CN111671922A - Amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent and preparation method thereof - Google Patents
Amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent and preparation method thereof Download PDFInfo
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- CN111671922A CN111671922A CN202010717453.1A CN202010717453A CN111671922A CN 111671922 A CN111671922 A CN 111671922A CN 202010717453 A CN202010717453 A CN 202010717453A CN 111671922 A CN111671922 A CN 111671922A
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- contrast agent
- phospholipid
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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
- A61K49/225—Microparticles, microcapsules
-
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Acoustics & Sound (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention belongs to the field of ultrasonic image diagnosis, and particularly relates to a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent and a preparation method thereof. The invention provides a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, which comprises a shell and an inner core, wherein the shell is a phospholipid-like amphiphilic comb-shaped block graft copolymer polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine), and the inner core is an ultrasonic responder. The particle size distribution of the ultrasonic contrast agent prepared by the invention is more uniform, the stability is obviously superior to that of a phospholipid-based ultrasonic contrast agent, and the ultrasonic contrast agent is more suitable for ultrasonic diagnosis and treatment 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 comb-shaped block graft 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 comb-shaped block graft 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 comb-shaped block graft copolymer-based ultrasonic contrast agent, which comprises a shell and an inner core, wherein the shell is a phospholipid-like amphiphilic comb-shaped block graft copolymer polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) (PCL-b- (PBrCL-g-PMPC)), and the inner core is an ultrasonic responder.
Further, the phospholipid-like amphiphilic comb-shaped block graft copolymer is a copolymer obtained by carrying out block and graft copolymerization on-Caprolactone (CL) and 2-Methacryloyloxyethyl Phosphorylcholine (MPC).
Further, the phospholipid-like amphiphilic comb-shaped block graft copolymer is prepared by adopting the following method:
(1) preparation of macromolecular backbone PBrCLp-b-PCLmFirstly, cyclohexanone is used as raw material to react with liquid bromine to obtain α -bromocyclohexanone, then α -bromocyclohexanone reacts with m-chloroperoxybenzoic acid to obtain α -bromo-caprolactone (α BrCL), and then α BrCL and-CL are used as monomers, stannous octoate is used as catalyst, ring-opening block polymerization is carried out to obtain macromolecular main chain PBrCLp-b-PCLmWherein p is the number of α BrCL contained in the main chain of the copolymer on average, and m is the polymerization degree of CL;
(2) preparing a phospholipid-like amphiphilic comb-shaped block graft copolymer PCL-b- (PBrCL-g-PMPC): with the PBrCL obtained in the step (1)p-b-PCLmIs a macromolecular main chain, 2-Methacryloyloxyethyl Phosphorylcholine (MPC) is a monomer, and a phospholipid-like amphiphilic comb-shaped block graft copolymer PCL-b- (PBrCL-g-PMPC) is obtained by side chain polymerization by an atom transfer radical polymerization (ARGET ATRP) method of an electron transfer regenerated catalyst.
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) 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).
Preferably, the core of the ultrasound contrast agent is gaseous perfluorocarbon or liquid perfluorocarbon that can be phase-changed.
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 comb-shaped block graft copolymer-based ultrasonic contrast agent is prepared from polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) and an ultrasonic response substance in a self-assembly mode to obtain a nano-emulsion, and the obtained nano-emulsion is subjected to phase change to form the phospholipid-like amphiphilic comb-shaped block graft copolymer coated gaseous perfluorocarbon ultrasonic contrast agent.
Further, when the core of the ultrasonic contrast agent is gaseous perfluorocarbon, the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent is prepared from polycaprolactone-b- (polybromocaprolactone-g-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 comb-shaped block graft copolymer-based ultrasonic contrast agent is 5 +/-0.13 mu m.
Further, after the phospholipid-like amphiphilic comb-shaped block graft 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 invention is to provide a preparation method of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, wherein the preparation method comprises the following steps: the ultrasonic contrast agent of polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) coated ultrasonic response substance is prepared by self-assembling polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) and the ultrasonic response substance.
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 comb-graft copolymer-based ultrasound contrast agent comprises the following steps: polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) and an ultrasonic responder are subjected to direct ultrasonic vibration or high-speed oscillation to prepare the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent.
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 comb-graft copolymer-based ultrasound contrast agent comprises the following steps: polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) and an ultrasonic responder are prepared into the nanoemulsion in a self-assembly mode, and the obtained nanoemulsion is subjected to temperature-induced phase change or sound-induced phase change to form the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent. When the shell layer uses phospholipid-like amphiphilic comb-shaped block graft copolymer, high-temperature hydrophobic liquid perfluorocarbon which can change phase is wrapped inside 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 wrapping gaseous perfluorocarbon which can be ultrasonically contrasted 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 comb-shaped block graft copolymer polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine);
(2) preparing a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent: dissolving the phospholipid-like amphiphilic comb-shaped block graft copolymer and the modified substance M obtained in the step (1) by a mixed solvent, mixing with an ultrasonic corresponding substance, preparing a nano-emulsion in a self-assembly mode, separating and purifying, and preparing the phospholipid-like amphiphilic comb-shaped block graft 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.
Further, in the step (2), the method for preparing the phospholipid-like amphiphilic comb-shaped block graft 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 comb-shaped block graft copolymer-based nanoemulsion suspension by using a water bath at 60-80 ℃ (preferably 70 ℃) for 5-15 min (preferably 10min) to obtain a phospholipid-like amphiphilic block comb-shaped graft copolymer-based ultrasonic contrast agent;
the second method comprises the following steps: subjecting the prepared phospholipid-like amphiphilic comb-shaped block graft copolymer-based nanoemulsion suspension to ultrasonic action by an ultrasonic therapeutic apparatus to obtain the phospholipid-like amphiphilic comb-shaped block graft 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), 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) Distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-carboxyl cross-linked substance (DSPE-PEG5000-COOH), distearoyl phosphatidyl ethanolamine-polyAt least one of ethylene glycol 2000-carboxyl cross-linked substance (DSPE-PEG2000-COOH) or distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-hydroxyl cross-linked substance (DSPE-PEG 5000-OH).
Further, the phospholipid-like amphiphilic comb-shaped block graft copolymer prepared in the step (1) is prepared by adopting the following method:
(1) preparation of macromolecular backbone PBrCLp-b-PCLmFirstly, cyclohexanone is used as raw material to react with liquid bromine to obtain α -bromocyclohexanone, then α -bromocyclohexanone reacts with m-chloroperoxybenzoic acid to obtain α -bromo-caprolactone (α BrCL), and then α BrCL and-CL are used as monomers, stannous octoate is used as catalyst, ring-opening block polymerization is carried out to obtain macromolecular main chain PBrCLp-b-PCLmWherein p is the number of α BrCL contained in the main chain of the copolymer on average, and m is the polymerization degree of CL;
(2) preparing a phospholipid-like amphiphilic comb-shaped block graft copolymer PCL-b- (PBrCL-g-PMPC): the PBrCL obtained in (1)p-b-PCLmIs a macromolecular main chain, 2-Methacryloyloxyethyl Phosphorylcholine (MPC) is a monomer, and a phospholipid-like amphiphilic comb-shaped block graft copolymer PCL-b- (PBrCL-g-PMPC) is obtained by side chain polymerization by an atom transfer radical polymerization (ARGET ATRP) method of an electron transfer regenerated catalyst.
The third technical problem to be solved by the invention is to provide the application of the phospholipid-like amphiphilic comb-shaped block graft 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 comb-shaped block graft 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 Sonovue contrast agent), and is more suitable for ultrasonic diagnosis and therapeutic research.
(2) The phospholipid-like amphiphilic block comb-shaped graft 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 invention utilizes the phospholipid-like amphiphilic comb-shaped block graft copolymer to coat the phase-change type ultrasonic contrast agent prepared by liquid fluorocarbon (namely PCL-b- (PBrCL-g-PMPC) is taken as a shell, and the liquid fluorocarbon is taken as a core), and explores the feasibility of the phase-change type ultrasonic contrast agent; in vitro and in vivo experiments show that the PCL-b- (PBrCL-g-PMPC) based ultrasonic contrast agent has good echogenic characteristics under various ultrasonic parameter conditions. More importantly, the imaging time of the PCL-b- (PBrCL-g-PMPC) based ultrasonic contrast agent is longer than that of the phospholipid based ultrasonic contrast agent under the same ultrasonic parameters and concentrations, and the PCL-b- (PBrCL-g-PMPC) based ultrasonic contrast agent has great potential as a novel contrast agent in ultrasonic imaging.
(4) The phospholipid-like amphiphilic comb-shaped graft copolymer in the phospholipid-like amphiphilic comb-shaped block graft 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 comb-shaped block graft copolymer PCL-b- (PBrCL-g-PMPC) and a preparation schematic diagram of an ultrasonic contrast agent.
FIG. 2 shows the polymer PCL obtained in the example34、PCL34-b-PBrCL5And PCL34-b-(PBrCL5-g-PMPC5×5) Nuclear magnetic spectrum of (1).
FIG. 3 shows the polymer PCL obtained in example34、PCL34-b-PBrCL5And PCL34-b-(PBrCL5-g-PMPC5×5) An infrared spectrum of (1).
FIG. 4 shows the polymer PCL obtained in the example43And PCL34-b-(PBrCL5-g-PMPC5×5) The DSC temperature increase curve (a) and temperature decrease curve (b) of (1).
FIG. 5 shows the phospholipid-like amphiphilic block comb-shaped graft copolymer PCL obtained in the example34-b-(PBrCL5-g-PMPC5×5) CLSM picture (a), size distribution map (b) and size and zeta data (c) based on ultrasound contrast agent.
FIG. 6 shows the phospholipid-like amphiphilic comb-shaped block graft copolymer PCL obtained in the example34-b-(PBrCL5-g-PMPC5×5) The 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 comb-shaped block graft copolymer PCL obtained in the example34-b-(PBrCL5-g-PMPC5×5) Variation 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 comb-shaped block graft copolymer PCL obtained in the example34-b-(PBrCL5-g-PMPC5×5) The change of the contrast effect in the animal body of the base ultrasonic contrast agent along with the time is an ultrasonic contrast map (a) and a corresponding gray value (b); wherein the red circles represent the kidney visualization area of the rabbits.
Detailed Description
The first technical problem to be solved by the invention is to provide a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, which comprises a shell and an inner core, wherein the shell is a phospholipid-like amphiphilic comb-shaped block graft copolymer polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) (PCL-b- (PBrCL-g-PMPC)), and the inner core is an ultrasonic responder.
The second technical problem to be solved by the invention is to provide a preparation method of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, wherein the preparation method comprises the following steps: the ultrasonic contrast agent of polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) coated ultrasonic response substance is prepared by self-assembling polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) and the ultrasonic response substance.
The third technical problem to be solved by the invention is to provide the application of the phospholipid-like amphiphilic comb-shaped block graft 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. Preparation of phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent
1)PCL34-b-(PBrCL5-g-PMPC5×5) Preparation of the copolymer:
① α Synthesis and characterisation of bromocyclohexanone A flask (500mL) was charged with cyclohexanone (31.00g,0.3159mol) and deionised water (200.0mL) and stirred with a magnetic rotor, then liquid bromine (50.61g, 0.3167mol) was added dropwise over 5h, during which time the temperature was kept at 25-30 deg.C, after the addition was complete stirring was continued until the reaction mixture was colourless (about 1h), the lower organic layer was separated from the aqueous layer and separated with anhydrous MgSO4Dried and distilled to give pure α -bromocyclohexanone (26.7g, 47% yield).
② α Synthesis and characterization of bromo-caprolactone (α BrCL) 3-chloroperoxybenzoic acid (36.80g, 0.1599mol, 75%) was added to the CH of α -bromocyclohexanone (26.70g, 0.1508mol)2Cl2(200.0mL) in solution; stirring at room temperature for 48h, and then placing the reaction flask in a refrigerator for 3h to precipitate 3-chlorobenzoic acid generated in the reaction; the solution was then filtered and washed with Na2S2O3Saturated solution (50.00mL) was washed 3 times with NaHCO3The solution (50.00mL) was washed 3 times and finally with deionized water until pH 7.0; the organic phase was over anhydrous MgSO4Drying overnight; filter off MgSO4Thereafter, the solvent CH was removed by rotary evaporation2Cl2(ii) a Dissolving the crude product in petroleum ether and ethyl acetate: (V/V, 10/3) and passing through a silica gel column prepared with the same solvent, collecting a second fraction; the solvent was removed by rotary evaporation and the white solid was dried in vacuo at room temperature overnight. Yield: 20.20g (69%).
③PCL34Synthesis and characterization of (2): mixing lauryl alcohol (0.82g,0.004mol), -CL (10.13g,0.089mol) and Sn (Otc)2(4.05g,0.01mol) was added to the reaction vessel; removing O therefrom2And residual water; reacting for 24 hours at 120 ℃ under vacuum; the crude product was dissolved in CH2Cl2And purified by cold methanol precipitation to obtain PCL34。
④PCL34-b-PBrCL5Synthesis and characterization of (2) PCL obtained in ③34(5.00g) was charged to a Schlenk flask with α BrCL (0.9670g, 5.010mmol) from ② (0.9670g, 5.010mmol) and stirred, then stannous octoate catalyst (5.200mg, 0.1 wt%) was added to the previous mixture, and after deoxygenation the mixture was N2The reaction was carried out at 120 ℃ for 24 hours. The crude product was dissolved in CH2Cl2And using cold methanol to form a precipitate; obtaining the final product PCL34-b-PBrCL5。
⑤PCL34-b-(PBrCL5-g-PMPC5×5) Synthesis and characterization of (2): synthesis of comb-shaped block-graft copolymer PCL by ARGET (Electron transfer regeneration activator) ATRP method34-b-(PBrCL5-g-PMPC5×5) Wherein PMPC5×5Medium 5 × 5 indicates a degree of polymerization of PMPC side chains of 5 and an average number of PMPC side chains on the copolymer backbone of 5, Me6TREN(20μL)，CuBr2A mixed solution of (8.200mg) and THF/MeOH (V/V ═ 6/6mL) was added to a Schlenk flask and stirred; then, in N2Add MPC (1.117g), PCL34-b-PBrCL5(0.3615g) and vitamin C (6.500 mg); three freeze-vacuum-thaw cycles were performed with the mixture in N2Reacting for 24 hours at 35 ℃; dialyzing the crude solution for 72 h; then, the resulting suspension was freeze-dried and a comb-shaped block-graft copolymer PCL was obtained34-b-(PBrCL5-g-PMPC5×5)。
2) Preparing an ultrasonic contrast agent:
the preparation process comprises the following steps: 3mg of PCL was weighed separately34-b-(PBrCL5-g-PMPC5×5) The copolymer and 1mg of dppe-mPEG 5000 were dissolved in 567 μ L of Tetrahydrofuran (THF) and 283 μ L of methanol (MeOH) (2:1, v/v), and after sufficient dispersion in water bath with sonication, 2ml of pbs buffer (0.01M, pH 7.4) was added, followed by 150 μ L of perfluoropentane (PFP); ultrasonically emulsifying by using a probe under an ice-water bath environment, wherein ultrasonic parameters are as follows: frequency of 24KHz, power of 35W, ultrasonic on for 3s and then off for 6s, diameter of a sound vibration probe of 3mm, processing time of 3min to form milky suspension, centrifuging for 5min under centrifugal force of 3000g, discarding supernatant, and adding 6ml PBS for heavy suspension; and (3) carrying out water bath action on the obtained copolymer-based nano-emulsion suspension for 10min at 70 ℃ to prepare the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent.
2. Characterization of phospholipid-like amphiphilic comb-block graft copolymers
FIG. 2 shows a polymer PCL34、PCL34-b-PBrCL5And PCL34-b-(PBrCL5-g-PMPC5×5) The nuclear magnetic spectrum of (1), characteristic peak of segment of PCL (═ 4.1 ppm; 2.3 ppm; 1.7 ppm; 1.4ppm) appeared on the nuclear magnetic spectrum, indicating the successful synthesis of PCL, and the polymerization degree of PCL is calculated by the area ratio of the peaks; according to calculation, the invention successfully obtains the copolymer with the polymerization degree of 40; the appearance of a characteristic peak, indicating that the PMPC segment was successfully grafted onto the PCL 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-1There are successive methylene absorption peaks. It can be found that PCL34-b-(PBrCL5-g-PMPC5×5) The characteristic absorption peak (1090 cm) related to the structure of the phosphatidylcholine appears in the infrared spectrum-1And 1230cm-1，-POCH2-；970cm-1，N+(CH3)3) Also shown is PCL34-b-(PBrCL5-g-PMPC5×5) Successful synthesis of the copolymer.
The thermal properties of the synthesized polymer were measured by DSC, and the results are shown in fig. 4; as can be seen from FIG. 4, the PCL homopolymer has data such as the highest crystallization temperature, melting temperature and crystallinity; PCL in contrast to PCL34-b-(PBrCL5-g-PMPC5×5) With the introduction of an amorphous PMPC chain segment, when PCL is crystallized, the regular arrangement of the chain segment is hindered, thereby reducing various data.
3. Characterization of microvesicles
PCL after PBS and PFP addition34-b-(PBrCL5-g-PMPC5×5) The copolymer solution is changed into opaque milky white suspension after the sound vibration treatment, and is still milky white suspension after the phase change of PFP is triggered at temperature after the organic solvent is removed; the particle size of the copolymer-based ultrasound contrast agent changes after the phase transition, as shown in fig. 5. PCL34-b-(PBrCL5-g-PMPC5×5) The copolymer and the ultrasonic contrast agent have narrow particle size distribution range, and PCL is measured by a Malvern nanometer particle size analyzer34-b-(PBrCL5-g-PMPC5×5) The average particle size of the copolymer-based ultrasound contrast agent is about 5 μm.
3. Stability test
The concentrations of the copolymer-based ultrasonic contrast agent obtained by the invention and a reference phospholipid-based ultrasonic contrast agent (a commercial sononovue 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 observed under a sampling microscope after standing for 24 hours, 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 is obviously higher than that of the phospholipid-based ultrasonic contrast agent.
4. In vitro radiography and blasting experiment
Adding 0.3mL of the copolymer-based ultrasound contrast agent prepared in step 1 into an agarose gel model, observing in a Cadence Contrast Agent Imaging (CCAI) mode (MI ═ 0.21) using a Siemens color imaging system (SIEMENS Acuson Antares), wherein the contrast agent has an optimal development frequency of 5.71MHz (see FIG. 6), with a sharp point-like echo in the model, and a change in echo intensity from 4.0MHz to 10.0MHz is observed; 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 male New Zealand white rabbit with the body weight of 2.5-3 kg is fixed on an experiment table, an indwelling needle is placed in an ear edge vein, after the right waist and back are unhaired, a method of ear edge vein bolus injection is adopted to respectively inject SonoVue and PCL34-b-(PBrCL5-g-PMPC5×5) Co-polymerizing the contrast agent to obtain an ultrasound 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. SonoVue and PCL were injected intravenously, respectively, compared to PBS control group34-b-(PBrCL5-g-PMPC5×5) After the copolymer contrast agent, significant contrast enhancement begins to occur in the liver region. Clear ultrasound images of the liver indicate PCL34-b-(PBrCL5-g-PMPC5×5) The copolymer contrast agent successfully passes through the pulmonary capillaries during blood circulation, which is necessary for the safety properties of the intravenous contrast agent. As can be seen from FIG. 8, the ultrasound signal intensity of the SonoVue contrast agent is substantially similar to that of PCL at 20s34-b-(PBrCL5-g-PMPC5×5) Contrast image intensity of copolymer contrast agents is comparable; however, at 40s, the ultrasound signal of the SonoVue microbubbles decreased rapidly, and at 80s, the PCL disappeared substantially34-b-(PBrCL5-g-PMPC5×5) The copolymer contrast agent still had a contrast signal that could be observed after 80 s. This indicates that the PCL34-b-(PBrCL5-g-PMPC5×5) Compared with a SouoVue contrast agent, the block graft copolymer ultrasound contrast agent has longer in vivo duration, and proves that the block graft copolymer ultrasound contrast agent has great potential as an ultrasound contrast agent with novel diagnostic and therapeutic effects.
Example 2
PCL preparation by shearing method in a different manner from example 134-b-(PBrCL5-g-PMPC5×5) The contrast agent is prepared by the following specific steps:
3mg of PCL was weighed separately34-b-(PBrCL5-g-PMPC5×5) Copolymer and 1mg DPPE-mPEG 5000 were dissolved in 567 μ L Tetrahydrofuran (THF) and 283 μ L methanol (MeOH) (2:1, v/v), and after sufficient dispersion in water bath with sonication, 2ml pbs buffer (0.01M, pH 7.4) was added, followed by 150 μ L perfluoropentane (PFP); under the condition of ice-water bath, adding liquid perfluoropentane, adopting an electric internal cutting homogenizing method, wherein the homogenizing rotating speed is 12000-30000 rpm, the homogenizing time is 1-3min, and obtaining milky graft polymer mixed liquid wrapping the perfluoropentane after homogenizing.
Subjecting the mixture to phase change with therapeutic ultrasound to form PCL34-b-(PBrCL5-g-PMPC5×5) Phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, wherein the parameter of an ultrasonic instrument is 3W/cm2The duty ratio is 50%, and the action time is 3 min.
Example 3
Preparation of PCL by lyophilization34-b-(PBrCL5-g-PMPC5×5) The contrast agent is prepared by the following specific steps: 3mgPCL was weighed separately34-b-(PBrCL5-g-PMPC5×5) The copolymer and 1mg of DPPE-mPEG 5000 were dissolved in 567 μ L of Tetrahydrofuran (THF) and 283 μ L of methanol (MeOH) (2:1, v/v), and after sufficient dispersion in a water bath with sonication, 2ml of pbs buffer (0.01M, pH 7.4) was added, followed by 150 μ L of perfluoropentane (PFP). Performing ultrasonic sound vibration under the condition of an ice salt water bath, wherein the probe frequency is 24KHz, the power is 35W, the ultrasonic is turned on for 3s and turned off for 6s, the ultrasonic action is performed for 3min to form milky suspension, then the milky suspension is centrifuged for 5min under the action of 3000g centrifugal force, the supernatant is discarded, and 6mL of sucrose solution containing 10 percent of weight 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 gas such as perfluoropropane, perfluorobutane and sulfur hexafluoride into the freeze-dried powder through a ventilation device, recombining the freeze-dried powder with 2mL of physiological saline after ventilation to obtain milky copolymer suspension, and mechanically oscillating the recombined microbubble suspension for 1minThe frequency is 75Hz, and PCL is prepared34-b-(PBrCL5-g-PMPC5×5) Phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent.
According to the above three examples, the PCL prepared by different preparation methods is adopted in the invention34-b-(PBrCL5-g-PMPC5×5) The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent is spherical in shape, good in dispersion, smooth and transparent in surface, uniform in size (see 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 drive the contrast agent may reflect the echo signal more strongly, whereas when MI is too large it may cause rupture of the contrast agent and loss of contrast effect; PCL in contrast to phospholipid-based contrast agents34-b-(PBrCL5-g-PMPC5×5) The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent can tolerate the action of higher ultrasonic mechanical index and keep an integral morphological structure.
PCL after ultrasonic irradiation34-b-(PBrCL5-g-PMPC5×5) The change of the copolymer-based ultrasonic contrast signal intensity along with the time is realized by respectively acquiring PCL at different time points34-b-(PBrCL5-g-PMPC5×5) Ultrasound images of the copolymer-based ultrasound contrast agent and the SonoVue contrast agent, and the change in the mean gray value of the copolymer-based ultrasound contrast agent and the SonoVue contrast image were analyzed, and the results are shown in fig. 7. As can be seen from FIG. 7, with the time being prolonged, the brightness of the ultrasonic image generated by the SonoVue microbubble within 1-3min is rapidly darkened, the intensity of the ultrasonic signal is rapidly reduced, and the contrast effect is almost lost after 4 min. And PCL34-b-(PBrCL5-g-PMPC5×5) The brightness of the ultrasonic image of the copolymer-based ultrasonic contrast agent shows good stability, the brightness of the image slowly becomes dark between 1 and 3min, the intensity of an ultrasonic signal slowly decreases to be stable after 6min, and the ultrasonic image 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; but PCL34-b-(PBrCL5-g-PMPC5×5) The ultrasonic signal intensity of the 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. PCL34-b-(PBrCL5-g-PMPC5×5) The greater stability of the copolymer-based ultrasound contrast agent is attributed to the PCL34-b-(PBrCL5-g-PMPC5×5) The copolymer can form a stable core-shell structure in aqueous solution, which provides a strong barrier for keeping PFP gas from diffusing and dissolving in the inner core under ultrasonic radiation, while the lipid shell layer of the SonoVue ultrasonic contrast agent has a thickness of only about 4nm, and the gas in the micro-bubbles can quickly diffuse out through the shell layer under ultrasonic radiation, so that the contrast capability of the SonoVue ultrasonic contrast agent is rapidly reduced. In addition, PCL34-b-(PBrCL5-g-PMPC5×5) Gaseous PFP in copolymer-based ultrasound contrast agents compared to SF in SonoVue ultrasound contrast agents6Gas, with lower water solubility, which also contributes to PCL enhancement34-b-(PBrCL5-g-PMPC5×5) Stability of copolymer-based ultrasound contrast agents. Such that the PCL34-b-(PBrCL5-g-PMPC5×5) The copolymer-based ultrasound contrast agents have longer cycle times, providing a longer imaging time window for clinical diagnosis.
From the above, the phospholipid-like amphiphilic comb-shaped block graft 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 molecular weight/mL and the mechanical index MI are 0.2, obtaining an ultrasonic contrast image in a frequency range of 4-10 MHz, analyzing the change of the average gray value of the contrast image of the copolymer ultrasonic contrast agent along with the frequency, and due to the difference between the resonance frequency of the ultrasonic contrast agent and the ultrasonic driving frequency, after the frequency is increased from 4MHz to 5.71MHz, partially PCL-b- (PBrCL-g-PMPC) copolymer-based ultrasonic manufacturingThe intensity of the ultrasound contrast image of the contrast agent increases. 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 comb-shaped block graft 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 comb-shaped block graft copolymer-based ultrasonic contrast agent under a high mechanical index is prolonged compared with that of a phospholipid-based contrast agent.
Claims (10)
1. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent is characterized by comprising a shell and an inner core, wherein the shell is phospholipid-like amphiphilic comb-shaped block graft copolymer polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine), and the inner core is an ultrasonic responder.
2. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent as claimed in claim 1, wherein the phospholipid-like amphiphilic comb-shaped block graft copolymer is a copolymer obtained by block and graft copolymerization of caprolactone and 2-methacryloyloxyethyl phosphorylcholine;
further, the shell in the ultrasound contrast agent further comprises a modifying substance M, 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;
still further, the modifying substance M is selected from: dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-azidopolyethylene glycol 5000, distearoylphosphatidylethanolamine-azidopolyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol-thiol cross-links, distearoylphosphatidylethanolamine-polyethylene glycol 2000-thiol cross-links, distearoylphosphatidylethanolamine-polyethylene glycol 5000-amino cross-links, distearoylphosphatidylethanolamine-polyethylene glycol 2000-amino cross-links, distearoylphosphatidylethanolamine-polyethylene glycol 5000-carboxyl cross-links, distearoylphosphatidylethanolamine-polyethylene glycol 2000-carboxyl cross-links, or distearoylphosphatidylethanolamine-polyethylene glycol 5000-hydroxypolyethylene glycol At least one of the group-crosslinked substances.
3. The phospholipid-like amphiphilic comb-block graft copolymer-based ultrasound contrast agent according to claim 1 or 2, wherein the core of the ultrasound contrast agent is gaseous perfluorocarbon or liquid perfluorocarbon which can be phase-changed;
further, the inner core of the ultrasound contrast agent comprises at least one of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, or perfluoroheptane.
4. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent as claimed in claim 3, wherein when the core of the ultrasonic contrast agent is liquid perfluorocarbon capable of changing phase, the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent is prepared from polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) and an ultrasonic responder in a self-assembly manner to obtain a nanoemulsion, and the obtained nanoemulsion forms the ultrasonic contrast agent with gaseous perfluorocarbon wrapped by the phospholipid-like amphiphilic comb-shaped block graft copolymer through phase change;
further, when the core of the ultrasonic contrast agent is gaseous perfluorocarbon, the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent is prepared from polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) and an ultrasonic response substance in a self-assembly manner;
further, the self-assembly mode is one of the following modes: high shear homogenization, high pressure homogenization, high speed oscillation or ultrasonic sound vibration.
5. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent as defined in any one of claims 1 to 4, wherein the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent is diluted with physiological saline, and observed under a laser confocal microscope, and has a spherical shape, good dispersion and a smooth and bright surface.
6. The preparation method of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent as defined in any one of claims 1 to 5, wherein the preparation method comprises the following steps: the ultrasonic contrast agent of polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) coated ultrasonic response substance is prepared by self-assembling polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) and the ultrasonic response substance.
7. The method for preparing the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent according to claim 6, wherein the self-assembly is carried out in one of the following ways: high-speed shearing homogenate, high-pressure homogenate, high-speed oscillation or ultrasonic sound vibration;
further, the ultrasonic responder is liquid perfluorocarbon or gaseous perfluorocarbon which can change phase;
further, when the ultrasound response substance in the ultrasound contrast agent is gaseous perfluorocarbon, the preparation method of the phospholipid-like amphiphilic comb-graft copolymer-based ultrasound contrast agent comprises the following steps: preparing a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent from polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) and an ultrasonic responder in a direct ultrasonic vibration mode or a high-speed oscillation mode;
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 comb-graft copolymer-based ultrasound contrast agent comprises the following steps: polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine) and an ultrasonic responder are prepared into the nanoemulsion in a self-assembly mode, and the obtained nanoemulsion is subjected to temperature-induced phase change or sound-induced phase change to form the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent.
8. The preparation method of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent as claimed in claim 7, wherein when the ultrasonic responder in the ultrasonic contrast agent is liquid perfluorocarbon capable of phase transition, the preparation method comprises the following steps:
(1) preparing a phospholipid-like amphiphilic comb-shaped block graft copolymer polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethyl phosphorylcholine);
(2) preparing a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent: dissolving the phospholipid-like amphiphilic comb-shaped block graft copolymer and the modified substance M obtained in the step (1) by a mixed solvent, mixing with an ultrasonic corresponding substance, preparing a nano-emulsion in a self-assembly mode, separating and purifying, and preparing the phospholipid-like amphiphilic comb-shaped block graft 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; preferably, in the step (2), the mixed solvent is a mixture of solvents with a volume ratio of 2:1 of a mixed solvent of tetrahydrofuran and methanol;
further, in the step (2), the method for preparing the phospholipid-like amphiphilic comb-shaped block graft 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: the prepared phospholipid-like amphiphilic comb-shaped block graft copolymer based nanoemulsion suspension is subjected to water bath at the temperature of 60-80 ℃ for 5-15 min, and the phospholipid-like amphiphilic block comb-shaped graft copolymer based ultrasonic contrast agent is prepared;
the second method comprises the following steps: subjecting the prepared phospholipid-like amphiphilic comb-shaped block graft copolymer-based nanoemulsion suspension to ultrasonic action by an ultrasonic therapeutic apparatusObtaining the phospholipid-like amphiphilic comb-shaped block graft 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.
9. The preparation method of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent according to claim 8, wherein the amphiphilic comb-shaped block graft copolymer polycaprolactone-b- (polybromocaprolactone-g-polymethacryloxyethylphosphonium choline) is prepared by the following method:
(1) preparation of macromolecular backbone PBrCLp-b-PCLm：
Firstly, reacting cyclohexanone serving as a raw material with liquid bromine to obtain α -bromocyclohexanone, reacting α -bromocyclohexanone with m-chloroperoxybenzoic acid to obtain α -bromo-caprolactone, then reacting α -bromo-caprolactone with-caprolactone serving as a monomer, and using stannous octoate as a catalyst to carry out ring-opening block polymerization to obtain a macromolecular main chain PBrCLp-b-PCLmWherein p is the average number of α -bromo-caprolactone in the main chain of the copolymer, and m is the polymerization degree of caprolactone;
(2) preparing a phospholipid-like amphiphilic comb-shaped block graft copolymer: with the PBrCL obtained in the step (1)p-b-PCLmIs a macromolecular main chain, 2-methacryloyloxyethyl phosphorylcholine is a monomer, and a phospholipid-like amphiphilic comb-shaped block graft copolymer is obtained by side chain polymerization by an atom transfer radical polymerization method of an electron transfer regenerated catalyst.
10. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent can be used in the fields of ultrasonic image diagnosis and treatment or in-vitro agarose model contrast imaging; or the medicine is carried and then used for in vivo ultrasonic contrast imaging and treatment; wherein the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent is the ultrasonic contrast agent as defined in any one of claims 1 to 5 or the ultrasonic contrast agent prepared by the preparation method as defined in any one of claims 6 to 9.
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