WO2021115070A1 - 一种用于近红外二区荧光检测的吲哚菁绿脂质体及其制备方法和用途 - Google Patents

一种用于近红外二区荧光检测的吲哚菁绿脂质体及其制备方法和用途 Download PDF

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WO2021115070A1
WO2021115070A1 PCT/CN2020/129545 CN2020129545W WO2021115070A1 WO 2021115070 A1 WO2021115070 A1 WO 2021115070A1 CN 2020129545 W CN2020129545 W CN 2020129545W WO 2021115070 A1 WO2021115070 A1 WO 2021115070A1
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indocyanine green
liposome
liposomes
test tube
infrared
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English (en)
French (fr)
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郑海荣
盛宗海
耿晓瑞
高笃阳
胡德红
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
    • A61K49/0084—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion liposome, i.e. bilayered vesicular structure

Definitions

  • the invention belongs to the field of pharmaceutical preparations, and specifically discloses an indocyanine green liposome and a preparation method and application thereof.
  • the fatality rate of tumor is the second leading cause of death in the world after cardiovascular and cerebrovascular diseases, and it is a serious threat to human health.
  • the incidence of common types of cancer in my country is about 137-174 per 100,000, but the mortality rate is more than 117 per 100,000.
  • the vast majority of cancer patients have reached the middle and advanced stages when they see a doctor, and the tumor resection rate is only 10% to 30%.
  • Related studies have reported that the size of the tumor and the clinical stage at the time of diagnosis are closely related to the survival rate of tumor patients. If the tumor is diagnosed in the early stage, it will greatly reduce the tumor mortality.
  • NIR fluorescence imaging is an emerging non-invasive biomedical imaging mode, which is compatible with several other imaging modes, including magnetic resonance imaging (MRI), X-ray, computer tomography (CT), and positron emission tomography.
  • MRI magnetic resonance imaging
  • CT computer tomography
  • US ultrasonography
  • the absorption and scattering of light by tissue and blood within the wavelength range of NIR-II are significantly reduced, and there is almost no autofluorescence interference from organisms, making the NIR-II fluorescent probes have a deeper Tissue penetration and spatial resolution.
  • the materials with fluorescence emission spectra in the second region of the near-infrared mainly include inorganic materials (nanotubes, quantum dots, rare earth nanoparticles) and organic materials (polymers, organic small molecule dyes).
  • organic small molecule fluorescent probes with low toxicity and rapid metabolism are the most ideal choice.
  • most NIR-II organic small molecule fluorophores have disadvantages such as poor water solubility, low light stability, and low quantum yield. Therefore, the development of a new type of near-infrared two-zone fluorescent molecular probe is particularly important.
  • ICG Indocyanine green
  • FDA Food and Drug Administration
  • ICG has fluorescence emission in the NIR-II window, and it has the application potential of near-infrared two-zone fluorescence imaging.
  • ICG is very unstable in aqueous solution and is easily cleared quickly in the blood circulation, and it is easy to form dimers between molecules to cause fluorescence quenching.
  • liposomes are mainly composed of cholesterol and natural phospholipids, which can be biodegraded when entering the body and will not accumulate in the body. They are non-toxic, non-pyrogenic, and non-immunogenic. Liposomes are proven to be an effective drug delivery carrier, which has the advantages of targeting and lymphatic orientation; slow release, delaying renal excretion and metabolism, prolonging the action time; reducing drug toxicity; improving stability and other advantages. More than a dozen liposomal drug carriers have been used in clinical medicine.
  • the liposomes are very close to the natural cell membrane, they still cannot completely avoid immune clearance.
  • the natural cell membrane is used to camouflage the micro/nano carrier, and the obtained bionic micro/nano carrier not only has the physical and chemical properties of the micro/nano carrier itself, but also has biological properties similar to natural cells.
  • the research of cell membrane camouflaged micro/nano carriers is still in its infancy, and the development of more new cell membrane pseudo-carriers is of great significance to the field of biomedicine.
  • the present invention utilizes the hydrophobic interaction between indocyanine green and small phospholipid molecules to synthesize liposomes in which indocyanine green is embedded in the bimolecular shell of phospholipids.
  • Indocyanine green avoids the self-quenching caused by the interaction with water.
  • the fluorescence intensity in the second infrared zone is significantly improved.
  • the cell membrane is modified by physical extrusion to form a biomimetic liposome on the nanoparticle, which has biological properties similar to natural cells.
  • the invention helps to promote the development of near-infrared two-zone fluorescence imaging, and provides new theories and new ideas for the clinical diagnosis of cancer.
  • the materials with fluorescence emission spectra in the second region of the near-infrared mainly include inorganic materials (nanotubes, quantum dots, rare earth nanoparticles) and organic materials (polymers, organic small molecule dyes).
  • organic small molecule fluorescent probes with low toxicity and rapid metabolism are the most ideal choice.
  • most NIR-II organic small molecule fluorophores have disadvantages such as poor water solubility, low light stability, and low quantum yield. Therefore, the development of a new type of near-infrared two-zone fluorescent molecular probe is particularly important.
  • the present invention uses indocyanine green, a near-infrared fluorescent contrast agent approved by the U.S. Food and Drug Administration (FDA) for clinical use, and uses its hydrophobic interaction with phospholipid small molecules
  • Indocyanine green is synthesized into liposomes in which indocyanine green is embedded in the liposome shell. Indocyanine green avoids self-quenching caused by interaction with water, and the fluorescence intensity of the near-infrared two-region is significantly improved.
  • the cell membrane is modified by physical extrusion to form a biomimetic liposome on the nanoparticle, which has biological properties similar to natural cells.
  • the compound has the characteristics of good biocompatibility, and the modification of different cell membranes can make this high-brightness near-infrared two-zone fluorescent probe be widely used.
  • the invention helps to promote the development of near-infrared two-zone fluorescence imaging, and provides new theories and new ideas for the clinical diagnosis of cancer.
  • the materials with fluorescence emission spectra in the second region of the near-infrared mainly include inorganic materials (nanotubes, quantum dots, rare earth nanoparticles) and organic materials (polymers, organic small molecule dyes).
  • Inorganic materials have certain toxicity and slow metabolism.
  • Most NIR-II organic small molecule fluorophores have disadvantages such as poor water solubility, low light stability, and low quantum yield. Considering the clinical transformation of NIR-II imaging agents, organic small molecule fluorescent probes with low toxicity and rapid metabolism are the most ideal choice.
  • the invention discloses a method for synthesizing a high-brightness near-infrared two-zone indocyanine green biomimetic liposome.
  • the method utilizes the hydrophobic interaction between indocyanine green and small lipid molecules to synthesize indocyanine green embedded lipid
  • indocyanine green avoids the self-quenching caused by the interaction with water, and the fluorescence intensity of the near-infrared two-zone is significantly improved.
  • the cell membrane is modified by physical extrusion to form a biomimetic liposome on the nanoparticle, which has biological properties similar to natural cells.
  • the compound has the characteristics of good biocompatibility, and the modification of different cell membranes can make this high-brightness near-infrared two-zone fluorescent probe be widely used.
  • the invention helps to promote the development of near-infrared two-zone fluorescence imaging, and provides theories and ideas for the clinical diagnosis of cancer.
  • One aspect of the present invention provides an indocyanine green liposome having lipid bilayers and indocyanine green embedded between the lipid bilayers.
  • the indocyanine green embedded between the lipid bilayers is formed by mixing the indocyanine green and the lipid bilayer materials in the organic phase and uniformly dispersing them into a thin film, and then adding the water phase Formed by hydration to form liposomes.
  • the lipid bilayer of the indocyanine green liposome is also embedded with cell membrane proteins.
  • the phospholipid bilayer is composed of phospholipids, or a mixture of phospholipids and cholesterol.
  • the phospholipid is selected from dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoylphosphatidylcholine, 1-palmitoyl- 2-Olecithin, Soy Lecithin, Hydrogenated Soy Lecithin, Dilauroyl Lecithin, Dimyristoyl Lecithin, Dimyristoyl Lecithin, Dilauroylphosphatidylglycerol, Dipalmitoylphosphatidic Acid, Two Myristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, dipalmitoylphosphatidyldisserine, dipalmitoylphosphatidylcholine, brain phosphatidylserine,
  • the phospholipid bilayer is composed of dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline, dioleoyl lecithin and cholesterol, preferably, the molar ratio of which is dipalmitoyl phosphatidyl choline.
  • Acyl choline: distearoyl phosphatidyl choline: dioleoyl lecithin: cholesterol 5:3:1:1.
  • the molar ratio of the phospholipid bilayer to indocyanine green is 25-1000:1, preferably 100-500:1, more preferably 200-300:1.
  • Another aspect of the present invention provides a method for preparing indocyanine green liposomes, which comprises the following steps:
  • the preparation method of indocyanine green liposomes further includes step 3) embedding cell membrane proteins on the surface of the indocyanine green liposomes.
  • the mechanical force dispersion refers to dispersion by means of ultrasonic dispersion and repeated extrusion by liposome extruder.
  • the method of removing the solvent in step 1) is selected from the group consisting of removing the solvent under reduced pressure or removing the solvent under normal pressure.
  • inert gas is used for protection during the preparation process.
  • the mass ratio of indocyanine green liposomes to cell membrane proteins is 200-400:1, preferably 250-300:1.
  • the material of the phospholipid bilayer is selected from phospholipids, or a mixture of phospholipids and cholesterol.
  • the cell membrane protein is selected from cancer cell membrane protein, red blood cell membrane protein, neutrophil membrane protein, platelet cell membrane protein, macrophage membrane protein, natural killer cell membrane protein, or dimer or tetramer Glucose transporter 1, in the form of body Neurothelin/HT7, serum gamma glutamyl transpeptidase, P-glycoprotein, PD-1 ligand PDL1/PDL2, Fas ligand FasL, immune costimulatory protein B7-H4, membrane-associated complement regulatory protein CRRY, and non-classical MHC Class I molecules.
  • the method of step 3) is to uniformly disperse the membrane protein and liposomes, and then extrude them with a liposome extruder to prepare liposomes with cell membrane proteins.
  • the method for removing free cell membrane proteins is dialysis or ultracentrifugation filtration.
  • the liposomes of the present invention are used in the preparation of near-infrared two-zone fluorescence detection reagents.
  • the near-infrared two-zone fluorescence detection reagent can be applied to aqueous solutions, phosphate buffers, culture media, or body fluids or blood in and out of human or animal bodies.
  • the near-infrared second zone refers to the light wave zone with a wavelength of 1000-1700 nm.
  • the near-infrared second zone is selected from the light wave zone with a wavelength of 1000-1400 nm.
  • Another aspect of the present invention provides a near-infrared two-zone detection reagent, which includes the liposome of the present invention.
  • the compound has the characteristics of good biocompatibility, and the modification of different cell membranes can make this high-brightness near-infrared two-zone fluorescent probe be widely used.
  • the advantages of this probe are embodied in high brightness, low dose, mild reaction conditions, good reproducibility, low toxicity, monodisperse, good biocompatibility, rapid metabolism, etc.
  • the present invention helps to promote the near-infrared second zone
  • the development of fluorescence imaging provides theories and ideas for the clinical diagnosis of cancer.
  • Figure 1 is a schematic cross-sectional view of liposomes formed by indocyanine green and phospholipids after hydrophobic interaction.
  • FIG. 2 is a solution diagram of Example 1 to Example 7.
  • FIG. 2 is a solution diagram of Example 1 to Example 7.
  • FIG. 3 Comparison of fluorescence brightness in the near-infrared zone.
  • LIPO ICG is liposome: indocyanine green 2
  • LIPO-ICG is indocyanine green liposome 3
  • ICG-INPUT is the same amount of indocyanine green 4
  • ICG-OD is Indocyanine green 5 with the same UV absorption
  • PBS is phosphate buffer 6
  • CULTURE is serum-free medium solution 7
  • 10% FBS 10% fetal bovine serum solution).
  • Figure 4 Fluorescence brightness change diagram in the near-infrared zone.
  • LIPO-ICG means indocyanine green biomimetic liposome 3
  • SAME INPUT means the same amount of input Indocyanine green 4
  • SAME OD is indocyanine green 5 with the same ultraviolet absorption
  • PBS is phosphate buffer 6
  • CULTURE is serum-free medium solution 7
  • 10% FBS is 10% fetal bovine serum solution).
  • Figure 5 shows the near-infrared two-zone fluorescence spectrum of liposomes prepared by mixing indocyanine green dissolved in anhydrous methanol and liposome raw materials to prepare a film blown film.
  • Figure 6 is a quantitative graph of near-infrared two-zone fluorescence intensity in phosphate buffer solution.
  • Figure 7 is a quantitative graph of near-infrared two-zone fluorescence intensity in a cell serum-free medium solution solution.
  • Figure 8 is a quantitative graph of near-infrared two-zone fluorescence intensity in a 10% fetal bovine serum solution.
  • Figure 9 is a schematic diagram of vascular imaging of the right leg of mice in different treatment groups, in which ICG-INPUT, ICG-OD are free indocyanine green (the same amount of input and the same ultraviolet absorption) LIPO-ICG is indocyanine green liposome, RLIPO-ICG is an indocyanine green liposome modified with erythrocyte membrane, and the blood circulation time in the body is greatly improved compared with free indocyanine green.
  • the molar ratio of liposome raw material to indocyanine green is 25:1 Heat the mixed solution in a 65°C water bath and mix thoroughly, vortex on a vortexer, and at the same time fill the test tube at a moderate rate and a stable flow rate Until the organic solution evaporates to form a uniform film, and there is no liquid residue in the test tube.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes in the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside world. Place the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to completely volatilize the organic solvent in the test tube.
  • the solution is equipped with 200
  • the liposome extruder with nm polycarbonate membrane was repeatedly extruded 20 times to obtain indocyanine green liposomes.
  • the indocyanine green liposomes were dialyzed for 12 hours at 4°C and protected from light to remove unloaded indocyanine green and cell membrane proteins, and purified Indocyanine green liposomes.
  • the molar ratio of liposome raw material to indocyanine green is 50:1 Heat the mixed solution in a 65°C water bath and mix thoroughly, vortex on a vortexer, and at the same time fill the test tube at a moderate rate and a stable flow rate Until the organic solution evaporates to form a uniform film, and there is no liquid residue in the test tube.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes in the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside world. Place the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to completely volatilize the organic solvent in the test tube.
  • the solution was repeatedly extruded 20 times with a liposome extruder equipped with a 200 nm polycarbonate membrane to obtain indocyanine green liposomes, which were dialyzed for 12 hours at 4°C under dark conditions to remove unloaded indocyanine green. , To obtain purified indocyanine green liposomes.
  • the molar ratio of liposome raw material to indocyanine green is 100:1 Heat the mixed solution in a 65°C water bath and mix thoroughly, vortex on a vortexer, and at the same time fill the test tube at a moderate rate and a stable flow rate Until the organic solution evaporates to form a uniform film, and there is no liquid residue in the test tube.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes in the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside world. Place the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to completely volatilize the organic solvent in the test tube.
  • the solution was repeatedly extruded 20 times with a liposome extruder equipped with a 200 nm polycarbonate membrane to obtain indocyanine green liposomes, which were dialyzed for 12 hours at 4°C under dark conditions to remove unloaded indocyanine green. , To obtain purified indocyanine green liposomes.
  • the molar ratio of liposome raw material to indocyanine green is 250:1 Heat the mixed solution in a 65°C water bath and mix thoroughly, vortex on a vortexer, and at the same time fill the test tube at a moderate rate and a stable flow rate Until the organic solution evaporates to form a uniform film, and there is no liquid residue in the test tube.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes in the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside world. Place the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to completely volatilize the organic solvent in the test tube.
  • the solution was repeatedly extruded 20 times with a liposome extruder equipped with a 200 nm polycarbonate membrane to obtain indocyanine green liposomes, which were dialyzed for 12 hours at 4°C under dark conditions to remove unloaded indocyanine green. , To obtain purified indocyanine green liposomes.
  • the molar ratio of liposome raw material to indocyanine green is 500:1 Heat the mixed solution in a 65°C water bath and mix thoroughly, vortex on a vortexer, and at the same time fill the test tube at a moderate rate and a stable flow rate Until the organic solution evaporates to form a uniform film, and there is no liquid residue in the test tube.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes in the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside world. Place the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to completely volatilize the organic solvent in the test tube.
  • the solution was repeatedly extruded 20 times with a liposome extruder equipped with a 200 nm polycarbonate membrane to obtain indocyanine green liposomes, which were dialyzed for 12 hours at 4°C under dark conditions to remove unloaded indocyanine green. , To obtain purified indocyanine green liposomes.
  • the molar ratio of liposome raw material to indocyanine green is 750:1 Heat the mixed solution in a 65°C water bath and mix thoroughly, vortex on a vortexer, and at the same time fill the test tube at a moderate rate and a stable flow rate Until the organic solution evaporates to form a uniform film, and there is no liquid residue in the test tube.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes in the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside world. Place the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to completely volatilize the organic solvent in the test tube.
  • the solution was repeatedly extruded 20 times with a liposome extruder equipped with a 200 nm polycarbonate membrane to obtain indocyanine green liposomes, which were dialyzed for 12 hours at 4°C under dark conditions to remove unloaded indocyanine green. , To obtain purified indocyanine green liposomes.
  • the molar ratio of liposome raw material to indocyanine green is 1000:1 Heat the mixed solution in a 65°C water bath and mix thoroughly, vortex on a vortexer, and at the same time fill the test tube at a moderate rate and a stable flow rate Until the organic solution evaporates to form a uniform film, and there is no liquid residue in the test tube.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes on the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside. Put the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to make the organic solvent in the test tube volatilize completely.
  • the solution was repeatedly extruded 20 times with a liposome extruder equipped with a 200 nm polycarbonate membrane to obtain indocyanine green liposomes, which were dialyzed for 12 hours at 4°C under dark conditions to remove unloaded indocyanine green. , To obtain purified indocyanine green liposomes.
  • the molar ratio of liposome raw material to indocyanine green is 250:1 Heat the mixed solution in a 65°C water bath and mix thoroughly, vortex on a vortexer, and at the same time fill the test tube at a moderate rate and a stable flow rate Until the organic solution evaporates to form a uniform film, and there is no liquid residue in the test tube.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes on the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside. Put the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to make the organic solvent in the test tube volatilize completely.
  • the solution is equipped with 200
  • the liposome extruder with nm polycarbonate membrane was repeatedly extruded 20 times to obtain ordinary indocyanine green liposomes.
  • the molar ratio of liposome raw material to indocyanine green is 250:1 Heat the mixed solution in a 65°C water bath and mix thoroughly, vortex on a vortexer, and at the same time fill the test tube at a moderate rate and a stable flow rate Until the organic solution evaporates to form a uniform film, and there is no liquid residue in the test tube.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes in the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside world. Place the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to completely volatilize the organic solvent in the test tube.
  • the solution is equipped with 200
  • the liposome extruder with nm polycarbonate membrane was repeatedly extruded 20 times to obtain ordinary indocyanine green liposomes.
  • the molar ratio of liposome raw material to indocyanine green is 250:1 Heat the mixed solution in a 65°C water bath and mix thoroughly, vortex on a vortexer, and at the same time fill the test tube at a moderate rate and a stable flow rate Until the organic solution evaporates to form a uniform film, and there is no liquid residue in the test tube.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes in the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside world. Place the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to completely volatilize the organic solvent in the test tube.
  • the solution is equipped with 200
  • the liposome extruder with nm polycarbonate membrane was repeatedly extruded 20 times to obtain ordinary indocyanine green liposomes.
  • test tube Seal the mouth of the test tube with a sealing film, and poke holes on the sealing film with a sharp tool to ensure that the gas in the test tube can communicate with the outside. Put the test tube in a vacuum vessel to avoid light and vacuum for 4 hours to make the organic solvent in the test tube volatilize completely.
  • the solution is equipped with 200
  • the liposome extruder with nm polycarbonate membrane was repeatedly extruded 20 times, and the unloaded indocyanine green was removed by dialyzing for 12 hours at 4°C under dark conditions to obtain purified indocyanine green liposomes.
  • Examples 1-7 and Comparative Examples 1-2 were detected by ultraviolet spectroscopy and fluorescence spectroscopy respectively.
  • the ultraviolet spectra of the comparative example and the comparative example 1 show that the ultraviolet spectra of the liposomes of the present invention have a red shift with respect to the comparative examples 1-2.
  • the fluorescence spectrum of the embodiment of the present invention has a red shift of about 15 nm in the near-infrared one-region window, and the fluorescence intensity is increased.
  • the fluorescence spectrum of the window in the near-infrared zone is consistent with that of free indocyanine green, and the fluorescence intensity is reduced. It can be known from the experimental results that the liposome prepared by the method of the present invention can enhance the fluorescence emission of indocyanine green.
  • the reason may be that the hydrophobic bilayer restricts the self-aggregation of indocyanine green, which leads to a cumulative increase in fluorescence intensity.
  • the method of Comparative Example 1 prepares liposomes. Since indocyanine green is restricted to the water-soluble core of liposomes, the interaction with water causes the fluorescence quenching of self-aggregation, and the same is true for free indocyanine green Face the same problem.
  • the fluorescence spectrum of the indocyanine green liposome prepared by the method of the present invention is red-shifted so that more fluorescent signals enter the near-infrared two-region window, and the increase in the fluorescence intensity of the near-infrared one
  • the fluorescence spectrum of the indocyanine green liposome prepared by the method of the present invention is raised in the near-infrared two-zone window as a whole, so that the near-infrared two-zone window shows significantly improved fluorescence intensity.
  • the liposome indocyanine green of the present invention can be used as a high-brightness imaging probe in the second region of near-infrared fluorescence.
  • the experimental phenomenon reveals: within a certain range, the fluorescence intensity increases with the increase of the concentration of indocyanine green, but the fluorescence exceeds the range. The intensity decreases as the concentration of indocyanine green increases, which indicates that there is an optimal upper limit for the dose of indocyanine green embedded in the phospholipid membrane bilayer in the liposome of the present invention.
  • the specific experimental methods and parameters are the same as those in Effect Example 2.
  • the samples in the 96-well plate are stored with tin foil and protected from light, and the changes in the fluorescence intensity of the near-infrared zone are detected within 60 days.
  • fluorescence intensity detection see Fig. 4
  • it can be known that the liposome prepared by the present invention can decay very slowly in the near-infrared two-zone fluorescence intensity in different media within 60 days, and maintain a certain fluorescence stability.
  • RLIPO-ICG is an indocyanine green liposome group modified with erythrocyte membrane, that is, the liposome of Example 9.
  • the ICG-INPUT group is the free indocyanine green group with the same amount of input
  • the ICG-OD is the free indocyanine green group with the same ultraviolet absorption
  • the LIPO-ICG is the indocyanine green liposome group.
  • the concentration of free indocyanine green with the same amount of input was 44 ⁇ g/mL, and the concentration of free indocyanine green with the same UV absorption was 132 ⁇ g/mL.
  • the synthetic injection concentration of indocyanine green liposomes is the synthetic stock solution concentration. Use a near-infrared fluorescence two-zone imager for detection. Experimental results prove that the blood circulation time in the body is greatly improved compared with free indocyanine green and liposomes that are not coated with red blood cell membranes.

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Abstract

一种用于近红外二区荧光检测的吲哚菁绿脂质体及其制备方法和用途，其具有脂质双分子层以及嵌在脂质双分子层之间的吲哚菁绿，其通过1)将制备脂质双分子层的材料与吲哚菁绿混合形成均匀溶液，去除有机溶剂获得分散的薄膜；2)加入水相进行水化，机械力分散形成吲哚菁绿脂质体。所述脂质体制备方法简单，近红外二区荧光强度高，稳定性好。

Description

一种用于近红外二区荧光检测的吲哚菁绿脂质体及其制备方法和用途 技术领域
本发明属于药物制剂领域，具体公开了一种吲哚菁绿脂质体及其制备方法和用途。
 背景技术
当前，肿瘤的致死率是仅次于心脑血管疾病的全球第二大致死疾病，严重威胁人类的健康。我国常见癌症种类发病约为 137-174 人/10 万人，但死亡率超过 117 人/10 万人，绝大多数肿瘤患者就诊时已达中晚期，肿瘤切除率仅为 10%~30%。相关研究报道肿瘤的尺寸大小和诊断时的临床分期与肿瘤患者生存率密切相关。若在肿瘤早期进行诊断将会极大降低肿瘤病死率。
近红外（NIR）荧光成像是一种新兴的非侵入性生物医学成像模式，与其他几种成像模式，包括磁共振成像（MRI）、X 射线、计算机断层扫描（CT）、正电子发射断层扫描（PET）、超声波检查(US)等相比，具有敏感性高、成本低、高时空分辨的优点，在肿瘤诊疗中具有极大的应用潜力。近红外二区 (NIR-II，1 000 ～1 700 nm)波长范围内组织和血液对光的吸收和散射显著降低，并且几乎无生物体自发荧光干扰，使得近红外二区荧光探针具有更深的组织穿透力和空间分辨率。目前荧光发射光谱在近红外二区的材料主要有无机材料（纳米管、量子点、稀土纳米粒子）和有机材料（聚合物、有机小分子染料）。考虑到 NIR-II 成像剂的临床转化，具有低毒性，快速代谢性质的有机小分子荧光探针是最理想的选择。但多数 NIR-II 有机小分子荧光团存在水溶性差、光稳定性低、量子产率低等缺点。因此开发新型的近红外二区荧光的分子探针尤为重要。
吲哚菁绿（ICG）是目前被美国食品药品监督管理局（FDA）批准用于临床的近红外荧光造影剂。有研究表明ICG在NIR-II窗口有荧光发射,具有近红外二区荧光成像的应用潜力。然而，ICG 在水溶液中很不稳定、在血液循环中容易被快速清除，分子间容易形成二聚体导致荧光淬灭。这些不足严重限制了ICG 在近红外二区诊疗方面的应用。
近年来，受到天然细胞的启发，人们尝试模仿天然细胞，构建仿生微纳米药物运输载体。其中，脂质体（Liposome）主要由胆固醇和天然磷脂组成，进入体内可被生物降解，不会在体内蓄积，无毒性、无致热原性、无免疫原性。脂质体被证明是一种行之有效的药物运输载体，它具有靶向性和淋巴定向性；缓慢释放，延缓肾排泄和代谢，延长作用时间；降低药物毒性；提高稳定性等优点。现已经有十几种脂质体药物载体被应用于临床医疗。虽然构成脂质体非常接近天然的细胞膜，但仍然无法完全躲避免疫清除。利用天然细胞膜伪装微纳米载体，获得的仿生微纳米载体不但具有微纳米载体自身的理化性能，而且具有类天然细胞的生物性能。细胞膜伪装的微纳米载体的研究尚处于起步阶段，开发更多的新型细胞膜伪装载体对于生物医学领域具有重要的意义。
目前，荧光增强的仿生吲哚菁绿脂质在近红外二区荧光成像的应用还未见有公开报道。本发明利用吲哚菁绿与磷脂小分子的疏水相互作用，合成吲哚菁绿被嵌入磷脂双分子壳层的脂质体，吲哚菁绿避免了与水相互作用导致的自淬灭，近红外二区荧光强度显著提高。将细胞膜通过物理挤压修饰在纳米颗粒上形成仿生脂质体，具有类天然细胞的生物性能。本发明有助于推动近红外二区荧光成像的发展，为癌症的临床诊断提供新理论和新思路。
目前荧光发射光谱在近红外二区的材料主要有无机材料（纳米管、量子点、稀土纳米粒子）和有机材料（聚合物、有机小分子染料）。考虑到 NIR-II 成像剂的临床转化，具有低毒性，快速代谢性质的有机小分子荧光探针是最理想的选择。但多数 NIR-II 有机小分子荧光团存在水溶性差、光稳定性低、量子产率低等缺点。因此开发新型的近红外二区荧光的分子探针尤为重要。为解决现有技术的缺点和不足之处，本发明采用被美国食品药品监督管理局（FDA）批准用于临床的近红外荧光造影剂吲哚菁绿，利用它与磷脂小分子的疏水相互作用，合成吲哚菁绿被嵌入脂质体壳层的脂质体，吲哚菁绿避免了与水相互作用导致的自淬灭，近红外二区荧光强度显著提高。将细胞膜通过物理挤压修饰在纳米颗粒上形成仿生脂质体，具有类天然细胞的生物性能。该化合物具有生物相容性好的特点，修饰不同的细胞膜可使这种高亮度的近红外二区荧光探针得到广泛应用。本发明有助于推动近红外二区荧光成像的发展，为癌症的临床诊断提供新理论和新思路。
 技术解决方案
目前荧光发射光谱在近红外二区的材料主要有无机材料（纳米管、量子点、稀土纳米粒子）和有机材料（聚合物、有机小分子染料）。无机材料具有一定的毒性且代谢较为缓慢，多数 NIR-II 有机小分子荧光团存在水溶性差、光稳定性低、量子产率低等缺点。考虑到 NIR-II 成像剂的临床转化，具有低毒性，快速代谢性质的有机小分子荧光探针是最理想的选择。
本发明公开了一种高亮度的近红外二区吲哚菁绿仿生脂质体的合成方法，该方法利用吲哚菁绿与脂质小分子的疏水相互作用，合成吲哚菁绿被嵌入脂质双分子层的脂质体，吲哚菁绿避免了与水相互作用导致的自淬灭，近红外二区荧光强度显著提高。将细胞膜通过物理挤压修饰在纳米颗粒上形成仿生脂质体，具有类天然细胞的生物性能。该化合物具有生物相容性好的特点，修饰不同的细胞膜可使这种高亮度的近红外二区荧光探针得到广泛应用。本发明有助于推动近红外二区荧光成像的发展，为癌症的临床诊断提供理论和思路。
本发明一个方面提供了一种吲哚菁绿脂质体，其具有脂质双分子层以及嵌在脂质双分子层之间的吲哚菁绿。
在本发明的技术方案中，嵌在脂质双分子层之间的吲哚菁绿是通过将吲哚菁绿和脂质双分子层的材料在有机相混合均匀分散成薄膜后，加入水相水化形成脂质体而形成的。
在本发明的技术方案中，所述的吲哚菁绿脂质体脂质双分子层还嵌入有细胞膜蛋白。
在本发明的技术方案中，所述的磷脂双分子层由磷脂，或者磷脂和胆固醇的混合物构成。
在本发明的技术方案中，所述磷脂选自二棕榈酰磷脂酰胆碱、二硬脂酰基磷脂酰胆碱、二油酰基卵磷脂、二肉豆蔻酰磷脂酰胆碱、1-棕榈酰基-2-油酰基卵磷脂、大豆卵磷脂、氢化大豆卵磷脂、二月桂酰卵磷脂、二肉豆蔻酰卵磷脂、二肉豆蔻酰卵磷脂、二月桂酰磷脂酰甘油、二棕榈酰磷脂酸、二肉豆蔻酰磷脂酰乙醇胺、二棕榈酰磷脂酰乙醇胺、二肉豆蔻酰磷脂酰丝氨酸、二棕榈酰磷脂酰二丝氨酸、二棕榈酰磷脂酰胆碱、脑磷脂酰丝氨酸、脑神经鞘磷脂、二棕榈酰神经鞘磷脂、二硬脂酰神经鞘磷脂、二硬脂酰磷脂酰乙醇胺中的一种或几种的组合。
在本发明的技术方案中，磷脂双分子层由二棕榈酰磷脂酰胆碱、二硬脂酰基磷脂酰胆碱、二油酰基卵磷脂和胆固醇构成，优选地，其摩尔比例为二棕榈酰磷脂酰胆:二硬脂酰基磷脂酰胆碱:二油酰基卵磷脂:胆固醇=5:3:1:1。
在本发明的技术方案中，磷脂双分子层与吲哚菁绿的摩尔量比为25-1000:1，优选地为100-500:1，更优选为200-300:1。
本发明另一个方面提供了吲哚菁绿脂质体的制备方法，其包括如下步骤：
1）将制备磷脂双分子层的材料与吲哚菁绿在有机相混合形成均匀溶液，去除有机溶剂获得分散的薄膜；
2）加入水相进行水化，机械力分散形成吲哚菁绿脂质体。
在本发明的技术方案中，吲哚菁绿脂质体的制备方法还包括步骤3）在吲哚菁绿脂质体的表面镶嵌细胞膜蛋白。
在本发明的技术方案中，机械力分散指通过超声分散、脂质体挤出器反复挤出的方式进行分散。
在本发明的技术方案中，步骤1）中去除溶剂的方法选自，以减压方式去除溶剂或在常压下气体吹拂的方式去除。
在本发明的技术方案中，制备过程中以惰性气体进行保护。
在本发明的技术方案中，吲哚菁绿脂质体与细胞膜蛋白的质量比为200-400:1，优选为250-300:1。
在本发明的技术方案中，磷脂双分子层的材料选自磷脂，或者磷脂和胆固醇的混合物。
在本发明的技术方案中，所述的细胞膜蛋白选自癌细胞膜蛋白、红细胞膜蛋白、中性粒细胞膜蛋白、血小板细胞膜蛋白、巨噬细胞膜蛋白、自然杀伤细胞膜蛋白或以二聚体或四聚体的形式存在的葡萄糖转运蛋白1、 Neurothelin/HT7、血清γ谷氨酰转肽酶、P-糖蛋白、PD-1配体 PDL1/PDL2、Fas配体FasL、免疫共刺激蛋白B7-H4、膜相关补体调控蛋白CRRY以及非经典MHC I类分子。
在本发明的技术方案中，步骤3）的方法为将膜蛋白与脂质体分散均匀，然后以脂质体挤出器进行挤出，制备具细胞膜蛋白的脂质体。
在本发明的技术方案中，去除游离细胞膜蛋白的方法为透析或超速离心过滤。
本发明所述的脂质体在制备近红外二区荧光检测试剂中的用途。
在本发明的技术方案中，近红外二区荧光检测试剂能够应用于水溶液、磷酸盐缓冲液、培养基或人体或动物体体内外体液或血液中。
在本发明的方案中，所述的近红外二区为是指波长位于1000-1700nm的光波区段。
优选地，近红外二区选自波长位于1000-1400nm的光波区段。
本发明再一个方面提供了一种近红外二区检测试剂，其包括本发明所述的脂质体。
 有益效果
该化合物具有生物相容性好的特点，修饰不同的细胞膜可使这种高亮度的近红外二区荧光探针得到广泛应用。这种探针的优点体现在高亮度、低剂量、反应条件温和、重现性好、低毒、单分散的、生物相容性好、快速代谢等，本发明有助于推动近红外二区荧光成像的发展，为癌症的临床诊断提供理论和思路。
 附图说明
图1为吲哚菁绿与磷脂疏水作用后形成脂质体的横截面示意图。
图2为实施例1至实施例7的溶液图。
图3 近红外二区荧光亮度对比图。图注（1、LIPO:ICG为脂质体：吲哚菁绿 2、LIPO-ICG为吲哚菁绿脂质体 3、ICG-INPUT为相同投入量的吲哚菁绿 4、ICG-OD为相同紫外吸收的吲哚菁绿 5、PBS为磷酸盐缓冲液 6、CULTURE为无血清培养基溶液 7、10%FBS为10%胎牛血清溶液）。
图4 近红外二区荧光亮度变化图。图注（1、LIPO:ICG=250:1为脂质体：吲哚菁绿摩尔比为250:1  2、LIPO-ICG为吲哚菁绿仿生脂质体 3、SAME INPUT为相同投入量的吲哚菁绿 4、SAME OD为相同紫外吸收的吲哚菁绿 5、PBS为磷酸盐缓冲液 6、CULTURE为无血清培养基溶液 7、10%FBS为10%胎牛血清溶液）。
图5为溶解在无水甲醇的吲哚菁绿与脂质体原材料混合吹膜制备的脂质体的近红外二区荧光光谱。
图6为在磷酸盐缓冲溶液中近红外二区荧光强度定量图。
图7为在细胞无血清培养基溶液溶液中近红外二区荧光强度定量图。
图8为在10%的胎牛血清溶液中近红外二区荧光强度定量图。
图9为不同处理组小鼠右腿血管成像示意图，其中ICG-INPUT，ICG-OD为游离的吲哚菁绿（相同投入量和相同紫外吸收）LIPO-ICG为吲哚菁绿脂质体，RLIPO-ICG为修饰了红细胞膜的吲哚菁绿脂质体，在体内的血液循环时间相较于游离的吲哚菁绿大大提高。
 本发明的实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
实施例1：
1、精确称取0.3152mg二棕榈酰磷脂酰胆碱、0.072mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108mg 胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），准确称取0.434112mg 吲哚菁绿溶于无水甲醇。（脂质体原材料与吲哚菁绿摩尔量比为25:1）将混合溶液于 65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空 4h，使试管中的有机溶剂挥发完全。
3、用2mL 0.01M PBS（pH=7.4-7.5）在 65℃水浴条件下对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温 65℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，得吲哚菁绿脂质体，4℃避光条件下透析12h除去未装载的吲哚菁绿和细胞膜蛋白，得到纯化的吲哚菁绿脂质体。
实施例2：
1、精确称取0.3289mg二棕榈酰磷脂酰胆碱、0.072mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108mg 胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），准确称取0.217056mg 吲哚菁绿溶于无水甲醇。（脂质体原材料与吲哚菁绿摩尔量比为50:1）将混合溶液于 65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空 4h，使试管中的有机溶剂挥发完全。
3、用2mL 0.01M PBS（pH=7.4-7.5）在 65℃水浴条件下对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温 65℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行 5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，得吲哚菁绿脂质体，4℃避光条件下透析12h除去未装载的吲哚菁绿，得到纯化的吲哚菁绿脂质体。
实施例3：
1、精确称取0.3357mg二棕榈酰磷脂酰胆碱、0.072mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108mg 胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），准确称取0.108528mg 吲哚菁绿溶于无水甲醇。（脂质体原材料与吲哚菁绿摩尔量比为100:1）将混合溶液于 65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空 4h，使试管中的有机溶剂挥发完全。
3、用 2mL 0.01M PBS（pH=7.4-7.5）在 65℃水浴条件下对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温 65 ℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行 5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，得吲哚菁绿脂质体，4℃避光条件下透析12h除去未装载的吲哚菁绿，得到纯化的吲哚菁绿脂质体。
实施例4：
1、精确称取0.33988 mg二棕榈酰磷脂酰胆碱、0.072 mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108 mg 胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），准确称取0.043411mg 吲哚菁绿溶于无水甲醇。（脂质体原材料与吲哚菁绿摩尔量比为250:1）将混合溶液于 65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空 4h，使试管中的有机溶剂挥发完全。
3、用 2mL 0.01M PBS（pH=7.4-7.5）在 65℃水浴条件下对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温 65℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行 5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，得吲哚菁绿脂质体，4℃避光条件下透析12h除去未装载的吲哚菁绿，得到纯化的吲哚菁绿脂质体。
实施例5：
1、精确称取0.3412mg二棕榈酰磷脂酰胆碱、0.072mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108mg 胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），准确称取0.021705mg 吲哚菁绿溶于无水甲醇。（脂质体原材料与吲哚菁绿摩尔量比为500:1）将混合溶液于 65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空 4h，使试管中的有机溶剂挥发完全。
3、用 2mL 0.01M PBS（pH=7.4-7.5）在 65℃水浴条件下对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温 65℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行 5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，得吲哚菁绿脂质体，4℃避光条件下透析12h除去未装载的吲哚菁绿，得到纯化的吲哚菁绿脂质体。
实施例6：
1、精确称取0.3417mg二棕榈酰磷脂酰胆碱、0.072mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108mg 胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），准确称取0.014108mg 吲哚菁绿溶于无水甲醇。（脂质体原材料与吲哚菁绿摩尔量比为750:1）将混合溶液于 65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空 4h，使试管中的有机溶剂挥发完全。
3、用 2mL 0.01M PBS（pH=7.4-7.5）在 65℃水浴条件下对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温 65℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行 5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，得吲哚菁绿脂质体，4℃避光条件下透析12h除去未装载的吲哚菁绿，得到纯化的吲哚菁绿脂质体。
实施例7：
1、精确称取0.3419mg二棕榈酰磷脂酰胆碱、0.072mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108mg 胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），准确称取0.010852mg 吲哚菁绿溶于无水甲醇。（脂质体原材料与吲哚菁绿摩尔量比为1000:1）将混合溶液于 65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空4h，使试管中的有机溶剂挥发完全。
3、用 2mL 0.01M PBS（pH=7.4-7.5）在 65℃水浴条件下对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温 65℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行 5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，得吲哚菁绿脂质体，4℃避光条件下透析12h除去未装载的吲哚菁绿，得到纯化的吲哚菁绿脂质体。
实施例8：
1、精确称取0.33988mg二棕榈酰磷脂酰胆碱、0.072mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108mg 胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），准确称取0.043411mg 吲哚菁绿溶于无水甲醇。（脂质体原材料与吲哚菁绿摩尔量比为250:1）将混合溶液于 65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空4h，使试管中的有机溶剂挥发完全。
3、用 2mL 0.01M PBS（pH=7.4-7.5）在 65℃水浴条件下对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温 65℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行 5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，得到普通的吲哚菁绿脂质体。
6、加入SW1990胰腺癌细胞膜蛋白（蛋白与脂质体质量比为1:300），移液枪吹打混合均匀后用装有 200 nm 和100nm聚碳酸酯膜的脂质体挤出器反复挤压 20 次，4℃避光条件下透析12h除去未装载的吲哚菁绿和细胞膜蛋白，得到吲哚菁绿仿生脂质体。
实施例9：
1、精确称取0.33988mg二棕榈酰磷脂酰胆碱、0.072mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108mg 胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），准确称取0.043411mg 吲哚菁绿溶于无水甲醇。（脂质体原材料与吲哚菁绿摩尔量比为250:1）将混合溶液于 65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空 4h，使试管中的有机溶剂挥发完全。
3、用 2mL 0.01M PBS（pH=7.4-7.5）在 65℃水浴条件下对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温 65℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行 5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，得到普通的吲哚菁绿脂质体。
6、加入红细胞膜蛋白（蛋白与脂质体质量比为1:300），移液枪吹打混合均匀后用装有 200 nm 和100nm聚碳酸酯膜的脂质体挤出器反复挤压20 次，4℃避光条件下透析12h除去未装载的吲哚菁绿和细胞膜蛋白，得到吲哚菁绿仿生脂质体。
实施例10：
1、精确称取0.33988mg二棕榈酰磷脂酰胆碱、0.072mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108mg 胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），准确称取0.043411mg 吲哚菁绿溶于无水甲醇。（脂质体原材料与吲哚菁绿摩尔量比为250:1）将混合溶液于65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空 4h，使试管中的有机溶剂挥发完全。
3、用 2mL 0.01M PBS（pH=7.4-7.5）在 65℃水浴条件下对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温65℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行 5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，得到普通的吲哚菁绿脂质体。
6、加入中性粒细胞膜蛋白（蛋白与脂质体质量比为1:300），移液枪吹打混合均匀后用装有 200 nm 和100nm聚碳酸酯膜的脂质体挤出器反复挤压 20 次，4℃避光条件下12h透析除去未装载的吲哚菁绿和细胞膜蛋白，得到吲哚菁绿仿生脂质体。
对比例1
1、精确称取0.33988mg二棕榈酰磷脂酰胆碱、0.072mg二硬脂酰基磷脂酰胆碱、0.216mg二油酰基卵磷脂DOPC 和0.108mg胆固醇分别溶于三氯甲烷中（摩尔量比例为5:3:1:1），将混合溶液于65℃水浴加热并充分混匀，在涡旋器上涡旋，同时向试管中充入速率适中且流速稳定的氮气，直至有机溶液挥发完全形成均匀薄膜，试管内无液体残留。
2、用封口膜将试管口封口，并用利器在封口膜上戳孔，保证试管内气体可与外界连通，将试管置于真空器皿中避光抽真空4h，使试管中的有机溶剂挥发完全。
3、准确称取0.043411mg 吲哚菁绿溶于2mL 0.01M PBS（pH=7.4-7.5）中获得吲哚菁绿PBS溶液，在 65℃水浴条件下将吲哚菁绿PBS溶液对薄膜进行水化至试管壁上的脂质膜完全脱落后，将试管置于超声清洗机中（水温 65℃）水浴超声5 min。
4、在液氮和65℃环境水浴条件下反复冻融，循环进行 5 次。
5、溶液用装有 200 nm 聚碳酸酯膜的脂质体挤出器反复挤压 20 次，4℃避光条件下透析12h除去未装载的吲哚菁绿，得到纯化的吲哚菁绿脂质体。
对比例2
准确称取吲哚菁绿溶于2mL 0.01M PBS中获得与实施例4相同浓度的吲哚菁绿溶液。
效果例1光谱检测
分别对实施例1-7，对比例1-2的样品进行紫外光谱，荧光光谱检测。
通过检测发现，对比实施例以及对比例1的紫外光谱可知，本发明实施例脂质体的紫外光谱相对于对比例1-2发生了红移。
对比实施例与比例2以及对比对比例1-2的荧光光谱可知，本发明实施例在近红外一区窗口荧光光谱红移了约15nm，且荧光强度提高。对比例1在近红外一区窗口荧光光谱与游离吲哚菁绿保持一致，荧光强度降低。通过实验结果可知，本发明方法制备的脂质体能够增强吲哚菁绿的荧光发射，原因可能为疏水的双分子层限制了吲哚菁绿自聚集，导致荧光强度实现了累积增强的效果。而对比例1的方法制备获得脂质体，由于吲哚菁绿是限制在脂质体水溶性核心的，因此与水发生相互作用导致自聚集的荧光猝灭，同理游离的吲哚菁绿也面临相同的问题。
相对于游离的吲哚菁绿溶液，本发明方法制备获得的吲哚菁绿脂质体荧光光谱发生红移使更多的荧光信号进入近红外二区窗口，近红外一区荧光强度的提高让本发明方法制备获得的吲哚菁绿脂质体在近红外二区窗口的荧光光谱整体抬高，从而近红外二区窗口显示显著提高的荧光强度。综上所述，本发明的脂质体吲哚菁绿在近红外荧光二区可作为一种高亮度的成像探针。
效果例2 荧光强度检测
将不同比例的脂质体吲哚菁绿加入96孔板，以相同投入量的吲哚菁绿或相同紫外吸收的吲哚菁绿为对照，每孔体积100 mL 在近红外二区荧光成像仪上检测荧光强度，（仪器参数采用1000的滤光片，高度为220，激发光为808nm，曝光时间为15ms）实验结果参见图3，6-8，可知本发明制备脂质体的近红外二区荧光强度远高于游离的吲哚菁绿，在不同的溶剂中仍保持相当的稳定亮度。
通过对比不同的磷脂膜材与吲哚菁绿的合成比例的近红外二区荧光强度，实验现象揭示：一定范围内，荧光强度随着吲哚菁绿浓度的增加而上升，但超出范围后荧光强度随着吲哚菁绿浓度的增加而降低，这说明本发明的脂质体中的磷脂膜双层中嵌入的吲哚菁绿剂量存在最佳的上限。
效果例3 荧光强度稳定性检测
具体实验方法及参数与效果例2相同，96孔板里的样品用锡纸避光保存，在60天内去检测近红外二区荧光强度的变化。通过荧光强度检测实验结果（参见图4），可知本发明制备脂质体可以在不同介质中60天内近红外二区荧光强度衰减很慢，保持一定的荧光稳定。
效果例4 体内实验
对不同组小鼠进行体内实验，检测其荧光强度和维持时间，其中，RLIPO-ICG为修饰了红细胞膜的吲哚菁绿脂质体组，即实施例9的脂质体。ICG-INPUT组为相同投入量游离的吲哚菁绿组，ICG-OD为相同紫外吸收的游离的吲哚菁绿组，LIPO-ICG为吲哚菁绿脂质体组。小鼠尾静脉注射200μl不同处理组的试剂，相同投入量的游离吲哚菁绿浓度为44μg/mL，相同紫外吸收的游离吲哚菁绿浓度为132μg/mL。吲哚菁绿脂质体合成注射浓度为合成原液浓度。用近红外荧光二区成像仪进行检测。实验结果证明，在体内的血液循环时间相较于游离的吲哚菁绿以及未包覆红细胞膜的脂质体大大提高。

Claims (10)

  1. 一种用于近红外二区荧光检测的吲哚菁绿脂质体，其具有磷脂双分子层以及嵌在磷脂双分子层之间的吲哚菁绿，所述的吲哚菁绿脂质体外侧还可附有细胞膜蛋白。
  2. 根据权利要求1所述的吲哚菁绿脂质体，磷脂双分子层与吲哚菁绿的摩尔量比为25-1000:1，更优选为100-500:1。
  3. 根据权利要求1所述的吲哚菁绿脂质体，所述的磷脂双分子层由磷脂，或者磷脂和胆固醇的混合物构成；优选地，所述磷脂选自二棕榈酰磷脂酰胆碱、二硬脂酰基磷脂酰胆碱、二油酰基卵磷脂、二肉豆蔻酰磷脂酰胆碱、1-棕榈酰基-2-油酰基卵磷脂、大豆卵磷脂、氢化大豆卵磷脂、二月桂酰卵磷脂、二肉豆蔻酰卵磷脂、二肉豆蔻酰卵磷脂、二月桂酰磷脂酰甘油、二棕榈酰磷脂酸、二肉豆蔻酰磷脂酰乙醇胺、二棕榈酰磷脂酰乙醇胺、二肉豆蔻酰磷脂酰丝氨酸、二棕榈酰磷脂酰二丝氨酸、二棕榈酰磷脂酰胆碱、脑磷脂酰丝氨酸、脑神经鞘磷脂、二棕榈酰神经鞘磷脂、二硬脂酰神经鞘磷脂、二硬脂酰磷脂酰乙醇胺中的一种或几种的组合；
    更优选地，磷脂双分子层由二棕榈酰磷脂酰胆碱、二硬脂酰基磷脂酰胆碱、二油酰基卵磷脂和胆固醇构成。
  4. 根据权利要求2-3任一项所述的吲哚菁绿脂质体，吲哚菁绿脂质体与细胞膜蛋白的质量比为200-400:1，
    优选，所述的细胞膜蛋白选自癌细胞膜蛋白、红细胞膜蛋白、中性粒细胞膜蛋白、凝血调节蛋白、以二聚体或四聚体的形式存在的葡萄糖转运蛋白1、 Neurothelin/HT7、血清γ谷氨酰转肽酶、P-糖蛋白、PD-1配体 PDL1/PDL2、Fas配体FasL、免疫共刺激蛋白B7-H4、膜相关补体调控蛋白CRRY以及非经典MHC I类分子。
  5. 权利要求1-4任一项所述的吲哚菁绿脂质体的制备方法，以薄膜分散法制备，其包括如下步骤：
    1）将制备磷脂双分子层的材料与溶于有机溶剂的吲哚菁绿混合形成均匀溶液，去除有机溶剂获得分散的薄膜；
    2）加入水相进行水化，机械力分散形成吲哚菁绿脂质体；
    任选地，吲哚菁绿脂质体的制备方法还包括步骤3）在吲哚菁绿脂质体的表面附着细胞膜蛋白。
  6. 根据权利要求5所述的制备方法，机械力分散指通过超声分散、脂质体挤出器反复挤出的方式进行分散。
  7. 根据权利要求5所述的制备方法，磷脂双分子层的材料选自磷脂，或者磷脂和胆固醇的混合物；
    优选地，磷脂双分子层的材料与吲哚菁绿的摩尔量比为25-1000:1，更优选为100-500:1。
  8. 根据权利要求5所述的制备方法，步骤3）的方法为将膜蛋白与脂质体分散均匀，然后以脂质体挤出器进行挤出，分离具细胞膜蛋白的脂质体；优选地，分离具细胞膜蛋白的脂质体的方法为透析或离心。
  9. 权利要求1-4任一项所述的吲哚菁绿脂质体在制备近红外二区荧光检测试剂中的用途。
  10. 一种近红外二区用检测试剂，其包括权利要求1-4任一项所述的吲哚菁绿脂质体。
PCT/CN2020/129545 2019-12-12 2020-11-17 一种用于近红外二区荧光检测的吲哚菁绿脂质体及其制备方法和用途 WO2021115070A1 (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114432265A (zh) * 2022-02-17 2022-05-06 湖南万欧科技有限公司 负载日蟾蜍他灵的仿生纳米递送系统及其制备方法和应用
CN114778838A (zh) * 2022-05-31 2022-07-22 西南交通大学 一种快速广谱检测细菌的试剂盒、制备方法及其检测方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110960694B (zh) * 2019-12-12 2021-09-17 深圳先进技术研究院 一种用于近红外二区荧光检测的吲哚菁绿脂质体及其制备方法和用途
CN111676011B (zh) * 2020-05-09 2021-10-26 厦门大学 一种吲哚菁绿-磷脂复合物及其制备方法和应用
CN112843258A (zh) * 2021-01-28 2021-05-28 厦门大学附属翔安医院 一种乳腺癌靶向分子探针及其制备方法和应用
CN112999200B (zh) * 2021-03-03 2024-04-09 北京昊澄生物科技有限公司 一种经皮肤给药层
CN113521315A (zh) * 2021-07-28 2021-10-22 深圳先进技术研究院 一种多模态微泡造影剂及其制备方法和应用
WO2023102877A1 (zh) * 2021-12-10 2023-06-15 深圳先进技术研究院 吲哚菁绿脂质体及其大规模制备方法和应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130323178A1 (en) * 2012-02-23 2013-12-05 Canon Kabushiki Kaisha Indocyanine green-containing particle and contrast agent for photoacoustic imaging having the particle
CN103690486A (zh) * 2013-12-27 2014-04-02 深圳先进技术研究院 一种吲哚菁绿纳米靶向脂质体及其制备方法和应用
CN103874482A (zh) * 2011-10-07 2014-06-18 国立大学法人鸟取大学 脂质体复合物
KR101499143B1 (ko) * 2013-10-24 2015-03-05 전북대학교산학협력단 형광색소를 봉입한 만노오스화된 리포좀을 포함하는 림프절 영상제제
CN105854030A (zh) * 2015-02-08 2016-08-17 苏州同力生物医药有限公司 脂质纳米颗粒及其应用
CN110960694A (zh) * 2019-12-12 2020-04-07 深圳先进技术研究院 一种用于近红外二区荧光检测的吲哚菁绿脂质体及其制备方法和用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130336889A1 (en) * 2012-06-14 2013-12-19 National Taiwan University Nanoparticle and method for detecting or treating a tumor using the same
CN110025576A (zh) * 2019-04-23 2019-07-19 上海市第六人民医院 一种用于荧光成像介导的光热肿瘤治疗的光热试剂的制备方法及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103874482A (zh) * 2011-10-07 2014-06-18 国立大学法人鸟取大学 脂质体复合物
US20130323178A1 (en) * 2012-02-23 2013-12-05 Canon Kabushiki Kaisha Indocyanine green-containing particle and contrast agent for photoacoustic imaging having the particle
KR101499143B1 (ko) * 2013-10-24 2015-03-05 전북대학교산학협력단 형광색소를 봉입한 만노오스화된 리포좀을 포함하는 림프절 영상제제
CN103690486A (zh) * 2013-12-27 2014-04-02 深圳先进技术研究院 一种吲哚菁绿纳米靶向脂质体及其制备方法和应用
CN105854030A (zh) * 2015-02-08 2016-08-17 苏州同力生物医药有限公司 脂质纳米颗粒及其应用
CN110960694A (zh) * 2019-12-12 2020-04-07 深圳先进技术研究院 一种用于近红外二区荧光检测的吲哚菁绿脂质体及其制备方法和用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KRAFT JOHN C., HO RODNEY J. Y.: "Interactions of Indocyanine Green and Lipid in Enhancing Near-Infrared Fluorescence Properties: The Basis for Near-Infrared Imaging in Vivo", BIOCHEMISTRY, vol. 53, no. 8, 4 March 2014 (2014-03-04), pages 1275 - 1283, XP055819860, ISSN: 0006-2960, DOI: 10.1021/bi500021j *
XU HE-LIN, SHEN BI-XIN, LIN MENG-TING, TONG MENG-QI, ZHENG YA-WEN, JIANG XUE, YANG WAI-GENG, YUAN JIAN-DONG, YAO QING, ZHAO YING-Z: "Homing of ICG-loaded liposome inlaid with tumor cellular membrane to the homologous xenografts glioma eradicates the primary focus and prevents lung metastases through phototherapy", BIOMATERIALS SCIENCE, R S C PUBLICATIONS, GB, vol. 6, no. 9, 1 January 2018 (2018-01-01), GB, pages 2410 - 2425, XP055819857, ISSN: 2047-4830, DOI: 10.1039/C8BM00604K *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114432265A (zh) * 2022-02-17 2022-05-06 湖南万欧科技有限公司 负载日蟾蜍他灵的仿生纳米递送系统及其制备方法和应用
CN114432265B (zh) * 2022-02-17 2023-09-05 湖南万欧科技有限公司 负载日蟾蜍他灵的仿生纳米递送系统及其制备方法和应用
CN114778838A (zh) * 2022-05-31 2022-07-22 西南交通大学 一种快速广谱检测细菌的试剂盒、制备方法及其检测方法

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