CO release carrier composition, carrier and preparation method, release method and application thereof

Abstract

The invention provides a CO release carrier composition, a carrier as well as a preparation method, a release method and application of the CO release carrier composition. The CO release carrier composition comprises a surfactant, inert fluoride liquid and CO gas, the surfactant wraps the inert fluoride liquid; the CO gas is dissolved in the inert fluoride liquid; the surface active agent comprises a surface active agent A and a surface active agent B, and the mass ratio of the surface active agent A to the surface active agent B is 25: (1-50). The prepared CO release carrier has the advantages of being high in loading capacity, controllable in release, long in circulation time, accurate in targeting and the like, and an innovative solution is provided for safe and effective delivery of CO gas.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a CO release carrier composition, a carrier and a preparation method, a release method and use thereof. Background Art

[0002] Carbon monoxide (CO), as an endogenous signaling molecule, has multiple biological activities such as anti-inflammatory, antioxidant, anti-apoptotic and inhibition of tumor cell proliferation, and has shown broad application prospects in the treatment of cardiovascular diseases, inflammatory responses, organ transplant rejection and tumors. However, the high affinity of CO for hemoglobin can seriously interfere with the delivery of oxygen to tissues, leading to fatal consequences such as coma, convulsions, and respiratory depression. How to safely and effectively deliver CO to target tissues has become the focus and difficulty of research in the field of CO therapy.

[0003] In the prior art, treatment is usually performed by directly inhaling a mixed gas containing CO or injecting CO-releasing molecules. CO-releasing molecules can release CO under specific conditions, but face problems such as difficult to control the release rate, insufficient targeting, and poor stability in the body. In addition, direct delivery of CO poses a toxicity risk, so innovative methods are very necessary to improve the safety and effectiveness of CO in the body.

[0004] In recent years, researchers have proposed a variety of design strategies for CO release carriers. These CO release carriers can control the release of CO under specific stimuli in vitro or in vivo (such as light, heat, pH changes, etc.). However, these methods still have some problems in practical applications, such as complex preparation, limited CO loading, and uneven release. In particular, there are still huge challenges in precisely controlling the temporal and spatial distribution of CO release.

[0005] In summary, there is an urgent need for a CO release carrier with high safety, large carrier gas volume, long in vivo circulation time, and strong controllability of dosage and release to promote the development of CO therapy. Summary of the invention

[0006] In view of this, the purpose of the present invention is to overcome the problems existing in the prior art that biosafety, high loading, controllable dosage and release behavior cannot be taken into account during CO treatment, and a CO release carrier composition, a CO release carrier containing the CO release carrier composition and a preparation method thereof are provided. The CO release carrier composition and / or CO release carrier of the present invention can be used in conjunction with ultrasonic stimulation, which is beneficial to the delivery of CO in deep tissues, and the release time and spatial distribution of CO can be controlled under the condition of taking biosafety into account, thereby improving the treatment effect and reducing side effects.

[0007] The inventors of the present invention have found that when the CO release carrier composition of the present invention is used to deliver CO in vivo, the CO gas is dissolved in the fluoride liquid, which can load more CO gas and circulate in the body for a longer time (>2h), so that it is easier to control the release of CO only in the target tissue. In addition, the CO release carrier composition of the present invention wraps the fluoride liquid with a surfactant having a hydrophilic end and a lipophilic end, has good biocompatibility and stability, and reduces unnecessary consumption of CO gas during transportation and toxic side effects on non-target tissues. Therefore, the CO release carrier composition of the present invention has the advantages of high biosafety, large gas carrier volume, long in vivo circulation time, and strong controllability of dosage and release.

[0008] The first aspect of the present invention provides a CO release carrier composition, comprising a surfactant, an inert fluoride liquid, and CO gas; the surfactant encapsulates the inert fluoride liquid; and the CO gas is dissolved in the inert fluoride liquid.

[0009] Wherein, the surfactant includes surfactant A and surfactant B, and the mass ratio is 25: (1-50).

[0010] In the present invention, the term "inert fluoride liquid" refers to a fluoride (such as perfluoropentane, etc.) that has a high solubility in CO gas, is non-polar, and is not easy to react after entering the human body. It has high biological safety and is discharged through the lungs after vaporization. Different fluoride liquids can be selected according to the dosage and release position of CO.

[0011] In the present invention, CO gas is dissolved in an inert fluoride liquid, the dosage of loaded CO is increased, and the stability of the CO-releasing carrier composition is enhanced.

[0012] In the present invention, the surfactant A is a surfactant that can encapsulate perfluorinated liquid, has a hydrophilic end and a lipophilic end, and is non-toxic and harmless to organisms. Different surfactant A agents can be selected according to the target tissue of CO delivery, the in vivo circulation time, and the characteristics of the disease.

[0013] In the present invention, the surfactant B agent is used in combination with the surfactant A agent to prevent the aggregation and fusion of the CO releasing carrier composition during the emulsification process, increase the stability and uniformity of the CO releasing carrier composition, adjust the dispersibility, and enhance the biocompatibility. It can be freely selected according to the CO delivery location, disease type and surfactant A agent.

[0014] The CO release carrier composition of the present invention comprises CO gas dissolved in an inert fluoride liquid, a surfactant A having a hydrophilic end and a lipophilic end wrapping the perfluorinated liquid, and has a high gas carrying capacity, good biocompatibility and targeting, and the surfactant B agent modification makes the composition have good stability and uniformity, can circulate in the body for a long time (>2h), and can controllably release CO in deep tissues when used in conjunction with ultrasonic stimulation.

[0015] The solubility of CO in the inert fluoride liquid of the present invention under standard conditions is ≥5 ml / 100 ml.

[0016] In the present invention, the standard condition is a standard condition at a temperature of 0° C. (273.15 K) and a pressure of 101.325 kPa (1 standard atmosphere, 760 mmHg).

[0017] Preferably, the inert fluoride liquid includes one or more of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorooctane bromide, perfluorodecalin, and perfluorotripropylamine.

[0018] Preferably, when one or more of the inert fluorides are mixed and used, the boiling point is ≥37° C. under a pressure of 0.5 MPa.

[0019] Preferably, the inert fluoride liquid is selected from one or more of perfluoropentane, perfluorohexane and perfluorooctane bromide.

[0020] Preferably, the inert fluoride liquid comprises fluoride A and fluoride B. Preferably, fluoride A is perfluoropropane or perfluorobutane, and fluoride B is one or more of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorooctane bromide, perfluorodecalin, and perfluorotripropylamine. Preferably, the volume ratio of fluoride A to fluoride B is (1:9)-(9:1).

[0021] The specific ratio of the surfactant A and the surfactant B of the present invention can achieve a good effect of the CO release carrier composition. In order to further increase the CO loading and maintain or improve the stability, the mass ratio of the surfactant A to the surfactant B is preferably 20: (1-30); more preferably 10: (1-13). Preferably, the absolute value of the zeta potential of the surfactant A is between 20mV and 60mV (pH=7).

[0022] In a specific embodiment of the present invention, the surfactant A agent is one or more of lipids, proteins, Tween surfactants, Span surfactants or polymer surfactants. In order to exert a better synergistic effect with other components in the CO release carrier composition of the present invention, more preferably, the lipid is selected from one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid or cationic lipids; the protein is deoxyhemoglobin (Hb); the Tween surfactant is selected from one or more of Tween 20, Tween 60, and Tween 80; the Span surfactant is one or more of Span 80, Span 60, and Span 85; the polymer surfactant is one or more of polylactic acid-glycolic acid copolymer (PLGA), PLGA-COOH, PLGA-NH2, PLGA-maleimide, and polylactic acid-glycolic acid-polycaprolactone (PLGA-PCL).

[0023] More preferably, the phosphatidylcholine is selected from 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DEPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DEPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC ... The phosphatidylethanolamine is selected from one or more of dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), and dioleoylphosphatidylethanolamine (DOPE); the phosphatidylglycerol is selected from 1,2-distearoylphosphatidylglycerol (DSPG) and / or 1,2-dipalmitoylphosphatidylglycerol (DSPG). The phosphatidic acid is selected from one or more of 1,2-dioleoylphosphatidic acid (DOPA), 1,2-distearoylphosphatidic acid (DSPA), and 1,2-dipalmitoylphosphatidic acid (DPPA); the phosphatidylserine is selected from one or more of 1,2-dioleoylphosphatidylserine (DOPS), 1,2-distearoylphosphatidylserine (DSPS), and 1,2-dipalmitoylphosphatidylserine (DPPS); the cationic lipid One or more selected from 1,2-dioleoyl-3-trimethylammonium chloride propionamide (DOTAP), N-[1-(2,3-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride methylamine salt (DOTMA), distearyl-3-trimethylammonium chloride propionamide (DSTAP), N,N-dimethyl distearyl propane ammonium chloride (DODMA), N-(4-hydroxybutyl)-N,N-dimethyl-2,3-dipalmitoyl propylamine chloride (DMRIE). Preferably, the mass ratio of lactic acid to glycolic acid in the PLGA is 100:(0-100), more preferably (75-100):(25-0), more preferably (75-90):(25-10).

[0024] In a preferred embodiment of the present invention, the surfactant A agent is at least one of DPPC, DSPC, DOPC and DBPC.

[0025] In a preferred embodiment of the present invention, the surfactant A agent is a combination of at least one of DPPC, DSPC, DOPC, and DBPC and at least one of DSPG, DPPG, DOPA, DSPA, DPPA, DOPS, DSPS, DPPS, DOTAP, and DOTMA in a mass ratio of 10:(1-7).

[0026] In a preferred embodiment of the present invention, the surfactant A agent is a combination of at least one of DPPC, DSPC, DOPC, and DBPC, and at least one of DPPE, DOPE, and DSPE, and at least one of DSPG, DPPG, DOPA, DSPA, DPPA, DOPS, DSPS, DPPS, DOTAP, and DOTMA, in a mass ratio of 10:(2-10):(1-7).

[0027] In a preferred embodiment of the present invention, the surfactant A is at least one of DMPC, DPPC, DSPC, and DBPC, and is combined with deoxyhemoglobin in a mass ratio of 2: (1-10).

[0028] In a preferred embodiment of the present invention, the surfactant A is a combination of at least one of Tween 20, Tween 60, and Tween 80 and at least one of Span 80, Span 60, and Span 85 in a mass ratio of 4:(1-7).

[0029] In a preferred embodiment of the present invention, the surfactant A agent is at least one of PLGA and PLGA-PCL.

[0030] In a specific embodiment of the present invention, the surfactant B agent is one or more of cholesterol, polyethylene glycol (PEG) derivatives, poloxamer surfactants or fatty alcohol polyoxyethylene ether surfactants. In order to prevent the aggregation and fusion of the CO releasing carrier composition during the emulsification process, increase the stability and uniformity of the CO releasing carrier composition, and enhance the biocompatibility, more preferably, the polyethylene glycol (PEG) derivative is selected from one or more of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), polyethylene glycol methyl ether-polylactic acid-polyglycolic acid copolymer (PEG2000-PLGA), polyethylene glycol 4000 (PEG4000), and polyethylene glycol 2000 (PEG2000); the poloxamer surfactant is selected from one of Pluronic F68, Pluronic F108, and Pluronic F127; and the fatty alcohol polyoxyethylene ether surfactant is polyoxyethylene ether 23 lauryl alcohol (Brij35) or polyoxyethylene ether 20 stearyl alcohol (Brij78). Preferably, the mass ratio of lactic acid to glycolic acid in the PEG2000-PLGA is 100:(0-100), more preferably (75-100):(25-0), and more preferably (75-90):(25-10).

[0031] In a preferred embodiment of the present invention, the surfactant B agent is at least one of DSPE-PEG2000, PEG2000-PLGA, PEG4000, and PEG2000.

[0032] In a preferred embodiment of the present invention, the surfactant B agent is a combination of at least one of DSPE-PEG2000, PEG2000-PLGA, PEG4000, and PEG2000 and at least one of Pluronic F68, PluronicF108, and Pluronic F127 in a mass ratio of 5:(1-5).

[0033] In a preferred embodiment of the present invention, the surfactant B agent is at least one of DSPE-PEG2000, PEG2000-PLGA, PEG4000, and PEG2000, in combination with cholesterol at a mass ratio of 1: (1-5).

[0034] In a preferred embodiment of the present invention, the surfactant B agent is at least one of DSPE-PEG2000, PEG2000-PLGA, PEG4000, and PEG2000, in combination with Brij35 or Brij78 at a mass ratio of 2: (1-10).

[0035] The types and combinations of the above-mentioned surfactant A agent and surfactant B agent are further preferred, which can further improve the loading effect, stability and biosafety of the CO release carrier composition of the present invention and prolong its circulation time in the body.

[0036] In a preferred embodiment of the present invention, the surfactant A agent is a lipid, and the lipid is selected from one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid or cationic lipids, and the surfactant B agent is a combination of polyethylene glycol (PEG) derivatives, poloxamer surfactants and fatty alcohol polyoxyethylene ether surfactants. Preferably, the mass ratio of polyethylene glycol (PEG) derivatives, poloxamer surfactants and fatty alcohol polyoxyethylene ether surfactants is 5:(1-5):(2-20).

[0037] In a preferred embodiment of the present invention, the surfactant A agent is one of DPPC, DSPC, DOPC, and DBPC, and the surfactant B agent is DSPE-PEG2000.

[0038] In a preferred embodiment of the present invention, the surfactant A agent is a combination of one of DPPC, DSPC, DOPC, DBPC and one of DSPG, DPPG, DOPA, DSPA, DPPA, DOPS, DSPS, DPPS, DOTAP, DOTMA in a mass ratio of 10: (1-7), and the surfactant B agent is DSPE-PEG2000.

[0039] In a preferred embodiment of the present invention, the surfactant A agent is a combination of one of DPPC, DSPC, DOPC, DBPC and Hb in a mass ratio of 10:(1-20), and the surfactant B agent is DSPE-PEG2000.

[0040] In a preferred embodiment of the present invention, the surfactant A agent is one of DPPC, DSPC, DOPC, and DBPC, and the surfactant B agent is a combination of DSPE-PEG2000 and cholesterol in a mass ratio of 1: (1-5).

[0041] In a preferred embodiment of the present invention, the surfactant A agent is a combination of one of DPPC, DSPC, DOPC, DBPC and one of DSPG, DPPG, DOPA, DSPA, DPPA, DOPS, DSPS, DPPS, DOTAP, DOTMA in a mass ratio of 10: (1-7), and the surfactant B agent is a combination of DSPE-PEG2000 and cholesterol in a mass ratio of 1: (1-5).

[0042] In a preferred embodiment of the present invention, the surfactant A agent is a combination of one of DPPC, DSPC, DOPC, DBPC and Hb in a mass ratio of 1: (1-9), and the surfactant B agent is a combination of DSPE-PEG2000 and cholesterol in a mass ratio of 1: (1-5).

[0043] In a preferred embodiment of the present invention, the surfactant A agent is one of DPPC, DSPC, DOPC, and DBPC, and the surfactant B agent is a combination of DSPE-PEG2000 and one of Pluronic F68, Pluronic F108, and Pluronic F127 in a mass ratio of 5:(1-5).

[0044] In a preferred embodiment of the present invention, the surfactant A agent is a combination of one of DPPC, DSPC, DOPC, DBPC and one of DSPG, DPPG, DOPA, DSPA, DPPA, DOPS, DSPS, DPPS, DOTAP, DOTMA in a mass ratio of 10: (1-7), and the surfactant B agent is a combination of DSPE-PEG2000 and one of Pluronic F68, Pluronic F108, Pluronic F127 in a mass ratio of 5: (1-5).

[0045] In a preferred embodiment of the present invention, the surfactant A agent is a combination of one of DPPC, DSPC, DOPC, DBPC and Hb in a mass ratio of 1: (1-9), and the surfactant B agent is a combination of DSPE-PEG2000 and one of Pluronic F68, Pluronic F108, Pluronic F127 in a mass ratio of 5: (1-5).

[0046] In a preferred embodiment of the present invention, the surfactant A agent is one of DPPC, DSPC, DOPC, and DBPC, and the surfactant B agent is a combination of DSPE-PEG2000 and Brij35 (or Brij78) in a mass ratio of 2: (1-10).

[0047] In a preferred embodiment of the present invention, the surfactant A agent is a combination of one of DPPC, DSPC, DOPC, DBPC and one of DSPG, DPPG, DOPA, DSPA, DPPA, DOPS, DSPS, DPPS, DOTAP, DOTMA in a mass ratio of 10: (1-7), and the surfactant B agent is a combination of DSPE-PEG2000 and Brij35 (or Brij78) in a mass ratio of 2: (1-10).

[0048] In a preferred embodiment of the present invention, the surfactant A agent is a combination of one of DPPC, DSPC, DOPC, DBPC and Hb in a mass ratio of 1: (1-9), and the surfactant B agent is a combination of DSPE-PEG2000 and Brij35 (or Brij78) in a mass ratio of 2: (1-10).

[0049] In a preferred embodiment of the present invention, the surfactant A agent is a combination of one of Tween 20, Tween 60, Tween 80 and one of Span 80, Span 60, Span 85 in a mass ratio of 4: (1-7), and the surfactant B agent is one of DSPE-PEG2000, PEG4000, and PEG2000.

[0050] In a preferred embodiment of the present invention, the surfactant A agent is one of PLGA and PLGA-PCL, and the surfactant B agent is one of PEG2000-PLGA, PEG4000, and PEG2000.

[0051] The types and combinations of the above-mentioned inert fluoride liquid, surfactant A agent and surfactant B agent are further preferred, which can further improve the ultrasonic release performance and CO loading capacity of the CO releasing carrier composition of the present invention.

[0052] In a preferred embodiment of the present invention, the inert fluoride liquid comprises one or more of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorooctane bromide, perfluorodecalin, and perfluorotripropylamine, the solubility of CO of the inert fluoride liquid under standard conditions is ≥5ml / 100ml, the surfactant A agent is a combination of one of DPPC, DSPC, DOPC, and DBPC and one of DSPG, DPPG, DOPA, DSPA, DPPA, DOPS, DSPS, DPPS, DOTAP, DOTMA, and Hb in a mass ratio of 10: (1-90), and the surfactant B agent is DSPE-PEG2000, or a combination of DSPE-PEG2000 and one or more of cholesterol, Pluronic F68, PluronicF108, Pluronic F127, Brij35, and Brij78 in a mass ratio of 5: (1-25).

[0053] In a preferred embodiment of the present invention, the inert fluoride liquid is perfluoropentane, the surfactant A agent is a combination of one of DPPC, DSPC, DOPC, DBPC and one of DSPG, DPPG, DOPA, DSPA, DPPA, DOPS, DSPS, DPPS, DOTAP, DOTMA, Hb in a mass ratio of 10: (1-90), and the surfactant B agent is a combination of DSPE-PEG2000 with Brij35 and Pluronic F68 in a mass ratio of 5: (1-5): (2-20).

[0054] In a preferred embodiment of the present invention, the inert fluoride liquid is perfluoropentane, the surfactant A agent is a combination of DBPC and DSPA in a mass ratio of 10: (1-90), and the surfactant B agent is a combination of DSPE-PEG2000, Brij35 and Pluronic F68 in a mass ratio of 5: (1-5): (2-20).

[0055] In a preferred embodiment of the present invention, the inert fluoride liquid is perfluoropentane, the surfactant A is a combination of Span 60 and Tween 80 in a mass ratio of (1-5): (5-1), and the surfactant B is a combination of PEG 2000 and Pluronic F68 in a mass ratio of (1-5): (5-1).

[0056] In a preferred embodiment of the present invention, the inert fluoride liquid is perfluoropentane, the surfactant A agent is PLGA, and the surfactant B agent is PEG2000-PLGA.

[0057] In a preferred embodiment of the present invention, the inert fluoride liquid is perfluoropentane, the surfactant A agent is a combination of DSPC and DSPA in a mass ratio of 10: (1-90), and the surfactant B agent is a combination of DSPE-PEG2000, Brij35 and Pluronic F68 in a mass ratio of 5: (1-5): (2-20).

[0058] In a preferred embodiment of the present invention, the inert fluoride liquid is a combination of one of perfluoropropane, perfluorobutane and one or more of perfluoroheptane, perfluorooctane, perfluorooctane bromide, perfluorodecalin, and perfluorotripropylamine. Under a pressure of 0.5 MPa, the boiling point of the inert fluoride liquid is ≥37°C, the surfactant A agent is one or more of lipids, proteins, Tween surfactants, Span surfactants or polymer surfactants, and the surfactant B agent is selected from one or more of cholesterol, polyethylene glycol (PEG) derivatives, poloxamer surfactants or fatty alcohol polyoxyethylene ether surfactants.

[0059] In a preferred embodiment of the present invention, the inert fluoride liquid is a combination of one of perfluoropropane, perfluorobutane and one or more of perfluoroheptane, perfluorooctane, perfluorooctane bromide, perfluorodecalin, and perfluorotripropylamine. Under a pressure of 0.5 MPa, the boiling point of the inert fluoride liquid is ≥37°C, the surfactant A agent is one or more of Tween 20, Span 85, DOPC, DSPC, DOTAP, DBPC, and Hb, and the surfactant B agent is one or more of PEG4000, Brij78, DSPE-PEG2000, cholesterol, PluronicF108, and Pluronic F68.

[0060] The second aspect of the present invention provides a CO releasing carrier, comprising the CO releasing carrier composition described in the first aspect of the present invention.

[0061] In the present invention, the CO release carrier may include other components, such as drugs used in combination, or other functional additives, in addition to the CO release carrier composition described in the first aspect of the present invention. The CO release carrier provided in the second aspect of the present invention has all the technical effects of the CO release carrier composition of the first aspect of the present invention, which will not be described in detail here.

[0062] In the present invention, the CO releasing carrier is a carrier that releases CO under ultrasonic stimulation.

[0063] In a specific embodiment of the present invention, the CO release carrier is a spherical droplet, CO gas is dissolved in the internal fluoride liquid, and the carrier function of storing CO gas is realized. Under ultrasonic stimulation, the fluoride liquid vaporizes and explodes to release CO gas, and the function of releasing CO is realized.

[0064] Preferably, the response frequency range of the acoustic stimulation is 0.1-30 MHz.

[0065] Preferably, the power range of the acoustic stimulation is 0.1-5 W / cm 2 .

[0066] Preferably, the in vivo circulation time of the CO-releasing carrier is >2h.

[0067] Preferably, the CO release carrier concentration is greater than 1×10 9 Pieces / ml.

[0068] Preferably, the structure of the CO release carrier is a spherical droplet, preferably a nanosphere.

[0069] In the present invention, the spherical droplet structure of the CO release carrier is CO / inert liquid core-surfactant shell from the inside to the outside.

[0070] Preferably, the volume weighted average particle size of the CO release carrier is 0.05-2 μm, more preferably 50-500 nm, more preferably 50-200 nm.

[0071] Preferably, at least 60% of the particle size of the CO release carrier is distributed in the range of 0.05-2μm, and more preferably at least 80% is distributed in the range of 50-500nm. Further preferably, at least 80% of the particle size of the CO release carrier is distributed in the range of 0.05-2μm, and more preferably at least 90% is distributed in the range of 50-500nm. Further preferably, at least 100% of the particle size of the CO release carrier is distributed in the range of 0.05-2μm, and more preferably at least 100% is distributed in the range of 50-500nm. The particle size of the CO release carrier can better prolong its circulation time in the body and improve the tissue penetration depth, and can also regulate the CO loading dose and further enhance the comprehensive effect of the CO release carrier.

[0072] The polydispersity index of the CO-releasing carrier according to the invention is preferably less than 0.2, preferably less than 0.1.

[0073] In the present invention, the volume weighted average particle size, particle size distribution and polydispersity index can be measured by dynamic light scattering (DLS).

[0074] Preferably, the carrier gas volume of the CO release carrier is 10000ppm-2000000ppm CO per milliliter of carrier, more preferably 100000-1000000ppm. The carrier gas volume of the CO release carrier is preferably not only to improve the treatment effect for different diseases, but also to adjust the stability, particle size distribution and carrier circulation time of the CO release carrier.

[0075] In the present invention, the term "carrier gas volume" refers to the volume of CO released by 1 ml of CO release carrier after complete explosion, which can be measured by high performance gas chromatography. In the present invention, the carrier gas volume of CO release carrier is 1×10 10 The samples were measured at 100 μg / ml.

[0076] Preferably, the absolute value of the zeta potential of the CO releasing carrier of the present invention at pH=7 is less than 70 mV, more preferably 30-65 mV, and further preferably 45-65 mV.

[0077] The activation efficiency of the CO-releasing carrier of the present invention is greater than 40%, preferably greater than 50%, and more preferably greater than 60%.

[0078] The activation efficiency was calculated by measuring the concentration of CO release carriers before and after ultrasonic activation. Measurement conditions: The initial CO release carrier concentration was 1×10 9 / ml, ultrasonic stimulation power is 2W / cm 2 , frequency is 1MHz and duration is 2min.

[0079] The third aspect of the present invention provides a method for preparing the CO release carrier according to the second aspect of the present invention, comprising the following steps: (a) adding surfactant A and surfactant B in a certain ratio to solvent A to obtain solution I, and mixing to obtain solution II; (b) contacting an inert fluoride liquid with CO to obtain a CO-saturated fluoride solution III; (c) Solution II is contacted with solution III and mixed.

[0080] In step (a) of the present invention, the solution A is not particularly limited in its selection, as long as it can dissolve the surfactant described in the first aspect of the present invention without causing any chemical reaction.

[0081] Preferably, the solution A is one or more of pure water, ion buffer, glycerol, propylene glycol, acetone, ether, ethanol, methanol, chloroform, dichloromethane or ethyl acetate.

[0082] Preferably, the ionic buffer is PBS buffer.

[0083] Preferably, the solution A is a combination of ethanol and methanol, or ethanol and dichloromethane. Further preferably, the volume ratio of ethanol to methanol, or ethanol to dichloromethane is (1-2): (1-2).

[0084] In step (a) of the present invention, the solution I is mixed. The mixing method is not particularly limited and can be performed using conventional mixing methods in the art.

[0085] Preferably, in order to improve the performance of the CO release carrier described in the second aspect of the present invention, the solution I is mixed by one or more of a stirring method, a high temperature method, and a thin film hydration method.

[0086] In a specific embodiment of the present invention, the stirring method comprises the following steps: heating the solution I to 35-120° C., keeping the temperature and stirring continuously for 0.5-12 hours to obtain a solution II.

[0087] Preferably, the heating temperature of the stirring method is 40-95° C., and the stirring time is 2-8 h.

[0088] In a specific embodiment of the present invention, the high temperature method comprises the following steps: heating the solution I to 35-110° C., and keeping the temperature for 10-60 minutes until the solution becomes transparent, to obtain a solution II.

[0089] Preferably, the heating temperature of the high temperature method is 40-90°C, and the insulation time is 15-40 min. More preferably, the heating temperature is 45-80°C, and the insulation time is 20-30 min.

[0090] In a specific embodiment of the present invention, the thin film hydration method comprises the following steps: rotary evaporating the solution I in a water bath until the solvent is completely evaporated to form a film, adding solution B to slowly hydrate and continuously stir to make the lipid film fall off the bottle wall to obtain solution II.

[0091] The solution B is not particularly limited in its selection, as long as it can remove and hydrate the film formed after the rotary evaporation of the solution I in a water bath without causing any chemical reaction.

[0092] Preferably, the solution B is one or more of glycerol, propylene glycol, pure water, and an ion buffer. The ion buffer is preferably a PBS buffer.

[0093] Preferably, the temperature of the water bath rotary evaporation is preferably 35-80°C, and the pressure is preferably 0.01-0.1Mpa; more preferably, the temperature of the water bath rotary evaporation is preferably 45-80°C, and the pressure is preferably 0.03-0.08Mpa.

[0094] Solution I is mixed by one or more of stirring method, high temperature method and thin film hydration method, and the emulsification conditions are continuously optimized to make solution II more uniform, which is convenient for the subsequent preparation of CO release carrier, and the particle size of CO release carrier is made more uniform, the carrier concentration is increased, and the ability of CO gas loading is improved, and the stability is improved.

[0095] In a preferred embodiment of the present invention, the volume content of glycerol in the solution B is 5-40%, more preferably 5-30%; the volume content of propylene glycol is 2-20%, more preferably 5-15%. The type of solution B is preferably able to reduce the aggregation between CO release carriers, improve the stability of CO release carriers, and help form a uniform liquid phase system.

[0096] In a preferred embodiment of the present invention, the volume contents of glycerol and propylene glycol in the solution B are both 10%.

[0097] In step (b) of the present invention, the fluoride solution is subjected to CO saturation treatment. The saturation method is not particularly limited and can be carried out by a conventional gas saturation method in the art.

[0098] Preferably, in order to improve the performance of the CO releasing carrier described in the second aspect of the present invention, the CO saturation treatment of the fluoride solution adopts a high pressure method and / or a purge method.

[0099] Preferably, when the high pressure method is used for CO saturation, CO gas is introduced into the sealed container twice. Preferably, when the high pressure method is used for CO saturation, before the fluoride liquid is added to the sealed container, a step of replacing the gas in the sealed container with CO gas is included, and after the fluoride liquid is added to the sealed container, a step of injecting CO gas and increasing the pressure in the sealed container is included. The preferred method of introducing CO gas is conducive to controlling the stability and uniformity of the prepared CO release carrier.

[0100] Preferably, after the fluoride liquid is added to the sealed container, CO gas is injected to make the system pressure reach 0.2-1 MPa. More preferably, the system pressure is 0.5 MPa, and the pressure is maintained for more than 0.5 hours, preferably for more than 1 hour.

[0101] In a specific embodiment of the present invention, the high pressure method comprises the following steps: adding the fluoride liquid into a sealed container, replacing the gas in the container with CO in advance, and injecting CO gas to make the system pressure reach 0.2-1 MPa.

[0102] In a specific embodiment of the present invention, the purging method comprises the following steps: adding the fluoride liquid into an oxygen-free sealed container, the container having an air inlet and an air outlet, the air inlet extending into the fluoride liquid, and continuously introducing CO gas into the fluoride liquid for a ventilation time of 0.1-2h.

[0103] In step (c) of the present invention, the mixing of solution II and solution III means that solution II and solution III are mixed at high speed under the action of external force to form a spherical CO release carrier with a liquid core. The mixing method is not specifically limited, and it is sufficient to obtain a spherical CO release carrier with a liquid core.

[0104] In a preferred embodiment of the present invention, the mixing of the solution II and the solution III is carried out by one or more of ultrasonic cavitation, extrusion or homogenization.

[0105] Preferably, the process conditions of the ultrasonic cavitation method include: power of 60-550W, duty cycle of 10%-75%, time of 1-10min. Further preferably: power of 60-300W, duty cycle of 25%-60%, time of 2-6min.

[0106] Preferably, the process conditions of the extrusion method include: the membrane pore size is 0.2-5 μm, and the number of extrusions is 10-40 times. More preferably: 0.2-2 μm, and the number of extrusions is 10-25 times.

[0107] Preferably, the process conditions of the homogenization method include: a homogenization pressure of 100-2000 bar, a treatment number of 1-15 times, and a 1-10% (w / v%) PVA solution of the aqueous phase solution. Further preferably: a homogenization pressure of 500-1500 bar, a treatment number of 1-15 times, and a 1-5% (w / v%) PVA solution of the aqueous phase solution.

[0108] The CO releasing carrier prepared by the above-mentioned preparation method of the CO releasing carrier of the present invention has the same performance and effect as the CO releasing carrier described in the second aspect of the present invention. In addition, the CO releasing carrier obtained by adding some other steps to the above-mentioned preparation method, such as adding certain drugs to form a complex, or adding certain functional additives is also within the protection scope of the present invention.

[0109] The fourth aspect of the present invention provides a pharmaceutical composition, comprising the CO releasing carrier composition described in the first aspect of the present invention, or the CO releasing carrier described in the second aspect of the present invention, or the CO releasing carrier obtained by the preparation method described in the third aspect of the present invention, and optionally a drug.

[0110] In the present invention, the type of the drug is not specifically limited, and conventional drugs that do not chemically react with CO gas can be selected.

[0111] In the present invention, the CO release carrier can load drugs to form a CO-drug composition carrier, which can release drugs while releasing CO, thereby improving the targeting, controllability and tissue penetration ability of drug delivery and reducing the toxic side effects of drugs.

[0112] The fifth aspect of the present invention provides a method for inducing CO release by acoustic stimulation, wherein a CO releasing carrier releases CO in a controllable manner under ultrasonic stimulation in a characteristic response frequency range and / or a specific power range; wherein the CO releasing carrier is one or more of the CO releasing carrier composition described in the first aspect of the present invention, or the CO releasing carrier described in the second aspect of the present invention, or the CO releasing carrier obtained by the preparation method described in the third aspect of the present invention.

[0113] The inventors of the present invention have found that compared with light stimulation, acoustic stimulation has deeper tissue penetration ability and higher targeting than thermal stimulation, and has the advantages of safety and easy operation. In the acoustic stimulation-induced CO release method of the present invention, the CO release carrier vaporizes and ruptures under the action of a specific ultrasonic field, thereby achieving controllable CO release and significantly improving the targeting of CO release. In addition, the CO release carrier will produce mechanical effects, thermal effects, and biological effects during the rupture process, which will help deliver CO to deep tissues and enhance the therapeutic effect of CO.

[0114] Therefore, the acoustic stimulation-induced CO release method of the present invention can deliver CO gas in a controllable, targeted and more precise manner. The ultrasound-induced CO targeted controllable release method will greatly promote the development of the CO treatment field and has far-reaching significance for its promotion and application in clinical practice.

[0115] In a specific embodiment of the present invention, the characteristic response frequency range is 0.1-30 MHz.

[0116] In a specific embodiment of the present invention, the specific power range is 0.1-5 W / cm 2 .

[0117] The sixth aspect of the present invention provides a use of a CO releasing carrier in the preparation of a drug for treating tumor diseases, wherein the CO releasing carrier is one or more of the CO releasing carrier composition described in the first aspect of the present invention, or the CO releasing carrier described in the second aspect of the present invention, or the CO releasing carrier obtained by the preparation method described in the third aspect of the present invention. According to the present invention, in the treatment of tumor diseases, the CO releasing carrier of the present invention is used at a concentration of 1×10 6 -1×10 9 The drug is provided in the form of a unit dose of 1000 g / kg body weight and is administered once every 0.5-7 days during the administration cycle. This administration method not only ensures the dosage of CO, but also makes the delivery of CO safe and effective.

[0118] The present invention adopts the above technical scheme to have the following beneficial effects: the CO releasing carrier obtained by the CO releasing carrier composition of the present invention can have both safety, biocompatibility and high CO loading dose, which can not only ensure that the CO releasing carrier has a long in vivo circulation time (>2 hours), but also can realize the precise and controllable release of CO in the target tissue through ultrasonic stimulation, thereby improving the delivery efficiency of CO and the targeting and effectiveness of treatment.

[0119] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1 Shown is an optical microscope image of the CO-releasing carrier obtained in Example A1.

[0121] Figure 2 Shown is the particle size distribution of the CO releasing carrier obtained in Example A1. DETAILED DESCRIPTION

[0122] The present invention will be described in detail below by way of examples. The examples described in the present invention are only a part of the examples of the present invention, rather than all of the examples. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0123] Unless otherwise specified, the materials used in the following examples were all commercially available analytically pure.

[0124] Preparation Example 1.

[0125] The PBS buffer preparation process includes: weighing 0.097 g of KCl, 4.005 g of NaCl, 1.145 g of Na2HPO4·H2O and 0.096 g of KH2PO4 into a 1 L beaker, adding deionized water to make the volume to 500 ml to prepare an ion buffer for use.

[0126] Preparation Example 2.

[0127] The preparation process of solution B includes: weighing 80 ml of the PBS buffer obtained in Preparation Example 1, adding 10 ml of glycerol and 10 ml of propylene glycol and mixing evenly to prepare solution B.

[0128] Example A1.

[0129] (I) preparing a CO release carrier composition, which is denoted as I1, comprising: Surfactant A: 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DBPC), 1,2-distearoylphosphatidic acid (DSPA); Surfactant B: distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), Brij35, Pluronic F68; Inert fluoride liquid: perfluoropentane; CO gas.

[0130] (II) Prepare a CO-releasing carrier, which is designated as III1.

[0131] (a) Preparation of Solution II: 15 mg of surfactant (DBPC, DSPA, DSPE-PEG2000, Brij35, Pluronic F68 in a mass ratio of 6:1.5:1:1:0.5) was added to 3 mL of solvent A (ethanol to methanol in a volume ratio of 1:1) to obtain Solution I. The solution I was transferred to a round-bottom flask and evaporated in a water bath at 80°C and 0.08 MPa until the solvent was completely evaporated. 4 ml of Solution B (PBS ion buffer, glycerol, propylene glycol in a volume ratio of 8:1:1) was added and slowly rotated to hydrate until the film fell off the flask wall to obtain Solution II.

[0132] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluoropentane into the autoclave, seal the container and replace the internal air with CO2, inject CO2 gas into the autoclave at a pressure of 0.5 MPa using a high-pressure pump, and maintain the pressure for 1 hour to ensure that CO2 is completely dissolved to obtain CO2-saturated fluoride solution III.

[0133] (c) Preparation of CO release carrier composition: 1 mL of solution II was mixed with 100 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0134] The power of the ultrasonic device was set to 100 W, the duty cycle to 50%, and the ultrasonic time to 5 minutes. Ultrasonic cavitation was used to fully mix solution II and solution III to form a spherical CO release carrier with a liquid core.

[0135] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0136] Example A2.

[0137] (I) preparing a CO release carrier composition, which is denoted as I2, comprising: Surfactant A: Span60, Tween80; Surfactant B: PEG2000, Pluronic F68; Inert fluoride liquid: perfluoropentane; CO gas.

[0138] (II) Prepare a CO-releasing carrier, which is designated as II2.

[0139] (a) Preparation of solution II: 5 g of surfactant (mass ratio of Span 60: Tween 80: PEG2000: Pluronic F68 = 15:35:10:15) was added to 50 mL of solvent A (PBS buffer) and heated in an autoclave at 110 °C for 30 min until the solution was completely homogeneous and transparent to obtain solution II.

[0140] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluoropentane into the autoclave, seal the container and replace the internal air with CO2, inject CO2 gas into the autoclave at a pressure of 0.5 MPa using a high-pressure pump, and maintain the pressure for 1 hour to ensure that CO2 is completely dissolved to obtain CO2-saturated fluoride solution III.

[0141] (c) Preparation of CO release carrier composition: 5 mL of solution II was mixed with 500 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0142] The power of the ultrasonic device was set to 100 W, the duty cycle to 50%, and the ultrasonic time to 5 minutes. Ultrasonic cavitation was used to fully mix solution II and solution III to form a spherical CO release carrier with a liquid core.

[0143] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0144] Example A3.

[0145] (I) preparing a CO release carrier composition, which is denoted as I3, comprising: Surfactant A: PLGA (lactic acid and glycolic acid mass ratio of 75:25); Surfactant B: PEG2000-PLGA (mass ratio of lactic acid and glycolic acid is 50:50); Inert fluoride liquid: perfluoropentane; CO gas.

[0146] (II) Prepare a CO-releasing carrier, which is designated as II3.

[0147] (a) Preparation of Solution II: PLGA and PEG2000-PLGA were dissolved in solvent A (the volume ratio of dichloromethane to ethanol was 1:1) at a mass ratio of 5:1, with a concentration of 8 mg / ml. The mixture was stirred magnetically at 40°C for 2-3 h until the solution was completely homogeneous and transparent, thereby obtaining Solution II.

[0148] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluoropentane into the autoclave, seal the container and replace the internal air with CO2, inject CO2 gas into the autoclave at a pressure of 0.5 MPa using a high-pressure pump, and maintain the pressure for 1 hour to ensure that CO2 is completely dissolved to obtain CO2-saturated fluoride solution III.

[0149] (c) Preparation of colostrum: 5 mL of solution II was mixed with 500 μL of CO2-saturated fluoride solution III, and the mixture was treated with an ultrasonic probe (power of 100 W, duty cycle of 50%, and ultrasonic time of 5 min) in an ice bath to form colostrum IV.

[0150] (d) Preparation of aqueous phase: Dissolve PVA in deionized water to obtain a 1-2% (w / v) aqueous phase solution V, and precool (4°C).

[0151] (e) Preparation of CO-releasing carrier composition: Colostrum IV was slowly added dropwise to 20 ml of aqueous solution V, and further treated using a high-pressure homogenizer at a pressure of 1000 bar for 5 times.

[0152] After homogenization, the obtained mixture is allowed to stand, and after stratification, the upper layer is removed, and the CO release carrier in the lower layer is collected.

[0153] Example A4.

[0154] (I) preparing a CO release carrier composition, which is denoted as I4, comprising: Surfactant A: DSPC, DSPA; Surfactant B: DSPE-PEG2000, Brij35, Pluronic F68; Inert fluoride liquid: perfluoropentane; CO gas.

[0155] (II) Preparation of a CO-releasing carrier, denoted as II4 (a) Preparation of solution II: 15 mg of surfactant (DSPC, DSPA, DSPE-PEG2000, Brij35, Pluronic F68 in a mass ratio of 6:1.5:1:1:0.5) was added to 3 mL of solvent A (ethanol to methanol volume ratio of 1:1) to obtain solution I. The solution I was transferred to a round-bottom flask and evaporated in a water bath at 70°C and 0.08 MPa until the solvent was completely evaporated. 4 ml of solution B (PBS ion buffer, glycerol, propylene glycol in a volume ratio of 8:1:1) was added and slowly rotated to hydrate until the film fell off the flask wall to obtain solution II.

[0156] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluoropentane into the autoclave, seal the container and replace the internal air with CO2, inject CO2 gas into the autoclave at a pressure of 0.5 MPa using a high-pressure pump, and maintain the pressure for 1 hour to ensure that CO2 is completely dissolved to obtain CO2-saturated fluoride solution III.

[0157] (c) Preparation of CO release carrier composition: 1 mL of solution II and 100 μL of CO saturated fluoride solution III were transferred to the two syringes of the extruder respectively, a membrane with a pore size of 0.8 μm was selected, and extrusion was performed 20 times.

[0158] After extrusion, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0159] Example A5.

[0160] (I) preparing a CO release carrier composition, which is denoted as I5, comprising: Surfactant A: DPPC; Surfactant B: PEG2000-PLGA (mass ratio of lactic acid and glycolic acid is 75:25); Inert fluoride liquid: perfluorohexane; CO gas.

[0161] (II) Prepare a CO-releasing carrier, which is designated as II5.

[0162] (a) Preparation of Solution II: 12 mg of surfactant (DPPS and PEG2000-PLGA in a mass ratio of 9:4) was added to 3 mL of solvent A (ethanol) to obtain Solution I. The solution I was transferred to a round-bottom flask and subjected to rotary evaporation in a water bath at 60°C and 0.08 MPa until all the solvent was evaporated. 4 ml of Solution B (ionic buffer and propylene glycol in a volume ratio of 9:1) was added and slowly rotated to hydrate until the film fell off the flask wall to obtain Solution II.

[0163] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorohexane into a beaker and continue purging CO for 2 h to obtain CO2-saturated fluoride solution III.

[0164] (c) Preparation of CO release carrier composition: 1 mL of solution II was mixed with 100 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0165] The power of the ultrasonic device was set to 150 W, the duty cycle to 50%, and the ultrasonic time to 5 minutes. Ultrasonic cavitation was used to fully mix solution II and solution III to form a spherical CO release carrier with a liquid core.

[0166] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0167] Example A6.

[0168] (I) preparing a CO release carrier composition, which is denoted as I6, comprising: Surfactant A: DEPC; Surfactant B: PEG2000, Pluronic F127; Inert fluoride liquid: perfluorooctyl bromide; CO gas.

[0169] (II) Prepare a CO-releasing carrier, which is designated as II6.

[0170] (a) Preparation of solution II: 14 mg of surfactant (DEPS, PEG2000, Pluronic F127 in a mass ratio of 10:3:1) was added to 3 mL of solvent A (PBS buffer, glycerol, propylene glycol in a volume ratio of 7:1:2), and the mixture was slowly heated to 70 °C for 30 min until completely dissolved to obtain solution II.

[0171] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorooctane bromide into a beaker and continue purging CO for 2 h to obtain CO2-saturated fluoride solution III.

[0172] (c) Preparation of CO release carrier composition: 1 mL of solution II was mixed with 150 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0173] The power of the ultrasonic device was set to 180 W, the duty cycle was set to 50%, and the ultrasonic time was set to 5 min. Ultrasonic cavitation was used to treat the solution II and the solution III to be fully mixed to form a spherical CO release carrier with a liquid core.

[0174] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0175] Example A7.

[0176] (I) preparing a CO release carrier composition, which is denoted as I7, comprising: Surfactant A: DEPC, DPPE, DSPG; Surfactant B: PEG4000, cholesterol; Inert fluoride liquid: perfluorooctyl bromide; CO gas.

[0177] (II) Prepare a CO-releasing carrier, which is designated as II7.

[0178] (a) Preparation of Solution II: Add 10 mg of surfactant A (DEPC, DPPE, DSPG in a mass ratio of 3:1:1) and 4 mg of surfactant B (PEG4000, cholesterol in a mass ratio of 1:3) to 4 mL of solvent A (PBS buffer, glycerol, propylene glycol in a volume ratio of 8:1:1), slowly heat to 65°C, and stir for 30 min until completely dissolved to obtain Solution II.

[0179] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorooctane bromide into a beaker and continue purging CO for 2 h to obtain CO2-saturated fluoride solution III.

[0180] (c) Preparation of CO release carrier composition: 1 mL of solution II and 150 μL of CO saturated fluoride solution III were transferred to the two syringes of the extruder respectively, a membrane with a pore size of 1 μm was selected, and extrusion was performed 15 times.

[0181] After extrusion, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0182] Example A8.

[0183] (I) preparing a CO release carrier composition, which is denoted as I8, comprising: Surfactant A: DEPC, Hb; Surfactant B: DSPE-PEG2000, Pluronic F68; Inert fluoride liquid: perfluorohexane; CO gas.

[0184] (II) Prepare a CO-releasing carrier, which is designated as II8.

[0185] (a) Preparation of solution II: 15 mg of surfactant (DEPC, DSPE-PEG2000, Pluronic F68 in a mass ratio of 10:4:1) was added to 4 mL of solvent A (PBS buffer and propylene glycol in a volume ratio of 7:3), slowly heated to 60 °C, stirred for 20 min, 5 mg of Hb was added, heated to 40 °C, and stirred for 20 min to obtain solution II.

[0186] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorohexane into a beaker and continue purging CO for 2 h to obtain CO2-saturated fluoride solution III.

[0187] (c) Preparation of CO release carrier composition: 1 mL of solution II was mixed with 100 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0188] The power of the ultrasonic device was set to 150 W, the duty cycle to 50%, and the ultrasonic time to 5 minutes. Ultrasonic cavitation was used to fully mix solution II and solution III to form a spherical CO release carrier with a liquid core.

[0189] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0190] Example A9.

[0191] (I) preparing a CO release carrier composition, which is denoted as I9, comprising: Surfactant A: Tween 20, Span 85; Surfactant B: PEG4000, Brij78; Inert fluoride liquids: perfluorobutane and perfluoroheptane; CO gas.

[0192] (II) Prepare a CO-releasing carrier, which is designated as II9.

[0193] (a) Preparation of solution II: 2 g of surfactant A (Tween 20 and Span 85 in a mass ratio of 1:1) and 0.5 g of surfactant B (PEG4000 and Brij78 in a mass ratio of 1:2) were added to 50 mL of solvent A (PBS buffer and propylene glycol in a volume ratio of 9:1), and the mixture was heated to 120°C in an autoclave for 20 min to obtain solution II.

[0194] (b) Preparation of CO2-saturated fluoride liquid: 1 mL of perfluoroheptane and perfluorobutane (3:2 by volume at 0.5 MPa) was added to an autoclave. The container was sealed and the air inside was replaced with CO2. CO2 gas was injected into the autoclave at a pressure of 0.5 MPa using a high-pressure pump. The pressure was maintained for 1 hour to ensure that CO2 was completely dissolved, thereby obtaining CO2-saturated fluoride solution III.

[0195] (c) Preparation of CO2-releasing carrier composition: 1 mL of solution II was mixed with 120 μL of CO2-saturated fluoride solution III and transferred to an ultrasonic device.

[0196] The power of the ultrasonic device was set to 150 W, the duty cycle to 30%, and the ultrasonic time to 3 minutes. Ultrasonic cavitation was used to fully mix solution II and solution III to form a spherical CO release carrier with a liquid core.

[0197] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0198] Example A10.

[0199] (I) preparing a CO release carrier composition, which is denoted as I10, comprising: Surfactant A: DSPC, Hb; Surfactant B: DSPE-PEG2000; Inert fluoride liquid: perfluorohexane; CO gas.

[0200] (II) Prepare a CO-releasing carrier, which is designated as II10.

[0201] (a) Preparation of solution II: 15 mg of surfactant (DSPC, DSPE-PEG2000 in a mass ratio of 3:1) was added to 5 mL of solvent A (PBS buffer and propylene glycol in a volume ratio of 9:1), slowly heated to 80 °C, stirred for 20 min, 5 mg of Hb was added, heated to 40 °C, and stirred for 20 min to obtain solution II.

[0202] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorohexane into the autoclave, seal the container and replace the internal air with CO2, inject CO2 gas into the autoclave at a pressure of 0.3 MPa using a high-pressure pump, and maintain the pressure for 1 hour to ensure that CO2 is completely dissolved, thereby obtaining CO2-saturated fluoride solution III.

[0203] (c) Preparation of CO release carrier composition: 1 mL of solution II was mixed with 100 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0204] The power of the ultrasonic device was set to 150 W, the duty cycle to 40%, and the ultrasonic time to 3 minutes. Ultrasonic cavitation was used to fully mix solution II and solution III to form a spherical CO release carrier with a liquid core.

[0205] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0206] Example A11.

[0207] (I) preparing a CO release carrier composition, which is denoted as I11, comprising: Surfactant A: DOPC; Surfactant B: DSPE-PEG2000 and cholesterol; Inert fluorinated liquids: perfluoropropane and perfluorodecalin; CO gas.

[0208] (II) Prepare a CO-releasing carrier, which is designated as II11.

[0209] (a) Preparation of Solution II: 12 mg of surfactant (DOPC, DSPE-PEG2000 and cholesterol in a mass ratio of 4:1:1) was added to 2 mL of solvent A (dichloromethane) to obtain Solution I. Solution I was transferred to a round-bottom flask and subjected to rotary evaporation in a water bath at 70°C and 0.06 MPa until all the solvent was evaporated. 3 ml of Solution B (ionic buffer and glycerol in a volume ratio of 9:1) was added and slowly rotated to hydrate until the film fell off the flask wall to obtain Solution II.

[0210] (b) Preparation of CO2-saturated fluoride liquid: 1 mL of perfluorodecalin and perfluoropropane (1:4 by volume at 0.5 MPa) was added into an autoclave. The container was sealed and the air inside was replaced with CO2. CO2 gas was injected into the autoclave at a pressure of 0.5 MPa using a high-pressure pump. The pressure was maintained for 1 h to ensure that CO2 was completely dissolved, thereby obtaining CO2-saturated fluoride solution III.

[0211] (c) Preparation of CO release carrier composition: 1 mL of solution II and 100 μL of CO saturated fluoride solution III were transferred to the two syringes of the extruder respectively, a membrane with a pore size of 1 μm was selected, and extrusion was performed 10 times.

[0212] After extrusion, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0213] Example A12.

[0214] (I) preparing a CO release carrier composition, which is recorded as I12, comprising: Surfactant A: DSPC, DOTAP; Surfactant B: DSPE-PEG2000 and cholesterol; Inert fluoride liquids: perfluoropropane and perfluorotripropylamine; CO gas.

[0215] (II) Prepare a CO-releasing carrier, which is designated as II12.

[0216] (a) Preparation of Solution II: 12 mg of surfactant A (DSPC and DOTAP in a mass ratio of 5:1) and 5 mg of surfactant B (DSPE-PEG2000 and cholesterol in a mass ratio of 1:2) were added to 3 mL of solvent A (chloroform) to obtain Solution I. The solution I was transferred to a round-bottom flask and evaporated in a water bath at 80°C and 0.09 MPa until the solvent was completely evaporated. 3.5 ml of Solution B (PBS buffer and glycerol in a volume ratio of 8:2) was added and slowly rotated to hydrate until the film fell off the flask wall to obtain Solution II.

[0217] (b) Preparation of CO2-saturated fluoride liquid: 1 mL of perfluorotripropylamine and perfluoropropane (volume ratio of 1:3 under 0.5 MPa pressure) were added into an autoclave. The container was sealed and the air inside was replaced with CO2. CO2 gas was injected into the autoclave at a pressure of 0.5 MPa using a high-pressure pump. The pressure was maintained for 1 hour to ensure that CO2 was completely dissolved, thereby obtaining CO2-saturated fluoride solution III.

[0218] (c) Preparation of CO release carrier composition: 1 mL of solution II was mixed with 100 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0219] The power of the ultrasonic device was set to 120 W, the duty cycle was set to 50%, and the ultrasonic time was set to 5 minutes. Ultrasonic cavitation was used to mix solution II and solution III thoroughly to form a spherical CO release carrier with a liquid core.

[0220] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0221] Example A13.

[0222] (I) preparing a CO release carrier composition, which is recorded as I13, comprising: Surfactant A: DPPC, Hb; Surfactant B: DSPE-PEG2000, cholesterol; Inert fluoride liquid: perfluoropentane; CO gas.

[0223] (II) Prepare a CO-releasing carrier, which is designated as II13.

[0224] (a) Preparation of solution II: Surfactant A (DPPC) and surfactant B (DSPE-PEG2000 and cholesterol at a mass ratio of 1:2) were dissolved in solvent A (PBS buffer) at a mass ratio of 4:1 to a concentration of 7 mg / ml to obtain solution I. Solution I was transferred to a beaker, magnetically stirred at 80°C for 1 h, Hb (concentration of 2 mg / ml) was added, and magnetically stirred at 40°C for 1 h to obtain solution II.

[0225] (b) Preparation of CO2-saturated fluoride liquid: Add 5 mL of perfluoropentane into a beaker and continue purging CO for 1.5 h to obtain CO2-saturated fluoride solution III.

[0226] (c) Preparation of CO2-releasing carrier composition: 1 mL of solution II was mixed with 120 μL of CO2-saturated fluoride solution III and transferred to an ultrasonic device.

[0227] The power of the ultrasonic device was set to 100 W, the duty cycle to 30%, and the ultrasonic time to 5 min. Ultrasonic cavitation was used to fully mix solution II and solution III to form a spherical CO release carrier with a liquid core.

[0228] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0229] Example A14.

[0230] (I) preparing a CO release carrier composition, which is recorded as I14, comprising: Surfactant A: DBPC; Surfactant B: DSPE-PEG2000 and PluronicF108; Inert fluoride liquids: perfluorobutane, perfluorodecalin; CO gas.

[0231] (II) Prepare a CO-releasing carrier, which is designated as II14.

[0232] (a) Preparation of solution II: 9 mg of surfactant A (DBPC) and 5 mg of surfactant B (DSPE-PEG2000 and Pluronic F108 at a mass ratio of 3:1) were added to 8 mL of solvent A (PBS buffer) to obtain solution I. The solution I was transferred to a beaker and magnetically stirred at 80° C. for 3 h to obtain solution II.

[0233] (b) Preparation of CO2-saturated fluoride liquid: 1 mL of perfluorodecalin and perfluorobutane (1:3 by volume at 0.5 MPa) was added to an autoclave. The container was sealed and the air inside was replaced with CO2. CO2 gas was injected into the autoclave at a pressure of 0.5 MPa using a high-pressure pump. The pressure was maintained for 1 h to ensure that CO2 was completely dissolved, thereby obtaining CO2-saturated fluoride solution III.

[0234] (c) Preparation of CO release carrier composition: 1 mL of solution II and 130 μL of CO saturated fluoride solution III were transferred to the two syringes of the extruder respectively, a membrane with a pore size of 0.5 μm was selected, and extrusion was performed 10 times.

[0235] After extrusion, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0236] Example A15.

[0237] (I) preparing a CO release carrier composition, which is recorded as I15, comprising: Surfactant A: DPPC, DOTMA; Surfactant B: DSPE-PEG2000 and Pluronic F68; Inert fluoride liquid: perfluorooctyl bromide; CO gas.

[0238] (II) Prepare a CO-releasing carrier, which is designated as II15.

[0239] (a) Preparation of Solution II: 10 mg of surfactant A (DPPC, DOTMA at a mass ratio of 4:1) and 5 mg of surfactant B (DSPE-PEG2000 and Pluronic F68 at a mass ratio of 3:1) were added to 3 mL of solvent A (ethanol) to obtain Solution I. The solution I was transferred to a round-bottom flask and evaporated in a water bath at 70°C and 0.09 MPa until the solvent was completely evaporated. 3.5 ml of Solution B (PBS buffer, glycerol, propylene glycol at a volume ratio of 8:1:1) was added and slowly rotated to hydrate until the film fell off the flask wall to obtain Solution II.

[0240] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorooctane bromide into the autoclave, seal the container and replace the air inside with CO2. Inject CO2 gas into the autoclave at a pressure of 0.5 MPa using a high-pressure pump and maintain the pressure for 1 hour to ensure that CO2 is completely dissolved, thereby obtaining CO2-saturated fluoride solution III.

[0241] (c) Preparation of CO release carrier composition: 1 mL of solution II was mixed with 100 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0242] The power of the ultrasonic device was set to 130 W, the duty cycle to 50%, and the ultrasonic time to 3 min. Ultrasonic cavitation was used to fully mix solution II and solution III to form a spherical CO release carrier with a liquid core.

[0243] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0244] Example A16.

[0245] (I) preparing a CO release carrier composition, which is recorded as I16, comprising: Surfactant A: DOPC, Hb; Surfactant B: DSPE-PEG2000, Pluronic F68; Inert fluoride liquids: perfluorobutane, perfluorotripropylamine; CO gas (II) Prepare a CO-releasing carrier, which is designated as II16.

[0246] (a) Preparation of solution II: 7 mg of surfactant A (DOPC) and 6 mg of surfactant B (DSPE-PEG2000 and Pluronic F68 in a mass ratio of 3:2) were added to 2 mL of solvent A (ethanol to dichloromethane in a volume ratio of 3:1) to obtain solution I. The solution I was transferred to a round-bottom flask and evaporated in a water bath at 75°C and 0.08 MPa until the solvent was completely evaporated. 4 mg of Hb and 3.5 ml of solution B (PBS buffer and propylene glycol in a volume ratio of 9:1) were added and slowly rotated to hydrate until the film fell off the flask wall to obtain solution II.

[0247] (b) Preparation of CO2-saturated fluoride liquid: 1 mL of perfluorotripropylamine and perfluorobutane (1:2 by volume at 0.5 MPa) were added to an autoclave. The container was sealed and the air inside was replaced with CO2. CO2 gas was injected into the autoclave at a pressure of 0.5 MPa using a high-pressure pump. The pressure was maintained for 1 h to ensure that CO2 was completely dissolved, thereby obtaining CO2-saturated fluoride solution III.

[0248] (c) Preparation of CO release carrier composition: 1 mL of solution II was mixed with 100 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0249] The power of the ultrasonic device was set to 200 W, the duty cycle was set to 25%, and the ultrasonic time was set to 5 minutes. Ultrasonic cavitation was used to mix solution II and solution III thoroughly to form a spherical CO release carrier with a liquid core.

[0250] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0251] Example A17.

[0252] (I) preparing a CO release carrier composition, which is recorded as I17, comprising: Surfactant A: DPPC; Surfactant B: DSPE-PEG2000 and Brij78; Inert fluoride liquid: perfluorohexane; CO gas.

[0253] (II) Prepare a CO-releasing carrier, which is designated as II17.

[0254] (a) Preparation of solution II: 8 mg of surfactant A (DPPC) and 5 mg of surfactant B (DSPE-PEG2000 and Brij78 at a mass ratio of 5:1) were added to 6 mL of solvent A (the volume ratio of PBS buffer to propylene glycol was 9:1) to obtain solution I. The solution I was transferred to a beaker and magnetically stirred at 65° C. for 4 h to obtain solution II.

[0255] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorohexane into the autoclave, seal the container and replace the internal air with CO2, inject CO2 gas into the autoclave at a pressure of 0.4 MPa using a high-pressure pump, and maintain the pressure for 1 hour to ensure that CO2 is completely dissolved to obtain CO2-saturated fluoride solution III.

[0256] (c) Preparation of CO release carrier composition: 1 mL of solution II was mixed with 100 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0257] The power of the ultrasonic device was set to 100 W, the duty cycle to 50%, and the ultrasonic time to 5 minutes. Ultrasonic cavitation was used to fully mix solution II and solution III to form a spherical CO release carrier with a liquid core.

[0258] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0259] Example A18.

[0260] (I) preparing a CO release carrier composition, which is recorded as I18, comprising: Surfactant A: DBPC, Hb; Surfactant B: DSPE-PEG2000, Brij35; Inert fluoride liquid: perfluorohexane; CO gas.

[0261] (II) Prepare a CO-releasing carrier, which is designated as II18.

[0262] (a) Preparation of solution II: 6 mg of surfactant A (DBPC) and 4 mg of surfactant B (DSPE-PEG2000 and Brij35 in a mass ratio of 1:1) were added to 2 mL of solvent A (ethanol) to obtain solution I. The solution I was transferred to a round-bottom flask and evaporated in a water bath at 80°C and 0.08 MPa until the solvent was completely evaporated. 4 mg of Hb and 2.5 ml of solution B (PBS buffer and propylene glycol in a volume ratio of 9:1) were added and slowly rotated to hydrate until the film fell off the flask wall to obtain solution II.

[0263] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorohexane into the autoclave, seal the container and replace the internal air with CO2, inject CO2 gas into the autoclave at a pressure of 0.5 MPa using a high-pressure pump, and maintain the pressure for 1 hour to ensure that CO2 is completely dissolved to obtain CO2-saturated fluoride solution III.

[0264] (c) Preparation of CO release carrier composition: 1 mL of solution II was mixed with 100 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0265] The power of the ultrasonic device was set to 200 W, the duty cycle was set to 25%, and the ultrasonic time was set to 5 minutes. Ultrasonic cavitation was used to mix solution II and solution III thoroughly to form a spherical CO release carrier with a liquid core.

[0266] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0267] Example A19.

[0268] (I) preparing a CO release carrier composition, which is recorded as I19, comprising: Surfactant A: Hb; Surfactant B: Pluronic F68; Inert fluoride liquid: perfluorohexane; CO gas.

[0269] (II) Prepare a CO-releasing carrier, which is designated as II19.

[0270] (a) Preparation of Solution II: 16 mg of surfactant A (Hb) and 4 mg of surfactant B (Pluronic F68) were added to 5 mL of solvent A (the volume ratio of PBS buffer to propylene glycol was 9:1) to obtain Solution I. The Solution I was transferred to a small beaker and magnetically stirred at 40° C. for 4 h to obtain Solution II.

[0271] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorohexane into the autoclave, seal the container and replace the internal air with CO2, inject CO2 gas into the autoclave at a pressure of 0.5 MPa using a high-pressure pump, and maintain the pressure for 1 hour to ensure that CO2 is completely dissolved to obtain CO2-saturated fluoride solution III.

[0272] (c) Preparation of CO release carrier composition: 1 mL of solution II and 100 μL of CO saturated fluoride solution III were transferred to the two syringes of the extruder respectively, a membrane with a pore size of 0.8 μm was selected, and extrusion was performed 20 times.

[0273] After extrusion, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0274] Example A20.

[0275] (I) preparing a CO release carrier composition, which is recorded as I20, comprising: Surfactant A: PLGA-PCL (mass ratio of lactic acid and glycolic acid is 75:25); Surfactant B agent: Brij78; Inert fluoride liquid: perfluorohexane; CO gas.

[0276] (II) Prepare a CO-releasing carrier, which is designated as II3.

[0277] (a) Preparation of Solution II: PLGA-PCL and Brij78 were dissolved in solvent A (the volume ratio of ethyl acetate to chloroform was 1:1) at a mass ratio of 2:1, with a concentration of 20 mg / ml. The mixture was stirred magnetically at 60°C for 2-3 h until the solution was completely homogeneous and transparent, thereby obtaining Solution II.

[0278] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorohexane into the autoclave, seal the container and replace the internal air with CO2, inject CO2 gas into the autoclave at a pressure of 0.3 MPa using a high-pressure pump, and maintain the pressure for 1 hour to ensure that CO2 is completely dissolved, thereby obtaining CO2-saturated fluoride solution III.

[0279] (c) Preparation of colostrum: 2 mL of solution II was mixed with 100 μL of CO2-saturated fluoride solution III, and the mixture was treated with an ultrasonic probe (power of 100 W, duty cycle of 30%, and ultrasonic time of 5 min) under ice bath conditions to form colostrum IV.

[0280] (d) Preparation of aqueous phase: Dissolve PVA in deionized water to obtain a 1-2% (w / v) aqueous phase solution V, and precool (4°C).

[0281] (e) Preparation of CO-releasing carrier composition: Colostrum IV was slowly added dropwise to 20 ml of aqueous solution V, and further treated by ultrasonic treatment (power of 100 W, duty cycle of 30%, and ultrasonic time of 3 minutes).

[0282] After homogenization, the obtained mixture is allowed to stand, and after stratification, the upper layer is removed, and the CO release carrier in the lower layer is collected.

[0283] Example A21.

[0284] (I) preparing a CO release carrier composition, which is recorded as I21, comprising: Surfactant A: Span80, Tween20; Surfactant B: PEG4000, Brij78; Inert fluoride liquid: perfluorooctyl bromide; CO gas.

[0285] (II) Prepare a CO-releasing carrier, which is designated as II21.

[0286] (a) Preparation of solution II: 3 g of surfactant (Span 80: Tween 20: PEG4000: Brij78 = 2:2:2:2 by mass ratio) was added to 50 mL of solvent A (PBS buffer) and heated in an autoclave at 110 °C for 30 min until the solution was completely homogeneous and transparent to obtain solution II.

[0287] (b) Preparation of CO2-saturated fluoride liquid: Add 1 mL of perfluorooctane bromide into a beaker and continue purging CO for 1 h to obtain CO2-saturated fluoride solution III.

[0288] (c) Preparation of CO release carrier composition: 15 mL of solution II was mixed with 1000 μL of CO saturated fluoride solution III and transferred to an ultrasonic device.

[0289] The power of the ultrasonic device was set to 200 W, the duty cycle to 50%, and the ultrasonic time to 2 minutes. Ultrasonic cavitation was used to fully mix solution II and solution III to form a spherical CO release carrier with a liquid core.

[0290] After ultrasonic treatment, the resulting mixture was centrifuged to collect the CO2-releasing carrier.

[0291] Example A22.

[0292] This example was carried out according to the method of Example A1, except that the surfactant A was DBPC, the surfactant B was DSPE-PEG2000, Brij35, and Pluronic F68, and the mass ratio of DBPC: DSPE-PEG2000: Brij35: Pluronic F68 was 6:1:1:0.5. The CO release carrier was denoted as II22.

[0293] Example A23.

[0294] This example was carried out according to the method of Example A1, except that the surfactant A agent was DBPC and DSPA, the surfactant B agent was DSPE-PEG2000, and the mass ratio of DBPC: DSPA: DSPE-PEG2000 was 6:1.5:1. The CO release carrier was denoted as II23.

[0295] Example A24.

[0296] This example was carried out according to the method of Example A1, except that the surfactant A agent was DBPC and DSPA, the surfactant B agent was DSPE-PEG2000 and Brij35, and the mass ratio of DBPC: DSPA: DSPE-PEG2000: Brij35 was 6:1.5:1:1. The CO release carrier was denoted as II24.

[0297] Example A25.

[0298] This example was carried out according to the method of Example A1, except that the surfactant A was DBPC and DSPA, the surfactant B was DSPE-PEG2000 and Pluronic F68, and the mass ratio of DBPC: DSPA: DSPE-PEG2000: Pluronic F68 was 6:1.5:1:0.5. The CO release carrier was denoted as II25.

[0299] Example A26.

[0300] This example was carried out according to the method of Example A1, except that the surfactant A agent was DBPC, the surfactant B agent was DSPE-PEG2000, and the mass ratio of DBPC: DSPE-PEG2000 was 6: 1. The CO release carrier was denoted as II26.

[0301] Example A27.

[0302] This example was carried out according to the method of Example A1, except that the CO2-saturated fluoride liquid was prepared by a purge method: 1 mL of perfluoropentane was added to a beaker, and CO2 was continuously purged for 2 h to obtain a CO2-saturated fluoride solution III. The CO2-releasing carrier was denoted as II27.

[0303] Example A28.

[0304] This example is carried out according to the method of Example A1, except that the preparation of the CO release carrier composition adopts a homogenization method: 5 mL of solution II is mixed with 500 μL of CO saturated fluoride solution III, and the mixture is treated with an ultrasonic probe under ice bath conditions (power is 100 W, duty cycle is 50%, and ultrasonic time is 5 minutes) to form colostrum IV; PVA is dissolved in deionized water to obtain a 1-2% (w / v) aqueous phase solution V, and precooled (4°C); colostrum IV is slowly added dropwise to 20 ml of aqueous phase solution V, and further treated with a high-pressure homogenizer at a pressure of 1000 bar for 5 times. After homogenization, the obtained mixture is allowed to stand, and the upper layer is removed after stratification, and the lower layer CO release carrier is collected. The CO release carrier is recorded as II28.

[0305] Example A29.

[0306] This example was carried out according to the method of Example A1, except that the CO release carrier composition was prepared by extrusion: 1 mL of solution II and 100 μL of CO saturated fluoride solution III were transferred to two syringes of the extruder respectively, a membrane with a pore size of 0.5 μm was selected, and extrusion was performed 20 times. After the extrusion treatment, the obtained mixture was collected by a low temperature centrifuge to collect the CO release carrier. The CO release carrier was recorded as II29.

[0307] Example A30.

[0308] This example was carried out according to the method of Example A1, except that the surfactant A was DSPC, the surfactant B was DSPE-PEG2000, and the mass ratio of DSPC:DSPE-PEG2000 was 4:5. The CO release carrier was denoted as II30.

[0309] Example A31.

[0310] This example was carried out according to the method of Example A1, except that the surfactant A was DBPC, the surfactant B was Pluronic F68 and DSPE-PEG2000, and the mass ratio of DBPC: Pluronic F68: DSPE-PEG2000 was 2:3:3. The CO release carrier was denoted as II31.

[0311] Example A32.

[0312] This example was carried out according to the method of Example A1, except that the surfactant A agent was DPPC, the surfactant B agent was DSPE-PEG2000 and DSPE-PEG2000-maleimide, and the mass ratio of DPPC: DSPE-PEG2000: DSPE-PEG2000-maleimide was 85:7:8. The CO release carrier was denoted as II32.

[0313] Example A33.

[0314] This example was carried out according to the method of Example A3, except that the mass ratio of lactic acid to glycolic acid in PLGA in the surfactant A was 100:0. The CO releasing carrier was denoted as II33.

[0315] Example A34.

[0316] This example was carried out in accordance with the method of Example A3, except that the surfactant A was PLGA-maleamide, and 10 mg of camptothecin-SH was added after the surfactant was added to the solvent A. The CO release carrier was designated as II34.

[0317] Comparative example 1.

[0318] This example was carried out according to the method of Example A1, except that the surfactant B agent was not included. The CO releasing carrier was denoted as D1.

[0319] Comparative example 2.

[0320] This example is carried out according to the method of Example A1, except that the mass ratio of DBPC, DSPA, DSPE-PEG2000, Brij35 and Pluronic F68 is 6:1.5:0.1:0.1:0.05. The CO release carrier is denoted as D2.

[0321] Test example.

[0322] The CO releasing carriers II1-34, D1 and D2 obtained above were selectively subjected to the following tests.

[0323] (1) Optical microscope observation: The obtained CO release carriers II1-34, D1, and D2 were observed under an optical microscope. The specific steps are as follows: dilute the CO release carrier stock solution to an appropriate concentration using PBS buffer, take 10 μL of the diluted solution and place it on a clean glass slide and cover it with a coverslip. Observe under objective lenses of different magnifications and take photos for record. Among them, the observation results of II1 are as follows: Figure 1 shown.

[0324] (2) Particle size, polydispersity index and Zeta potential measurement: The average particle size, size distribution, polydispersity index and Zeta potential of the obtained CO release carriers II1-34, D1 and D2 were detected by DLS. The specific operation steps are as follows: a. Dilute the newly prepared CO release carrier solution 1000 times with PBS solution; b. Transfer 1.5 ml of the diluted CO release carrier solution to a clean sample cell; c. Place the sample pool into the corresponding card slot of the instrument for measurement. Repeat the measurement three times for each sample.

[0325] Among them, the test results of II1 are as follows Figure 2 The measurement results of other CO release carriers are recorded in Table 1.

[0326] (3) CO release test: The CO release amount of the CO release carrier was detected by high performance gas chromatography. 1 mL of CO release carrier (concentration of 1×10 10 / ml), respectively, were placed in a 3ml container, and the gas in the container was replaced with perfluoropropane by the upward exhaust method, and the container was sealed. The container was placed in an ultrasonic cleaner for 12 hours to completely break the CO release carrier, and the CO release amount was detected. Among them, the measurement results of II1-34, D1, and D2 are recorded in Table 1.

[0327] (4) Cytotoxicity test: In order to explore the inhibitory effect of the CO release carriers in the above examples and comparative examples on the proliferation of cancer cells, the CCK-8 kit was used to test the survival rate of cells under the combined action of ultrasonic stimulation and CO release carriers. The specific steps are as follows: 100 μL (density: 10,000 cells / well) of 4T1 cell suspension was placed in a 96-well plate and cultured in an incubator for 24 hours. After the cells adhered, the original culture medium was discarded, and 400 μL of CO release carriers were added to different groups, and the dilution multiple of CO release carriers was 1000 times. The parameters of ultrasonic stimulation are: average intensity 0.6 W / cm 2, frequency 1 MHz, stimulation time 1 min. After 1 h of treatment, the culture medium was replaced. After 24 h of culture, the absorbance of each well at 450 nm was measured using an ELISA reader and the cell activity of different treatment groups was evaluated. The cell survival rate was calculated using the following formula:

[0328] Wherein, is the absorbance of the experimental group, is the absorbance of the negative control group, and is the absorbance of the culture medium. The test results of the CO release carriers No. II1-34, D1, and D2 are recorded in Table 1.

[0329] (5) Test of ultrasonic activation efficiency: In order to explore the activation efficiency of the CO release carriers of the above examples and comparative examples under ultrasonic stimulation, the concentration of the CO release carriers was measured before and after ultrasonic stimulation, and the activation efficiency was calculated. The specific steps are as follows: the concentration of the CO release carrier was measured and adjusted to 1×10 9 Transfer 5 ml of the above solution to a container with good acoustic permeability and apply ultrasonic stimulation (power 2 W / cm 2 , frequency is 1 MHz, duration is 2 min), the concentration of CO release carrier in the solution is measured again, and the activation efficiency is calculated as follows:

[0330] Among them, C 前 1×10 9 / ml, C 后 The test results of CO release carriers No. II1-34, D1, and D2 are recorded in Table 1.

[0331] Table 1 CO release carrier CO release carrier Zeta potential (pH=7) Volume weighted average particle size Polydispersity Index The proportion of particle size in the range of 0.05-2μm The proportion of particles in the range of 50-500nm <![CDATA[Amount of CO loaded per milliliter of CO-releasing carrier (×10 5 ppm)]]> Activation efficiency Cell viability II1 -62.4±0.50mV 163.26±13.23 nm 0.025 100% 100% 8.62±2.60 74.62% 11.07±2.632% II2 -6.8±1.23 mV 356.11±25.01nm 0.162 93.1% 75.2% 7.63±2.75 61.33% 16.65±5.334% II3 -15.3±0.86mV 341.23±64.69nm 0.136 94.3% 75.1% 9.23±6.31 63.20% 17.35±4.285% II4 -67.8±1.32mV 149.87±9.35nm 0.062 100% 100% 2.65±4.11 82.19% 18.67±1.02% II5 -6.2±1.01mV 386.70±48.77nm 0.086 89.1% 72.31% 6.21±2.03 91.71% 21.08±3.21% II6 2.0±0.56mV 423.51±42.30nm 0.203 62.1% 49.3% 0.38±1.84 92.36% 26.51±6.12% II7 -21.3±1.24mV 311.54±17.5nm 0.042 100% 96.44% 1.87±0.69 88.42% 21.03±4.22% II8 -19.5±2.69mV 214.61±8.4nm 0.090 96.7% 80.27% 15.71±4.41 95.37% 10.32±2.70% II9 -3.0±0.95mV 396.36±72.6nm 0.186 91.2% 69.38% 5.94±2.66 59.12% 11.97±7.6% II10 -18.2±3.01mV 348.91±15.66nm 0.086 90.4% 72.94% 12.94±3.37 96.60% 16.55±7.3% II11 -6.8±2.94mV 366.47±20.13nm 0.012 89.3% 70.33% 10.52±2.01 86.75% 23.22±6.85% II12 57.6±5.32mV 289.22±9.41nm 0.062 94.7% 81.57% 5.78±1.96 80.35% 17.94±9.02% II13 -11.63±2.84mV 346.89±16.55nm 0.087 90.8% 76.49% 14.67±2.08 90.81% 13.81±4.61% II14 -5.21±1.37mV 239.55±13.91nm 0.096 100% 86.65% 11.38±3.94 89.20% 15.89±6.51% II15 60.54±2.77mV 296.20±8.31nm 0.052 99.5% 90.30% 6.90±1.02 71.33% 14.51±3.92% II16 -9.21±1.89mV 323.51±6.72nm 0.082 92.9% 80.05% 16.11±2.28 68.58% 10.07±2.44% II17 -4.99±0.93mV 377.29±7.01nm 0.115 93.7% 78.50% 6.90±1.16 91.3% 11.34±2.08% II18 -14.76±2.55mV 310.54±5.67nm 0.054 95.2% 83.17% 15.31±2.09 65.21% 9.53±2.42% II19 -39.82±4.37mV 427.18±19.80nm 0.215 70.2% 59.04% 0.33±0.015 98.47% 36.61±4.96% II20 -12.68±3.18mV 487.25±29.44nm 0.227 68.7% 55.67% 0.18±0.03 80.12% 38.45±2.71% II21 -5.21±1.52mV 388.43±9.71nm 0.190 91.5% 72.80% 10.92±3.02 54.81% 15.24±4.28% II22 -8.23±2.76mV 240.21±18.94nm 0.034 100% 94.66% 8.45±4.20 80.31% 15.57±4.09% II23 -60.47±1.62mV 277.09±19.86nm 0.51 98.4% 88.07% 7.35±1.98 87.09% 17.33±4.17% II24 -61.24±1.13mV 210.88±13.90nm 0.030 100% 95.62% 8.18±1.06 78.51% 13.09±2.47% II25 -60.92±1.47mV 198.23±11.07nm 0.29 100% 96.01% 7.51±0.98 76.93% 12.62±2.05% II26 -6.48±2.75mV 310.57±21.97nm 0.144 91.1% 80.39% 1.27±0.15 89.77% 23.43±5.90% II27 -63.02±0.62mV 183.56±14.8nm 0.029 100% 100% 3.77±1.29 76.93% 14.70±3.412% II28 -29.74±5.29mV 378.66±21.94nm 0.121 93.07% 75.42% 1.33±0.20 81.97% 18.03±5.479% II29 -51.22±1.60mV 151.98±6.33nm 0.019 100% 100% 5.90±1.77 71.94% 9.81±2.198% II30 -1.97±0.60mV 360.98±27.61nm 0.139 86.4% 70.21% 0.85±0.21 92.71% 28.03±4.661% II31 -1.02±0.31mV <h2 style=";text-align:left;direction:ltr">368.11±27.45nm 0.143 83.8% 67.90% 0.83±0.34 92.09% 27.91±5.44% <h2 style=";text-align:left;direction:ltr"> II32 <h2 style=";text-align:left;direction:ltr"> -7.03±1.21mV <h2 style=";text-align:left;direction:ltr"> 315.90±20.79nm 0.129 92.3% 80.82% 1.91±0.62 88.64% 18.34±4.091% <h2 style=";text-align:left;direction:ltr"> II33 <h2 style=";text-align:left;direction:ltr"> -19.62±2.93mV <h2 style=";text-align:left;direction:ltr"> 280.04±13.45nm 0.803 95.2% 80.01% 10.15±1.73 50.08% 14.37±2.354% <h2 style=";text-align:left;direction:ltr"> II34 <h2 style=";text-align:left;direction:ltr"> -14.17±3.22mV <h2 style=";text-align:left;direction:ltr"> 391.53±20.31nm 0.133 92.0% 76.39% 9.05±2.07 68.92% 6.10±1.021% D1 <h2 style=";text-align:left;direction:ltr"> -18.67±3.88mV <h2 style=";text-align:left;direction:ltr"> 589.23±141.80nm 0.323 74.9% 62.81% 0.117±0.028 98.73% 38.99±8.50% D2 <h2 style=";text-align:left;direction:ltr"> -39.04±2.55mV <h2 style=";text-align:left;direction:ltr"> 397.25±22.90nm 0.103 87.02% 71.35% 2.01±0.52 89.40% 25.30±5.41%

[0332] It can be seen that, compared with the comparative example, the examples of the present invention have excellent CO release performance.

Claims

1. A CO release carrier composition, characterized in that: It comprises a surfactant, an inert fluoride liquid, and CO gas; the surfactant encapsulates the inert fluoride liquid; the CO gas is dissolved in the inert fluoride liquid; Wherein, the surfactant includes surfactant A agent and surfactant B agent, and the mass ratio is 25:(1-50).

2. The CO release carrier composition according to claim 1, characterized in that: Preferably, the solubility of CO in the inert fluoride liquid under standard conditions is ≥5ml / 100ml; preferably, the inert fluoride liquid includes one or more of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorooctane bromide, perfluorodecalin, and perfluorotripropylamine; preferably, when used alone or in combination, the inert fluoride liquid has a boiling point of ≥37°C under a pressure of 0.5Mpa; Preferably, the surfactant A agent has a hydrophilic end and a lipophilic end; preferably, the surfactant A agent is selected from one or more of lipids, proteins, Tween surfactants, Span surfactants or polymer surfactants; Preferably, the surfactant B agent is selected from one or more of cholesterol, polyethylene glycol (PEG) derivatives, poloxamer surfactants or fatty alcohol polyoxyethylene ether surfactants; Preferably, the lipid is selected from one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid or cationic lipids.

3. A CO release carrier, characterized in that: The invention comprises the CO releasing carrier composition as claimed in claim 1 or 2.

4. The carrier according to claim 3, characterized in that The CO release carrier is a carrier that regulates CO release under acoustic stimulation; Preferably, the response frequency range of the acoustic stimulation is 0.1-30 MHz; Preferably, the power range of the acoustic stimulation is 0.1-5 W / cm 2 ; Preferably, the in vivo circulation time of the CO-releasing carrier is >2h; Preferably, the CO release carrier concentration is greater than 1×10 9 Pieces / ml; Preferably, the structure of the CO release carrier is a spherical droplet, preferably a nanosphere; Preferably, the spherical droplet structure of the CO release carrier is CO / inert liquid core-surfactant shell from inside to outside; Preferably, the volume weighted average particle size of the CO release carrier is 0.05-2 μm, more preferably 50-500 nm, more preferably 50-200 nm; Preferably, at least 60% of the particle size of the CO release carrier is distributed in the range of 0.05-2 μm, and more preferably at least 80% is distributed in the range of 50-500 nm; Preferably, the polydispersity index of the CO-releasing carrier is less than 0.2, preferably less than 0.1; Preferably, the carrier gas volume of the CO release carrier is 10000ppm-2000000ppmCO per milliliter of carrier, more preferably 100000-1000000ppm; Preferably, the absolute value of the zeta potential of the CO releasing carrier at pH=7 is less than 70 mV, more preferably 30-65 mV, and further preferably 45-65 mV.

5. The method for preparing a CO release carrier according to claim 3 or 4, characterized in that: The following steps are involved: (a) adding surfactant A and surfactant B in a certain ratio to solvent A to obtain solution I, and mixing to obtain solution II; (b) contacting an inert fluoride liquid with CO to obtain a CO-saturated fluoride solution III; (c) Solution II is contacted with solution III and mixed.

6. The preparation method according to claim 5, characterized in that: In the step (a), the mixing is carried out by one or more of a stirring method, a high temperature method, and a thin film hydration method; Preferably, the stirring method comprises the following steps: heating the solution I to 35-120° C., keeping the temperature and stirring continuously for 0.5-12 hours to obtain a solution II; Preferably, the high temperature method comprises the following steps: heating the solution I to 35-110° C., and keeping the temperature for 10-60 min until the solution becomes transparent, to obtain a solution II; Preferably, the thin film hydration method comprises the following steps: rotary evaporating the solution I in a water bath until the solvent is completely evaporated to form a film, adding solution B to slowly hydrate and continuously stir to make the lipid film fall off the bottle wall to obtain solution II; wherein the temperature of the rotary evaporation in the water bath is preferably 35-80°C, and the pressure is preferably 0.01-0.1Mpa; Preferably, in step (b), CO saturation is carried out by high pressure method and / or purge method; Preferably, in step (c), the mixing is carried out by one or more of ultrasonic cavitation, extrusion or homogenization; Preferably, the process conditions of the ultrasonic cavitation method include: power of 60-550W, duty cycle of 10%-75%, time of 1-10min; Preferably, the process conditions of the extrusion method include: the membrane pore size is 0.2-5 µm, the number of extrusions is 10-40 times; Preferably, the process conditions of the homogenization method include: a homogenization pressure of 100-2000 bar, a treatment number of 1-15 times, and an aqueous phase solution of 1-10% (w / v%) PVA solution; Preferably, the solution A is one or more of pure water, ion buffer, acetone, ether, ethanol, methanol, chloroform, dichloromethane or ethyl acetate; Preferably, the solution B is one or more of glycerol, propylene glycol, pure water, and ion buffer.

7. A pharmaceutical composition, characterized in that The invention comprises one or more of the CO releasing carrier composition according to claim 1 or 2, the CO releasing carrier according to claim 3 or 4, or the CO releasing carrier obtained by the preparation method according to claim 5 or 6, and optionally a drug.

8. A method for inducing CO release by acoustic stimulation, characterized in that: The CO release carrier releases CO in a controllable manner under ultrasonic stimulation within a characteristic response frequency range and / or a specific power range; wherein the CO release carrier is the CO release carrier composition according to claim 1 or 2, or the CO release carrier according to claim 3 or 4, or the CO release carrier obtained by the preparation method according to claim 5 or 6; Preferably, the characteristic response frequency range is 0.1-30MHz; Preferably, the specific power range is 0.1-5 W / cm 2 .

9. Use of a CO releasing carrier in the preparation of a drug for treating tumor diseases, characterized in that: The CO releasing carrier is the CO releasing carrier composition according to claim 1 or 2, or the CO releasing carrier according to claim 3 or 4, or the CO releasing carrier obtained by the preparation method according to claim 5 or 6.

10. The use according to claim 9, characterized in that The CO release carrier is 1×10 6 -1×10 9 The drug is provided in the form of a unit dose of 100 g / kg body weight and is administered at a dosage regimen of once every 0.5-7 days during the administration cycle.

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