progression.
  • hypoxia makes solid tumors more resistant to radiotherapy and chemotherapy, resulting in lower patient survival rates.
  • hypoxia In view of the role of hypoxia in tumor progression and resistance to cancer treatment, people began to consider whether oxygen supply to tumor tissue can prevent cancer growth and development. It has been proven that tumor oxygenation can significantly improve chemotherapy and radiotherapy by increasing cell sensitivity and help overcome resistance to chemotherapy and radiotherapy.
  • tumor oxygenation is related to reduced cell proliferation, angiogenesis, and metastasis, and oxygenation may have a tumor suppressive effect in certain tumor subtypes.
  • Changes in the hypoxic tumor microenvironment can reduce the expression of HIF-1 ⁇ , thereby weakening the pathway driven by hypoxia.
  • oxygenation in tumors plays a role in epigenetic programming. After oxygenation, several hypoxia-related genes and tumor suppressor genes can be reprogrammed. This means that oxygenation can not only be used as an adjuvant for cancer treatment, but also has the potential to reverse the proteome and genomic changes in tumor sites caused by hypoxia.
  • Hyperbaric oxygen (HBO) therapy is a recognized method for the treatment of hypoxia-related diseases.
  • HBO therapy can effectively increase the oxygen content of the tumor and improve the results of chemotherapy and radiotherapy, but it still faces limitations such as high cost and poor patient experience.
  • oxygen cannot be transported deep in hypoxic tumors, which can impair clinical results in some cases. Due to the limitations of HBO therapy, scientists have tried to develop oxygen carriers that can transport large amounts of oxygen to tumors.
  • RBC Artificial red blood cells
  • PFC perfluorocarbon
  • HBOC cell-free hemoglobin-based oxygen carrier
  • lipid-based microbubbles are preferred for medical applications due to their good biocompatibility and biodegradability.
  • Microbubbles can carry large amounts of oxygen and can be used in combination with ultrasound for site-controlled oxygen release.
  • Oxygenated microbubbles have been shown to change the hypoxic microenvironment in the body and enhance the results of chemotherapy and radiotherapy.
  • the further development of oxygenated microbubbles still faces some challenges and limitations.
  • oxygenated microbubbles may encounter stability problems (such as dissolution and coalescence) when entering the circulatory system, and this is related to greater product loss, which may lead to reactive oxygen species (ROS) The overproduction.
  • ROS reactive oxygen species
  • microbubbles cannot pass through the vascular system due to their size limitations, and if combined with drug delivery, their effectiveness may be impaired.
  • Photodynamic therapy has become a successful clinical treatment method for cancer treatment, and it has been approved by the U.S. Food and Drug Administration (FDA).
  • PDT relies on a synergistic interaction between a photosensitizer and corresponding light. These two elements are respectively non-toxic, but when they work together, they can trigger a chemical reaction and achieve tumor cell apoptosis.
  • the photosensitizer can selectively enter the tumor site through the blood circulation, and when excited by light of the appropriate activation wavelength, it will generate a large amount of ROS.
  • ROS especially singlet oxygen free radicals
  • PDT In addition to producing ROS to directly kill cancer cells, PDT can also cause an immune response against cancer cells after treatment, and cut off nutrient supply through cancer-related vascular system damage to inhibit tumor growth. Due to its recognized advantages of safety, selectivity and reproducibility, PDT is not only used in the field of dermatology, but also as an adjuvant therapy for the treatment of lung, respiratory and urinary tract tumors. During PDT treatment, the availability of oxygen may be the limiting factor for efficacy, because the production of ROS depends on the concentration of oxygen. However, cancer tissues often exhibit hypoxia, which usually limits the effectiveness of PDT. Therefore, increasing the oxygen concentration at the tumor site is a good strategy to enhance the efficacy of PDT.
  • Oxygenated microbubbles have been used to enhance PDT, and its efficacy has been recognized in vitro and in vivo. For example, existing studies have proved that the presence of microbubbles during PDT can cause more singlet oxygen to be produced and enhance cell apoptosis. This means that oxygenated microbubbles together with photosensitizers can cause enhanced cytotoxicity. However, due to stability limitations and relatively large size, the application of oxygenated microbubbles is limited.
  • SDT sonodynamic therapy
  • PDT photodynamic therapy
  • the present invention provides surface-modified novel targeted GVs (such as PH-GV), lipid GVs, lipid targeted GVs, preparation methods and applications thereof.
  • the first aspect of the present invention provides a targeted GV that can be specifically targeted to tumor sites by surface modification.
  • the targeted GV of the present invention is surface-covered with biocompatible materials (such as polyethylene glycol (PEG), chitosan, polyurethane, polylactic acid, polyolefin, polysulfone, polycarbonate, polyacrylonitrile, etc.) or Any combination thereof) and/or GV modified by biological targeting materials (such as hyaluronic acid (HA), RGD peptide, folic acid, galactose, glucose, etc. or any combination thereof).
  • the molecular weight of the PEG used in the present invention is about 5000 Da.
  • the targeted GV is a GV whose surface is modified by PEG and/or HA, preferably a GV whose surface is modified by PEG and HA (PH-GV).
  • the targeted GV of the present invention such as PH-GV, has a cylindrical shape with a particle size of about 400 nm.
  • the particle size of the targeted GV is about 375-425 nm, and the zeta potential is about -23 to -31 mV.
  • the second aspect of the present invention provides a contrast agent or diagnostic agent, which contains the targeted GV such as PH-GV according to the first aspect of the present invention.
  • the concentration of the targeted GV in the contrast agent is about 250 pM-1 nM.
  • the third aspect of the present invention provides a diagnostic kit, which contains the contrast agent or diagnostic agent according to the second aspect of the present invention, and optionally instructions for use.
  • the targeted GV described in the first aspect of the present invention, the contrast agent or diagnostic agent described in the second aspect, and the kit described in the third aspect of the present invention can be used to diagnose diseases such as cancer.
  • the targeted GV or contrast agent can be used to image tumors to diagnose the presence or absence of cancer. Therefore, the present invention also provides the use of the GV-targeting agent described in the first aspect or the contrast agent or diagnostic agent described in the second aspect in preparing a diagnostic agent or diagnostic kit for diagnosing diseases such as cancer.
  • the present invention also provides the use of the targeted GV or contrast agent in the preparation of diagnostic reagents or kits for imaging tumors.
  • the fourth aspect of the present invention provides a diagnostic method, which comprises using the targeted GV according to the first aspect of the present invention, the contrast agent or diagnostic agent according to the second aspect, or the kit according to the third aspect To diagnose the presence or absence of cancer in the subject.
  • the diagnosis includes administering the targeted GV, contrast agent, or diagnostic agent to the subject or applying the kit to the subject.
  • the diagnostic method also includes ultrasound imaging of the subject to determine the presence or absence of cancer.
  • the targeted GV of the present invention can also be used as a drug carrier. Therefore, the fifth aspect of the present invention provides a drug carrier, which contains or consists of the targeted GV such as PH-GV according to the first aspect of the present invention.
  • the pharmaceutical carrier of the present invention further contains a drug for treating the disease to be treated, such as cancer.
  • the drug can be connected to the drug carrier in various ways, so that the drug can be delivered to a target site in the body by administering the drug carrier.
  • the method of linking the drug carrier and the drug together and the drugs used to treat specific diseases such as cancer are well known to those skilled in the art. Therefore, the pharmaceutical carrier of the present invention can also be used to treat diseases such as cancer.
  • the sixth aspect of the present invention provides a method of using the drug carrier of the fifth aspect to treat a disease such as cancer in a subject, including administering the drug carrier to the subject.
  • the method further includes the step of linking the drug carrier with a drug used to treat the disease, such as cancer, before administering the drug carrier to the subject.
  • the tumor or cancer is, for example, a CD44 high-expressing tumor, including but not limited to bladder cancer, lung cancer, kidney cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, melanoma and the like.
  • the seventh aspect of the present invention provides a method for preparing a targeted GV, which includes the steps: (a) linking the biological targeting material with the GV, for example, fixing the biological targeting material such as HA to the GV by covalent conjugation Protein shell.
  • the method further includes step (b) linking the biocompatible material with the product of step (a), for example chemically conjugating the biocompatible material, such as PEG, to step ( a) product.
  • a biological targeting material such as HA
  • a phosphate such as sodium phosphate
  • a3 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS)
  • EDC 1-(3-dimethylaminopropyl)-3-eth
  • the step (b) includes: (b1) dissolving the targeting material-GV conjugate (for example, the H-GV conjugate from step (a) above) in phosphate buffered saline (PBS ) And mix it with EDC in PBS and NHS in methanol; and (b2) adding biocompatible materials such as monomethoxy PEG-amine to the mixture in step (b1) to obtain PH-GV .
  • PBS phosphate buffered saline
  • biocompatible materials such as monomethoxy PEG-amine
  • the eighth aspect of the present invention provides a lipid GV whose surface is modified by lipid molecules.
  • the lipid molecule is distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dioleoylphosphatidylcholine (DOPC), PEG, DSPE, polyvinyl alcohol (PVA ), glycerol, glyceride, fatty acid, phospholipid or any combination thereof.
  • the lipid GV is a lipid GV whose surface is modified by DSPE-PEG and DOPC.
  • the lipid GV of the present invention is cylindrical, and its particle size can be, for example, about 290-330 nm, for example, about 300-330 nm, or about 310-330 nm.
  • the zeta potential of the lipid GV is about -21.3 to -19.3 mV.
  • the lipid GV of the present invention itself encapsulates certain gases such as air and oxygen in the lipid molecular membrane, but it can be designed to encapsulate/load/carry specific target gases, such as therapeutic target gases, such as oxygen, oxygen Nitrogen oxide (NO), hydrogen, etc. Therefore, the lipid GV of the present invention can be used as a carrier for delivering therapeutic gas into a subject. In this case, the target gas can be encapsulated in the protein shell of the GV by lipid molecules.
  • the ninth aspect of the present invention provides a gas-filled lipid GV, which includes the lipid GV and gas described in the eighth aspect of the present invention.
  • the gas may be any gas type, such as a therapeutic gas.
  • the gas is filled in the protein shell of GV and encapsulated by the lipid molecules.
  • the therapeutic gas described in the present invention includes any gas known in the art to help treat, alleviate, alleviate disease or disease symptoms or inhibit its progression, including but not limited to: oxygen, NO, hydrogen, etc., preferably oxygen.
  • the gas-filled lipid GV of the present invention includes but is not limited to oxygenated lipid GV, NO lipid GV, hydrogenated lipid GV, etc., preferably oxygenated lipid GV.
  • the tenth aspect of the present invention provides a therapeutic agent, which comprises or consists of the lipid GV of the eighth aspect of the present invention or the gas-filled lipid GV of the ninth aspect.
  • the eleventh aspect of the present invention provides a contrast agent or diagnostic agent, which comprises or consists of the lipid GV of the eighth aspect of the present invention or the gas-filled lipid GV of the ninth aspect.
  • the twelfth aspect of the present invention provides a kit comprising the lipid GV of the eighth aspect of the present invention, the gas-filled lipid GV of the ninth aspect, the therapeutic agent of the tenth aspect, or the tenth aspect of the present invention.
  • the lipid GV, gas-filled lipid GV, contrast agent or diagnostic agent, therapeutic agent or kit of the present invention can be used to diagnose or treat diseases, such as hypoxic diseases such as cancer. Therefore, the present invention also provides the lipid GV of the eighth aspect or the gas-filled lipid GV of the ninth aspect (such as oxygenated lipid GV) or the therapeutic agent of the tenth aspect in preparation for treating a subject The use of drugs for diseases such as cancer. The present invention also provides the lipid GV of the eighth aspect or the gas-filled lipid GV of the ninth aspect (such as oxygenated lipid GV) or the contrast agent or diagnostic agent of the eleventh aspect in preparation for use in the diagnosis of patients Use in diagnostic reagents for subjects’ diseases such as cancer.
  • diseases such as hypoxic diseases such as cancer. Therefore, the present invention also provides the lipid GV of the eighth aspect or the gas-filled lipid GV of the ninth aspect (such as oxygenated lipid GV) or the therapeutic agent of the tenth aspect in preparation for treating a
  • the present invention also provides the lipid GV of the eighth aspect or the gas-filled lipid GV of the ninth aspect (e.g., oxygenated lipid GV) or the contrast agent of the eleventh aspect in preparation for making a subject Use in diagnostic reagents for tumor imaging.
  • the lipid GV, gas-filled lipid GV or contrast agent is used for diagnosing diseases or imaging tumors, it also includes the application of contrast ultrasound to the subject to determine the presence or absence of cancer.
  • the drug may be combined with a second therapeutic agent, such as other cancer treatment drugs or cancer treatment methods, to treat cancer.
  • the cancer treatment method is, for example, chemotherapy, radiation therapy, preferably PDT or SDT.
  • the other cancer treatment drugs may be drugs well-known to those skilled in the art for the treatment of various cancers.
  • the thirteenth aspect of the present invention provides a method for treating a disease such as cancer in a subject, comprising administering the lipid GV of the eighth aspect or the gas-filled lipid GV of the ninth aspect to the subject Oxylipid GV) or the therapeutic agent of the tenth aspect or the kit according to the twelfth aspect is applied to the subject.
  • the method further comprises, after administering the lipid GV or gas-filled lipid GV or therapeutic agent to the subject (e.g., 1 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h after administration , 3h, 4h, 5h, 6h, or even 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, and any time point between the above values), the subject is administered the first Two therapeutic agents or treatments, such as other cancer treatment drugs or cancer treatment methods (such as chemotherapy, radiation therapy, preferably PDT or SDT) steps.
  • the other cancer treatment drugs may be drugs well-known to those skilled in the art for the treatment of various cancers.
  • the method further includes, after administering the lipid GV or gas-filled lipid GV or therapeutic agent to the subject and before applying other cancer treatment drugs or cancer treatment methods, to the recipient
  • the present invention also includes a carrier which comprises or consists of the above-mentioned lipid GV or gas-filled lipid GV (for example, oxygenated lipid GV).
  • the carrier may also include a drug for treating diseases such as cancer (e.g., drugs for treating cancer known in the art) or a gas such as any therapeutic gas (e.g., a therapeutic gas as defined above).
  • cancer e.g., drugs for treating cancer known in the art
  • a gas such as any therapeutic gas (e.g., a therapeutic gas as defined above).
  • the cancer described in the above aspect of the present invention can be any tumor that can cause hypoxia, such as breast cancer, bladder cancer, lung cancer, kidney cancer, gastric cancer, colorectal cancer, liver cancer, melanoma, and the like.
  • the GV solution is a PBS solution of GV.
  • the lipid molecule in step (a) is a mixture of DSPE-PEG and DOPC.
  • the step (b) further includes sonicating the formed solution.
  • the step (c) further includes adding HEPES buffer to the solution and incubating overnight.
  • the fifteenth aspect of the present invention provides a method for preparing a gas-filled lipid GV, which is similar to the method of the fourteenth aspect of the present invention, except that step (c) includes: adding the product of step (b) Before adding to the GV solution, aerate the GV solution (for example, oxygen) until saturation.
  • step (c) includes: adding the product of step (b) Before adding to the GV solution, aerate the GV solution (for example, oxygen) until saturation.
  • the sixteenth aspect of the present invention provides a lipid-targeted GV whose surface is modified by lipid molecules and biological targeting materials (and optionally, biocompatible materials).
  • the lipid molecule is selected from one or more of the following: DSPE-PEG, DOPC, PEG, DSPE, PVA, glycerol, glyceride, fatty acid, phospholipid;
  • the biological targeting material is selected from the following One or more: HA, RGD peptide, folic acid, galactose, glucose; and, optionally, the biocompatible material is selected from one or more of the following: PEG, chitosan, polyamino Ester, polylactic acid, polyolefin, polysulfone, polycarbonate, polyacrylonitrile.
  • the surface of the lipid-targeted GV is modified by DSPE-PEG, DOPC and HA, and more preferably, the surface of the lipid-targeted GV is modified by DSPE-PEG, DOPC, HA and PEG.
  • the present invention also provides a gas-filled lipid-targeted GV, which includes the lipid-targeted GV of the present invention and a gas.
  • the gas can be any gas type, such as a therapeutic gas. The gas is filled in the protein shell of GV and encapsulated by the lipid molecules.
  • the therapeutic gas described in the present invention includes any gas known in the art to help treat, alleviate, alleviate disease or disease symptoms or inhibit its progression, including but not limited to: oxygen, NO, hydrogen, etc., preferably oxygen.
  • the lipid-targeted GV or gas-filled (for example, oxygenated) lipid-targeted GV of the present invention can be used as cancer therapeutic agents, diagnostic agents, contrast agents and drug carriers. Therefore, the present invention also provides the use of the lipid-targeted GV or the gas-filled lipid-targeted GV of the present invention in the preparation of a medicine for treating cancer, or the use of a diagnostic reagent for diagnosing cancer.
  • the lipid-targeted GV or gas-filled lipid-targeted GV of the present invention can also be used in combination with other cancer treatment methods or cancer treatment drugs defined above in the present invention.
  • the cancer may be a cancer as defined herein above.
  • step (c) includes: in step (b) Before the product of) is added to the GV solution, the biological targeting material such as HA and GV are connected together.
  • the eighteenth aspect of the present invention provides a method for preparing gas-filled lipid-targeted GV, which is similar to the method described in the fifteenth aspect of the present invention, except that step (c) includes: Before aeration (for example, oxygen) in the GV solution, a biological targeting material such as HA and GV are connected together.
  • aeration for example, oxygen
  • a biological targeting material such as HA and GV are connected together.
  • the nineteenth aspect of the present invention provides a pharmaceutical composition comprising the targeted GV, lipid GV, gas-filled GV, targeted lipid GV or gas-filled targeted lipid GV of any aspect of the present invention.
  • the present invention also provides a method for treating cancer in a subject using the targeted GV, lipid GV, gas-filled GV, targeted lipid GV, gas-filled targeted lipid GV or pharmaceutical composition of any aspect of the present invention.
  • the present invention also provides the targeted GV, lipid GV, gas-filled GV, targeted lipid GV, gas-filled targeted lipid GV or pharmaceutical composition of any aspect of the present invention in the preparation of a medicament for the treatment of cancer in a subject Or its use with a second therapeutic agent or therapy in the preparation of a drug combination for treating cancer in a subject.
  • the targeted GV, lipid GV, gas-filled GV, targeted lipid GV or gas-filled targeted lipid GV of the present invention can also be used as a diagnostic agent, a contrast agent, a therapeutic agent or a drug carrier.
  • the method includes administering the aforementioned GV or pharmaceutical composition to the subject, and then administering to the subject a second therapeutic agent or therapy, such as other cancer treatment drugs or cancer treatment methods; optional
  • the method further comprises applying ultrasound to the subject before administering the second therapeutic agent or therapy.
  • the drug combination is implemented in the following manner: administering the above-mentioned GV or pharmaceutical composition to the subject, and then administering to the subject a second therapeutic agent or therapy such as other cancer treatment drugs or cancer Treatment method; and optionally, applying ultrasound to the subject before administering the second therapeutic agent or therapy.
  • a second therapeutic agent or therapy such as other cancer treatment drugs or cancer Treatment method
  • ultrasound to the subject before administering the second therapeutic agent or therapy.
  • the other cancer treatment drugs or cancer treatment methods are as defined above, and the cancer is as defined above.
  • the subject may be a human, a non-human primate, a mammal, a rodent, etc., preferably a human.
  • Figure 1 depicts the morphology, particle size distribution and zeta potential of natural GV and lipid GV.
  • A Image of 1nM GV.
  • B TEM image of GV showing its morphology (image is representative). Scale bar, 100nm.
  • C GV particle size distribution and zeta potential statistics.
  • D The relative particle size distribution of the two GV groups.
  • E Observe the stability of the GV group within 6 months by measuring its concentration.
  • F Observe the stability of the GV group within 6 months by measuring its size.
  • the data in (E) and (F) represent the mean ⁇ standard deviation of 3 independent experiments.
  • Figure 2 depicts the characterization of GV and PH-GV.
  • Figure 3 depicts the measurement results of GV's oxygen carrying capacity.
  • A The oxygen concentration in natural GV with different concentrations after oxygen is continuously supplied for one minute. The data represents the mean ⁇ standard deviation of 3 independent experiments. *p ⁇ 0.05 vs. control.
  • B The oxygen concentration in lipid GVs of different concentrations after oxygen was continuously supplied for one minute. The data represents the mean ⁇ standard deviation of 3 independent experiments. *p ⁇ 0.05 vs. control.
  • Figure 4 depicts the measurement results of the oxygen release kinetics of GV.
  • A The change of oxygen concentration in 5 ml of severe hypoxic solution after injection of 1 ml of 1 nM oxygenated-GV/oxygen-lipid-GV. The data represents the mean ⁇ standard deviation of 3 independent experiments.
  • B The change of oxygen concentration in 5 ml of severe hypoxic solution after injection of 1 ml of 1 nM oxygenated-GV/oxygen-lipid-GV, in which ultrasonic treatment was performed at 5 minutes for 10 seconds. The data represents the mean ⁇ standard deviation of 3 independent experiments. *p ⁇ 0.05 vs. control. **p ⁇ 0.01 vs. control.
  • Figure 5 depicts oxygenation-GV-mediated oxygen delivery in vitro and in vivo.
  • A Add 200 ⁇ l of oxygenated-PBS, oxygenated-lipid GV, and oxygenated-GV (final concentration of 1nM) to the culture medium, and incubate with the cells under hypoxia for 1 hour, then use hypoxia A representative image of hypoxia in cultured cells detected by the oxygen reagent, where the fluorescent signal indicates the occurrence of hypoxia.
  • B Quantification of hypoxia staining. The data represents the mean ⁇ standard deviation of 3 independent experiments. *p ⁇ 0.05 vs. control. Scale bar, 25 ⁇ m.
  • (C) Inject 200 ⁇ l of 5nM oxygenated-GV, oxygenated-PBS, oxygenated-lipid GV into the blood vessels of tumor-bearing mice by tail injection, and monitor the oxygen-Hb( Changes in the level of oxyhemoglobin and deoxy-Hb (deoxyhemoglobin) over time.
  • (C) shows representative photoacoustic images of tumor oxygen content at different time points. Red pixels: oxygen-Hb; blue pixels: deoxy-Hb.
  • Figure 6 depicts the toxicity of GV in vitro and in vivo.
  • A, B 200 ⁇ l of PBS, GV, and lipid GV (final concentration of 1nM) were added to the culture medium and cultured together with the cells. SCC-7 cells were measured at different time points by MTT and LDH assays. The cell proliferation and LDH toxicity. The data represents the mean ⁇ standard deviation of 3 independent experiments.
  • D H&E-stained histological images of major organs collected from GV-treated mice on day 7. Scale bar, 100 ⁇ m.
  • Figure 7 depicts the optical setup of the extracorporeal PDT.
  • Figure 8 depicts the cell viability of differently treated MCF-7 cells after PDT.
  • the in vitro cytotoxicity of PDT to MCF-7 cells was determined by CCK-8 assay.
  • Data represents the mean ⁇ SEM of 3 independent experiments. **p ⁇ 0.01 vs. control.
  • Figure 9 depicts the effects of different treatments on apoptosis.
  • the upper figure By flow cytometry, with the aid of annexin-V and propidium iodide (PI) double staining, the apoptosis after PDT was evaluated.
  • Figure 10 depicts the amount of ROS in cells after different treatments.
  • the intracellular ROS was stained by DCFH-DA and analyzed by flow cytometry.
  • Data represents the mean ⁇ SEM of three independent experiments. **p ⁇ 0.01 vs. control.
  • Figure 11 depicts the fold increase in SOSG fluorescence intensity of PBS (control), GV, PpIX and GV+PpIX after ultrasound irradiation.
  • the concentrations of PpIX and GV are 10 ⁇ M and 2nM, respectively.
  • Figure 13 depicts (a) cellular uptake of GV and PH-GV. Confocal microscope images of ICG-labeled GV and ICG-labeled PH-GV in SCC-7 cells. Scale bar, 20 ⁇ m. (b) The immune escape ability of GV and PH-GV. Under a fluorescence microscope, cell uptake in murine RAW 264.7 macrophages. (c, d) Measurement results of the effect of GV, collapsed GV and PH-GV at a concentration of 0.031-1 nM on SCC-7 cell viability after 24 hours (c) and 48 hours (d). Cells treated with PBS were used as controls.
  • Figure 14 depicts the distribution of GV and PH-GV after intravenous administration to tumor-bearing mice.
  • T/M Tumor/muscle
  • c In vitro fluorescence imaging of main organs and tumors taken from tumor-bearing nude mice 4, 12, 24, and 48 hours after injection of ICG-labeled GV and ICG-labeled PH-GV.
  • Figure 15 depicts the ultrasound contrast produced by GV at the tumor site.
  • Figure 16 depicts (a) Confocal images of tumor sections collected from mice 12 hours after injection of ICG-labeled GV and ICG-labeled PH-GV. The green signal and the red signal come from the fluorescence of ICG and CD31 staining vascular endothelial cells, respectively. (b) Representative H&E sections of major organs (heart, liver, spleen, lung, and kidney) and tumors 30 days after treatment. Scale bar, 100 ⁇ m. (c) Body weight measured under different conditions during the 30-day evaluation period for mice.
  • Figure 17 depicts the cavitation reaction experimental platform and results, (a) the top view of the GV-mediated cavitation reaction experimental platform; (b) the signal spectrum of GV and PBS solutions.
  • Figure 18 depicts a schematic diagram of the application of surface-modified targeted GV in tumor diagnosis, imaging and treatment according to some embodiments of the present invention.
  • GV can produce strong ultrasound contrast in the picomolar frequency range, exhibiting harmonic scattering, and thus can enhance the detection of relative in vivo background.
  • non-targeted tissues such as liver, spleen, and lung.
  • the pharmacokinetics and biodistribution of nanoparticles are partly determined by their own surface properties.
  • hydrophilic coatings are necessary for successful intravenous administration of nanoparticles.
  • chemical surface modification of GV with biocompatible materials can improve the pharmacokinetic properties and targeting of GV, thereby increasing its tumor accumulation in in vivo molecular imaging. This surface modification is also expected to enhance GV, making it a good carrier for drug delivery.
  • the present invention in order to improve stability and targeting, we used biomaterials to modify the surface functions of GV nanoparticles, and successfully synthesized double-modified GVs through the carboxyl reaction.
  • chemical modification of the surface of GV with biological materials greatly increases the accumulation of GV at the tumor site, so it can be used as an ultrasound contrast agent or imaging agent for tumor-specific ultrasound molecular imaging in vivo.
  • the modified GV can pass through the gap in the tumor endothelium and enter the gap of tumor tissue.
  • the surface of the GV is modified by a biomaterial with good biocompatibility and targeting, such as polyethylene glycol (PEG)-conjugated hyaluronic acid (HA).
  • PEG polyethylene glycol
  • HA hyaluronic acid
  • the present invention uses PEGylated HA to functionalize GV (PH-GV) particles, and uses ultrasound and fluorescence imaging, immunohistochemical sectioning and confocal microscopy techniques to indirectly and directly prove that modified GV enters tumors.
  • PH-GV GV
  • ultrasound and fluorescence imaging, immunohistochemical sectioning and confocal microscopy techniques to indirectly and directly prove that modified GV enters tumors.
  • the ability of the intracellular space of the tissue proves that the novel nano-scale imaging agent of the present invention can penetrate the gap of the tumor endothelial layer and enter the gap of the tumor tissue, so it can be applied to specific and effective ultrasound molecular imaging and cancer treatment.
  • oxygenated microbubbles can help deliver oxygen to the tissues of interest, thereby improving the therapeutic effect.
  • the application of existing oxygenated microbubbles is limited.
  • the inventors found that in PDT therapy, oxyGV, especially oxyGV modified by lipid molecules, can provide enhanced cytotoxicity to tumors, and has a significant enhancement effect on PDT in vivo and in vitro. Therefore, the novel nano-sized oxyGV of the present invention can be used as an effective oxygen carrier to improve the efficacy of oxygen-consuming PDT.
  • the present invention also studies the ability of oxyGV to improve the efficacy of sonodynamic therapy in vitro and in vivo, and proves that oxyGV, especially oxyGV modified by lipid molecules, can cause enhanced cavitation and overproduction of ROS in a cell-free system. And can enhance the production of ROS in vitro, which leads to increased cytotoxicity of SDT. Therefore, oxyGV can be used as a therapeutic agent to improve the efficacy of SDT.
  • natural GV refers to a nano-sized airbag isolated from nature, such as algae, without any modification
  • airbag is sometimes referred to as “nanoparticle” herein.
  • the GV involved in the present invention includes natural GV, lipid GV, PH-GV, oxygenated-GV, oxygenated-lipid GV and the like.
  • Lipid GV refers to GV that has been surface-modified with lipid molecules (such as DOPC, DSPE, PEG, etc.).
  • PH-GV and “PH-modified GV” can be used interchangeably and refer to GV that has been surface-modified with PEG and HA.
  • oxygenated-GV or "oxy-GV” are used interchangeably, and both refer to a GV filled with oxygen.
  • oxygenated-lipid GV oxygenated-lipid GV
  • oxyGV modified with lipid molecules or “oxy-lipid GV” are also used interchangeably, which refers to a lipid molecule filled with oxygen. GV with surface modification.
  • surface-modified GV refers to those that have specific functions (such as targeting, hydrophilicity, etc.) due to their surface modification (such as lipid molecules, targeting materials, biocompatible materials, etc.)
  • One type of GV for example, the PH-GV and lipid GV of the present invention belong to the surface modified GV.
  • Example 1 Preparation and characterization of GV and lipid-GV
  • the Anabaena algae (FACHB-1255, freshwater algae seed bank of the Institute of Hydrobiology, Chinese Academy of Sciences) was placed in a sterile BO-II medium under a 14-hour/10-hour bright/dark working cycle with fluorescent lighting Under cultivation.
  • GV was separated according to the method of Walsby (Buckland and Walsby, 1971). In short, hypertonic lysis achieved by rapidly adding a sucrose solution to a final concentration of 25% is used to dissolve the algae to release GV from the algae. Then, it was centrifuged at 600 g for 3 hours to separate GV. The separated GV can form a white milky layer on top of the solution, which can be collected by a syringe.
  • GV was purified three times with phosphate buffered saline (PBS) by centrifugation and stored in PBS at 4°C.
  • PBS phosphate buffered saline
  • GV concentration using a literature-based formula (450nM per OD500) (Walsby, 1994), where OD500 is an ultraviolet-visible spectrophotometer (2100Pro; GE Healthcare, Piscataway, New Jersey, USA) at a wavelength of 500 nm
  • OD500 is an ultraviolet-visible spectrophotometer (2100Pro; GE Healthcare, Piscataway, New Jersey, USA) at a wavelength of 500 nm
  • the optical density measured at.
  • a gas volume of approximately 8.4 ⁇ L/mg and a molar amount of 107 MDa were used to estimate the volume fraction.
  • the GV morphology was imaged using a transmission electron microscope (TEM) (JEOL 2100F; JEOL, Tokyo, Japan) operating at 200 kV.
  • TEM transmission electron microscope
  • the GV sample 0.5 nM
  • deionized water was deposited on a grid coated with carbon and dried overnight at room temperature.
  • the dynamic light scattering (DLS) method is used to obtain hydro
  • the liposome preparation solution with a volume of 0.5 mL was added to the GV solution. Also increase the volume to 1mL with 20mM HEPES buffer. The mixed solution was incubated overnight on a shaker. After that, the GV was washed three times by centrifugation (2.4 krpm, 10 minutes) and resuspended in Milli-Q water. Finally, the lipid GV was resuspended in PBS and ready for use.
  • Morphology analysis The size and morphology of lipid GV were determined by transmission electron microscope (TEM) at an operating voltage of 200 kV. A sample of lipid GV (OD 0.1) was deposited on a carbon-coated formvar grid and stained with 2% uranyl acetate.
  • Particle stability In order to measure the stability of the lipid GV formulation, the single particle size and concentration of the liposome-encapsulated GV formulation were measured on the first 1, 3, and 7 days after manufacture.
  • the lipid GV was characterized by particle size distribution and zeta potential. We found that these nanobubbles have an average diameter of about 300-330 nm and a uniform distribution (Figure 1C- Figure 1D).
  • the average particle size of lipid GV is about 290-330nm ( Figure 1C) or about 310-330nm (measured by DLS, Figure 1D), which is about 10nm larger than natural GV.
  • the zeta potential of lipid GV is about -21.3 to -19.3 mV, and its negative charge is lower than that of GV ( Figure 1C).
  • the HA is fixed to the protein shell of GV by covalent conjugation.
  • EDC 3.37 mM
  • NHS 2 mM
  • a HA solution 10 mg
  • the reaction mixture was stirred for another 24 hours at 4°C.
  • the resulting mixture was added to an ultrafiltration tube (50 mL), and centrifuged at 1.8 rpm for 5 minutes to remove free EDC, NHS and HA.
  • the resulting nanoparticles were stored in PBS buffer at 4°C.
  • H-GV conjugate 120 mg was dissolved in phosphate buffered saline (PBS, pH 7.2) and mixed with EDC (3.37 mg) in PBS and NHS (2 mg) in methanol. After slowly adding monomethoxy PEG-amine (73.5 mg), the mixture was stirred on an ice bath for 24 hours. The resulting solution was then centrifuged and washed 4 times with PBS to remove excess methanol and PEG.
  • PBS phosphate buffered saline
  • GV is labeled with NIR dye (ICG).
  • EDC 3.37 mg
  • NHS 2 mg
  • ICG solution 1 mM
  • the mixture was then shaken for 4 hours at room temperature, and then purified by centrifugation 4 times.
  • the resulting mixture was added to an ultrafiltration tube (50 mL), and centrifuged at 1.8 rpm for 5 minutes to remove free ICG.
  • the resulting nanoparticles were stored in PBS buffer.
  • the particle size and particle size distribution of PH-GV NP were measured by dynamic light scattering DLS (Varian, Palo Alto, USA). Perform zeta potential measurement on the Malvern Zeta Size-Nano Z instrument at 25°C.
  • the nanostructure and size of PH-GV were observed by transmission electron microscope (TEM) (Bruker, Germany). Observe the ultraviolet-visible light absorption spectra of GV and PH-GV by Multiskan Go microplate reader (Thermo Fisher Scientific, Massachusetts, USA).
  • a fluorescence spectrophotometer (Varian, Palo Alto, USA) was used to measure the fluorescence signal of ICG and ICG-labeled PH-GV.
  • Ultrasonic signal test of GV and PH-GV US imaging of PH-GV and GV containing the same amount of GV. Put PH-GV and GV into a dropper (5mL) before imaging, and immerse all the droppers in deionized water at the same depth. A high-frequency ultrasound system with LZ250D transducer was used to obtain ultrasound B-mode images and contrast-mode images of the GV solution. Set the center frequency and output energy level to 18MHz and 4%, respectively.
  • hyaluronic acid is a polysaccharide composed of repeating units of N-acetylglucosamine and glucuronic acid
  • EDC and NHS PEG-5000 is easily attached to the backbone of the HA conjugate through amide formation, which can prevent HA-GV from the host’s immune system by preventing non-specific interactions with plasma proteins.
  • TEM transmission electron microscopy
  • the particle size of GV is about 320-380nm, PH-GV is about 375-425nm; the zeta potential of GV is in the range of -40 to -50mV, and the potential of PH-GV is about -27 ⁇ 4mV ( Figure 2b).
  • PH-GV has a cylindrical morphology with a radius of about 400 nm, and all nanoparticles are cylindrical and show a relatively uniform size distribution.
  • the appropriate particle size and negative zeta potential of PH-GV nanoparticles ensure good tumor-targeted cargo delivery via the EPR effect, while reducing RES clearance.
  • GV from Anabaena algae was purified by high concentration (tonic) cell lysis and centrifugation assisted flotation, and operated at 21 MHz at different concentrations Use the Vevo 2100 imaging system (FUJIFILM VisualSonics, Toronto, Ontario, Canada; B mode) to image them (for imaging details, see the example below).
  • a concentration range of 250 pM to 1 nM Figures 2c-2d
  • a gas volume fraction of about 0.01% to 0.1% GV produced a strong contrast with the buffer control. The contrast is strongest at the highest concentration, and the scattering produced by Ana GV is 23.4 ⁇ 2.5 larger than the buffer control ( Figure 2e).
  • Example 3 Determination of oxygen carrying properties and oxygen release kinetics of GV and lipid GV
  • oximeter Use an oximeter (Portamess 913 OXY; Knick, Germany) to monitor the oxygen concentration in the solution, and record the data as mg/l. Different concentrations of GV and lipid GV were aerated with oxygen/nitrogen, and the oxygen concentration of different solutions was measured respectively.
  • Oxygen release without ultrasound The oxygen concentration of the PBS sealed in the vial is reduced to 0.8 mg/l (severe hypoxia) by continuous N 2 filling to simulate hypoxic conditions. Continue to fill with oxygen to saturate the PBS, GV and lipid GV with oxygen. Monitor the oxygen concentration of oxygen diffused from GV/lipid GV into anoxic solution over time. Before each experiment, the oximeter will be calibrated. All experiments were repeated three times.
  • Oxygen release under ultrasound In order to study the effect of ultrasound (US) on the oxygen release of GV/lipid GV, a US probe was used with an oscillation frequency of 1MHz and an average sound pressure distribution value of 2.4 ⁇ 0.2Mpa (nominal frequency) : 50Hz; nominal power: 30W). The sonication time is 10 seconds. Detect the change of oxygen concentration in the solution after ultrasound. All experiments were repeated three times.
  • US ultrasound
  • the oxygen-carrying capacity of lipid GV is comparable to that of GV, but it shows a slower release pattern, which can increase the oxygen concentration at the target site to a greater extent.
  • Example 4 Determination of the ability of oxygenated GV and oxygenated lipid GV to transport oxygen
  • the cells were cultured and passaged in a hypoxic incubator (1% oxygen, 5% CO 2 ).
  • Image-iT TM Red Hypoxia Reagent purchased from Thermo Fisher was used to monitor hypoxia conditions in the medium before/after the addition of oxygenated lipid GV.
  • mice Female Balb/c mice weighing 18-20g were provided by the Animal Resource Center of Hong Kong Polytechnic University. Mice were adapted to the room for one week after arrival and maintained a normal 12-hour light-dark cycle. The mice are housed in conventional cages (6 animals per cage), and under the conditions of no specific pathogens, a temperature of 24 ⁇ 2° C., and a relative humidity of 60-70%, they can freely obtain standard pellet feed and water. Standard wood chips for mice were used as litter.
  • 0.1ml of the SCC-7 cell line (1 * 107 cells / ml) were resuspended in 100 ⁇ L of Matrigel and injected subcutaneously implanted back Balb / c mice. Tumor formation occurs about two weeks after cell implantation, and when the volume reaches about 200 mm 3 , the tumor shows a significant degree of hypoxia.
  • mice After mice are injected with oxygenated lipid GV, they pass through a Vevo LAZR photoacoustic imager (Fujifilm Visualsonics, Amsterdam, Netherlands) to monitor oxygen-Hb and deoxy-Hb levels in subcutaneous tumors. Record and store pO 2 levels for later comparison between groups.
  • Vevo LAZR photoacoustic imager Flujifilm Visualsonics, Amsterdam, Netherlands
  • each GV group oxygenated GV and oxygenated-lipid GV
  • oxygenated GV and oxygenated-lipid GV were grown for 24 hours under hypoxic conditions, and use Image-iT Red Hypoxia Reagent (red hypoxia reagent) to monitor their hypoxia levels.
  • red hypoxia reagent Image-iT Red Hypoxia Reagent
  • Example 5 In vitro and in vivo toxicity detection
  • MCF-7 Human breast cancer cell line
  • MCF-7 cells were cultured in high glucose (4.5g/l) Dulbecco's modified Eagle's medium with L-glutamine. All media were supplemented with 10% (volume/volume) fetal bovine serum, penicillin (100U/mL) and streptomycin (100 ⁇ g/ml).
  • fetal bovine serum fetal bovine serum
  • penicillin 100U/mL
  • streptomycin 100 ⁇ g/ml
  • lipid GV 1 nM
  • LDH assay was performed using Pierce LDH Cytotoxicity Assay Kit (Life Invitrogen) according to the manufacturer's instructions. Cell viability and apoptosis were measured by MTT assay and apoptosis assay according to the manufacturer's instructions.
  • In vivo toxicity determination Perform autopsy on lipid GV-treated mice, and collect tissue samples (liver, lung, and kidney) for histological examination. Liver, lung, and kidney samples collected from the mouse bioassay were fixed in 10% formalin buffer, processed by conventional histological techniques, and stained with hematoxylin and eosin. Use an optical microscope (Olympus BX51) equipped with a camera (Olympus Q-Color-5) to perform microscopy, and use Image Pro-Express software to record the image in the computer.
  • mice were scored on a 30-point scale, and we observed that these indicators did not decrease during this time (Figure 6C).
  • H&E hematoxylin and eosin staining to determine the major organs (heart, liver, spleen, lung, and kidney) of mice one week after GV administration.
  • the tissue sections of the two GV groups did not show obvious pathological abnormalities or lesions ( Figure 6D). Therefore, we determined that GV and lipid GV were not cytotoxic to cells and did not cause any significant damage to the tested mice.
  • Example 6 The effect of the combination of oxyGV and PDT on cytotoxicity and apoptosis
  • Photodynamic therapy setting Expose the cells to the laser at a power of 100 mW/cm 2 for 5 minutes.
  • the optical setup for PDT processing is shown in FIG. 7.
  • a light source with a wavelength of 396nm is generated through an optical fiber, which is aligned with the aperture and irradiated to a 35mm cell culture dish.
  • the position of the cell culture plate is manually controlled by a dual-axis electric linear platform. After treatment, the cells were cultured in fresh medium for 4 hours and then ready for different analyses.
  • MCF-7 Human breast cancer cell line
  • DMEM high-glucose Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • MCF-7 cells were randomly divided into eight groups: (1) control, (2) laser only, (3) PpIX only, (4) PpIX plus laser (PDT), ( 5) PpIX+GV+laser (PDT+GV), (6) PpIX+oxyGV+laser (PDT+oxy-GV), (7) GV+laser, (8) oxyGV+laser.
  • PpIX treatment cells were incubated with 10 ⁇ M PpIX in DMEM medium supplemented with 10% FBS for a drug loading time of 4 hours.
  • Cell counting kit-8 (Sigma-Aldrich) was used to determine cell viability at different time points after PDT according to the manufacturer's instructions.
  • cells were seeded in a 96-well plate at a density of 5000 cells per well, and incubated in 100 ⁇ L of medium for 24 hours.
  • the cytotoxicity was determined by adding 10 ⁇ l of CCK-8 reagent per well for 1 hour at 37°C in 5% CO 2 .
  • the detection wavelength measure the absorbance of the treated sample to the blank control. The viability of the treated cells was determined by comparison with untreated cells in the control group.
  • Apoptosis test Inoculate the cells in a 6cm culture dish at a density of 5 ⁇ 10 5 cells and incubate for 24 hours.
  • the Alexa Fluor 488 Annexin V/Dead Apoptosis Kit (Thermo Fisher Scientific) was used to measure apoptosis according to the manufacturer's instructions.
  • the cells were collected and incubated with 5 ⁇ L of Annexin V conjugate and 1 ⁇ L of PI working solution for 15 minutes at room temperature.
  • the cells were analyzed by FACS Calibur flow cytometer and BD Accuri C6 software (Becton-Dickinson, USA).
  • Figure 8 depicts the effects of different treatments on cell viability.
  • the in vitro cytotoxicity of different treatments on MCF-7 cells was determined by CCK-8 assay.
  • Data represents the mean ⁇ SEM of 3 independent experiments. **p ⁇ 0.01 vs. comparison.
  • Example 7 The effect of OxyGV on ROS production during PDT
  • Intracellular ROS test DCFH-DA (Sigma-Aldrich) was used to measure intracellular ROS production. In short, 10 ⁇ M DCFH-DA diluted with PBS was added to MCF-7 cells at 37°C for 20 minutes. The cells were then washed three times with PBS. The labeled cells were trypsinized and analyzed by flow cytometry.
  • Example 8 Singlet oxygen generation in a cell-free system
  • the measured spatial peak time average intensity is 8W/cm 2 ; when the fixed pulse repetition frequency (PRF) is 1000 Hz, the duty cycle is 20%.
  • the overall sonication duration is 5 minutes.
  • the temperature rise of the cell culture test solution is controlled within 2.5°C. After sonication, the cells were cultured in fresh medium for 4 hours and then ready for different analyses.
  • Singlet oxygen generation SOSG (10 ⁇ M) is used to detect singlet oxygen generation.
  • the solution is exposed to ultrasound and protected from light during the exposure time.
  • the fluorescence intensity of SOSG was measured by a microplate reader at an excitation wavelength of 488nm and an emission wavelength of 530nm.
  • Passive cavitation detection is used to characterize the cavitation intensity (see Figure 17).
  • the ultrasound parameters remain the same as those used in cell culture and ROS characterization experiments.
  • the GV was loaded into a 5mm agar-gel cubic surface placed at the confocal area of the FUS transducer and the receiving transducer.
  • the RF data of the receiving transducer is obtained through an oscilloscope (LeCroy 715 Zi; LeCroy, Chestnut Ridge, New York, USA).
  • the acquired RF data is transmitted to the spectrum, and the inertial cavitation intensity is calculated through the integration in the broadband signal (4.8MHz-5.2MHz).
  • Example 11 Targeting and immune escape of GV and PH-GV (the following examples are all from P20914US)
  • GV and PH-GV Human squamous cell carcinoma cell line (SCC7 cells) and NIH-3T3 cells in an 8-well incubator at 37°C and 5% CO 2 Cultivate in Dulbecco's Modified Eagle Medium (DMEM)/high glucose medium with 10% FBS and 1% antibiotic solution. The next day, the two kinds of cells were washed with cold PBS and incubated with IGV for 4 hours at 37°C under a 5% CO 2 atmosphere. After incubation, all cells were washed thoroughly with cold PBS. Finally, fix the cells in cold ethanol for 15 minutes at -20°C, and then fix the cells in the dark for 10 minutes with a mounting medium containing DAPI.
  • SCC7 cells Human squamous cell carcinoma cell line
  • NIH-3T3 cells in an 8-well incubator at 37°C and 5% CO 2 Cultivate in Dulbecco's Modified Eagle Medium (DMEM)/high glucose medium with 10% FBS and 1% antibiotic solution.
  • ICG-labeled PH-GV and ICG-labeled GV were observed by a confocal microscope (Olympus, USA), and the excitation wavelength and emission wavelength of ICG were set to 780 nm and 800 nm, respectively.
  • the intracellular distribution of PH-GV nanoparticles is very different from that of GV solution.
  • a large amount of ICG fluorescence was distributed throughout the cytoplasm, while in SCC7 cells treated with ICG-labeled GV, no strong red fluorescence was observed in the cytoplasm.
  • H&E hematoxylin-eosin
  • mice Animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Hong Kong Polytechnic University (CC/ACUCC).
  • the subcutaneous part of athymic nude mice (seven weeks old, female, 20-24 g) was injected with a PBS suspension (80 ⁇ L) of 4 ⁇ 10 6 SCC7 cells.
  • the tumor size (in the area of the right leg) reached an average size of 120 mm 3
  • the mice were randomly divided into three groups: (a) Free ICG solution was injected into the tail vein of the mice; (b) ICG-labeled GV The solution was injected into the tail vein of the mouse; and (c) the ICG-labeled PH-GV solution was injected into the tail vein of the mouse.
  • IVIS Lumina II Caliper Life Sciences, USA; excitation filter: 780 nm, emission filter: 800 nm
  • SCC7 tumors and normal organs are collected and used to obtain fluorescence signal intensity.
  • ICG-labeled PH-GV produced a consistent fluorescent signal throughout the mouse for 8 hours, while there was no increase in high fluorescent signal in normal organs. And 8 hours after intravenous injection, obvious fluorescence was observed at the tumor site. The fluorescence of ICG-labeled PH-GV increased steadily and reached a peak at 12 hours. Subsequently, the fluorescence of the tumor site gradually decreased from 24 hours to 48 hours, indicating that PH-GV was continuously discharged from the tumor tissue. However, after administration of ICG and ICG-labeled GV, no strong fluorescent signal was found in the tumor tissue. This sharp difference may be attributed to the enhanced immune escape and specific tumor targeting ability of PH-GV.
  • Example 14 US imaging of cancer in vivo by PH-GV
  • mice Animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Hong Kong Polytechnic University (CC/ACUCC).
  • a PBS suspension (80 ⁇ L) of 4 ⁇ 10 6 SCC7 cells was injected into the subcutaneous site of nude mice (seven weeks old, female, 20-24 g).
  • the tumor size in the right leg area
  • the mice were randomly divided into two groups: (a) GV was injected into the tail vein of the mouse; (b) PH-GV was injected into the mouse’s tail vein In the tail vein.
  • US images of the tumor site were recorded on the Vevo 2100 imaging system.
  • a high-power US stimulation is performed, which can cause the fragmentation of the GV.
  • the Vevo 2100 imaging system is used to record US images and signal intensity before and after US stimulation for analysis. Use Vevo 2100 Workstation software to measure the signal strength of echo imaging.
  • the ICG fluorescence in the PH-GV group is much higher than that in the GV group, which indicates that modifying GV with PH can enhance tumor accumulation and retention. This is because there is a longer circulation time for PEG EPR effect and HA targeting. More importantly, we observed that a large number of ICG fluorescent signals are located far away from blood vessels, which indicates that PH-GV nanoparticles may penetrate through blood vessels around the tumor site and be internalized in tumor cells to achieve effective intratumor diffusion.
  • SCC7 cells were seeded in a 96-well plate at a density of 8000 cells per well, and cultured overnight at 37°C in a 5% CO 2 incubator. The next day, the cells were washed 3 times with PBS, and incubated with a series of concentrations of GV, broken GV, and PH-GV solutions under the same conditions for 24 hours and 48 hours. The cell viability was evaluated by CCK-8 assay kit. The optical density (OD) was measured at 450nm and recorded by a microplate reader.
  • mice treated with GV and PH-GV had no obvious signs of injury or toxicity.
  • Fig. 16c within 30 days after the treatment, similar changes in the body weight of each treatment group during the treatment period were observed. It is worth noting that no acute toxicity or adverse reactions of GV and PH-GV to mice were observed in our study.

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Abstract

本文提供了表面修饰的新型靶向GV(例如PH-GV)、脂质GV、脂质靶向GV、其制备方法及其在肿瘤的诊断、成像和治疗中的应用。

Description

表面修饰气囊及其制备和应用
相关申请的交叉引用
本发明要求2019年5月29日提交的、申请号为62/853,739、发明名称为“表面修饰的气囊”的美国临时专利申请；和2019年5月29日提交的、申请号为62/853,747、发明名称为“生物气囊及其制备和使用方法”的美国临时专利申请的优先权，上述临时申请的全部内容通过引用以其整体并入本文。
技术领域
本公开涉及表面修饰的靶向GV(例如PH-GV)、脂质GV、脂质靶向GV、其制备方法及其在肿瘤的诊断、成像和治疗中的应用。
背景技术
以充气微泡作为造影剂的超声造影(CEUS)在多种疾病的诊断和管理中起着重要作用。通过将结合配体附着到微泡壳上进行表面修饰的分子靶向造影剂有助于在分子水平上使过表达的生物标志物可视化并且显著地提高成像灵敏度和特异性。由于微泡的直径为几微米，因此其仅保留在血管腔隙内。这种特性使其特别适合于使在肿瘤新血管系统上表达的分子标志物可视化。
虽然微泡在成像和治疗上的应用均已显示出令人鼓舞的结果，但其在生物医学中的潜在应用受到了其较大流体动力学尺寸(1-8μm)的限制，因为较大流体动力学尺寸阻碍了其在静脉注射后向周围非血管组织的渗透。此外，这些微泡的气体内容物会迅速扩散到周围介质中，导致半衰期非常短(<20分钟)。
最近的研究已证实，粒径小于1000nm的纳米级超声造影剂表现出以下优点：分子量小、渗透力强和稳定性高。这种类型的造影剂包括碳氟乳液纳米滴、纳米气泡和纳米粒子。由于其纳米级尺寸，因此它们可以克服上述局限性，能够穿过肿瘤的渗漏 的有缺陷的血管系统，同时由于增强的渗透性和滞留(Enhanced Permeability and Retention，EPR)效应，它们也能够停留在健康组织的血管系统中。作为诊断成像的补充，这些纳米级超声造影剂具有作为活性药物载体进行局部药物递送的潜力，并且，由于对血管系统和细胞膜的渗透性的瞬时增加，纳米级超声造影剂还能够以空间和时间特异性的方式主动触发药物释放。尽管纳米级超声造影剂具有吸引人的益处，但将其用于诊断目的的临床转化并未取得很大的成功。纳米级超声造影剂的低气体密度和低对比度噪声比不允许在超声下以诊断频率进行高对比度成像。
最新报道的从浮游光合微生物中分离出来的气囊(gas vesicle，GV)作为一种新型超声成像纳米级造影剂具有巨大的潜力。GV在皮摩尔浓度的频率范围内产生强烈的超声造影，表现出谐波散射，因此能够增强体内相对于背景的检测。GV的非线性振荡会生成入射超声的谐波，该谐波可以被特异性地检测，以进一步增强对比度噪声比，从而提高灵敏度和特异性。GV的宽度为45-250nm并且长度为200-600nm，因此能够通过EPR效应进入组织间隙。与传统的超声造影剂不同(传统的超声造影剂会以不稳定的配置捕获预装气体)，GV的2nm厚的蛋白质壳排斥水，但容许气体从周围介质自由地扩散进出，从而使其在物理上稳定而不受其纳米尺寸的影响。此外，现有技术对GV进行基因工程改造可以设计出具有新的机械、声学、表面和功能特性的基于GV的造影剂，从而能够实现谐波、多路复用(multiplexed)和多模态超声成像以及细胞特异性分子靶向。由于具有上述优势，因此GV是超声(US)分子成像和癌症进一步治疗的非常有前途的纳米粒子。
然而，与其它纳米粒子相似，在静脉内施用后，大多数GV在非靶向组织(诸如，肝脏、脾脏和肺)中被摄取。现有发现认为网状内皮组织系统(reticuloendothelial system，RES)去除了84％的天然GV，这是由于吞噬细胞的捕获和GV在胆道系统中的破碎，因此在注射后2分钟的血液中没有GV残留。这限制了天然GV在肿瘤分子成像中的应用，因为GV的循环时间不会长到足以渗入到目标组织中的程度。此外，其不具有被特异性地内化到靶细胞中的能力，从而导致在细胞外相中释放大量的造影剂，这将进一步降低其成像和治疗的效率。因此，为了将GV转化为新型US分子成像剂，应将进一步的研究工作集中在改善GV的药代动力学特性上。
长期以来，肿瘤缺氧已被认为是肿瘤学中的关键问题。病理性缺氧可以引起肿瘤细胞内的蛋白质组变化和基因组变化以及肿瘤微环境的变化。这些变化可能会更改肿 瘤细胞的局部代谢并激活有助于肿瘤进展的适应性细胞反应。而且，缺氧的存在使实体瘤对放射疗法和化学疗法更具有抵抗力，从而导致患者的存活率较低。鉴于缺氧在肿瘤进展和抵抗癌症治疗中的作用，人们开始考虑为肿瘤组织供氧是否可以防止癌症生长和发展。已证明肿瘤氧合能够通过增加细胞敏感性来显著改善化学疗法和放射疗法并有助于克服对化学疗法和放射疗法的抗性。一些研究还暗示，肿瘤氧合与减少的细胞增殖、血管生成和转移有关，并且氧合在某些肿瘤亚型中可能具有肿瘤抑制效果。缺氧肿瘤微环境的变化可降低HIF-1α的表达，因而减弱缺氧驱动的途径。此外，据报道，肿瘤中的氧合作用在表观遗传程序化中起作用。在氧合后，可以重新编程几个缺氧相关基因和肿瘤抑制基因。这意味着氧合不仅可以作为癌症治疗的佐剂，而且具有逆转由缺氧引起的肿瘤部位蛋白质组变化和基因组变化的潜力。
考虑到氧气在肿瘤进展和对治疗的抗性方面具有重要作用，因此肿瘤氧合已被认为是抗癌治疗的辅助手段。高压氧(hyperbaric oxygen，HBO)疗法是治疗缺氧相关疾病的公认方法。HBO疗法可以有效提高肿瘤部位的含氧量，并且可以改善化学疗法和放射疗法的结果，但其仍面临费用高、患者体验不佳等局限性。此外，氧不能在缺氧肿瘤的深处输送，这在某些情况下可损害临床效果。由于HBO疗法的局限性，科学家已试图开发出可以将大量氧气运送到肿瘤的氧气载体。人工红细胞(artificial red blood cell，RBC)替代品(诸如，全氟化碳(PFC)乳液和无细胞血红蛋白基氧气载体(HBOC))已作为第一代氧气载体被开发。尽管如此，大多数产品仍无法达到足够的循环半衰期，也无法维持生理组织氧合作用。近来，随着新型含氧微粒的发展，纳米粒子成为一种合适的载体来到达感兴趣的组织并有效地输送氧气。
对于氧气载体，基于脂质的微泡由于其良好的生物相容性和生物降解性而优选用于医学应用。微泡能够携带大量氧气，并且可以与超声结合使用以用于定位控制(site-controlled)氧气释放。充氧微泡已被证明能够改变体内缺氧微环境并增强化学疗法和放射疗法的结果。充氧微泡的进一步发展仍面临一些挑战和局限性。首先，充氧微泡在进入循环系统时可能会遇到稳定性问题(诸如，溶解和聚结)，并且这与更大的产物损失有关，后者可能导致活性氧(reactive oxygen species，ROS)的过量产生。此外，微泡由于其尺寸限制而无法通过血管系统，如果与药物输送结合，则可能会损害其效果。
光动力疗法(photodynamic therapy，PDT)已成为一种用于癌症治疗的成功临床治 疗方法，其已经获得美国食品药品监督管理局(FDA)的批准。PDT依赖于光敏剂和对应光之间的协同相互作用。这两种元素分别是无毒的，但是当其一起工作时，则可以引发化学反应并实现肿瘤细胞凋亡。光敏剂可以通过血液循环选择性地进入肿瘤部位，并且当被适当激活波长的光激发时，会产生大量的ROS。ROS(尤其是单线态氧自由基)被认为是导致癌细胞坏死和凋亡的原因。除了产生ROS来直接杀死癌细胞外，PDT还可以在治疗后引起针对癌细胞的免疫反应，以及通过与癌症相关的血管系统损害切断营养供应来抑制肿瘤生长。由于具有公认的安全性、选择性和可重复性的优点，PDT不仅被应用于皮肤病学领域，而且还用作治疗肺、呼吸道和泌尿道肿瘤的辅助疗法。在PDT治疗期间，氧气的可用性可能是疗效的限制因素，因为ROS的产生取决于氧气的浓度。然而，癌组织经常表现出缺氧的情况，这通常限制了PDT的有效性。因此，提高肿瘤部位的氧浓度是增强PDT疗效的良好策略。充氧微泡已用于增强PDT，并且其疗效已在体外和体内得到认可。例如，已有的研究证明，在PDT期间微泡的存在可导致产生更多的单线态氧并增强细胞凋亡。这意味着充氧微泡与光敏剂一起可导致增强的细胞毒性。然而，由于稳定性的限制和相对较大的尺寸，充氧微泡的应用受到了限制。
与传统的癌症疗法(诸如，手术、化学疗法和放射疗法)相比，声动力疗法(sonodynamic therapy，SDT)在与癌症的持久战中已成为一种有前景的非侵入性治疗方法。SDT是一种超声疗法，在这种疗法中，通过施用声敏剂来提高超声对肿瘤的细胞毒性效应，同时使正常组织保持完好无损。SDT的概念与成熟的光动力疗法(PDT)非常相似，后者使用激光来激活光敏剂以产生毒性。与PDT(其由于激光的有限穿透力而仅限于体表肿瘤应用)相比，SDT具有能够治疗深层癌的优势，因为超声可以在三维空间中集中于深入组织内的单个点。SDT对癌症治疗的效果已在体外和体内得到了广泛证明。在声场的存在下，ROS的生成是造成SDT的细胞毒性作用的原因。超声引起的空化(cavitation)被认为与超声和敏化剂生成ROS的相互作用有关。空化是水性环境中气泡与超声的相互作用的表现。空化包括充气泡的成核、生长和内爆破碎的过程。惯性空化(在此期间，气泡在液体介质中迅速膨胀并猛地破裂)与ROS的过量产生密切相关。因此，在SDT的治疗应用中，微泡已被用作人工核以增强超声触发的惯性空化。微泡的添加可以降低空化阈值，从而增强ROS的产生和细胞毒性。然而，微泡在SDT疗法中的应用同样受限于其稳定性差和相对较大的尺寸。
因此，为了克服现有技术中癌症和其它一些疾病的诊断和治疗中存在的问题，亟需开发一种具有改善的药代动力学特性的新型GV，以提高肿瘤疾病诊断的精确性和治疗效果。
发明内容
为了解决现有技术中存在的问题，本发明提供了表面修饰的新型靶向GV(例如PH-GV)、脂质GV、脂质靶向GV、其制备方法及其应用。
本发明的第一方面提供了一种经表面修饰而能够特异性靶向肿瘤部位的靶向GV。本发明的靶向GV是表面被生物相容性材料(例如聚乙二醇(PEG)、壳聚糖、聚氨基酯、聚乳酸、聚烯烃、聚砜、聚碳酸酯、聚丙烯腈等或其任意组合)和/或生物靶向材料(例如透明质酸(HA)、RGD肽、叶酸、半乳糖、葡萄糖等或其任意组合)修饰的GV。在一些实施方案中，本发明中所使用的PEG的分子量为约5000Da。所述靶向GV是表面被PEG和/或HA修饰的GV，优选表面被PEG和HA修饰的GV(PH-GV)。
在一些实施方案中，本发明的靶向GV例如PH-GV为圆柱状，其粒径约为400nm。根据动态光散射(DLS)方法测定，所述靶向GV的粒径约为375-425nm，且ζ电位为约-23至-31mV。
本发明的第二方面提供了一种造影剂或诊断剂，其含有本发明第一方面所述的靶向GV例如PH-GV。在一些实施方案中，所述靶向GV在造影剂中的浓度为约250pM-1nM。
本发明的第三方面提供了一种诊断用试剂盒，其含有本发明第二方面所述的造影剂或诊断剂，以及任选地使用说明书。
在一些实施方案中，本发明第一方面所述的靶向GV、第二方面所述的造影剂或诊断剂以及第三方面所述的试剂盒可用于诊断疾病例如癌症。所述靶向GV或造影剂可用于使肿瘤成像，从而诊断癌症的存在或不存在。因此，本发明还提供了第一方面所述的靶向GV或第二方面所述的造影剂或诊断剂在制备用于诊断疾病例如癌症的诊断剂或诊断试剂盒中的用途。本发明还提供了所述靶向GV或造影剂在制备用于使肿瘤成像的诊断试剂或试剂盒中的用途。
本发明的第四方面提供了一种诊断方法，所述方法包括使用本发明第一方面所述的靶向GV、第二方面所述的造影剂或诊断剂或第三方面所述的试剂盒来诊断受试者中癌症的存在或不存在。在一些实施方案中，所述诊断包括将所述靶向GV、造影剂或诊断剂施用于所述受试者或者将所述试剂盒应用于所述受试者。所述诊断方法还包括对所述受试者进行超声成像，从而确定癌症的存在或不存在。
本发明的靶向GV还可用作药物载体。因此，本发明的第五方面提供了一种药物载体，其含有本发明第一方面所述的靶向GV例如PH-GV，或由其组成。在一些实施方案中，本发明所述的药物载体还包含用于治疗待治疗疾病例如癌症的药物。例如所述药物可以通过各种方式与所述药物载体连接在一起，从而通过施用药物载体将所述药物递送到体内的目标部位。将药物载体与药物连接在一起的方法以及用于治疗特定疾病例如癌症的药物均是本领域技术人员熟知的。因此，本发明的药物载体还可用于治疗疾病例如癌症。
本发明的第六方面提供了一种使用第五方面所述的药物载体治疗受试者的疾病例如癌症的方法，包括将所述药物载体施用于所述受试者。在一些实施方案中，所述方法还包括在将所述药物载体施用于所述受试者之前，将所述药物载体与用于治疗所述疾病例如癌症的药物连接在一起的步骤。
在本发明的上述方面中，所述肿瘤或癌症例如是CD44高表达型肿瘤，包括但不限于膀胱癌、肺癌、肾癌、胃癌、结直肠癌、肝癌、乳腺癌、黑色素瘤等。
本发明的第七方面提供了一种靶向GV的制备方法，包括步骤：(a)将生物靶向材料与GV连接在一起，例如通过共价缀合将生物靶向材料例如HA固定至GV的蛋白质壳。在一些实施方案中，所述方法还包括步骤(b)将生物相容性材料与步骤(a)的产物连接在一起，例如通过酰胺形成将生物相容性材料例如PEG化学缀合至步骤(a)的产物。
在一些实施方案中，所述步骤(a)包括：(a1)将生物靶向材料例如HA与磷酸盐例如磷酸钠混合形成溶液；(a2)将GV溶解在磷酸盐例如磷酸钠溶液中；(a3)将1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)加入到步骤(a1)的溶液中(优选pH＝7.4)；以及(a4)向步骤(a3)的混合物中加入步骤(a2)的溶液，从而形成靶向材料-GV(例如H-GV)缀合物。
在一些实施方案中，所述步骤(b)包括：(b1)将靶向材料-GV缀合物(例如来自上述步骤(a)的H-GV缀合物)溶解在磷酸盐缓冲液(PBS)中，并使其与PBS中的EDC和甲醇中的NHS混合；以及(b2)向步骤(b1)的混合物中加入生物相容性材料例如单甲氧基PEG-胺，从而得到PH-GV。
本发明的第八方面提供了一种表面被脂质分子修饰的脂质GV。在一些实施方案中，所述脂质分子是二硬脂酰磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)、二油酰磷脂酰胆碱(DOPC)、PEG、DSPE、聚乙烯醇(PVA)、甘油、甘油酯、脂肪酸、磷脂或其任意组合，优选地，所述脂质GV是表面经DSPE-PEG和DOPC修饰的脂质GV。在一些实施方案中，本发明的脂质GV为圆柱状，其粒径可以为例如约290-330nm，例如约300-330nm，约310-330nm。根据动态光散射(DLS)方法测定，所述脂质GV的ζ电位为约-21.3至-19.3mV。本发明的脂质GV本身在脂质分子膜内包封有一定的气体例如空气、氧气，但可以将其设计成包封/装载/携带特定的目标气体，例如治疗性目标气体，例如氧气、一氧化氮(NO)、氢气等。因此，本发明的脂质GV可用作向受试者体内输送治疗性气体的载体，在这种情况下，目标气体可被脂质分子包封在GV的蛋白质壳内。
因此，本发明的第九方面提供了一种充气的脂质GV，其包括本发明第八方面所述的脂质GV和气体，例如所述气体可以是任何气体种类，例如治疗性气体。所述气体被填充在GV的蛋白质壳中并被所述脂质分子包封。本发明所述的治疗性气体包括本领域已知的有助于治疗、缓解、减轻疾病或疾病症状或抑制其进展的任何气体，包括但不限于：氧气、NO、氢气等，优选氧气。因此，本发明所述的充气脂质GV包括但不限于充氧脂质GV、充NO脂质GV、充氢脂质GV等，优选充氧脂质GV。
本发明的第十方面提供了一种治疗剂，其包含本发明第八方面所述的脂质GV或第九方面所述的充气脂质GV或由其组成。
本发明的第十一方面提供了一种造影剂或诊断剂，其包含本发明第八方面所述的脂质GV或第九方面所述的充气脂质GV或由其组成。
本发明的第十二方面提供了一种试剂盒，其包括本发明第八方面所述的脂质GV、第九方面所述的充气脂质GV、第十方面所述的治疗剂或第十一方面所述的造影剂或诊断剂，以及任选地使用说明书。
本发明的脂质GV、充气脂质GV、造影剂或诊断剂、治疗剂或试剂盒均可用于诊断或治疗疾病，例如缺氧性疾病例如癌症。因此，本发明还提供了第八方面所述的脂质GV或第九方面的充气脂质GV(例如充氧脂质GV)或第十方面所述治疗剂在制备用于治疗受试者中的疾病例如癌症的药物中的用途。本发明还提供了第八方面所述的脂质GV或第九方面的充气脂质GV(例如充氧脂质GV)或第十一方面所述的造影剂或诊断剂在制备用于诊断受试者中的疾病例如癌症的诊断试剂中的用途。本发明还提供了第八方面所述的脂质GV或第九方面的充气脂质GV(例如充氧脂质GV)或第十一方面所述的造影剂在制备用于使受试者的肿瘤成像的诊断试剂中的用途。当将所述脂质GV、充气脂质GV或造影剂用于诊断疾病或使肿瘤成像的用途时，还包括对受试者应用超声造影，以确定癌症的存在或不存在。在一些实施方案中，所述药物可以与第二治疗剂例如其它癌症治疗药物或癌症治疗方法联合，以治疗癌症。在一些实施方案中，所述癌症治疗方法是例如化学疗法、放射疗法，优选PDT或SDT。所述其它癌症治疗药物可以是本领域技术人员熟知的用于治疗各种癌症的药物。
本发明的第十三方面提供了一种治疗受试者中的疾病例如癌症的方法，包括向所述受试者施用第八方面的脂质GV或第九方面的充气脂质GV(例如充氧脂质GV)或第十方面的治疗剂或将第十二方面所述的试剂盒应用于所述受试者。在一些实施方案中，所述方法还包括，在将所述脂质GV或充气脂质GV或治疗剂施用给受试者后(例如施用后1min、5min、10min、15min、30min、1h、2h、3h、4h、5h、6h、甚至1天、2天、3天、4天、5天、6天或7天，以及上述数值之间的任何时间点)，向所述受试者施用第二治疗剂或疗法，例如其它癌症治疗药物或癌症治疗方法(例如化学疗法、放射疗法，优选PDT或SDT)的步骤。所述其它癌症治疗药物可以是本领域技术人员熟知的用于治疗各种癌症的药物。在一些实施方案中，所述方法还包括，在将所述脂质GV或充气脂质GV或治疗剂施用给受试者后并在应用其它癌症治疗药物或癌症治疗方法之前，向所述受试者应用超声波，从而将所述脂质GV或充氧脂质GV破坏，使其中的气体释放出来。
本发明还包括一种载体，其包含上述脂质GV或充气脂质GV(例如充氧脂质GV)或由其组成。所述载体还可以包含用于治疗疾病例如癌症的药物(例如本领域已知的用于治疗癌症的药物)或气体例如任何治疗性气体(例如上文所定义的治疗性气体)。本发明上述方面所述的癌症可以是任何会导致缺氧状态的肿瘤，例如乳腺癌，膀胱癌、 肺癌、肾癌、胃癌、结直肠癌、肝癌、黑色素瘤等。
本发明的第十四方面提供了一种脂质GV的制备方法，包括以下步骤：(a)将脂质分子或脂质分子的混合物溶于氯仿中，并使其干燥；(b)向步骤(a)的产物中加入HEPES缓冲液(优选pH＝7.2)并搅拌，形成浑浊溶液；以及(c)将步骤(b)的产物加入到GV溶液中，从而形成脂质GV。在一些实施方案中，所述GV溶液是GV的PBS溶液。在一些实施方案中，步骤(a)中的所述脂质分子为DSPE-PEG和DOPC的混合物。在一些实施方案中，所述步骤(b)还包括对形成的溶液进行超声处理。在一些实施方案中，所述步骤(c)还包括向溶液中加入HEPES缓冲液，并温育过夜。
本发明的第十五方面提供了一种充气脂质GV的制备方法，该方法与本发明第十四方面的方法类似，不同之处在于步骤(c)包括：在将步骤(b)的产物加入到GV溶液中之前，向所述GV溶液中充气(例如氧气)直至饱和。
本发明的第十六方面提供了一种脂质靶向GV，其表面被脂质分子和生物靶向材料(以及任选地，生物相容性材料)修饰。例如，所述脂质分子选自以下中的一种或多种：DSPE-PEG、DOPC、PEG、DSPE、PVA、甘油、甘油酯、脂肪酸、磷脂；所述生物靶向材料选自以下中的一种或多种：HA、RGD肽、叶酸、半乳糖、葡萄糖；以及，任选地，所述生物相容性材料选自以下中的一种或多种：PEG、壳聚糖、聚氨基酯、聚乳酸、聚烯烃、聚砜、聚碳酸酯、聚丙烯腈。优选的，所述脂质靶向GV的表面通过DSPE-PEG、DOPC和HA进行修饰，更优选地，所述脂质靶向GV的表面被DSPE-PEG、DOPC、HA和PEG修饰。进一步的，本发明还提供了一种充气脂质靶向GV，其包括本发明所述的脂质靶向GV和气体，例如所述气体可以是任何气体种类，例如治疗性气体。所述气体被填充在GV的蛋白质壳中并被所述脂质分子包封。本发明所述的治疗性气体包括本领域已知的有助于治疗、缓解、减轻疾病或疾病症状或抑制其进展的任何气体，包括但不限于：氧气、NO、氢气等，优选氧气。本发明所述的脂质靶向GV或充气(例如充氧)脂质靶向GV可作癌症治疗剂、诊断剂、造影剂和药物载体。因此，本发明还提供了本发明所述的脂质靶向GV或充气脂质靶向GV在制备用于治癌症的药物中的用途、或制备用于诊断癌症的诊断试剂中的用途。本发明所述的脂质靶向GV或充气脂质靶向GV也可以与本发明上面定义的其它癌症治疗方法或癌症治疗药物联合应用。所述癌症可以是本文上面定义的癌症。
本发明的第十七方面提供了一种脂质靶向GV的制备方法，其与本发明第十四方 面所述的方法类似，不同之处在于在步骤(c)包括：在将步骤(b)的产物加入到GV溶液中之前，将生物靶向材料如HA与GV连接在一起。
本发明的第十八方面提供了一种充气脂质靶向GV的制备方法，其与本发明第十五方面所述的方法类似，不同之处在于在步骤(c)包括：在向所述GV溶液中充气(例如氧气)之前，将生物靶向材料如HA与GV连接在一起。
本发明的第十九方面提供了一种药物组合物，其包含本发明任何方面的靶向GV、脂质GV、充气GV、靶向脂质GV或充气靶向脂质GV。
本发明还提供了使用本发明任何方面的靶向GV、脂质GV、充气GV、靶向脂质GV、充气靶向脂质GV或药物组合物治疗受试者的癌症的方法。本发明还提供了本发明任何方面的靶向GV、脂质GV、充气GV、靶向脂质GV、充气靶向脂质GV或药物组合物在制备用于治疗受试者的癌症的药物中的用途；或者其与第二治疗剂或疗法在制备用于治疗受试者中的癌症的药物组合中的用途。本发明的靶向GV、脂质GV、充气GV、靶向脂质GV或充气靶向脂质GV还可用作诊断剂、造影剂、治疗剂或药物载体。在一些实施方案中，所述方法包括向所述受试者施用上述GV或药物组合物，然后向所述受试者施用第二治疗剂或疗法例如其它癌症治疗药物或癌症治疗方法；可选地，所述方法还包括在施用第二治疗剂或疗法之前向所述受试者应用超声波。在一些实施方案中，所述药物组合以如下方式实施：向所述受试者施用上述GV或药物组合物，然后向所述受试者施用第二治疗剂或疗法例如其它癌症治疗药物或癌症治疗方法；以及可选地，在施用第二治疗剂或疗法之前向所述受试者应用超声波。所述其它癌症治疗药物或癌症治疗方法如上文所定义，且所述癌症如上文所定义。
在本发明的任何方面，所述受试者可以是人类、非人灵长类动物、哺乳动物、啮齿动物等，优选人类。
附图说明
应理解，本文描述的附图仅用于示例目的。附图不一定按比例绘制，重点在于阐释本发明所教导的原理。附图不意味着以任何方式限制本发明的范围。
图1描绘了天然GV和脂质GV的形态、粒径分布和ζ电位。(A)1nM GV的图像。(B)GV的TEM图像，示出了其形态(图像是代表性的)。比例尺，100nm。(C) GV的粒径分布和ζ电位统计。(D)两个GV组的相对粒径分布。(E)通过测量其浓度，观察GV组在6个月内的稳定性。(F)通过测量其尺寸，观察GV组在6个月内的稳定性。(E)和(F)中的数据代表3个独立实验的平均值±标准偏差。
图2描绘了GV和PH-GV的表征。(a)GV和PH-GV的TEM图像。由于PH-HA对GV的包裹和叠加，可以发现GV的表面已被重物质包裹。比例尺，200nm。(b-c)在pH 7.4的PBS中，GV和PH-GV的ζ电位和粒径的动态光散射分析。(d)PBS缓冲液及不同浓度的GV和PH-GV(浓度范围为125-1000pM)的多普勒超声成像(dropper phantom)。如所指示的，在B模式和对比模式下获取图像。(e)d中图像的定量分析。(f)放置不同时间后GV和PH-GV的多普勒超声成像(GV浓度为500pM)。(g)f中图像的定量分析。
图3描绘了GV的携氧能力的测定结果。(A)在持续通入氧气一分钟后，不同浓度的天然GV中的氧浓度。数据代表3个独立实验的平均值±标准偏差。*p<0.05 vs.对照。(B)在持续通入氧气一分钟后，不同浓度的脂质GV中的氧浓度。数据代表3个独立实验的平均值±标准偏差。*p<0.05 vs.对照。
图4描绘了GV的氧气释放动力学的测定结果。(A)在注射1ml的1nM充氧-GV/充氧-脂质-GV后，在5ml严重缺氧溶液中氧浓度的变化。数据代表3个独立实验的平均值±标准偏差。(B)在注射1ml的1nM充氧-GV/充氧-脂质-GV后，在5ml严重缺氧溶液中氧浓度的变化，其中在5分钟时进行超声处理长达10秒钟。数据代表3个独立实验的平均值±标准偏差。*p<0.05对vs.对照。**p<0.01 vs.对照。
图5描绘了充氧-GV介导的体外和体内氧气输送。(A)将200μl的充氧-PBS、充氧-脂质GV、充氧-GV(最终浓度为1nM)分别加入到培养基中，并与细胞在缺氧条件下培养1小时后，使用缺氧试剂检测到的培养细胞中的缺氧状况的代表性图像，其中荧光信号表示缺氧的发生。(B)缺氧染色的定量。数据代表3个独立实验的平均值±标准偏差。*p<0.05 vs.对照。比例尺，25μm。(C)通过尾部注射分别将200μl的5nM充氧-GV、充氧-PBS、充氧-脂质GV注射到荷瘤小鼠的血管中，通过光声成像监测肿瘤部位中的氧-Hb(氧合血红蛋白)水平和脱氧-Hb(脱氧血红蛋白)水平随着时间的变化。(C)显示肿瘤含氧量在不同时间点的代表性光声图像。红色像素：氧-Hb；蓝色像素：脱氧-Hb。(D)肿瘤含氧量的定量。数据代表3个独立实验的平均值±标准偏差。*p<0.05 vs.对照。
图6描绘了GV在体外和体内的毒性。(A、B)将200μl的PBS、GV、和脂质GV(最终浓度分别为1nM)分别加入到培养基中并与细胞一起培养，分别通过MTT和LDH测定在不同时间点测量SCC-7细胞的细胞增殖和LDH毒性。数据代表3个独立实验的平均值±标准偏差。(C)在GV静脉注射前后小鼠健康状况的得分。以30分的量表对小鼠评分，该量表包含活动、体重和食物摄入，各10分。分别在注射前、注射后立即、注射后24小时、48小时和一周的时间点进行评估(N＝3，±标准偏差)。(D)在第7天从GV处理的小鼠收集的经H&E染色的主要器官的组织学图像。比例尺，100μm。
图7描绘了体外PDT的光学设置。
图8描绘了经不同处理的MCF-7细胞在PDT之后的细胞活性。通过CCK-8测定来确定PDT对MCF-7细胞的体外细胞毒性。数据代表3个独立实验的平均值±SEM。**p<0.01 vs.对照。
图9描绘了不同处理对细胞凋亡的影响。上面的图：通过流式细胞术，借助于膜联蛋白-V和碘化丙啶(PI)双重染色，对PDT后的细胞凋亡进行评估。下面的图：评估了早期凋亡细胞(膜联蛋白-V +/PI -)和晚期凋亡细胞(膜联蛋白-V +/PI +)占总细胞的百分比。数据代表3个独立实验的平均值±SEM。*p<0.05 vs.对照。
图10描绘了在不同处理后细胞内ROS的量。通过DCFH-DA对细胞内ROS染色并且通过流式细胞术对其进行分析。左图：流式细胞分析结果，横坐标表示荧光强度，纵坐标表示细胞数量；右图：左图所示结果的统计分析。数据代表三个独立实验的平均值±SEM。**p<0.01 vs.对照。
图11描绘了在超声辐照后PBS(对照)、GV、PpIX和GV+PpIX的SOSG荧光强度的增加倍数。数据表示为平均值±标准偏差(n＝3)。PpIX和GV的浓度分别为10μM和2nM。
图12描绘了在有和没有超声处理的情况下，用PBS(对照)、GV、PpIX、PpIX+GV处理后细胞内H 2DCFDA的荧光显微图像。比例尺＝20μm。激发光波长为488nm。PpIX和GV的浓度分别为10μM和2nM。
图13描绘了(a)GV和PH-GV的细胞摄取。SCC-7细胞中ICG标记的GV和ICG标记的PH-GV的共聚焦显微镜图像。比例尺，20μm。(b)GV和PH-GV的免疫逃逸 能力。在荧光显微镜下，在鼠RAW 264.7巨噬细胞中的细胞摄取。(c、d)以浓度为0.031-1nM的GV、崩塌的GV和PH-GV处理24小时(c)和48小时(d)后，对SCC-7细胞活力的影响的测定结果。将用PBS处理的细胞作为对照。
图14描绘了在对荷瘤小鼠进行静脉内施用后GV和PH-GV的分布。(a)在静脉分别注射ICG标记的GV和ICG标记的PH-GV后，在不同的时间(单位：小时)对SCC-7荷瘤小鼠进行的体内NIR荧光成像。圆形指示肿瘤位置。(b)SCC-7荷瘤小鼠模型在不同时间的肿瘤/肌肉(T/M)比。(c)在分别注射ICG标记的GV和ICG标记的PH-GV4、12、24和48小时后取自荷瘤裸鼠的主要器官和肿瘤的离体荧光成像。(d)ICG标记的GV和ICG标记的PH-GV在肿瘤和主要器官中的积聚的定量分析。误差条表示每组5只小鼠的标准偏差。*，P＜0.05。
图15描绘了GV在肿瘤部位产生的超声造影。(a)在静脉注射GV和PH-GV后肿瘤的体内超声图像。绿色表示由于GV引起的强度增强区域。(b)对(a)中每个感兴趣区域的超声信号进行的定量分析。(c)破坏性声波处理(insonation)(650kPa)引起的GV破碎及超声信号的消失。
图16描绘了(a)在注射ICG标记的GV和ICG标记的PH-GV后12小时从小鼠收集的肿瘤切片的共聚焦图像。绿色信号和红色信号分别来自ICG和CD31染色血管内皮细胞的荧光。(b)在处理30天后主要器官(心脏、肝脏、脾脏、肺和肾脏)和肿瘤的代表性H&E切片。比例尺，100μm。(c)在对小鼠进行的30天的评估期期间，在不同条件下测量的体重。
图17描绘了空化反应实验平台和结果，(a)GV介导空化反应实验平台俯视图；(b)GV和PBS溶液信号频谱。
图18描绘了根据本发明的一些实施方案的表面修饰的靶向GV在肿瘤的诊断、成像和治疗中的应用示意图。
具体实施方式
先前的研究已经成功地证明，GV可以在皮摩尔浓度的频率范围产生强烈的超声造影，表现出谐波散射，从而能够增强相对体内背景的检测。然而，与其它纳米粒子相似，在静脉内施用后，大多数GV在非靶向组织(诸如，肝脏、脾脏和肺)中被摄 取。纳米粒子的药代动力学和生物分布部分地是由其自身的表面特性决定的。而且，亲水性涂层对于纳米粒子成功的静脉内施用是必需的。本发明人发现，用生物相容性材料对GV进行化学表面修饰可以改善GV的药代动力学特性和靶向性，从而增加其在体内分子成像中的肿瘤积聚。这种表面修饰也有望增强GV，使其成为药物递送的良好载体。
在本发明中，为了提高稳定性和靶向性，我们用生物材料来修饰GV纳米粒子的表面功能，经由氨羧反应成功合成了双修饰的GV。出人意料地，发明人发现用生物材料对GV表面进行化学修饰大大提高了GV在肿瘤部位的积聚，因而可作为超声造影剂或成像剂用于体内肿瘤特异性超声分子成像。修饰的GV可以穿过肿瘤内皮层中的空隙并进入肿瘤组织间隙。这些结果表明，这些具有表面修饰的新型纳米级超声造影剂在肿瘤部位表现出优异的生物相容性、长血液循环和卓越的超声造影能力。本发明为将GV应用于肿瘤的有效血管外靶向超声分子成像以及肿瘤治疗提供了实验证据。
在一些实施方案中，通过具有良好生物相容性和靶向性的生物材料例如聚乙二醇(PEG)-缀合的透明质酸(HA)来修饰GV的表面。GV表面上的PEG有效地减少RES摄取并增加在血液中的循环时间，从而导致纳米粒子选择性地积聚到肿瘤部位。HA是一种生物相容性的天然材料，其是结缔组织的主要细胞外成分，并且对CD44阳性恶性癌细胞显示出内在的靶向性。本发明通过利用聚乙二醇化的HA来使GV(PH-GV)粒子功能化，并且应用超声和荧光成像、免疫组织化学切片和共聚焦显微镜技术等来间接地和直接地证明修饰GV进入肿瘤组织的细胞内间隙的能力，证明了本发明的新型纳米级成像剂可以穿过肿瘤内皮层的缝隙并进入肿瘤组织间隙，因而可应用于特异性的、有效的超声分子成像和癌症治疗。
如前所述，认为肿瘤氧合有助于癌症治疗，而充氧微泡可以帮助将氧气输送到感兴趣的组织中，从而改善治疗效果。然而，由于稳定性的限制和相对较大的尺寸，现有充氧微泡的应用受到了限制。发明人发现，在PDT疗法中，oxyGV，特别是经脂质分子修饰的oxyGV，能够提供对肿瘤增强的细胞毒性，在体内和体外对PDT的疗效均具有显著的增强效应。因此，本发明的新型纳米尺寸oxyGV可以作为有效的氧气载体来提高耗氧PDT的疗效。本发明还研究了oxyGV在体外和体内改善声动力疗法的功效的能力，证明了oxyGV，特别是经脂质分子修饰的oxyGV，可以在无细胞系 统中引起增强的空化和ROS的过量产生，并可以增强体外ROS的产生，从而导致SDT的细胞毒性增强。因此，oxyGV可用作治疗剂来提高SDT的疗效。
在本发明中，术语“天然GV”是指从自然界例如藻类中分离出来的没有经过任何修饰的纳米级气囊，而“气囊”在本文中有时也称为“纳米粒子”。本发明中涉及的GV包括天然GV、脂质GV、PH-GV、充氧-GV、充氧-脂质GV等类型。“脂质GV”是指经脂质分子(例如DOPC、DSPE、PEG等)进行表面修饰的GV。”PH-GV”与“PH修饰GV”可互换使用，是指经PEG和HA进行表面修饰的GV。术语“充氧-GV”或“oxy-GV”可互换使用，均指其中充有氧气的GV。类似地，“充氧-脂质GV”、“经脂质分子修饰的oxyGV”或“oxy-脂质GV”等也是可互换使用的，其指的是其中充有氧气的经脂质分子进行表面修饰的GV。
在本发明中，“表面修饰GV”是指因表面被(例如脂质分子、靶向性材料、生物相容性材料等)修饰而具有特定功能(例如靶向性、亲水性等)的一类GV，例如本发明的PH-GV、脂质GV均属于表面修饰GV。
实施例
统计分析
使用SPASS 17.0软件，经由独立样本单因素ANOVA测试来分析各组之间的比较。所有统计数据均使用两尾学生t测试和方差齐性检验来获得(p值<0.05认为是显著的)。
实施例1：GV和脂质-GV的制备和表征
GV的制备
在25℃，将水华鱼腥藻(FACHB-1255，中国科学院水生生物研究所的淡水藻种库)在无菌BO-II培养基中在14小时/10小时明/暗工作循环的荧光照明下进行培养。根据Walsby的方法(Buckland和Walsby，1971)来分离GV。简而言之，使用通过快速添加蔗糖溶液至最终浓度为25％而实现的高渗溶解(hypertonic lysis)来溶解藻类，以从藻类中释放出GV。然后，在600g离心3小时以分离GV。分离出的GV可以在溶液顶部形成白色乳状层，其可以通过注射器来收集。通过离心用磷酸盐缓冲盐水(PBS)纯化GV三次并在4℃储存在PBS中。
使用基于文献的公式(每OD500 450nM)(Walsby，1994)估算GV浓度，其中，OD500是用紫外-可见光分光光度计(2100Pro；GE Healthcare，美国新泽西州皮斯卡塔韦市)在500nm的波长处测得的光密度。如之前所描述(Walsby和Armstrong，1979)的，使用大约8.4μL/mg的气体体积和107MDa的摩尔量来估计体积分数。使用在200kV下操作的透射电子显微镜(TEM)(JEOL 2100F；JEOL，日本东京)对GV形态进行成像。将去离子水中的GV样本(0.5nM)沉积在涂有碳的栅格上，并在室温下干燥过夜。使用动态光散射(DLS)方法获得流体动力学尺寸。
脂质GV的制备
在25ml的圆底烧瓶中，将总量为1μmol的含有DOPC、DSPE-PEG(2000)(4:1)的脂质混合物溶于约100μL的氯仿中。然后使溶剂蒸发并使样本在真空旋转蒸发仪中干燥。然后将1mL的20mM HEPES缓冲液(pH＝7.2)加入至脂质干燥层，在剧烈搅拌后形成浑浊溶液。然后将混合物进行超声波处理(20W，15秒脉冲，持续20分钟，每个脉冲间隔30秒的时间)，持续3-5分钟，直到溶液变澄清为止。将所得脂质体溶液储存在4℃直至进一步使用。将体积为0.5mL的脂质体制备溶液加入至GV溶液中。同样用20mM HEPES缓冲液将体积增加至1mL。混合溶液在摇床上温育过夜。之后，通过离心(2.4krpm，10分钟)将GV洗涤三次并且重悬于Milli-Q水中。最后，将脂质GV重悬于PBS中并准备使用。
粒径分布和ζ电位确定：使用90Plus仪器(Brookhaven，美国纽约亨茨维尔)在90°固定角度和25℃的温度，通过激光散射来测定脂质GV的粒径分布和ζ电位。
形态分析：通过透射电子显微镜(TEM)在200kV的操作电压下测定脂质GV的尺寸和形态。将脂质GV(OD 0.1)的样本沉积在涂有碳的弗姆瓦(formvar)栅格上，并用2％的乙酸双氧铀染色。
粒子稳定性：为了测量脂质GV制剂的稳定性，分别在制造后的第1、3和7天测定脂质体包封的GV制剂的单粒径和浓度。
我们通过培养藻类水华鱼腥藻来生产用于本研究的GV，并通过离心分离GV。由于GV的壳对于气体分子是可渗透的，这可能会影响其氧气输送效率。因此，我们制备了两种GV：天然GV和使用脂质进行表面改性以减少气体交换的GV(脂质GV)(C.Zhang等,Colloids Surf.B Biointerfaces 160(2017)345–354；C.Zhang等,ACS Appl. Mater.Interfaces 10(1)(2018)1132–1146)。当在溶液中(图1A)或以通过TEM观察到的单个囊泡的形态呈现(图1B)时，天然GV和脂质GV没有可见的明显差异。接下来对脂质GV进行粒径分布和ζ电位的表征。我们发现，这些纳米气泡具有约300-330nm的平均直径和均匀的分布(图1C-图1D)。脂质GV的平均粒径约为290-330nm(图1C)或约310-330nm(DLS测量，图1D)，比天然GV大约10nm。脂质GV的ζ电位为约-21.3至-19.3mV，其负电荷较GV低(图1C)。我们还评估了GV在冷藏(4℃)中的稳定性，观察了两组(GV组和脂质GV组)在零到六个月的时间段中的浓度(由OD 500确定)和尺寸，发现两组均无显著变化(图1E-图1F)。因此，我们能够生产出纳米级的带负电荷的GV，其在溶液中储存能够长期保持稳定。
实施例2：HA-GV和PH-GV的制备和表征
HA-GV(H-GV)的制备
通过共价缀合将HA固定至GV的蛋白质壳。对于HA-GV合成，首先将EDC(3.37mM)和NHS(2mM)加入到0.1M磷酸钠中的HA溶液(10mg)中(pH＝7.4)。将溶液混合物在冰浴上搅拌2小时，并向其中滴加6mL溶解在磷酸钠中的GV(pH＝7.4)。将反应混合物在4℃再搅拌24小时。将所得混合物加入到超滤管(50mL)中，并以1.8rpm离心5分钟以去除游离的EDC、NHS和HA。将所得的纳米粒子储存在4℃的PBS缓冲液中。
聚乙二醇化HA-GV(PH-GV)的制备
通过在EDC和NHS的存在下改变PEG与HA的进料比，通过酰胺形成，将PEG化学缀合至H-GV缀合物。将H-GV缀合物(120mg)溶解在磷酸盐缓冲液(PBS，pH7.2)中，将其与PBS中的EDC(3.37mg)和甲醇中的NHS(2mg)混合。在缓慢加入单甲氧基PEG-胺(73.5mg)后，将混合物在冰浴上搅拌24小时。然后将所得溶液离心，并用PBS洗涤4次，以去除过量的甲醇和PEG。
对于细胞实验和动物成像测试，在加入HA和PEG之前的第一步，用NIR染料(ICG)标记GV。简而言之，将EDC(3.37mg)和NHS(2mg)加入到0.1M磷酸钠中的ICG溶液(1mM)中(pH＝7.4)。在室温下反应30分钟后，将该溶液加入到纯GV溶液中(摩尔比：ICG/GV＝1000/1)。然后在室温下将混合物摇动4小时，然后通过离心4次进行纯化。将所得混合物加入到超滤管(50mL)中，并以1.8rpm离心5分钟以去除游 离的ICG。将所得的纳米粒子储存在PBS缓冲液中。
通过动态光散射DLS(Varian，美国帕洛阿尔托)测量PH-GV NP的粒径和粒径分布。在25℃，在Malvern Zeta Size-Nano Z仪器上执行ζ电位测量。通过透射电子显微镜(TEM)(Bruker，德国)来观察PH-GV的纳米结构和尺寸。通过Multiskan Go酶标仪(Thermo Fisher Scientific，美国马萨诸塞州)来观察GV和PH-GV的紫外-可见光吸收光谱。使用荧光分光光度计(Varian，美国帕洛阿尔托)来测量ICG和ICG标记的PH-GV的荧光信号。
GV和PH-GV的超声信号测试：对含有相同GV量的PH-GV和GV进行US成像。在成像前将PH-GV和GV放入滴管(5mL)中，并将所有滴管以相同深度浸入去离子水中。使用带有LZ250D换能器的高频超声系统来获取GV溶液的超声B模式图像和对比模式图像。将中心频率和输出能量水平分别设定为18MHz和4％。
为了实现最佳稳定性和可靶向性，我们修饰了GV纳米粒子的表面功能。经由氨羧反应成功合成了双改性的GV。首先，用透明质酸(HA)修饰GV表面(透明质酸是一种由N-乙酰基葡萄糖胺和葡萄糖醛酸重复单元组成的多糖)，以提高GV的生物相容性和靶向性。然后在EDC和NHS的存在下通过酰胺形成将PEG-5000容易地附着到HA缀合物的骨架上，这可以通过阻碍与血浆蛋白的非特异性相互作用而使HA-GV从宿主的免疫系统中隐匿，因而可以通过减少网状内皮系统(RES)清除来延长其循环时间，并且可以经由亲水性PEG链的空间排斥来增强胶态分散体的稳定性。PH-GV的成功合成已通过透射电子显微镜(TEM)证实。如图2a所示，TEM显示了平均粒径为400nm的裸GV的圆柱状形态。由于PH-HA对GV的包绕和叠加，我们可以发现GV的表面已被重物质包裹(图2a)。根据动态光散射(DLS)报告，GV的粒径为约320-380nm，PH-GV约为375-425nm；GV的ζ电位在-40至-50mV的范围，PH-GV的电位为约-27±4mV(图2b)。
此外，通过利用PEG的涂层，聚乙二醇化HA-GV的ζ电位由于PEG层的屏蔽功能而降低。如图2a-b所示，PH-GV具有圆柱状形态，半径约为400nm，并且所有纳米颗粒均为圆柱状并显示出相对均匀的尺寸分布。PH-GV纳米颗粒的合适粒径和负ζ电位确保了经由EPR效应实现良好的肿瘤靶向货物输送，同时减少RES清除。
鉴于GV和PH-GV具有很强的超声特性，因此通过高浓度(tonic)细胞溶解和离心辅助浮选纯化了来自水华鱼腥藻(Ana)的GV，并在不同浓度下在21MHz下操作的 Vevo 2100成像系统(FUJIFILM VisualSonics，加拿大安大略省多伦多市；B模式)中使其成像(对于成像细节，参见下面实施例)。在浓度范围为250pM至1nM(图2c-2d)并且气体体积分数为约0.01％至0.1％的情况下，GV相对于缓冲液对照产生了强烈的对比。对比在最高浓度下最强，Ana GV产生的散射比缓冲液对照大23.4±2.5(图2e)。并且，在进行PEG和HA修饰后，GV的超声性能不受影响。在进行下一步试验之前，我们在PBS和胎牛血清(FBS)中鉴定了PH-GV的生物稳定性。GV和PH-GV的在放置超过一周的时间后仍具有很好的超声成像能力(图2f)，并且显示出良好的稳定性(图2g)。
实施例3：GV和脂质GV的携氧特性和氧气释放动力学的测定
使用血氧计(Portamess 913 OXY；Knick，德国)监测溶液中的氧浓度，并将数据记录为mg/l。用氧气/氮气对不同浓度的GV和脂质GV进行充气，并且分别测定不同溶液的氧浓度。
无超声作用下氧气释放：通过持续充入N 2将密封在小瓶中的PBS的氧浓度降低至0.8mg/l(严重缺氧)，以模拟缺氧条件。持续充入氧气使PBS、GV和脂质GV中达到氧气饱和。监测氧气从GV/脂质GV中扩散到缺氧溶液中的氧浓度随时间的变化。在每次实验之前，都会对血氧计进行校准。所有实验均重复三次。
超声作用下氧气释放：为研究超声(US)对GV/脂质GV的氧气释放的影响，使用了US探针，其振荡频率为1MHz并且平均声压分布值为2.4±0.2Mpa(标称频率：50Hz；标称功率：30W)。超声处理时间为10秒。检测超声作用后溶液中氧气浓度的变化。所有实验均重复三次。
我们比较了两个GV组(GV和脂质GV)的相对携氧特性。当对这些溶液进行持续充氧后，两组GV的氧浓度均持续增加并且最终变得稳定。我们发现GV的携氧能力远高于PBS和破碎的GV，并且总体携氧能力取决于GV浓度(图3A)。与PBS或破碎的GV相比，脂质GV也显示出相似的较好携氧能力，并且与浓度呈正相关(图3B)。
我们还检测了不同GV的氧气释放动力学。我们发现，与PBS相比，充氧-GV和充氧-脂质GV均可以显著提高严重缺氧溶液的氧浓度(图4A)。与充氧-GV相比，充氧-脂质GV在溶液中释放氧气的速度更慢。我们认为这是由于GV的表面修饰导致降低的氧气释放。此外，充氧-脂质GV的总氧气释放量更大(曲线下面积)，这是由于 GV表面脂质的存在，因为脂质减少了GV的氧气释放。该特性可以在体内长时间运输情况下提高氧气输送效率。因此，脂质GV的携氧能力可与GV媲美，但显示出更慢的释放模式，因而能够更大程度地提高目标部位的氧浓度。我们还添加了10秒钟的超声处理步骤，使用超声来触发更多的氧气释放。超声对PBS的氧气释放没有影响，但是却大大增加了GV组的氧气释放(图4B)。
实施例4：充氧GV和充氧脂质GV输送氧气的能力的测定
在缺氧培养箱(1％的氧气，5％的CO 2)中培养细胞并进行传代。使用购自Thermo Fisher的Image-iT TMRed Hypoxia Reagent(红色缺氧试剂)来监测加入充氧脂质GV之前/之后培养基中的缺氧条件。
体内实验：所有使用实验动物的程序均已获得香港特别行政区政府卫生署和香港理工大学动物受试对象伦理小组委员会的批准。18-20g的雌性Balb/c小鼠由香港理工大学动物资源中心提供。小鼠在到达后适应房间一周，并维持正常的12小时光-暗循环。将小鼠圈养在常规笼子中(每笼6只动物)，在无特定病原体、温度为24±2℃，相对湿度为60-70％的条件下，其可以自由获取标准颗粒饲料和水。将用于小鼠的标准木屑用作垫料。在经过1周的适应后，将0.1ml的SCC-7细胞系(1*10 7细胞/ml)重悬于100μL的基质胶中，并通过皮下注射植入Balb/c小鼠的后背。肿瘤形成发生在细胞植入后约两周，并且在体积达到约200mm 3的阶段，肿瘤表现出明显的缺氧程度。
使用光声成像测量氧/脱氧-Hb水平：小鼠注射充氧脂质GV后，通过具有混合US换能器(中心频率＝21MHz；空间分辨率＝75μm)的Vevo LAZR光声成像仪(Fujifilm Visualsonics，荷兰阿姆斯特丹)来监测皮下肿瘤中的氧-Hb和脱氧-Hb水平。记录并储存pO 2水平以供后面在各组之间进行比较。
进一步评估了各GV组(充氧GV和充氧-脂质GV)在体外和在体内肿瘤块中改变细胞的缺氧条件的能力。我们在缺氧条件下使人乳腺癌MCF-7细胞生长24小时，并使用Image-iT Red Hypoxia Reagent(红色缺氧试剂)监测其缺氧水平。与未经处理的对照和PBS处理组相比，充氧GV和充氧-脂质GV的加入显著降低了所观察到的缺氧水平(图5A-图5B)。
我们还执行了体内概念验证研究，以确定注入尾静脉的充氧-GV提高裸鼠中缺氧 皮下肿瘤氧合水平的能力。通过在处理前和处理后(0、5和15分钟)经由光声成像使氧合血红蛋白(氧-Hb)和脱氧血红蛋白(脱氧-Hb)的水平可视化来监测肿瘤氧合。充氧-GV组和充氧-脂质GV组的注射均导致肿瘤中的氧-Hb水平升高，但PBS对照则未升高(图5C)。此外，与对照相比，加入充氧-GV使肿瘤中的氧饱和度(sO 2)提高20％，但充氧-脂质GV的加入使sO 2提高50％(图5D)。
因此，在我们的初步实验中，在体内环境中，在增加含氧量的情况下，用脂质对GV的表面改性显示出明显更好的结果，证明其稳定性和氧气输送能力增强。
实施例5：体外和体内毒性检测
细胞活力和LDH测定：从中国科学院在上海的细胞库获得人乳腺癌细胞系(MCF-7)。按照标准细胞培养说明，将MCF-7细胞在带有L-谷氨酰胺的高葡萄糖(4.5g/l)杜氏改良伊格尔培养基中进行培养。所有培养基均补充有10％(体积/体积)的胎牛血清、青霉素(100U/mL)和链霉素(100μg/ml)。在37℃，在5％的CO 2和95％的空气气氛中，使细胞生长直至70％-80％汇合，然后进行胰蛋白酶化和收集，以用于细胞培养和体内研究。之后，在不同的时间点(24小时、48小时和72小时)向细胞培养基中加入不同剂量的脂质GV(1nM)。之后，使用Pierce LDH细胞毒性测定试剂盒(Life Invitrogen)根据制造商的说明来进行LDH测定。根据制造商的说明通过MTT测定和凋亡测定来测定细胞活力和凋亡。
体内毒性确定：对经脂质GV处理的小鼠执行尸检，并收集组织样本(肝脏、肺和肾脏)以进行组织学检查。将从小鼠生物测定收集的肝脏、肺和肾脏样本固定在10％的福尔马林缓冲液中，通过常规组织学技术进行处理，并用苏木精和曙红染色。使用配备有照相机(Olympus Q-Color-5)的光学显微镜(Olympus BX51)执行显微镜检查，并使用Image Pro-Express软件将图像记录在计算机中。
为了测试两个GV组(GV和脂质GV)的毒性和生物安全性，我们使用了体外LDH和MTT测定，发现两个GV组均未产生明显的细胞毒性。将GV或脂质GV(最终浓度＝1nM)加入至培养基，使其温育24、48或72小时。与对照相比，GV处理下均未观察到LDH或MTT水平的显著升高(图6A-图6B)。接下来，我们通过观察在GV注射之前、以及在GV注射之后24、48和72小时小鼠健康的三个基本量度(活动、体重和食物摄入)来测试各GV组的体内生物安全性。以30分的量表对小鼠评分，我们观察到这些指标在这段时间内没有减少(图6C)。我们还使用苏木精和曙红(H&E)染色在 GV施用后一周测定小鼠的主要器官(心脏、肝脏、脾脏、肺和肾脏)。与对照组相比，两个GV组的组织切片均未显示出明显的病理异常或病变(图6D)。因此，我们确定GV和脂质GV对细胞没有细胞毒性，并且没有对所测试的小鼠造成任何明显损害。
实施例6：oxyGV与PDT的组合对细胞毒性和细胞凋亡的影响
光动力疗法设置：使细胞以100mW/cm 2的功率暴露于激光，持续5分钟。在图7中示出了用于PDT处理的光学设置。通过光纤生成波长为396nm的光源，将其对准孔径并照射至35mm的细胞培养皿。细胞培养板的位置由双轴电动线性平台手动控制。在处理后，将细胞在新鲜培养基中培养4小时，然后准备用于不同分析。
细胞培养：从上海细胞库获得人乳腺癌细胞系(MCF-7)。在37℃，在5％的CO 2湿润气氛中，使MCF-7细胞在高葡萄糖杜氏改良伊格尔培养基(DMEM，4.5g/L的D-葡萄糖)中生长，该培养基补充有100U/ml含10％胎牛血清(FBS)的青霉素-链霉素。
细胞活力测试：在预备试验中，将MCF-7细胞随机分为八组：(1)对照，(2)仅使用激光，(3)仅使用PpIX，(4)PpIX加激光(PDT)，(5)PpIX+GV+激光(PDT+GV)，(6)PpIX+oxyGV+激光(PDT+oxy-GV)，(7)GV+激光，(8)oxyGV+激光。对于PpIX处理，将细胞与10μM的PpIX在补充有10％的FBS的DMEM培养基中温育4小时的药物加载时间。使用细胞计数试剂盒-8(Sigma-Aldrich)根据制造商的说明来测定在PDT后的不同时间点的细胞活力。简而言之，将细胞以每孔5000个细胞的密度接种在96孔板中，并在100μL的培养基中温育24小时。在37℃，在5％的CO 2中，通过每孔加入10μl的CCK-8试剂持续1小时来测定细胞毒性。在450nm，作为检测波长，测量经处理样本对空白对照的吸光度。通过与对照组中未经处理的细胞进行比较来确定经处理细胞的活力。
细胞凋亡测试：以5×10 5个细胞的密度将细胞接种在6cm培养皿中，温育24小时。使用Alexa Fluor 488膜联蛋白V/死亡细胞凋亡试剂盒(Thermo Fisher Scientific)根据制造商的说明来测量细胞凋亡。收集细胞，并在室温下用5μL的膜联蛋白V缀合物和1μL的PI工作溶液温育15分钟。通过FACS Calibur流式细胞仪和BD Accuri C6软件(Becton-Dickinson，USA)分析细胞。
图8描绘了不同处理对细胞活力的影响。通过CCK-8测定来确定不同处理对MCF-7细胞的体外细胞毒性。数据代表3个独立实验的平均值±SEM。**p<0.01 vs.对 照。
为了确定oxyGV是否可以提高PDT的疗效，如图8所示，在PDT处理后4h，使用CCK-8测定来确定ALA-PDT对MCF-7细胞系的细胞毒性。我们发现ALA-PDT(PpIX+激光)在PDT后4小时使MCF-7细胞的细胞活力明显降低至75％(图8，p<0.05)。在加入oxyGV的情况下，在ALA-PDT处理下，MCF-7细胞的细胞活力降至62％，而天然GV(不含氧气的GV)对ALA-PDT引起的细胞死亡影响很小。同时，其它组(包括仅使用PpIX、仅使用激光、GV+激光、oxyGV+激光)对细胞活力无影响。由于这些组的影响可忽略不计，因此在以下实验中将细胞分为四组：(1)对照，(2)PpIX+激光(PDT)，(3)PpIX+GV+激光(PDT+GV)，(4)PpIX+oxyGV+激光(PDT+oxy-GV)。
接下来，我们通过流式细胞术研究了oxyGV对ALA-PDT引起的细胞凋亡的影响，如图9所示。在图9上图中的每个小图中，左下象限中的细胞代表活细胞，右下象限和右上象限均显示凋亡细胞，并且坏死细胞出现在左上象限。如图9下面的图所示，在对照组、PDT组和PDT+GV组中分别有94.1％、74.7％和79.2％的活细胞。然而，PDT+oxyGV组中仅检测到66.4％的活细胞。这意味着在PDT期间加入oxyGV会导致明显更高的死亡和凋亡率。
实施例7：OxyGV在PDT期间对ROS产量的影响
细胞内ROS测试：使用DCFH-DA(Sigma-Aldrich)测量细胞内ROS产生。简而言之，在37℃，将用PBS稀释的10μM的DCFH-DA加入到MCF-7细胞中，持续20分钟。然后将细胞用PBS洗涤三次。使标记的细胞胰蛋白酶化，并通过流式细胞仪进行分析。
ROS的过量产生被认为是PDT期间造成肿瘤细胞的细胞毒性的原因。因此，我们使用流式细胞术进一步研究了在PDT后细胞内ROS是否增加，结果在图10中示出。正如预期的那样，在所有三个PDT处理组中均检测到过量的ROS产生，而与其它组相比，在加入oxyGV的情况下，总体细胞内ROS显著增加。
实施例8：无细胞系统中单线态氧的生成
SDT设置：在本研究中，使用直径为5cm的1MHz平面超声换能器(Olympus，日本东京)进行超声处理。使用超声发生器(AFG 3251；Tektronix Company，美国俄勒 冈州)和功率放大器(Model 500 A250 CAR；Souderton，美国宾夕法尼亚州)来生成超声脉冲。在25℃，将细胞暴露于距换能器10cm的超声中，其通过无气泡去离子水而耦合。通过水听器(HGL-200；Onda，美国加州森尼维尔市)来表征声强和声场。测得空间峰值时间平均强度为8W/cm 2；固定脉冲重复频率(PRF)为1000Hz时，占空比为20％。总体超声处理持续时间为5分钟。细胞培养实验溶液的温度升高控制在2.5℃内。在超声处理后，将细胞在新鲜培养基中培养4小时，然后准备用于不同分析。
单线态氧的生成：使用SOSG(10μM)来检测单线态氧的生成。将溶液暴露于超声中并在暴露时间内避光。通过酶标仪在488nm的激发波长和530nm的发射波长下测量SOSG的荧光强度。
为了研究GV是否可以增强SDT期间的ROS产生，我们使用SOSG荧光探针在超声辐照下测试了无细胞系统中单线态氧的产生，结果如图11所示。对于单线态氧检测，我们注意到在超声处理前后，单独使用PpIX(10μM)组、单独使用GV(2nM)组和PpIX加GV组的SOSG荧光强度的百分比显著增加。值得注意的是，与单独使用PpIX相比，与PpIX一起加入GV使得产生单线态氧的效率是其两倍以上。这些结果表明，在超声作用下GV和PpIX之间存在协同作用，从而提高单线态氧的产量。
实施例9：GV在SDT期间对ROS产量的影响
试验方法参照上述实施例7。
ROS的过量产生被认为是造成SDT期间对肿瘤细胞的细胞毒性的原因。因此，我们使用荧光成像进一步研究了在SDT后细胞内ROS是否增加，如图12所示。正如预期的那样，在所有三个SDT处理组中均检测到过量的ROS产生，而与其它组相比，在加入GV的情况下，总体细胞内ROS显著增加。
实施例10：空化表征
通过被动空化检测来表征空化强度(见图17)。将平面单元件超声换能器(中心频率＝5MHz，带宽＝4.8-5.2MHz)作为接收器垂直于治疗超声换能器放置并与其共聚焦对齐。使其与1MHz的治疗换能器同步。在每个实验期间，FUS换能器都会向有或没有GV(浓度＝2nM)的培养基发出超声脉冲。超声参数与细胞培养和ROS表征实验中使用的参数保持相同。将GV装入放置在FUS换能器和接收换能器的共聚焦区域处的5mm琼脂-凝胶立方曲面中。在每个FUS脉冲的超声处理期间，通过示波器(LeCroy  715 Zi；LeCroy，美国纽约Chestnut Ridge)获取接收换能器的RF数据。将所获取的RF数据传输到频谱，并通过宽带信号(4.8MHz–5.2MHz)内的积分来计算惯性空化强度。
实施例11：GV和PH-GV的靶向性和免疫逃逸(以下实施例均来自P20914US)
GV和PH-GV的细胞培养和细胞内化：在37℃和5％的CO 2下，将人类鳞状细胞癌细胞系(SCC7细胞)和NIH-3T3细胞在8孔培养箱中在补充有10％的FBS和1％的抗生素溶液的杜氏改良伊格尔培养基(Dulbecco's Modified Eagle Medium，DMEM)/高葡萄糖培养基中培养。第二天，用冷PBS洗涤两种细胞，并在5％的CO 2气氛下，在37℃，将其与IGV一起温育4小时。在温育后，所有细胞均用冷PBS彻底洗涤。最后，在-20℃，将细胞在冷乙醇中固定15分钟，然后用含有DAPI的封固剂将细胞在黑暗中封固10分钟。通过共聚焦显微镜(Olympus，USA)观察ICG标记的PH-GV和ICG标记的GV的细胞内化，并且将ICG的激发波长和发射波长分别设定为780nm和800nm。
为了研究PH-GV对CD44高表达肿瘤细胞的靶向效率，将其用NIR荧光团ICG标记，然后与SCC7癌细胞系和NIH3T3细胞一起进行温育。与低表达细胞系(NIH3T3)相比，该研究中使用的癌细胞已被证明在其表面上过表达CD44。然后使用共聚焦激光扫描显微镜(CLSM)检查在预定时间点固定的细胞。图13a显示了SCC7细胞在用PBS溶液、ICG标记的GV和ICG标记的PH-GV纳米粒子温育6小时后的CLSM图像。如CLSM图像所示，PH-GV纳米粒子的ICG细胞内分布与GV溶液有很大不同。在用ICG标记的PH-GV溶液温育6小时后，ICG的大量荧光分布在整个细胞质中，而在用ICG标记的GV处理的SCC7细胞中，则未在细胞质中观察到强烈的红色荧光。
通过荧光显微镜进一步评估了鼠RAW 264.7巨噬细胞中PH-GV和GV纳米粒子的细胞摄取(图13b)。PBS用作对照。捕获的图像显示，游离GV在具有强烈红色荧光的巨噬细胞中被广泛内化。然而，在PH-GV纳米粒子组中，来自巨噬细胞的红色荧光信号减少。FACS分析表明，与游离GV组相比，巨噬细胞中PH-GV纳米粒子的内化减少了约40％。因此，PH-GV纳米粒子的PEG表面修饰可以明显减少巨噬细胞的内化。
实施例12：GV和PH-GV的生物安全性
根据动物研究协议，当肿瘤体积达到2000mm 3时处死动物。为了对比处理效果，在注射2天后收集所有组中的一部分肿瘤用于苏木精-曙红(H&E)染色。
为了评估GV的生物安全性，在处理14天后收集主要器官(心脏、肝脏、脾脏、肺、肾脏)并通过H&E染色进行检查。
在SCC7癌细胞中在不同浓度(GV，0-1nM)下研究了GV和PH-GV纳米粒子对细胞毒性和细胞凋亡的影响。如图13c所示，在温育24小时后，即使在最高浓度(1nM)下，也几乎没有检测到游离GV和PH-GV纳米粒子对细胞的毒性。并且崩塌的GV也没有引起SCC7癌细胞的明显细胞毒性。
为了进一步研究GV和PH-GV的安全性，我们将SCC7细胞的温育时间延长至48小时。图13d表明，游离GV、崩塌的GV和PH-GV纳米粒子这三组未以浓度依赖方式引起明显的细胞死亡。因此，预期GV和PH改性GV在转移性SCC7癌细胞中的毒性是可忽略不计的。
实施例13：GV和PH-GV在裸鼠体内的生物分布
根据香港理工大学动物护理和使用委员会(CC/ACUCC)批准的协议来执行动物实验。向无胸腺裸鼠(七周龄，雌性，20-24g)的皮下部位注射4×10 6SCC7细胞的PBS悬浮液(80μL)。当肿瘤尺寸(在右腿区域)达到120mm 3的平均尺寸时，将小鼠随机分为三组：(a)将游离的ICG溶液注入小鼠的尾静脉中；(b)将ICG标记的GV溶液注入小鼠的尾静脉中；以及(c)将ICG标记的PH-GV溶液注入小鼠的尾静脉中。在注射后的0、0.5、1、2、4、8、12、24和48小时，使用IVIS Lumina II(Caliper Life Sciences，USA；激发滤光片：780nm，发射滤光片：800nm)来获取荧光图像。
在一剂注射后达到最高积聚量时，收集SCC7肿瘤和正常器官(心脏、肝脏、脾脏、肾脏、肺和肌肉)，将其用于获取荧光信号强度。
由于GV在体外实验展现出的良好的靶向功能和免疫逃逸的能力，我们将ICG标记的PH-GV注射入荷瘤小鼠内，使用实时NIRF成像技术进一步评估了纳米粒子的体内生物分布和肿瘤靶向特性。如图14a-b所示，对于游离的ICG组和ICG标记的GV组，在早期时间点(0.5-2小时)，在小鼠的正常器官(肝脏、肺、脾脏等)中检测到了大量的荧光信号，这是由于网状内皮系统(RES)在这些器官中的捕获所致，然后这些荧 光信号随时间逐渐减少。相反，ICG标记的PH-GV在整个小鼠体内产生一致的荧光信号达8小时，而正常器官中没有高荧光信号增加。并且在静脉注射后8小时在肿瘤部位观察到明显荧光。ICG标记的PH-GV荧光稳定增加，并在12小时达到峰值。随后，肿瘤部位的荧光从24小时到48小时逐渐减少，表明PH-GV从肿瘤组织中持续排出。然而，在施用ICG和ICG标记的GV后，在肿瘤组织中未发现强荧光信号。这种鲜明差异可能归因于PH-GV增强的免疫逃逸和特异性肿瘤靶向能力。
为了研究表面修饰后的GV是否可以有效减少肝脏摄取并改善在肿瘤中的积聚，在SCC7荷瘤模型中研究了表面修饰后的GV的体内靶向能力。如图14c-d所示，对于ICG标记的GV处理组，在体内注射后4小时在肝脏中检测到高荧光。即使在处理后12小时，肝脏中仍存在高水平的荧光。同时，在肿瘤和其它器官中没有出现明显的荧光。肿瘤中的低浓度和肝脏中的高含量表明，大多数GV被RES捕获，从而导致纳米粒子在血液中的迅速清除。相反，对于ICG标记的PH-GV处理的小鼠，在48小时的过程中，肿瘤中的荧光水平比其它器官强得多。肿瘤中最大的荧光信号发生在处理后12小时，然后荧光信号随时间略有下降。另一方面，在肝脏和脾脏中检测到低水平的荧光。综上所述，PH修饰显著降低了PH-GV的RES摄取，并改善了血液循环和及肿瘤分布情况。PH-GV的优异肿瘤积聚能力将有利于其在超声分子成像中的进一步应用，并在几乎没有副作用的情况下广泛提高治疗效果。
实施例14：通过PH-GV进行体内癌症的US成像
根据香港理工大学动物护理和使用委员会(CC/ACUCC)批准的协议来执行动物实验。向裸鼠(七周龄，雌性，20-24g)的皮下部位注射4×10 6SCC7细胞的PBS悬浮液(80μL)。当肿瘤尺寸(在右腿区域)达到120mm 3的平均尺寸时，将小鼠随机分为两组：(a)将GV注入小鼠的尾静脉中；(b)将PH-GV注入小鼠的尾静脉中。在注射后，在0、0.5、1、2、4、8、12、24和48小时，在Vevo 2100成像系统上记录肿瘤部位的US图像。
在一剂注射后达到最高积聚量时，执行高功率的US刺激，这可导致GV的破碎。并且，应用Vevo 2100成像系统来记录US刺激前后的US图像和信号强度，以进行分析。使用Vevo 2100 Workstation软件来测量回声成像的信号强度。
除了PH-GV的优异肿瘤积聚性外，我们还向SCC7荷瘤模型静脉注射GV和PH-GV，以证明表面修饰GV能够在体内产生超声造影，并且显示良好的非线性信号(图 15a)。首先，我们在SCC7荷瘤裸鼠的尾静脉中注入GV或PH-GV(200μL，浓度20.0nM)，并使用Vevo 2100成像系统获取非线性超声图像(以18MHz传输)。在图15a中呈现了在注射后0、0.5、1、2、4、8、12、24和48小时显示肿瘤部位的超声图像。这些图像中的绿色突出了在静脉注射后肿瘤内部超声信号增强的区域。在PH-GV组中，我们可以观察到8小时后肿瘤部位的超声信号强度大大增强。与注射PH-GV之前的信号相比，在注射后(p.i.)4、8、12和24小时，肿瘤中的US信号分别增加了1.05±0.13、1.2±0.41、1.51±0.25和1.35±0.12倍(图15b)。该积聚也在注射后12小时达到峰值，如荧光成像所显示的，并在注射后12小时后开始减少。然而，在GV处理的小鼠中未发现回声强度的显著变化。为了确认PH-GV是造成所观察到的造影的原因，我们在超崩塌压力(supra-collapse pressure)(650kPa)下施加了超声脉冲，从而导致造影消失(图15c)。含有GV的目的区域表现出的反向散射信号比注射缓冲液的对照强60±14％(p＝0.008)；该差异在GV破碎后消失(p＝0.23)。
实施例15：PH-GV在荷瘤小鼠中的滞留
我们研究了静脉注射后实体瘤内GV和PH-GV的间质渗透。在静脉注射ICG标记的GV或ICG标记的PH-GV后12小时从小鼠提取的肿瘤切片用DAPI(蓝色)和抗CD31抗体(红色)染色，以进行共聚焦成像(图16a)。对于ICG标记的GV组，如图16a所示，我们在肿瘤切片中未发现ICG荧光信号(绿色)，因为体内GV的快速清除速度导致血液循环时间不足，因此游离GV很难渗透通过肿瘤组织周围的血管。与之形成鲜明对比的是，PH-GV组中的ICG荧光远远高于GV组，这表明用PH修饰GV可以增强肿瘤的积聚和滞留，这是因为有更长的循环时间用于PEG的EPR效应和HA的靶向性。更重要的是，我们观察到大量ICG荧光信号远离血管定位，这表明PH-GV纳米粒子可能会渗透通过肿瘤部位周围的血管并在肿瘤细胞内化，以实现有效的肿瘤内扩散。
实施例16：GV和PH-GV的细胞毒性
将SCC7细胞以每孔8000个细胞的密度接种在96孔板中，并在5％CO 2的温育箱中于37℃过夜培养。第二天，将细胞用PBS洗涤3次，并与一系列浓度的GV、破碎的GV和PH-GV溶液在相同条件下温育24小时和48小时。通过CCK-8测定试剂盒评估细胞活力。在450nm下测量光密度(OD)，并通过酶标仪进行记录。
还通过3'6'-二(O-乙酰基)-4'5'-二[N,N-二(羧甲基)氨甲基]荧光素、四乙酰氧基甲酯 (钙黄绿素AM)/碘化丙啶(PI)染色(Sangon Biotech，中国上海)来研究细胞毒性。将U87细胞以1×10 5个细胞的密度接种在6孔板中，并使其生长至80-90％汇合。用平行浓度的GV、破碎GV和PH-GV将SCC7细胞温育24小时。在用PBS洗涤几次并浸入新鲜培养基(1mL)中后，将黑暗中的对照组在新鲜DMEM培养基中温育。在去除新鲜DMEM培养基后，将PBS中的钙黄绿素AM(4μmol/L)和PI溶液(4μmol/L)加入到SCC7细胞中，并在5％的CO 2下，在37℃，将其温育30分钟。最后，使用PBS将细胞清洗三次。通过荧光显微镜获得细胞的荧光图像。
为了研究GV的体内毒性，收集正常器官并进行H&E染色。如图16b所示，从心脏、肝脏、脾脏、肺和肾脏的病理分析来看，用GV和PH-GV处理的小鼠没有明显的损伤或毒性信号。如图16c所示，在处理后30天内观察到各个处理组在处理期间体重变化的相似的变化趋势。值得注意的是，在我们的研究中未观察到GV和PH-GV对小鼠的急性毒性或不良反应。
上述对实施例的描述是为方便本技术领域的普通技术人员能理解和应用本发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于本文公开的具体实施例，本领域技术人员根据本发明的原理，在不脱离本发明范畴的情况下所做出的改进和修改都应该在本发明的保护范围之内。

Claims (21)

  1. 一种经表面修饰而能够特异性靶向肿瘤部位的靶向GV，其表面被生物靶向材料和/或生物相容性材料修饰。
  2. 根据权利要求1所述的靶向GV，其中所述生物靶向材料选自以下中的一种或多种：HA、RGD肽、叶酸、半乳糖、葡萄糖，优选HA；和/或，所述生物相容性材料选自以下中的一种或多种：PEG、壳聚糖、聚氨基酯、聚乳酸、聚烯烃、聚砜、聚碳酸酯、聚丙烯腈，优选PEG。
  3. 根据权利要求1所述的靶向GV，其是表面被HA和PEG修饰的PH-GV。
  4. 一种制备权利要求1-3中任一项所述的靶向GV的方法，包括：
    (a)将生物靶向材料与GV连接在一起；和/或
    (b)将生物相容性材料与步骤(a)的产物连接在一起。
  5. 一种表面被脂质分子修饰的脂质GV，优选地，所述脂质分子选自以下中的一种或多种：DSPE-PEG、DOPC、PEG、DSPE、PVA、甘油、甘油酯、脂肪酸、磷脂。
  6. 根据权利要求5所述的脂质GV，其是表面被DSPE-PEG和DOPC修饰的脂质GV。
  7. 一种充气脂质GV，其包含权利要求5或6所述的脂质GV和治疗性气体，优选氧气。
  8. 一种脂质靶向GV，其表面被脂质分子和生物靶向材料以及任选地生物相容性材料修饰，优选地
    所述脂质分子选自以下中的一种或多种：DSPE-PEG、DOPC、PEG、DSPE、PVA、甘油、甘油酯、脂肪酸、磷脂；
    所述生物靶向材料选自以下中的一种或多种：HA、RGD肽、叶酸、半乳糖、葡萄糖；
    所述生物相容性材料选自以下中的一种或多种：PEG、壳聚糖、聚氨基酯、聚乳酸、聚烯烃、聚砜、聚碳酸酯、聚丙烯腈。
  9. 根据权利要求8所述的脂质靶向GV，其是表面被DSPE-PEG、DOPC和HA以及任选地PEG修饰的GV。
  10. 一种充气脂质靶向GV，其包含权利要求8-9中任一项所述的脂质靶向GV和治疗性气体，优选氧气。
  11. 一种制备权利要求5或6所述的脂质GV的方法，包括：
    (a)将脂质分子或脂质分子的混合物溶于氯仿中，并使其干燥；
    (b)向步骤(a)的产物中加入HEPES缓冲液并搅拌，形成浑浊溶液；以及
    (c)将步骤(b)的产物加入到GV溶液中，从而形成脂质GV。
  12. 一种制备权利要求8或9所述的脂质靶向GV的方法，包括：
    (a)将脂质分子或脂质分子的混合物溶于氯仿中，并使其干燥；
    (b)向步骤(a)的产物中加入HEPES缓冲液并搅拌，形成浑浊溶液；以及
    (c)将生物靶向材料与GV连接在一起，然后将步骤(b)的产物加入到GV溶液中，从而形成脂质靶向GV。
  13. 根据权利要求11或12所述的方法，还包括：
    在步骤(c)中，在将步骤(b)的产物加入到GV溶液中之前，向所述GV溶液中充气例如氧气直至饱和，从而形成权利要求7所述的充气脂质GV或权利要求10所述的充气脂质靶向GV。
  14. 一种造影剂或诊断剂，其含有权利要求1-3中任一项所述的靶向GV、权利要求5或6所述的脂质GV、权利要求7所述的充气脂质GV、权利要求8-9中任一项所述的脂质靶向GV或权利要求10所述的充气脂质靶向GV。
  15. 一种治疗剂，其含有权利要求5或6所述的脂质GV、权利要求7所述的充气脂质GV、权利要求8-9中任一项所述的脂质靶向GV或权利要求10所述的充气脂质靶向GV。
  16. 一种药物载体，其含有权利要求1-3中任一项所述的靶向GV、权利要求5或6所述的脂质GV、权利要求7所述的充气脂质GV、权利要求8-9中任一项所述的脂质靶向GV或权利要求10所述的充气脂质靶向GV，以及任选地用于治疗待治疗疾病的药物。
  17. 一种药物组合物，其含有权利要求15所述的治疗剂或权利要求16所述的药物载体。
  18. 权利要求1-3中任一项所述的靶向GV、权利要求5或6所述的脂质GV、权利要求7所述的充气脂质GV、权利要求8-9中任一项所述的脂质靶向GV、权利要求10所述的充气脂质靶向GV、权利要求14所述的造影剂或诊断剂在制备用于诊断受试者的癌症的诊断试剂或诊断试剂盒中的用途。
  19. 权利要求5或6所述的脂质GV、权利要求7所述的充气脂质GV、权利要求8-9中任一项所述的脂质靶向GV、权利要求10所述的充气脂质靶向GV、权利要求15所述的治疗剂、权利要求16所述的药物载体或权利要求17所述的药物组合物在制备用于治疗受试者的癌症的药物中的用途。
  20. 权利要求5或6所述的脂质GV、权利要求7所述的充气脂质GV、权利要求8-9中任一项所述的脂质靶向GV、权利要求10所述的充气脂质靶向GV、权利要求15所述的治疗剂、权利要求15所述的药物载体或权利要求17所述的药物组合物与第二治疗剂或疗法在制备用于治疗受试者的癌症的药物组合中的用途，其中所述药物组合以如下方式施用：
    向所述受试者施用权利要求5或6所述的脂质GV、权利要求7所述的充气脂质GV、权利要求8-9中任一项所述的脂质靶向GV、权利要求10所述的充气脂质靶向GV、权利要求15所述的治疗剂、权利要求15所述的药物载体或权利要求17所述的药物组合物；然后
    向所述受试者施用所述第二治疗剂或疗法例如PDT和SDT疗法，
    其中任选地，在向所述受试者施用所述第二治疗剂或疗法之前，向所述受试者应用超声波。
  21. 根据权利要求18-20中任一项所述的用途，其中所述癌症包括但不限于：膀胱癌、肺癌、肾癌、胃癌、结直肠癌、肝癌、乳腺癌、黑色素瘤。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060216810A1 (en) * 1996-11-12 2006-09-28 Lu-Kwang Ju Method for using semi-synthetic gas vesicles as ultrasound contrast agent
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

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020039594A1 (en) * 1997-05-13 2002-04-04 Evan C. Unger Solid porous matrices and methods of making and using the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060216810A1 (en) * 1996-11-12 2006-09-28 Lu-Kwang Ju Method for using semi-synthetic gas vesicles as ultrasound contrast agent
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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANUPAMA LAKSHMANAN, LU GEORGE J, FARHADI ARASH, NETY SUCHITA P, KUNTH MARTIN, LEE-GOSSELIN AUDREY, MARESCA DAVID, BOURDEAU RAYMOND: "Preparation of Biogenic Gas Vesicle Nanostructures for Use as Contrast Agents for Ultrasound and MRI", NATURE PROTOCOLS, vol. 10, no. 12, 1 October 2017 (2017-10-01), pages 2050, XP055757758, ISSN: 1754-2189, DOI: 10.1038/nprot.2017.081 *
WANG GUOHAO; SONG LIN; HOU XUANDI; KALA SHASHWATI; WONG KIN FUNG; TANG LIYA; DAI YUNLU; SUN LEI: "Surface-modified GVs as Nanosized Contrast Agents for Molecular Ultrasound Imaging of Tumor", BIOMATERIALS, vol. 236, 28 January 2020 (2020-01-28), pages 1 - 11, XP086041808, ISSN: 0142-9612, DOI: 10.1016/j.biomaterials.2020.119803 *

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