CN111729094B - Phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent and preparation method thereof - Google Patents
Phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent and preparation method thereof Download PDFInfo
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- CN111729094B CN111729094B CN202010717482.8A CN202010717482A CN111729094B CN 111729094 B CN111729094 B CN 111729094B CN 202010717482 A CN202010717482 A CN 202010717482A CN 111729094 B CN111729094 B CN 111729094B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F230/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
- C08F230/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
- C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
- G01N29/02—Analysing fluids
- G01N29/032—Analysing fluids by measuring attenuation of acoustic waves
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
- G01N29/04—Analysing solids
- G01N29/06—Visualisation of the interior, e.g. acoustic microscopy
- G01N29/0654—Imaging
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2438/00—Living radical polymerisation
- C08F2438/01—Atom Transfer Radical Polymerization [ATRP] or reverse ATRP
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- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention belongs to the field of ultrasonic image diagnosis, and particularly relates to a phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent and a preparation method thereof. The invention provides a phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent, which comprises a shell and an inner core, wherein the shell is an amphiphilic block copolymer polycaprolactone-b-polymethacryloxyethyl phosphorylcholine, and the inner core is an ultrasonic responder. The phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent prepared by the method has uniform particle size distribution, has stability obviously superior to that of the phospholipid-based ultrasonic contrast agent, and is more suitable for ultrasonic diagnosis and therapeutic research; and the defects of pressure resistance and mechanical index change resistance of the conventional ultrasonic contrast agent can be overcome, and the application range of the ultrasonic contrast agent is expanded.
Description
Technical Field
The invention belongs to the field of ultrasonic image diagnosis, and particularly relates to a phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent and a preparation method thereof.
Background
The ultrasonic contrast agent can enhance the contrast effect of a super image, remarkably improve the ultrasonic diagnosis precision, be widely applied to the field of clinical diagnosis and have great application potential in ultrasonic-mediated therapy. However, most of the currently applied ultrasound contrast agents in clinical use are composed of small-molecule phospholipid or albumin encapsulated inert gas, which has the disadvantages of large polydispersity or short half-life (<10min), and so on, and limits further application in imaging and therapy.
To enhance the stability of ultrasound contrast agents, materials with higher stiffness than phospholipids have been developed to stabilize the gaseous core in ultrasound contrast agents, which are referred to as hard shell contrast agents. The hard shell contrast agent exhibits little volume expansion and remains intact under low sound pressure conditions. But above a certain pressure threshold, the shell of the hard shell ultrasound contrast agent will also rupture. The polymer has higher rigidity than phospholipid, and the polymer-based ultrasonic contrast agent prepared based on the polymer shell can greatly improve the acoustic behavior of the ultrasonic contrast agent. Furthermore, by adjusting the chemical composition and relative molecular weight of the polymer, the acoustic properties of polymer-based ultrasound contrast agents can also be controlled. The polymer shell ultrasound contrast agent not only has better acoustic stability, but also has greatly improved pressure resistance and mechanical index change resistance under ultrasound. In addition, the grafted or encapsulated therapeutic drug for drug delivery can also be used for ultrasound image-guided diagnosis and treatment integrated preparation, so that the versatility of the multi-modal ultrasound contrast agent is increased, and the application range of the ultrasound contrast agent is further expanded.
Disclosure of Invention
The invention aims to provide a novel polymer-based ultrasonic contrast agent and a preparation method thereof, wherein the obtained contrast agent is a phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent, so that the defects of insufficient pressure resistance and mechanical index change resistance of the conventional ultrasonic contrast agent are solved, and the application range of the ultrasonic contrast agent is expanded.
The technical scheme of the invention is as follows:
the first technical problem to be solved by the invention is to provide a phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent, which comprises an outer shell and an inner core, wherein the outer shell is an amphiphilic block copolymer polycaprolactone-b-polymethacryloxyethyl phosphorylcholine (PCL-b-PMPC), and the inner core is an ultrasonic responder.
Further, the amphiphilic block copolymer is a copolymer obtained by block copolymerization of epsilon-Caprolactone (CL) and 2-Methacryloyloxyethyl Phosphorylcholine (MPC). Further, the arrangement of the hydrophilic segment/hydrophobic segment (A/B) in the amphiphilic block copolymer is AB type.
Further, the shell in the ultrasound contrast agent further comprises a modifying substance M, wherein the modifying substance M is: the shell is provided with a substance containing PEG chain segments which can avoid being cleared by the immune system in vivo, so as to increase the circulation time of the ultrasonic contrast agent.
Further, the modifying substance M is selected from: dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000(DPPE-mPEG 5000), dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000(DPPE-mPEG2000), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-MPEG2000), distearoylphosphatidylethanolamine-azidopolyethylene glycol 5000(DSPE-PEG 5000-N)3) Distearoyl phosphatidyl ethanolamine-azido polyethylene glycol 2000(DSPE-PEG 2000-N)3) Distearoyl phosphatidyl ethanolamine-polyethylene glycol-sulfhydryl cross-linked substance (DSPE-PEG5000-SH), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-sulfhydryl cross-linked substance (DSPE-PEG2000-SH), distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-amino cross-linked substance (DSPE-PEG 5000-NH)2) Distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino cross-linked complex (DSPE-PEG 2000-NH)2) Hard and hardAt least one of a phosphatidylethanolamine-polyethylene glycol 5000-carboxyl cross-linked complex (DSPE-PEG5000-COOH), a distearoylphosphatidylethanolamine-polyethylene glycol 2000-carboxyl cross-linked complex (DSPE-PEG2000-COOH), or a distearoylphosphatidylethanolamine-polyethylene glycol 5000-hydroxyl cross-linked complex (DSPE-PEG 5000-OH).
Preferably, the core of the ultrasound contrast agent is a liquid perfluorocarbon or a gaseous perfluorocarbon that is phase-changeable.
Further, the inner core of the ultrasound contrast agent comprises at least one of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, or perfluoroheptane.
Further, when the core of the ultrasonic contrast agent is liquid perfluorocarbon capable of changing phase, the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is prepared into a nano-emulsion by polycaprolactone-b-polymethacryloxyethyl phosphorylcholine and an ultrasonic response substance in a self-assembly mode, and the obtained nano-emulsion is subjected to phase change to form the phospholipid-like amphiphilic block copolymer coated gaseous perfluorocarbon ultrasonic contrast agent.
Further, when the inner core of the ultrasonic contrast agent is gaseous perfluorocarbon, the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is prepared from polycaprolactone-b-polymethacryloxyethyl phosphorylcholine and an ultrasonic response substance in a self-assembly mode.
Further, the self-assembly mode is one of the following modes: high shear homogenization, high pressure homogenization, high speed oscillation or ultrasonic sound vibration.
Further, the average particle size distribution range of the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is 5 +/-0.13 mu m.
Further, after the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is diluted by physiological saline, the shape is spherical, the dispersion is good, and the surface is smooth and bright when the ultrasonic contrast agent is observed under a laser confocal microscope.
The second technical problem to be solved by the present invention is to provide a preparation method of the phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent, wherein the preparation method comprises: the ultrasonic contrast agent of the polycaprolactone-b-polymethacryloxyethyl phosphorylcholine coated ultrasonic responder is prepared by self-assembling the polycaprolactone-b-polymethacryloxyethyl phosphorylcholine and the ultrasonic responder.
Further, the self-assembly is performed in one of the following ways: high shear homogenization, high pressure homogenization, high speed oscillation or ultrasonic sound vibration.
Further, the ultrasonic responder is liquid perfluorocarbon or gaseous perfluorocarbon which can change phase; preferably a liquid perfluorocarbon. The liquid phase-changeable perfluorocarbon is selected, has better stability than gas, has longer storage time, and can be selected to be subjected to the phase change step before use; the direct use of gaseous preparations has a low success rate and is not conducive to storage.
Further, when the ultrasound response substance in the ultrasound contrast agent is gaseous perfluorocarbon, the preparation method of the phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent comprises the following steps: the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is prepared by directly performing ultrasonic vibration or high-speed oscillation on polycaprolactone-b-polymethacryloxyethyl phosphorylcholine and an ultrasonic responder.
Further, when the ultrasound response substance in the ultrasound contrast agent is liquid perfluorocarbon capable of phase transition, the preparation method of the phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent comprises the following steps: the polycaprolactone-b-polymethacryloxyethyl phosphorylcholine and the ultrasonic response substance are prepared into the nano-emulsion in a self-assembly mode, and the nano-emulsion is subjected to temperature-induced phase change or sound-induced phase change to form the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent. When the shell layer uses phospholipid-like amphiphilic block copolymer, high-temperature hydrophobic liquid perfluorocarbon which can change phase is wrapped in the shell layer, nano-emulsion, also called nano liquid particles, is prepared in a self-assembly mode by utilizing a similar compatibility principle and hydrophilic-hydrophobic interaction, and then the ultrasonic contrast agent of phospholipid-like amphiphilic copolymer wrapped with gaseous perfluorocarbon which can be subjected to ultrasonic contrast is formed through phase change.
Further, when the ultrasound response substance in the ultrasound contrast agent is liquid perfluorocarbon capable of phase change, the preparation method comprises the following steps:
(1) preparing a phospholipid-like amphiphilic block copolymer polycaprolactone-b-polymethacryloxyethyl phosphorylcholine (PCL-b-PMPC);
(2) preparing a phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent: dissolving the phospholipid-like amphiphilic block copolymer PCL-b-PMPC obtained in the step (1) and the modified substance M in a mixed solvent, mixing with an ultrasonic corresponding substance, preparing a nano-emulsion in a self-assembly mode, separating and purifying, and performing temperature-induced phase change or sound-induced phase change to prepare the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent; wherein the modifying substance M is: providing the shell with a material comprising a PEG segment that avoids clearance by the immune system in vivo; the mixed solvent is a mixed solvent of tetrahydrofuran and methanol, or: a mixed solvent of chloroform and methanol.
Further, in the step (2), the method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent after the nanoemulsion is separated and purified and undergoes temperature-induced phase change or sound-induced phase change comprises one of the following modes:
the first method is as follows: preparing the phospholipid-like amphiphilic block copolymer-based nano-emulsion suspension by using a water bath at 60-80 ℃ (preferably 70 ℃) for 5-15 min (preferably 10min) to obtain a phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent;
the second method comprises the following steps: subjecting the prepared phospholipid-like amphiphilic block copolymer-based nanoemulsion suspension to ultrasonic action by an ultrasonic therapeutic apparatus to obtain the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent, wherein the ultrasonic power is 1-3W/cm2(preferably 3W/cm)2) The duty ratio is 20-80% (preferably 50%), and the action time is 2-5 min (preferably 3 min).
Preferably, in the step (2), the mixed solvent is a mixture of solvents with a volume ratio of 2:1 of tetrahydrofuran and methanol.
Further, in the step (2) of the above method, the modifying substance M is selected from: dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000(DPPE-mPEG 5000), dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000(DPPE-mPEG2000), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-MPEG2000), dipalmitoylphosphatidylethanolamineStearoyl phosphatidyl ethanolamine-azidopolyethylene glycol 5000(DSPE-PEG 5000-N)3) Distearoyl phosphatidyl ethanolamine-azido polyethylene glycol 2000(DSPE-PEG 2000-N)3) Distearoyl phosphatidyl ethanolamine-polyethylene glycol-sulfhydryl cross-linked substance (DSPE-PEG5000-SH), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-sulfhydryl cross-linked substance (DSPE-PEG2000-SH), distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-amino cross-linked substance (DSPE-PEG 5000-NH)2) Distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino cross-linked complex (DSPE-PEG 2000-NH)2) At least one of distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-carboxyl cross-linked complex (DSPE-PEG5000-COOH), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-carboxyl cross-linked complex (DSPE-PEG2000-COOH) or distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-hydroxyl cross-linked complex (DSPE-PEG 5000-OH).
Further, the method for preparing the phospholipid-like amphiphilic block copolymer PCL-b-PMPC in the step (1) comprises the following steps: epsilon-Caprolactone (CL) and 2-Methacryloyloxyethyl Phosphorylcholine (MPC) are polymerized by an atom transfer radical polymerization (ARGET ATRP) method of an electron transfer regeneration catalyst to obtain a phospholipid-like amphiphilic block copolymer PCL-b-PMPC.
Furthermore, the method for preparing the phospholipid-like amphiphilic block copolymer PCL-b-PMPC in the step (1) comprises the following steps:
1) preparing a macroinitiator PCL-Br:
taking epsilon-CL as a monomer and stannous octoate as a catalyst, carrying out ring-opening polymerization to obtain a homopolymer PCL, and then reacting the PCL with bromoisobutyryl bromide to obtain PCL-Br;
2) preparation of phospholipid-like amphiphilic block copolymer PCL-b-PMPC:
using PCL-Br obtained in 1) as a macroinitiator and MPC as a monomer, and polymerizing by an atom transfer radical polymerization (ARGET ATRP) method of an electron transfer regeneration catalyst to obtain a phospholipid-like amphiphilic block copolymer PCL-b-PMPC.
The third technical problem to be solved by the invention is to provide the application of the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent, which can be applied to the field of ultrasonic image diagnosis and treatment and is used for in-vitro agarose model contrast imaging; or the medicine is used for in-vivo ultrasonic contrast imaging and treatment after being loaded with the medicine.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent prepared by the method has uniform particle size distribution, has stability obviously superior to that of a phospholipid-based ultrasonic contrast agent (a commercial Sonowei SonoVue contrast agent), and is more suitable for ultrasonic diagnosis and therapeutic research.
(2) The phase-change phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent prepared by the invention has a remarkable image enhancement effect in a high mechanical index mode of ultrasonic contrast, has a longer duration time than that of a phospholipid shell contrast agent, improves the short duration time of the phospholipid-based ultrasonic contrast agent in the high mechanical index contrast, and overcomes the defects that the shells of ultrasonic contrast agents prepared by partial high polymer materials are hard and difficult to develop.
(3) The phase-change type ultrasonic contrast agent prepared by coating liquid fluorocarbon with phospholipid-like amphiphilic block copolymer PCL-b-PMPC (namely PCL-b-PMPC is a shell and liquid fluorocarbon is a core) is explored to be feasible as the ultrasonic contrast agent; in vitro and in vivo experiments show that the PCL-b-PMPC-based ultrasonic contrast agent has good echo characteristics under various ultrasonic parameter conditions. More importantly, the imaging time of the PCL-b-PMPC-based ultrasound contrast agent is longer than that of the phospholipid-based ultrasound contrast agent under the same ultrasound parameters and concentrations, and the PCL-b-PMPC-based ultrasound contrast agent has great potential as a novel contrast agent in ultrasound imaging.
(4) The amphiphilic block copolymer in the phase-change amphiphilic phospholipid block copolymer-based ultrasonic contrast agent has the advantages of controllable structure, safety, no toxicity, obviously lower cost than synthetic phospholipid, and simple and convenient preparation process. In addition, the preparation method can be widely applied in the field of clinical ultrasonic imaging, can also be used as a carrier of various therapeutic drugs, and has a good clinical application prospect.
Description of the drawings:
FIG. 1 is a synthesis scheme of the phospholipid-like amphiphilic block copolymer PCL-b-PMPC and a schematic diagram of preparation of an ultrasonic contrast agent.
FIG. 2 shows the polymer PCL obtained in example 143、PCL43-Br and PCL43-b-PMPC25Nuclear magnetic spectrum of (1).
FIG. 3 shows the polymer PCL obtained in example 143、PCL43-Br and PCL43-b-PMPC25An infrared spectrum of (1).
FIG. 4 shows the polymer PCL obtained in example 143And PCL43-b-PMPC25DSC (a) temperature rise curve and (b) temperature fall curve of (a).
FIG. 5 shows the phospholipid-like amphiphilic block copolymer PCL obtained in example 143-b-PMPC25Ultrasound contrast agent based (a) CLSM pictures, (b) size distribution maps, and (c) size and zeta data.
FIG. 6 shows the phospholipid-like amphiphilic block copolymer PCL obtained in example 143-b-PMPC25The contrast agent is based on a contrast map of different ultrasound parameters in vitro and corresponding gray values-frequency (a, d), gray values-mechanical index MI (b, e) and gray values-concentration (c, f).
FIG. 7 shows the phospholipid-like amphiphilic block copolymer PCL obtained in example 143-b-PMPC25Variation of the in vitro contrast effect of the base ultrasound contrast agent over time ultrasound contrast map (a) and corresponding grey values (b).
FIG. 8a shows the phospholipid-like amphiphilic block copolymer PCL obtained in example 143-b-PMPC25The results of the in vivo contrast effect of animals based on ultrasound contrast agent, PBS and SonoVue contrast agent as a function of time, wherein the circles represent the liver imaging area of rabbits; FIG. 8b shows the phospholipid-like amphiphilic block copolymer PCL obtained in example 143-b-PMPC25The gray values corresponding to the base ultrasound contrast agent and the SonoVue contrast agent.
Detailed Description
The first technical problem to be solved by the invention is to provide a phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent, which comprises an outer shell and an inner core, wherein the outer shell is an amphiphilic block copolymer polycaprolactone-b-polymethacryloxyethyl phosphorylcholine (PCL-b-PMPC), and the inner core is an ultrasonic responder.
The second technical problem to be solved by the present invention is to provide a preparation method of the phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent, wherein the preparation method comprises: the ultrasonic contrast agent of the polycaprolactone-b-polymethacryloxyethyl phosphorylcholine coated ultrasonic responder is prepared by self-assembling the polycaprolactone-b-polymethacryloxyethyl phosphorylcholine and the ultrasonic responder.
The third technical problem to be solved by the invention is to provide the application of the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent, which can be applied to the field of ultrasonic image diagnosis and treatment and is used for in-vitro agarose model contrast imaging; or the medicine is used for in-vivo ultrasonic contrast imaging and treatment after being loaded with the medicine.
The following examples are given to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.
Example 1
1. The preparation method of the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent comprises the following specific steps:
1) preparation of amphiphilic Block copolymer Polymer PCL43-b-PMPC25：
Firstly, preparing PCL: lauryl alcohol (0.82g,0.004 mol.), epsilon-CL (10.13g,0.089mol) and Sn (Otc)2(4.05g,0.01mol) was added to the reaction vessel. Removing O therefrom2And residual water. The reaction was carried out at 120 ℃ for 24 hours under vacuum. The crude product was dissolved in CH2Cl2And purified by cold methanol precipitation to obtain PCL.
Secondly, preparing PCL-Br: dissolving the PCL-OH (7.00g,0.002mol) obtained in the step (i) and triethylamine (0.96mL, 0.007mmol) in Tetrahydrofuran (THF); in N22-bromoisobutyryl bromide (0.85 mL,0.0069mmol) was added dropwise to the above solution at-20 deg.C; and (3) carrying out reaction at room temperature for 48-72 hours, passing through a neutral alumina column to remove quaternary ammonium salt, and precipitating and evaporating a small amount of THF-containing crude product in cold methanol to obtain white powdery polymer PCL-Br.
③ Final preparation of PCL43-b-PMPC25: the PCL obtained in step (2)-Br (1.99g,0.596mmol), MPC (6.15g, 0.021mol) and Bpy (0.28g,1.79mmol) were dissolved in CH2Cl2Performing the following steps; general formula (N)2After 30 minutes, at N2CuBr (0.13g,0.894mmol) was added to the atmosphere; after 24 hours of reaction, purifying by a neutral alumina column; and further purified by dialysis with ultrapure water. The product PCL is obtained by freeze-drying43-b-PMPC25。
2) Preparing a phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent: 3mg of PCL was weighed separately43-b-PMPC25Dissolving the block copolymer and 1mg of DPPE-mPEG 5000 in 567. mu.L of Tetrahydrofuran (THF) and 283. mu.L of methanol (MeOH) (2:1, v/v), dissolving by ultrasonic dispersion in a water bath, adding 2ml of PBS buffer (0.01M, pH 7.4, pure water or physiological saline is acceptable), and then adding 150. mu.L of perfluoropentane (PFP); ultrasonically emulsifying by using a probe under an ice-water bath environment, wherein ultrasonic parameters are as follows: the frequency is 24KHz, the power is 35W, the ultrasonic is switched on for 3s and then switched off for 6s, the diameter of a sound vibration probe is 3mm, the treatment time is 3min, a milky white suspension is formed, then the suspension is centrifuged for 5min under the centrifugal force of 3000g, the supernatant is discarded, and 6mL of PBS is added for heavy suspension. And (3) performing water bath action on the obtained copolymer-based nano-emulsion suspension for 10min at 70 ℃ to prepare the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent.
2. Characterization of phospholipid-like amphiphilic Block copolymers
FIG. 2 shows the synthesis of PCL43-b-PMPC25In nuclear magnetic spectra of various polymers in the process of the copolymer, characteristic peaks (delta is 4.1ppm, delta is 2.3ppm, delta is 1.7ppm and delta is 1.4ppm) of segments of the PCL all appear on the nuclear magnetic spectra, which indicates that the PCL is successfully synthesized, and the polymerization degree of the PCL is calculated by the area ratio of the peaks. Through calculation, the invention successfully obtains homopolymers with the polymerization degrees of 43 respectively; the appearance of a characteristic peak indicates that the PMPC chain segment is successfully grafted to the PCL chain segment; meanwhile, the degree of polymerization of the PMPC segment is determined by the area ratio of the peaks.
The structure of the polymer was further characterized by infrared, with the results shown in figure 3. 1728cm-1The peak at (A) is a stretching vibration peak of C ═ O, 2910cm-1Are continuous methylene absorption peaks; it can be found that PCL-b-PMPC is in the infraredThe spectrogram shows a characteristic absorption peak (1090 cm) related to the structure of the phosphatidylcholine-1And 1230cm-1，-POCH2-；970cm-1， N+(CH3)3) Successful synthesis of PCL-b-PMPC copolymers was also demonstrated.
The thermal properties of the synthesized polymer were measured by DSC, and the results are shown in fig. 4. The PCL homopolymer has data such as highest crystallization temperature, melting temperature and crystallinity; compared with PCL, the PCL-b-PMPC copolymer is introduced with an amorphous PMPC chain segment, and when the PCL is crystallized, the regular arrangement of the chain segment is hindered, so that various data are reduced.
3. Characterization of microvesicles
The PCL-b-PMPC copolymer solution added with PBS and PFP becomes opaque milky white suspension after the sound vibration treatment; after the organic solvent is removed, the phase change of the PFP is triggered at the temperature, and the PFP is still a milky white suspension solution; the particle size change of the copolymer-based ultrasound contrast agent after phase transition is shown in fig. 5; as can be seen from FIG. 5, the particle size distribution of the PCL-b-PMPC copolymer and the ultrasound contrast agent is narrow, and the average particle size of the PCL-b-PMPC copolymer-based ultrasound contrast agent measured by a Malvern nanometer particle size analyzer is about 5 μm.
3. Stability test
The concentrations of the copolymer-based ultrasonic contrast agent and a reference phospholipid-based ultrasonic contrast agent (a commercial Sonowei SonoVue contrast agent)) measured by Coulter are diluted to ensure that the copolymer-based ultrasonic contrast agent and the phospholipid-based ultrasonic contrast agent have the same concentration, and the copolymer-based ultrasonic contrast agent and the phospholipid-based ultrasonic contrast agent are placed in a refrigerator at 4 ℃ and are kept still for 24 hours and then are observed under a sampling microscope, so that the concentration of the phospholipid-based ultrasonic contrast agent is lower than that of the copolymer-based ultrasonic contrast agent, and the stability of the copolymer-based ultrasonic contrast agent obtained by the invention is obviously higher than that of the phospholipid-based ultrasonic contrast.
4. In vitro radiography and blasting experiment
Adding 0.3mL of the copolymer-based ultrasonic contrast agent prepared in the step 2) into an agarose gel model, observing in a Cadence Contrast Agent Imaging (CCAI) mode (MI ═ 0.21) by using a Siemens color imaging system (SIEMENS Acuson Antares), wherein the echo in the model is obviously enhanced and is in a dense fine-dot-shaped echo, and observing the change of the echo intensity from the ultrasonic frequency of 4.0MHz to 10.0MHz, wherein the optimal developing frequency of the contrast agent is 5.71MHz (shown in figure 6); the contrast agent is broken by starting a blasting (MI is 0.67) mode, the echo in the model is instantaneously reduced, and the number of the fine-point echoes is sharply reduced.
5. New Zealand white rabbit liver angiography
The method comprises the steps of fixing a male New Zealand white rabbit with the body weight of 2.5-3 kg on an experiment table, placing an indwelling needle into an ear edge vein, after the lumbar and the back of the right side are unhaired, respectively injecting a commercial Sonowev SonoVue contrast agent (a contrast sample) and the PCL-b-PMPC copolymer contrast agent obtained by the invention by adopting an ear edge vein bolus injection method, so as to obtain an ultrasonic contrast image of the liver. In the control experiment, only PBS buffered saline was injected, with dark areas in the ultrasound image, and the results are shown in FIG. 8. In contrast to the PBS control group, significant contrast enhancement began to occur in the liver region following intravenous injection of SonoVue and PCL-b-PMPC copolymer contrast agents, respectively. Bright ultrasound images of the liver indicate that PCL-b-PMPC copolymer contrast agent successfully passes through the pulmonary capillaries during blood circulation, which is necessary for the safety properties of intravenous contrast agents. As can be seen from FIG. 8, the ultrasound signal intensity of the SonoVue contrast agent is substantially comparable to the contrast image intensity of the PCL-b-PMPC copolymer contrast agent at 20 s; however, at 40s, the ultrasound signal of the SonoVue microbubbles decreased rapidly, and at 80s, the contrast agent substantially disappeared, while the PCL-b-PMPC copolymer contrast agent still had a contrast signal that was observable after 80 s. This shows that the PCL-b-PMPC phospholipid block polymer nano ultrasound contrast agent has longer in vivo duration compared with a SouoVue contrast agent, and proves that the PCL-b-PMPC phospholipid block polymer nano ultrasound contrast agent has great potential as an ultrasound contrast agent with novel diagnostic and therapeutic effects.
Example 2
A method different from that of example 1 was used to prepare a PCL-b-PMPC contrast medium by a shear method.
The specific preparation process of the PCL-b-PMPC phospholipid block polymer nano ultrasonic contrast agent prepared by the shearing method comprises the following steps:
3mg of PCL-b-PMPC copolymer and 1mg of DPPE-mPEG 5000 were each dissolved in 567. mu.L of Tetrahydrofuran (THF) and 283. mu.L of methanol (MeOH) (2:1, v/v), and after sufficient dispersion in a water bath, 2mL of PBS buffer (0.01M, pH 7.4) was added, followed by 150. mu.L of perfluoropentane (PFP). Under the condition of ice-water bath, adding liquid perfluoropentane, adopting an electric internally tangent homogenate method, wherein the homogenate rotating speed is 12000-30000rpm, and the homogenate time is 1-3min, and obtaining milky block polymer mixed solution wrapping the perfluoropentane after homogenate.
The mixed solution is subjected to phase change by a therapeutic ultrasonic instrument to form the PCL-b-PMPC phospholipid amphiphilic block copolymer-based ultrasonic contrast agent, and the parameter of the ultrasonic instrument is 3W/cm2The duty ratio is 50%, and the action time is 3 min.
EXAMPLE 3 Freeze-drying preparation of PCL-b-PMPC contrast media
The preparation process of the PCL-b-PMPC phospholipid amphiphilic block copolymer-based ultrasonic contrast agent by the freeze-drying method comprises the following steps:
3mg of PCL-b-PMPC copolymer and 1mg of DPPE-mPEG 5000 were each dissolved in 567. mu.L of Tetrahydrofuran (THF) and 283. mu.L of methanol (MeOH) (2:1, v/v), and after sufficient dispersion in a water bath, 2mL of PBS buffer (0.01M, pH 7.4) was added, followed by 150. mu.L of perfluoropentane (PFP). Performing ultrasonic vibration under ice salt water bath condition, wherein the probe frequency is 24KHz, the power is 35W, the ultrasonic is turned on for 3s and turned off for 6s, the action is performed for 3min, a milky white suspension is formed, then the suspension is centrifuged for 5min under the action of 3000g centrifugal force, the supernatant is discarded, and 6mL of sucrose solution containing 10 wt% is added respectively for heavy suspension. Subpackaging penicillin bottles with the specification of 10mL according to the volume of 2mL per bottle, placing the bottles in a refrigerator with the temperature of-20 ℃ for pre-freezing overnight, and freeze-drying the bottles for 24 hours by a freeze dryer, wherein the temperature of a cold trap is set to be-80 ℃.
Filling the prepared freeze-dried powder with gas such as perfluoropropane, perfluorobutane and sulfur hexafluoride through a ventilation device, recombining with 2mL of physiological saline after ventilation to obtain milky copolymer suspension, and mechanically oscillating the recombined microbubble suspension for 1min at the oscillation frequency of 75Hz to obtain the PCL-b-PMPC phospholipid amphiphilic block copolymer-based ultrasound contrast agent.
According to the three examples, the PCL-b-PMPC phospholipid amphiphilic block copolymer-based ultrasonic contrast agent prepared by different preparation methods is spherical in shape, good in dispersion, smooth and transparent in surface, uniform in size (shown in figure 5), and 5 +/-0.13 mu m in average particle size distribution range.
The mechanical index value (MI) has a great influence on the ultrasound contrast image, which substantially determines the intensity of the ultrasound because with higher energy ultrasound drives the contrast agent may reflect the echo signal more strongly, whereas when the MI is too large it may cause the destruction of the contrast agent and the loss of the contrast effect. Compared with the phospholipid-based contrast agent, the PCL-b-PMPC phospholipid amphiphilic block copolymer-based ultrasonic contrast agent can tolerate the action of higher ultrasonic mechanical index and keep an integral morphological structure.
The change of the PCL-b-PMPC copolymer-based ultrasound contrast signal intensity with time after ultrasound irradiation is shown in fig. 7 by acquiring ultrasound images of the PCL-b-PMPC copolymer-based ultrasound contrast agent and the SonoVue contrast agent at different time points, and analyzing the change of the average gray level of the copolymer-based ultrasound contrast agent and the SonoVue contrast image. As can be seen from the attached figure 7, the brightness of the ultrasonic image generated by the SonoVue microbubble within 1-3min is rapidly darkened along with the time, the intensity of the ultrasonic signal is rapidly reduced, and the contrast effect is almost lost after 4 min; the brightness of the ultrasonic contrast agent based on the PCL-b-PMPC copolymer shows good stability, the brightness of the image slowly becomes dark between 1 and 3min, the intensity of an ultrasonic signal slowly decreases, the ultrasonic signal tends to be stable after 6min, and the ultrasonic contrast agent still has a certain contrast effect in vitro for 20 min. However, the ultrasound signal generated by SonoVue ultrasound contrast agents drops sharply over a period of about 3min, down to about 20% of its initial intensity, with only 2.5% of the initial intensity at 20 min. However, the ultrasonic signal intensity of the PCL-b-PMPC copolymer-based ultrasonic contrast agent can be maintained for about 20min, and is reduced to 23% of the initial intensity at most and only 50% of the initial intensity at least compared with the initial ultrasonic signal intensity. The stronger stability of the PCL-b-PMPC copolymer-based ultrasonic contrast agent is attributed to the fact that the PCL-b-PMPC copolymer can form a stable core-shell structure in aqueous solution, which is to keep PFP gas in an inner core from being in a non-core state under ultrasonic radiationWill diffuse and dissolve providing a strong barrier, whereas the lipid shell of SonoVue ultrasound contrast agents is only about 4nm thick, and the gas within the microbubbles will diffuse out quickly through this shell under ultrasound radiation, thus rapidly reducing the contrast capabilities of SonoVue ultrasound contrast agents. Furthermore, gaseous PFP in PCL-b-PMPC copolymer-based ultrasound contrast agents compared to SF in SonoVue ultrasound contrast agents6Gas, has lower water solubility, which also helps to enhance the stability of PCL-b-PMPC copolymer-based ultrasound contrast agents. This allows the PCL-b-PMPC copolymer-based ultrasound contrast agents to have longer cycle periods, providing a longer imaging time window for clinical diagnosis.
From the above, the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent obtained by the invention is applied to in-vitro model ultrasonic contrast imaging, and the concentration of the microbubbles is set to be 5.0 × 108After the volume/mL and the mechanical index MI are 0.2, an ultrasonic contrast image is obtained in a frequency range of 4-10 MHz, the change of the average gray value of the contrast image of the copolymer ultrasonic contrast agent along with the frequency is analyzed, and due to the difference between the resonance frequency of the ultrasonic contrast agent and the ultrasonic driving frequency, the brightness of the ultrasonic contrast image of part of the PCL-b-PMPC copolymer-based ultrasonic contrast agent is increased along with the increase of the frequency from 4MHz to 5.71 MHz. When the frequency is increased by 6.67MHz, the brightness of all the ultrasonic contrast images is rapidly reduced; the brightness of the image remains substantially unchanged by 7.27 MHz. When the ultrasound frequency is chosen at 5.71MHz, the contrast image gray value of the ultrasound contrast agent is maximal. Copolymer-based ultrasound contrast agents have a longer duration than SonoVue contrast agents. The phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is applied to in-vivo ultrasonic contrast imaging of New Zealand white rabbits, and shows that the ultrasonic contrast agent has a certain in-vivo contrast effect, and the contrast time of the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent under a high mechanical index is prolonged compared with that of a phospholipid-based contrast agent.
Claims (17)
1. The phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is characterized by comprising an outer shell and an inner shellA core, the shell is an amphiphilic block copolymer polycaprolactone-b-polymethacryloxyethylphosphocholine, the core being an ultrasound-responsive substance, the ultrasound-responsive substance being a liquid perfluorocarbon or a gaseous perfluorocarbon which is phase-changeable.
2. The phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent according to claim 1, wherein the shell in the ultrasound contrast agent further comprises a modifying substance M, wherein the modifying substance M is: the shell is provided with a material containing a PEG segment that avoids clearance by the immune system in vivo.
3. The phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent according to claim 2, wherein the modifying substance M is selected from the group consisting of: dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-azidopolyethylene glycol 5000, distearoylphosphatidylethanolamine-azidopolyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol-mercapto, at least one of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-sulfhydryl, distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-amino, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino, distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-carboxyl, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-carboxyl or distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-hydroxyl.
4. The phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent according to claim 1, wherein the core of the ultrasound contrast agent comprises at least one of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, or perfluoroheptane.
5. The phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent according to claim 1, wherein the core of the ultrasound contrast agent is liquid perfluorinated when the core is phase-changeableWhen carbon is used, the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is prepared from polycaprolactone-bPreparing the polymethacryloxyethyl phosphorylcholine and the ultrasonic response substance into the nano-emulsion in a self-assembly mode, and forming the ultrasonic contrast agent with the phospholipid amphiphilic block copolymer wrapping gaseous perfluorocarbon through phase change.
6. The phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent as claimed in claim 1, wherein the phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent is prepared from polycaprolactone-when the core of the ultrasound contrast agent is gaseous perfluorocarbonbThe polymethacryloxyethyl phosphorylcholine and the ultrasonic responder are prepared into the ultrasonic contrast agent by a self-assembly mode.
7. The phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent according to claim 6, wherein the self-assembly is in one of the following manners: high shear homogenization, high pressure homogenization, high speed oscillation or ultrasonic sound vibration.
8. The phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent according to claim 1 or 2, wherein the phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent is diluted with physiological saline, and observed under a laser confocal microscope, the phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent is spherical in shape, good in dispersion and smooth and bright in surface.
9. The preparation method of the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent as defined in any one of claims 1 to 8, wherein the preparation method comprises the following steps: polycaprolactone-bThe poly (methacryloyloxyethyl phosphorylcholine) and the ultrasonic responder are prepared into polycaprolactone-b-an ultrasound contrast agent in which polymethacryloxyethylphosphocholine encapsulates an ultrasound responder.
10. The method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent according to claim 9, wherein the self-assembly is performed in one of the following ways: high shear homogenization, high pressure homogenization, high speed oscillation or ultrasonic sound vibration.
11. The method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent according to claim 9, wherein when the ultrasonic response substance in the ultrasonic contrast agent is gaseous perfluorocarbon, the method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent comprises the following steps: polycaprolactone-bThe phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is prepared from polymethacryloxyethyl phosphorylcholine and an ultrasonic responder in a direct ultrasonic sound vibration or high-speed oscillation mode.
12. The method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent according to claim 9, wherein when the ultrasonic response substance in the ultrasonic contrast agent is phase-changeable liquid perfluorocarbon, the method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent comprises the following steps: polycaprolactone-bThe poly (methacryloyloxyethyl phosphorylcholine) and the ultrasonic responder are prepared into the nano-emulsion in a self-assembly mode, and the nano-emulsion is subjected to temperature-induced phase change or sound-induced phase change to form the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent.
13. The method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent according to claim 12, wherein when the ultrasonic responder in the ultrasonic contrast agent is a phase-changeable liquid perfluorocarbon, the method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent comprises the following steps:
(1) preparation of phospholipid-like amphiphilic block copolymer polycaprolactone-b-polymethacryloxyethylphosphocholine;
(2) preparing a phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent: the phospholipid-like amphiphilic block copolymer polycaprolactone obtained in the step (1)bPolymethacryloxyethylphosphocholine and modifying substances MDissolving the mixture in a mixed solvent, mixing the mixture with an ultrasonic response substance, preparing a nano-emulsion in a self-assembly mode, separating and purifying, and then preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent after temperature-induced phase change or sound-induced phase change; wherein the modifying substance M is: providing the shell with a material comprising a PEG segment that avoids clearance by the immune system in vivo; the mixed solvent is a mixed solvent of tetrahydrofuran and methanol, or: a mixed solvent of chloroform and methanol.
14. The method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent according to claim 13, wherein in the step (2), the method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent after the temperature-induced phase change or the sound-induced phase change of the separated and purified nano-emulsion is one of the following modes:
the first method is as follows: the prepared phospholipid-like amphiphilic block copolymer based nano-emulsion suspension is subjected to water bath at the temperature of 60-80 ℃ for 5-15 min to prepare the phospholipid-like amphiphilic block copolymer based ultrasonic contrast agent;
the second method comprises the following steps: subjecting the prepared phospholipid-like amphiphilic block copolymer-based nanoemulsion suspension to ultrasonic action by an ultrasonic therapeutic apparatus to obtain the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent, wherein the ultrasonic power is 1-3W/cm2The duty ratio is 20% -80%, and the action time is 2-5 min.
15. The method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent according to claim 14, wherein in the step (2), the volume ratio of the mixed solvent is 2:1 of tetrahydrofuran and methanol.
16. The method for preparing the phospholipid-like amphiphilic block copolymer-based ultrasound contrast agent as claimed in claim 14, wherein the phospholipid-like amphiphilic block copolymer polycaprolactone-based material prepared in the step (1)bThe method of polymethacryloxyethylphosphocholine is as follows: regeneration of epsilon-caprolactone and 2-methacryloyloxyethyl phosphorylcholine by electron transferAtom transfer radical polymerization method of catalyst is used for polymerization to obtain the amphiphilic block copolymer polycaprolactone-like material for preparing phospholipidb-polymethacryloxyethylphosphocholine.
17. The phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is used for in-vitro agarose model contrast imaging; wherein the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent is the phospholipid-like amphiphilic block copolymer-based ultrasonic contrast agent as defined in any one of claims 1 to 8 or the ultrasonic contrast agent prepared by the preparation method as defined in any one of claims 9 to 16.
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