CN111671922A - Amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of ultrasonic imaging diagnosis, and in particular 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, the ultrasonic contrast agent comprises a shell and an inner core, and the shell is a phospholipid-like amphiphilic comb-shaped block graft copolymer polymer. Caprolactone-b-(polybromocaprolactone-g-polymethacryloyloxyethyl phosphorylcholine), the inner core is an ultrasonic responsive substance. The ultrasonic contrast agent prepared by the method has a more uniform particle size distribution, and the stability is obviously better than that of the phospholipid-based ultrasonic contrast agent, and is more suitable for ultrasonic diagnosis and treatment research; and can solve the pressure resistance and mechanical index of the current ultrasonic contrast agent. The lack of changes has expanded the application scope of ultrasound contrast agents.

Description

Amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent and preparation method thereof

technical field

The invention belongs to the field of ultrasonic imaging diagnosis, and in particular relates to a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent and a preparation method thereof.

Background technique

Ultrasound contrast agents can enhance the contrast effect of ultra-images and significantly improve the accuracy of ultrasound diagnosis. They are widely used in the field of clinical diagnosis and have great application potential in ultrasound-mediated therapy. However, the ultrasound contrast agents currently used in clinics are mostly composed of small molecular phospholipids or albumin-coated inert gases, which have shortcomings such as high polydispersity or short half-life (<10min), which limit their further application in imaging and therapy. application.

In order 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 called hard-shell contrast agents. The hard-shell contrast medium exhibits little volume expansion and remains intact under low acoustic pressure conditions. But beyond a certain pressure threshold, the shell of the hard-shell ultrasound contrast medium also ruptures. The polymer stiffness is higher than that of phospholipids, and the polymer-based ultrasound contrast agent prepared based on the polymer shell can greatly improve the acoustic behavior of the ultrasound contrast agent. Moreover, 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-shelled ultrasound contrast agent not only has better acoustic stability, but also its pressure resistance and resistance to mechanical index changes under ultrasound are also greatly improved. In addition, by grafting or encapsulating therapeutic drugs for drug delivery, they can also be used in ultrasound image-guided diagnosis and treatment integrated preparations, increasing the versatility of multimodal ultrasound contrast agents and further expanding the use of ultrasound contrast agents. Scope of application.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel polymer-based ultrasound contrast agent and a preparation method thereof, the obtained contrast agent is a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent, so as to solve the problem of the current ultrasound contrast agent. The pressure resistance and mechanical index change are insufficient to expand the application range of ultrasound contrast agents.

Technical scheme of the present invention:

The first technical problem to be solved by the present invention is to provide a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, the ultrasonic contrast agent includes an outer shell and an inner core, and the outer shell is a phospholipid-like amphiphilic Comb block graft copolymer polycaprolactone-b-(polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) (PCL-b-(PBrCL-g-PMPC)) , the inner core is an ultrasonic responder.

Further, the phospholipid-like amphiphilic comb-shaped block graft copolymer is obtained by block and graft copolymerization of ε-caprolactone (CL) and 2-methacryloyloxyethyl phosphorylcholine (MPC). the copolymer.

Further, the phospholipid-like amphiphilic comb block graft copolymer is obtained by the following method:

(1) Preparation of macromolecular main chain PBrCL _p -b-PCL _m : First, cyclohexanone is used as raw material to react with liquid bromine to obtain α-bromocyclohexanone, and then α-bromocyclohexanone is mixed with m-chloroperoxy α-Bromo-ε-caprolactone (αBrCL) was obtained by the reaction of benzoic acid, and then αBrCL and ε-CL were used as monomers, and stannous octoate was used as a catalyst for ring-opening block polymerization to obtain the macromolecular main chain PBrCL _p -b -PCL _m , where p is the average number of αBrCL contained in the main chain of the copolymer, and m is the degree of polymerization of CL;

(2) Preparation of phospholipid amphiphilic comb block graft copolymer PCL-b-(PBrCL-g-PMPC): using PBrCL _p -b-PCL _m obtained in step (1) as the macromolecular backbone, 2-Methacryloyloxyethylphosphorylcholine (MPC) was used as monomer, and phospholipid-like amphiphilic comb-shaped block grafts were obtained by side chain polymerization of atom transfer radical polymerization (ARGET ATRP) method of electron transfer regeneration catalyst. Copolymer PCL-b-(PBrCL-g-PMPC).

Further, the shell in the ultrasonic contrast agent further includes a modified substance M, and the modified substance M is: providing the shell with a substance containing PEG segments that can avoid being cleared by the immune system in the body, so as to increase the circulation of the ultrasonic contrast agent time.

Further, the modified substance M is selected from: dipalmitoyl phosphatidyl ethanolamine-methoxy polyethylene glycol 5000 (DPPE-mPEG 5000), dipalmitoyl phosphatidyl ethanolamine-methoxy polyethylene glycol 2000 (DPPE-mPEG 5000) -mPEG2000), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000), distearoyl phosphatidyl ethanolamine-azide polyethylene glycol 5000 (DSPE-PEG5000-N ₃ ), distearyl Phosphatidyl phosphatidyl ethanolamine-azide polyethylene glycol 2000 (DSPE-PEG2000-N ₃ ), distearoyl phosphatidyl ethanolamine-polyethylene glycol-thiol cross-linker (DSPE-PEG5000-SH), distearoyl Phosphatidylethanolamine-polyethylene glycol 2000-thiol cross-linker (DSPE-PEG2000-SH), distearoylphosphatidylethanolamine-polyethylene glycol 5000-amino cross-linker (DSPE-PEG5000-NH ₂ ), two Stearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino cross-linker (DSPE-PEG2000-NH ₂ ), distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-carboxyl cross-linker (DSPE-PEG5000-COOH) ), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-carboxyl cross-linker (DSPE-PEG2000-COOH) or distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-hydroxyl cross-linker (DSPE-PEG5000 -OH) at least one.

Preferably, the inner core of the ultrasound contrast agent is a gaseous perfluorocarbon or a phase-changeable liquid perfluorocarbon.

Further, the inner core of the ultrasound contrast agent includes at least one of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane or perfluoroheptane.

Further, when the inner core of the ultrasonic contrast agent is a phase-changeable liquid perfluorocarbon, the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent is composed of polycaprolactone-b-( Nanoemulsion prepared by self-assembly of polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) and ultrasonic responder, and the obtained nanoemulsion undergoes phase transition to form a phospholipid-like amphiphilic comb Ultrasound contrast agent encapsulating gaseous perfluorocarbons by morphological block-grafted copolymers.

Further, when the inner core of the ultrasonic contrast agent is a gaseous perfluorocarbon, the phospholipid amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent is composed of polycaprolactone-b-(polybromohexanone). Lactone-g-polymethacryloyloxyethylphosphoryl choline) and ultrasound responsive substance to prepare ultrasound contrast agent through self-assembly.

Further, the self-assembly method is one of the following methods: high-speed shear homogenization, high-pressure homogenization, high-speed oscillation or ultrasonic acoustic vibration.

Further, the average particle size distribution range of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent is 5±0.13 μm.

Further, after 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, the shape is spherical, well dispersed, and the surface is smooth and translucent.

The second technical problem to be solved by the present invention is to provide a preparation method of the above-mentioned phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, the preparation method is: polycaprolactone-b-(poly Preparation of polycaprolactone-b-(polybromocaprolactone-g-polymethacrylate by self-assembly of bromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) and ultrasonic responders Acyloxyethylphosphorylcholine) is an ultrasound contrast agent that encapsulates an ultrasound responder.

Further, the self-assembly adopts one of the following methods: high-speed shear homogenization, high-pressure homogenization, high-speed oscillation or ultrasonic acoustic vibration.

Further, the ultrasonic responder is a phase-changeable liquid perfluorocarbon or gaseous perfluorocarbon; preferably, it is a liquid perfluorocarbon. Choosing a liquid phase-changeable perfluorocarbon has better stability and longer storage time than gas, and you can choose to perform the phase change step before use; while the direct use of gaseous preparation has a low success rate and is not conducive to store.

Further, when the ultrasonic responsive substance in the ultrasonic contrast agent is gaseous perfluorocarbon, the preparation method of the phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent is: polycaprolactone-b -(Polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) and ultrasonic responsive materials prepared by direct ultrasonic sonication or high-speed oscillation to obtain phospholipid-like amphiphilic comb block grafts Copolymer-based ultrasound contrast agent.

Further, when the ultrasonic responsive substance in the ultrasonic contrast agent is a phase-changeable liquid perfluorocarbon, the preparation method of the phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent is: Nanoemulsion prepared by self-assembly of lactone-b-(polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) and ultrasonic responder, and the obtained nanoemulsion is then subjected to temperature-induced phase transition Or sono-induced phase transformation to form a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent. When the shell layer uses a phospholipid-like amphiphilic comb-shaped block graft copolymer, a high-temperature hydrophobic phase-changeable liquid perfluorocarbon is encapsulated inside, and is prepared by self-assembly using the similar compatibility principle and hydrophilic-hydrophobic interaction. Nanoemulsion, also known as nano-liquid particles, is produced, and then through phase transition, a phospholipid-like amphiphilic copolymer capable of ultrasound contrast is formed to encapsulate a gaseous perfluorocarbon ultrasound contrast agent.

Further, when the ultrasonic responder in the ultrasonic contrast agent is a phase-changeable liquid perfluorocarbon, the above preparation method includes the following steps:

(1) preparation of phospholipid-like amphiphilic comb block graft copolymer polycaprolactone-b-(polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine);

(2) Preparation of phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent: the phospholipid-like amphiphilic comb-shaped block graft copolymer obtained in (1) and the modified substance M were mixed with solvent After dissolving, the nanoemulsion is prepared by self-assembly after mixing with ultrasonic corresponding substances, and then separated and purified. A copolymer-based ultrasound contrast agent; wherein, the modified substance M is: a substance containing a PEG segment for the shell that can avoid being cleared by the immune system in the body; the mixed solvent is a mixed solvent of tetrahydrofuran and methanol, or: three A mixed solvent of methyl chloride and methanol.

Further, in step (2), the method for preparing the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent after the separation and purification of the nanoemulsion is subjected to temperature-induced phase transition or acoustic phase transition as follows: One of the ways:

Method 1: The prepared phospholipid-like amphiphilic comb-shaped block graft copolymer-based nanoemulsion suspension is prepared in a water bath at 60-80 °C (preferably 70 °C) for 5-15 min (preferably 10 min) to prepare phospholipid-like Amphiphilic block comb graft copolymer-based ultrasound contrast agent;

Method 2: the prepared phospholipid-like amphiphilic comb-shaped block graft copolymer-based nanoemulsion suspension is subjected to ultrasonic action with an ultrasonic therapeutic apparatus to obtain a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, Wherein, the ultrasonic power is 1-3W/cm ² (preferably 3W/cm ² ), the duty ratio is 20%-80% (preferably 50%), and the action time is 2-5min (preferably 3min).

Preferably, in step (2), the mixed solvent is a mixed solvent of tetrahydrofuran and methanol with a volume ratio of 2:1.

Further, in step (2) of the above method, the modified substance M is selected from: dipalmitoyl phosphatidyl ethanolamine-methoxy polyethylene glycol 5000 (DPPE-mPEG 5000), dipalmitoyl phosphatidyl ethanolamine-methyl Oxypolyethylene glycol 2000 (DPPE-mPEG2000), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000), distearoyl phosphatidyl ethanolamine-azide polyethylene glycol 5000 (DSPE- PEG5000-N ₃ ), distearoyl phosphatidyl ethanolamine-azide polyethylene glycol 2000 (DSPE-PEG2000-N ₃ ), distearoyl phosphatidyl ethanolamine-polyethylene glycol-mercapto cross-linker (DSPE- PEG5000-SH), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-mercapto cross-linker (DSPE-PEG2000-SH), distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-amino cross-linker ( DSPE-PEG5000-NH ₂ ), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino cross-linker (DSPE-PEG2000-NH ₂ ), distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-carboxyl Crosslinker (DSPE-PEG5000-COOH), distearoylphosphatidylethanolamine-polyethylene glycol 2000-carboxyl crosslinker (DSPE-PEG2000-COOH) or distearoylphosphatidylethanolamine-polyethylene glycol 5000 - At least one of hydroxyl crosslinkers (DSPE-PEG5000-OH).

Further, in step (1), the phospholipid-like amphiphilic comb-shaped block graft copolymer is prepared by the following method:

(1) Preparation of macromolecular main chain PBrCL _p -b-PCL _m : First, cyclohexanone is used as raw material to react with liquid bromine to obtain α-bromocyclohexanone, and then α-bromocyclohexanone is mixed with m-chloroperoxy α-Bromo-ε-caprolactone (αBrCL) was obtained by the reaction of benzoic acid, and then αBrCL and ε-CL were used as monomers, and stannous octoate was used as a catalyst for ring-opening block polymerization to obtain the macromolecular main chain PBrCL _p -b -PCL _m , where p is the average number of αBrCL contained in the main chain of the copolymer, and m is the degree of polymerization of CL;

(2) Preparation of phospholipid amphiphilic comb block graft copolymer PCL-b-(PBrCL-g-PMPC): Taking PBrCL _p -b-PCL _m obtained in (1) as the macromolecular backbone, 2 - Methacryloyloxyethylphosphorylcholine (MPC) as the monomer, by the atom transfer radical polymerization (ARGET ATRP) method of electron transfer regeneration catalyst side chain polymerization to obtain phospholipid-like amphiphilic comb block graft copolymerization The compound PCL-b-(PBrCL-g-PMPC).

The third technical problem to be solved by the present invention is to provide the application of the above-mentioned phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, which can be applied in the fields of ultrasonic imaging diagnosis and treatment, and used in the in vitro agarose model Contrast imaging; or it can be used for in vivo contrast-enhanced ultrasound imaging and treatment after drug loading.

Compared with the prior art, the present invention has the following beneficial technical effects:

(1) The particle size distribution of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent prepared by the present invention is relatively uniform, and the stability is obviously better than that of the phospholipid-based ultrasonic contrast agent (commercial SonoVue contrast agent), It is more suitable for ultrasonic diagnosis and treatment research.

(2) The phospholipid-like amphiphilic block comb-shaped graft copolymer-based ultrasound contrast agent prepared by the present invention has a significant image enhancement effect in the high mechanical index mode of ultrasound contrast, and the duration is compared with that of the contrast agent with the phospholipid shell The long duration improves the short contrast time of the phospholipid-based ultrasonic contrast agent in high mechanical index, and overcomes the shortcomings of the ultrasonic contrast agent prepared by some polymer materials, which are hard and difficult to develop.

(3) The present invention utilizes phospholipid-like amphiphilic comb-shaped block graft copolymer to coat liquid fluorocarbon phase-change ultrasonic contrast agent (ie PCL-b-(PBrCL-g-PMPC) as shell, liquid fluorine In vitro and in vivo experiments show that PCL-b-(PBrCL-g-PMPC)-based ultrasound contrast agents have good echogenicity under various ultrasonic parameters. More importantly, under the same ultrasound parameters and concentrations, PCL-b-(PBrCL-g-PMPC)-based ultrasound contrast agent showed longer imaging time than phospholipid-based ultrasound contrast agent. -g-PMPC)-based ultrasound contrast agents have great potential as novel contrast agents in ultrasound imaging.

(4) The phospholipid-like amphiphilic comb-shaped graft copolymer in the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent obtained by the present invention has the advantages of controllable structure, safety and non-toxicity, and the cost is obviously lower than that of synthetic Phospholipids, and the preparation process is simple. In addition, the preparation method can be widely used in the field of clinical contrast-enhanced ultrasound and can also be used as a carrier of various therapeutic drugs, and has a good clinical application prospect.

Description of drawings:

Fig. 1 is the synthetic route diagram of the phospholipid-like amphiphilic comb block graft copolymer PCL-b-(PBrCL-g-PMPC) and the schematic diagram of the preparation of the ultrasonic contrast agent of the present invention.

Fig. 2 is the nuclear magnetic spectrum of the polymers PCL ₃₄ , PCL ₃₄ -b-PBrCL ₅ and PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) obtained in Example.

3 is the infrared spectrum of the polymers PCL ₃₄ , PCL ₃₄ -b-PBrCL ₅ and PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) obtained in the Example.

FIG. 4 shows the DSC heating curve (a) and cooling curve (b) of the polymer polymers PCL ₄₃ and PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) obtained in the Example.

₅ is a _{CLSM photograph} (a) and a size distribution diagram ( b) and size and zeta data (c).

FIG. 6 is the contrast images of different ultrasound parameters in vitro of the phospholipid amphiphilic comb-shaped block graft copolymer PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 )-based ultrasound contrast agent obtained in the Example and the corresponding contrast images Gray value - frequency (a, d), gray value - mechanical index MI (b, e) and gray value - concentration (c, f).

Fig. 7 is a contrast-enhanced ultrasound image of the in vitro contrast effect of the phospholipid amphiphilic comb-shaped block graft copolymer PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 )-based ultrasound contrast agent obtained in the Example as a function of time (a) and the corresponding grayscale values (b).

Fig. 8a shows the changes of the contrast effect of the phospholipid amphiphilic comb-shaped block graft copolymer PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 )-based ultrasound contrast agent in animals with time as a function of time Figure (a) and the corresponding gray value (b); where the red circle represents the developed area of the rabbit kidney.

Detailed ways

The first technical problem to be solved by the present invention is to provide a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, the ultrasonic contrast agent includes an outer shell and an inner core, and the outer shell is a phospholipid-like amphiphilic Comb block graft copolymer polycaprolactone-b-(polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) (PCL-b-(PBrCL-g-PMPC)) , 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 above-mentioned phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, the preparation method is: polycaprolactone-b-(poly Preparation of polycaprolactone-b-(polybromocaprolactone-g-polymethacrylate by self-assembly of bromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) and ultrasonic responders Acyloxyethylphosphorylcholine) is an ultrasound contrast agent that encapsulates an ultrasound responder.

The third technical problem to be solved by the present invention is to provide the application of the above-mentioned phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, which can be applied in the fields of ultrasonic imaging diagnosis and treatment, and used in the in vitro agarose model Contrast imaging; or it can be used for in vivo contrast-enhanced ultrasound imaging and treatment after drug loading.

The specific embodiments of the present invention will be further described below with reference to the examples, but the present invention is not limited to the scope of the described examples.

Example 1

1. Preparation of phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent

1) Preparation of PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer:

①Synthesis and characterization of α-bromocyclohexanone: Add cyclohexanone (31.00g, 0.3159mol) and deionized water (200.0mL) to a flask (500mL), and stir with a magnetic rotor; then, dropwise over 5h Liquid bromine (50.61 g, 0.3167 mol) was added, during this period, the temperature was controlled to maintain at 25-30°C; after the dropwise addition was completed, stirring was continued until the reaction mixture was colorless (about 1 h). The lower organic layer was separated from the aqueous layer and dried over anhydrous _MgSO4 ; pure α-bromocyclohexanone (26.7 g, 47% yield) was obtained by distillation.

②Synthesis and characterization of α-bromo-ε-caprolactone (αBrCL): 3-chloroperoxybenzoic acid (36.80g, 0.1599mol, 75%) was added to α-bromocyclohexanone (26.70g, 0.1508mol) in CH ₂ Cl ₂ (200.0 mL) solution; after stirring at room temperature for 48 h, the reaction flask was placed in the refrigerator for 3 h to precipitate the 3-chlorobenzoic acid generated in the reaction; then the solution was filtered and washed with Na ₂ S ₂ O ₃ The saturated solution (50.00 mL) was washed 3 times, with NaHCO ₃ solution (50.00 mL) 3 times, and finally with deionized water until pH=7.0; the organic phase was dried with anhydrous MgSO ₄ overnight _; The solvent _CH2Cl2 was removed by rotary evaporation _; the crude product was dissolved in a mixture of petroleum ether and ethyl acetate (V/V, 10/3) and passed through a silica gel column prepared with the same solvent, and the second fraction was collected; by rotary evaporation The solvent was removed by evaporation and the white solid was dried in vacuo at room temperature overnight. Yield: 20.20 g (69%).

③Synthesis and characterization of PCL ₃₄ : Lauryl alcohol (0.82g, 0.004mol), ε-CL (10.13g, 0.089mol) and Sn(Otc) ₂ (4.05g, 0.01mol) were added to the reaction vessel; O ₂ and residual water; react under vacuum at 120 °C for 24 hours; the crude product was dissolved in CH ₂ Cl ₂ and purified by cold methanol precipitation to give PCL ₃₄ .

④Synthesis and characterization of PCL ₃₄ -b-PBrCL ₅ : PCL ₃₄ (5.00 g) obtained in ③ and αBrCL (0.9670 g, 5.010 mmol) obtained in ② were added to a Schlenk flask and stirred, then, stannous octoate catalyst was added (5.200 mg, 0.1 wt%) was added to the previous mixture; after deoxygenation operation, the mixture was reacted under _N2 at 120 °C for 24 h. The crude product was dissolved in _CH2Cl2 and precipitated using cold methanol _; the final product _PCL34 -b- _PBrCl5 was obtained.

⑤Synthesis and characterization of PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ): Synthesis of comb-shaped block-graft copolymer PCL ₃₄ -b-(PBrCL by ARGET (electron transfer regeneration activator) ATRP method ₅ -g-PMPC _5×5 ), wherein, _{5×5 in PMPC 5} ×5 indicates that the degree of polymerization of PMPC side chains is 5, and the average number of PMPC side chains on the main chain of the copolymer is 5; Me ₆ TREN (20 μL ), a mixed solution of CuBr ₂ (8.200 mg) and THF/MeOH (V/V=6/6 mL) was added to the Schlenk flask and stirred; then, MPC (1.117 g), PCL ₃₄ -b- was added under N _{2 PBrCL5} (0.3615 g) and vitamin C (6.500 mg); three freeze-vacuum-thaw cycles were performed, and the mixture was reacted under N at 35 °C for ₂₄ h; the crude solution was dialyzed for 72 h; then, the resulting suspension was freeze-dried and obtained Comb block-graft copolymer PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ).

2) Preparation of ultrasound contrast agent:

The specific preparation process is as follows: 3 mg of PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer and 1 mg of DPPE-mPEG 5000 were weighed and dissolved in 567 μL tetrahydrofuran (THF) and 283 μL methanol (MeOH) (2:1) , v/v), after fully dispersing and dissolving by water bath ultrasound, 2 mL PBS buffer (0.01M, pH=7.4) was added, and then 150 μL perfluoropentane (PFP) was added; in an ice-water bath environment, phacoemulsification was performed with a probe, wherein Ultrasonic parameters: frequency 24KHz, power 35W, ultrasonic on for 3s and then off for 6s, acoustic vibration probe diameter of 3mm, treatment time for 3min to form a milky white suspension, then centrifuged at 3000g for 5min, discard the supernatant, add 6mL PBS for reconstitution. Suspended; the obtained copolymer-based nanoemulsion suspension was treated in a 70° C. water bath for 10 min to prepare a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent.

2. Characterization of phospholipid-like amphiphilic comb block graft copolymers

Fig. 2 is the NMR spectra of the polymers PCL ₃₄ , PCL ₃₄ -b-PBrCL ₅ and PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ), the characteristic peaks of the segment of PCL (δ=4.1ppm; δ =2.3ppm; δ=1.7ppm; δ=1.4ppm) all appear on the nuclear magnetic spectrum, indicating the successful synthesis of PCL, and the polymerization degree of PCL is calculated from the area ratio of the peaks; it can be seen from the calculation that the present invention successfully obtained the polymerization The copolymers with a degree of 40 respectively; the appearance of characteristic peaks indicates that the PMPC segment was successfully grafted to the PCL segment; meanwhile, the degree of polymerization of the PMPC segment was determined by the area ratio of the peaks.

The structure of the polymer was further characterized by infrared, and the results are shown in Figure 3; the sharp peak at 1728 cm ^-1 is the stretching vibration peak of C=O, and the continuous methylene absorption peak at 2910 cm ^-1 . It can be found that PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) has characteristic absorption peaks (1090cm ^-1 and 1230cm ^-1 , -POCH ₂ -) related to the structure of phospholipid choline in the infrared spectrum; 970 cm ^-1 , N ⁺ (CH ₃ ) ₃ ), also indicating the successful synthesis of PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer.

The thermal properties of the synthesized polymers were tested by DSC, and the results are shown in Figure 4; it can be seen from Figure 4 that the PCL homopolymer has the highest crystallization temperature, melting temperature and crystallinity data; compared with PCL, PCL ₃₄ -b- (PBrCL ₅ -g-PMPC _5×5 ) copolymer With the introduction of amorphous PMPC segments, when PCL crystallizes, the regular arrangement of the segments is hindered, resulting in the decrease of various data.

3. Characterization of microbubbles

The PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer solution after adding PBS and PFP became an opaque milky white suspension after sonication treatment. After removing the organic solvent, PFP was triggered by temperature. After the phase transition, it is still a milky white suspension solution; after the phase transition, the particle size of the copolymer-based ultrasound contrast agent changes, as shown in Figure 5. The particle size distribution range of PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer and ultrasonic contrast agent is narrow, and PCL ₃₄ -b-(PBrCL ₅ -g The average particle size of the -PMPC _5×5 ) copolymer-based ultrasound contrast agent is about 5 μm.

3. Stability test

The Coulter-measured concentrations of the copolymer-based ultrasound contrast agent obtained in the present invention and the reference-like phospholipid-based ultrasound contrast agent (commercial Sonovue contrast agent) are diluted to make the copolymer-based ultrasound contrast agent and the phospholipid-based ultrasound contrast agent. With the same concentration, it was placed in a refrigerator at 4°C for 24 hours and then sampled and observed under a microscope. The results showed that the concentration of phospholipid-based ultrasound contrast agent was lower than that of copolymer-based ultrasound contrast agent, indicating that the stability of copolymer-based ultrasound contrast agent was significantly higher than that of phospholipid-based ultrasound contrast agent. contrast agent.

4. In vitro imaging and blasting experiments

0.3 mL of the copolymer-based ultrasound contrast agent prepared in step 1 was added to the agarose gel model, and the Siemens color imaging system (SIEMENS Acuson Antares) was used in the Cadence contrast agent imaging (CCAI) mode (MI=0.21) Observation, it can be seen that the echo in the model is obviously enhanced, and the echo is dense and fine, and the echo intensity changes from the ultrasonic frequency 4.0MHz to 10.0MHz. In this ultrasonic imaging system, the optimal development frequency of the contrast agent is 5.71MHz (see Figure 6); start the blasting (MI=0.67) mode to blast the contrast agent, the echo in the model decreases instantaneously, and the number of fine point echoes drops sharply.

5. New Zealand white rabbit liver imaging

Male New Zealand white rabbits, weighing 2.5-3 kg, were fixed on the experimental bench, and an indwelling needle was placed in the ear vein. After depilation of the right back and back, SonoVue and PCL ₃₄ -b- were injected by ear vein bolus injection. (PBrCL ₅ -g-PMPC _5×5 ) copolymer contrast agent to obtain a contrast-enhanced ultrasound image of the liver. In the control group experiment, only PBS buffered saline was injected, and the ultrasound image was a dark area. The results are shown in Fig. 8 . Compared with the PBS control group, after intravenous injection of SonoVue and PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer contrast agents, respectively, significant contrast enhancement began to appear in the liver region. Bright ultrasound images of the liver indicate that the PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer contrast agent successfully passes through the pulmonary capillaries during blood circulation, which is required for the safety of intravenous contrast agents. It can be seen from Figure 8 that the ultrasound signal intensity of SonoVue contrast agent is basically the same as that of PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer contrast agent at 20s; but at 40s, The ultrasound signal of SonoVue microbubbles decreased rapidly and disappeared at 80s, while PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer contrast agent still had an observable contrast signal after 80s. This indicates that PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) block graft copolymer ultrasound contrast agent has a longer in vivo duration than SouoVue contrast agent, proving its potential as a novel diagnostic therapy Great potential for the role of ultrasound contrast agents.

Example 2

A different method from Example 1 was used to prepare PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) contrast agent by shearing method, and the specific preparation process was as follows:

Weigh 3 mg PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer and 1 mg DPPE-mPEG 5000 and dissolve them in 567 μL tetrahydrofuran (THF) and 283 μL methanol (MeOH) (2:1, v/v, respectively). ), after fully dispersing and dissolving with water bath ultrasound, add 2 mL of PBS buffer (0.01M, pH=7.4), and then add 150 μL of perfluoropentane (PFP); under ice-water bath conditions, add liquid perfluoropentane, using an electrodynamic In the endo-homogenization method, the homogenizing speed is 12,000-30,000 rpm, and the homogenizing time is 1-3 min. After homogenizing, a milky white graft polymer mixed solution coated with perfluoropentane is obtained.

The above mixed solution was phase-transformed with a therapeutic ultrasound apparatus to form PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) phospholipid amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent, ultrasonically The parameters of the instrument are 3W/cm ² , the duty cycle is 50%, and the action time is 3min.

Example 3

PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _{5 ×5 )} contrast agent was prepared by freeze-drying method _. 1 mg of DPPE-mPEG 5000 was dissolved in 567 μL of tetrahydrofuran (THF) and 283 μL of methanol (MeOH) (2:1, v/v), and after the water bath was sonicated to fully disperse and dissolve, 2 mL of PBS buffer (0.01M, pH=7.4) was added, and then An additional 150 μL of perfluoropentane (PFP) was added. Under the condition of ice-salt bath, ultrasonic vibration was performed, the probe frequency was 24KHz, the power was 35W, the ultrasonic wave was turned on for 3s, turned off for 6s, and acted for 3 minutes to form a milky white suspension, and then centrifuged for 5 minutes under the action of 3000g centrifugal force, discard the supernatant. Add 6 mL of 10% wt sucrose solution for resuspension; use 10 mL vials of vials, divide them into 2 mL volumes, put them in a -20°C refrigerator to pre-freeze overnight, and freeze-dry them in a freeze dryer for 24 hours. Set to -80°C.

The prepared freeze-dried powder can be filled with perfluoropropane, perfluorobutane, sulfur hexafluoride and other gases through a ventilation device, and reconstituted with 2 mL of physiological saline after ventilation to obtain a milky white copolymer suspension. The microbubble suspension was mechanically shaken, the shaking action time was 1 min, and the shaking frequency was 75 Hz to prepare PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) phospholipid-like amphiphilic comb block graft copolymer based ultrasound contrast agent.

According to the above three examples, the PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) phospholipid amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent prepared by different preparation methods of the present invention , the shape is spherical, the dispersion is good, the surface is smooth and translucent, the size is relatively uniform (see Figure 5), and the average particle size distribution range is 5 ± 0.13 μm.

The mechanical index value (MI) has a great influence on the contrast-enhanced ultrasound image, which essentially determines the intensity of the ultrasound. Because of the ultrasound drive with higher energy, the contrast agent can reflect the echo signal more strongly, and when the MI is too large, It may cause the _rupture of _contrast agent and lose the contrast effect _; Ultrasound contrast agents can withstand the action of higher ultrasound mechanical index and maintain intact morphological structure.

The change of the signal intensity of PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer-based CEUS with time after ultrasonic irradiation was obtained by obtaining PCL ₃₄ -b-(PBrCL ₅ - g-PMPC _5×5 ) ultrasonic images of copolymer-based ultrasound contrast agent and SonoVue contrast agent, and the changes of the average gray value of copolymer-based ultrasound contrast agent and SonoVue contrast agent were analyzed. The results are shown in Figure 7. It can be seen from Figure 7 that with the prolongation of time, the brightness of the ultrasound image produced by SonoVue microbubbles between 1 and 3 minutes rapidly becomes darker, the intensity of the ultrasound signal drops sharply, and the contrast effect is almost lost after 4 minutes. On the other hand, the ultrasound images of PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer-based ultrasound contrast agent showed better stability, and the brightness of the images changed slowly between 1 and 3 minutes. dark, the ultrasonic signal intensity decreased slowly, became stable after 6 minutes, and still had a certain contrast effect in vitro for 20 minutes.

However, the ultrasound signal produced by the SonoVue ultrasound contrast agent dropped sharply to about 20% of its initial intensity in about 3 min, and was only 2.5% of its initial intensity at 20 min; but PCL ₃₄ -b-(PBrCL ₅ -g The ultrasonic signal intensity of the -PMPC _5×5 ) copolymer-based ultrasonic contrast agent can be maintained for about 20 minutes. Compared with its initial ultrasonic signal intensity, the highest is reduced to 23% of its initial intensity, and the lowest is only reduced to 50% of its initial intensity . The stronger stability of the PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer-based ultrasound contrast agent is attributed to the fact that the PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer can be A stable core-shell structure is formed in aqueous solution, which provides a strong barrier to keep PFP gas from diffusing and dissolving in the inner core under ultrasound irradiation, while the lipid shell layer of SonoVue ultrasound contrast agent is only about 4 nm thick, which can Under ultrasound irradiation, the gas in the microbubble will quickly diffuse out through this shell, so that the contrasting ability of SonoVue ultrasound contrast agent decreases rapidly. In addition, the gaseous PFP in the PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _{5×5 )} copolymer-based ultrasound contrast agent has lower water solubility than the SF gas in the SonoVue ultrasound contrast agent, which also has Helps to enhance the stability of PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer-based ultrasound contrast agent. In this way, the PCL ₃₄ -b-(PBrCL ₅ -g-PMPC _5×5 ) copolymer-based ultrasound contrast agent has a longer circulation period and provides a longer imaging time window for clinical diagnosis.

It can be seen from the above that the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent obtained by the present invention is applied to ultrasound contrast imaging in an in vitro model, and the concentration of microbubbles is set to be 5.0×10 ⁸ /mL and mechanical After the index MI=0.2, the ultrasound contrast image was obtained in the frequency range of 4-10MHz, and the average gray value of the contrast image of the copolymer ultrasound contrast agent was analyzed. There is a difference between the two, resulting in an increase in the brightness of the CEUS images of some PCL-b-(PBrCL-g-PMPC) copolymer-based ultrasound contrast agents as the frequency increases from 4MHz to 5.71MHz. When the frequency increased by 6.67MHz, the brightness of all contrast-enhanced ultrasound images decreased rapidly; when the frequency reached 7.27MHz, the brightness of the images basically remained unchanged. When the ultrasonic frequency is selected at 5.71MHz, the gray value of the contrast image of the ultrasonic contrast agent is the largest. The copolymer-based ultrasound contrast agent has a longer duration than the SonoVue contrast agent. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent is applied to in vivo ultrasound contrast imaging of New Zealand white rabbits, indicating that the ultrasound contrast agent has a certain in vivo contrast effect, and the phospholipid-like amphiphilic comb-shaped Compared with the phospholipid-based contrast agent, the block-grafted copolymer-based ultrasound contrast agent can prolong the contrast time at high mechanical index.

Claims (10)

1. a kind of phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, it is characterized in that, described ultrasonic contrast agent comprises shell and inner core, and described shell is phospholipid-like amphiphilic comb-shaped block grafting The copolymer polycaprolactone-b-(polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine), the inner core is an ultrasound responder. 2. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent according to claim 1, wherein the phospholipid-like amphiphilic comb-shaped block graft copolymer is ε-hexane A copolymer obtained by block and graft copolymerization of lactone and 2-methacryloyloxyethylphosphorylcholine; Further, the shell in the ultrasonic contrast agent further includes a modified substance M, and the modified substance M is: providing the shell with a substance containing PEG segments that can avoid being cleared by the immune system in vivo; Further, the modified substance M is selected from: dipalmitoyl phosphatidyl ethanolamine-methoxy polyethylene glycol 5000, dipalmitoyl phosphatidyl ethanolamine-methoxy polyethylene glycol 2000, distearoyl phosphatidyl Ethanolamine-polyethylene glycol 2000, distearoyl phosphatidyl ethanolamine-azide polyethylene glycol 5000, distearoyl phosphatidyl ethanolamine-azide polyethylene glycol 2000, distearoyl phosphatidyl ethanolamine-polyethylene glycol Diol-mercapto cross-linker, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-mercapto cross-linker, distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-amino cross-linker, distearoyl Phosphatidyl ethanolamine-polyethylene glycol 2000-amino cross-linker, distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-carboxyl cross-linker, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-carboxyl cross-linker At least one of a linker or a distearoylphosphatidylethanolamine-polyethylene glycol 5000-hydroxyl crosslinker. 3. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent according to claim 1 or 2, wherein the inner core of the ultrasound contrast agent is a gaseous perfluorocarbon or a phase-changeable of liquid perfluorocarbons; Further, the inner core of the ultrasound contrast agent includes at least one of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane or perfluoroheptane. 4. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent according to claim 3, wherein when the inner core of the ultrasound contrast agent is a phase-changeable liquid perfluorocarbon , the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent is composed of polycaprolactone-b-(polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) A nanoemulsion is prepared by self-assembly with an ultrasonic responder, and the obtained nanoemulsion undergoes a phase transition to form an ultrasonic contrast agent encapsulating gaseous perfluorocarbon by a phospholipid-like amphiphilic comb-shaped block graft copolymer; Further, when the inner core of the ultrasonic contrast agent is a gaseous perfluorocarbon, the phospholipid amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent is composed of polycaprolactone-b-(polybromohexanone). Lactone-g-polymethacryloyloxyethylphosphoryl choline) and ultrasound responsive substance to prepare ultrasound contrast agent by self-assembly; Further, the self-assembly method is one of the following methods: high-speed shear homogenization, high-pressure homogenization, high-speed oscillation or ultrasonic acoustic vibration. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent according to any one of claims 1 to 4, wherein the phospholipid-like amphiphilic comb-shaped block graft copolymerization The substance-based ultrasound contrast agent was diluted with physiological saline and observed under a laser confocal microscope. The shape was spherical, well dispersed, and the surface was smooth and translucent. 6. The preparation method of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent according to any one of claims 1 to 5, wherein the preparation method is: polycaprolactone- Self-assembly of b-(polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) and ultrasonic responders to prepare polycaprolactone-b-(polybromocaprolactone-g- Polymethacryloyloxyethylphosphorylcholine) is an ultrasound contrast agent that encapsulates an ultrasound responder. 7. The preparation method of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent according to claim 6, wherein the self-assembly adopts one of the following modes: high-speed shearing Homogenization, high-pressure homogenization, high-speed vibration or ultrasonic vibration; Further, the ultrasonic responder is a phase-changeable liquid perfluorocarbon or gaseous perfluorocarbon; Further, when the ultrasonic responsive substance in the ultrasonic contrast agent is gaseous perfluorocarbon, the preparation method of the phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent is: polycaprolactone-b -(Polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) and ultrasonic responsive materials prepared by direct ultrasonic sonication or high-speed oscillation to obtain phospholipid-like amphiphilic comb block grafts Copolymer-based ultrasound contrast agent; Further, when the ultrasonic responsive substance in the ultrasonic contrast agent is a phase-changeable liquid perfluorocarbon, the preparation method of the phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent is: Nanoemulsion prepared by self-assembly of lactone-b-(polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine) and ultrasonic responder, and the obtained nanoemulsion is then subjected to temperature-induced phase transition Or sono-induced phase transformation to form a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent. 8. The preparation method of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent according to claim 7, characterized in that, when the ultrasound responder in the ultrasound contrast agent is phase-changeable When liquid perfluorocarbon, the preparation method comprises the following steps: (1) preparation of phospholipid-like amphiphilic comb block graft copolymer polycaprolactone-b-(polybromocaprolactone-g-polymethacryloyloxyethylphosphorylcholine); (2) Preparation of phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent: the phospholipid-like amphiphilic comb-shaped block graft copolymer obtained in step (1) and the modified substance M are mixed After the solvent is dissolved, the nanoemulsion is prepared by self-assembly after mixing with the corresponding substances of ultrasonic, and then separated and purified. A branch copolymer-based ultrasound contrast agent; wherein, the modified substance M is: a substance containing a PEG segment for the shell that can be avoided to be cleared by the immune system in the body; the mixed solvent is a mixed solvent of tetrahydrofuran and methanol, or: A mixed solvent of chloroform and methanol; preferably, in step (2), the mixed solvent is a mixed solvent of tetrahydrofuran and methanol with a volume ratio of 2:1; Further, in step (2), the method for preparing the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent after the separation and purification of the nanoemulsion is subjected to temperature-induced phase transition or acoustic phase transition as follows: One of the ways: Method 1: The prepared phospholipid-like amphiphilic comb-shaped block graft copolymer-based nanoemulsion suspension is used in a water bath at 60 to 80°C for 5 to 15 minutes to prepare a phospholipid-like amphiphilic block comb-shaped graft copolymer. based ultrasound contrast agent; Method 2: the prepared phospholipid-like amphiphilic comb-shaped block graft copolymer-based nanoemulsion suspension is subjected to ultrasonic action with an ultrasonic therapeutic apparatus to obtain a phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasonic contrast agent, Wherein, the ultrasonic power is 1-3W/cm ² , the duty ratio is 20%-80%, and the action time is 2-5min. 9. The preparation method of the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent according to claim 8, wherein the amphiphilic comb-shaped block graft copolymer polycaprolactone Ester-b-(polybromocaprolactone-g-polymethacryloyloxyethyl phosphorylcholine) is obtained by the following method: (1) Preparation of macromolecular backbone PBrCL _p -b-PCL _m : First, cyclohexanone is used as raw material to react with liquid bromine to obtain α-bromocyclohexanone, and then α-bromocyclohexanone is reacted with m-chloroperoxybenzoic acid to obtain α-bromo-ε-caprolactone, and then Using α-bromo-ε-caprolactone and ε-caprolactone as monomers and stannous octoate as a catalyst, the macromolecular main chain PBrCL _p -b-PCL _m was obtained by ring-opening block polymerization, where p is The average number of α-bromo-ε-caprolactone contained in the main chain of the copolymer, m is the polymerization degree of caprolactone; (2) Preparation of phospholipid-like amphiphilic comb-shaped block graft copolymer: using PBrCL _p -b-PCL _m obtained in step (1) as the macromolecular backbone, 2-methacryloyloxyethyl phosphochol The base is used as the monomer, and the phospholipid-like amphiphilic comb-shaped block graft copolymer is obtained by the side chain polymerization of the atom transfer radical polymerization method of the electron transfer regeneration catalyst. 10. The phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent can be used in the field of ultrasound imaging diagnosis and treatment, or used for in vitro agarose model contrast imaging; or loaded with drugs for in vivo ultrasound contrast imaging and treatment; wherein, the phospholipid-like amphiphilic comb-shaped block graft copolymer-based ultrasound contrast agent is the ultrasound contrast agent according to any one of claims 1 to 5, or any one of claims 6 to 9. The ultrasonic contrast agent prepared by the preparation method.

Images