CN111760037B - Phospholipid-like amphiphilic comb graft copolymer-based ultrasonic contrast agent and preparation method thereof - Google Patents
Phospholipid-like amphiphilic comb graft copolymer-based ultrasonic contrast agent and preparation method thereof Download PDFInfo
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- CN111760037B CN111760037B CN202010727913.9A CN202010727913A CN111760037B CN 111760037 B CN111760037 B CN 111760037B CN 202010727913 A CN202010727913 A CN 202010727913A CN 111760037 B CN111760037 B CN 111760037B
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- Prior art keywords
- contrast agent
- phospholipid
- graft copolymer
- ultrasonic
- amphiphilic comb
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Abstract
The invention belongs to the field of ultrasonic image diagnosis, and particularly relates to a phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent and a preparation method thereof. The invention provides a phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent, which comprises a shell and an inner core, wherein the shell is amphiphilic comb-shaped graft copolymer polycaprolactone-g-polymethacryloxyethyl phosphorylcholine, and the inner core is an ultrasonic responder. The phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent prepared by the method has uniform particle size distribution, has stability obviously superior to that of the phospholipid-based ultrasonic contrast agent, and is more suitable for ultrasonic diagnosis and 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-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 greatly improves the pressure resistance and the 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 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 graft copolymer-based ultrasonic contrast agent, which comprises an outer shell and an inner core, wherein the outer shell is amphiphilic comb-shaped graft copolymer polycaprolactone-g-polymethacryloxyethyl phosphorylcholine (PCL-g-PMPC), and the inner core is an ultrasonic responder.
Further, the amphiphilic comb-shaped graft copolymer is a copolymer obtained by graft copolymerization of epsilon-Caprolactone (CL) and 2-Methacryloyloxyethyl Phosphorylcholine (MPC).
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) Distearoylphosphatidylethanolamine-polyethylene glycol 2000-amino cross-linked polymer (DSPE-P)EG2000-NH2) 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 a liquid perfluorocarbon or a gaseous perfluorocarbon that is phase-changeable.
Further, the inner core of the ultrasound contrast agent comprises at least one of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, or perfluoroheptane.
Further, when the core of the ultrasonic contrast agent is liquid perfluorocarbon capable of changing phase, the phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent is prepared into a nano-emulsion by polycaprolactone-g-polymethacryloxyethyl phosphorylcholine and an ultrasonic response substance in a self-assembly mode, and the obtained nano-emulsion is subjected to phase change to form the phospholipid-like amphiphilic comb-shaped graft copolymer-coated gaseous perfluorocarbon ultrasonic contrast agent.
Further, when the inner core of the ultrasonic contrast agent is gaseous perfluorocarbon, the phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent is prepared from polycaprolactone-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 acoustic oscillation.
Further, after the phospholipid-like amphiphilic comb-shaped 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 graft copolymer-based ultrasonic contrast agent, wherein the preparation method comprises the following steps: the ultrasonic contrast agent of the polycaprolactone-g-polymethacryloxyethyl phosphorylcholine coated ultrasonic responder is prepared by self-assembling the polycaprolactone-g-polymethacryloxyethyl phosphorylcholine and the ultrasonic responder.
Further, the self-assembly is performed in one of the following ways: high shear homogenization, high pressure homogenization, high speed oscillation or ultrasonic sound vibration.
Further, the ultrasonic responder is liquid perfluorocarbon or gaseous perfluorocarbon which can change phase; preferably liquid perfluorinated carbon. The liquid phase-changeable perfluorocarbon is selected, has better stability than gas, has longer storage time, and can be selected to perform the step of phase change 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-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 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-shaped graft copolymer-based ultrasound contrast agent comprises the following steps: polycaprolactone-g-polymethacryloxyethyl choline phosphate and an ultrasonic responder are prepared into nano-emulsion in a self-assembly mode, and the nano-emulsion is subjected to temperature-induced phase change or sound-induced phase change to form the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent. When the shell layer uses phospholipid-like amphiphilic comb-shaped 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 the interaction of hydrophilic and hydrophobic phases, and then the ultrasonic contrast agent of the phospholipid-like amphiphilic copolymer wrapped with gaseous perfluorocarbon which can be subjected to ultrasonic contrast is formed through the 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 graft copolymer polycaprolactone-g-polymethacryloxyethyl phosphorylcholine (PCL-g-PMPC);
(2) preparing a phospholipid-like amphiphilic comb graft copolymer-based ultrasonic contrast agent: dissolving the phospholipid-like amphiphilic comb-shaped graft copolymer PCL-g-PMPC obtained in the step (1) and the modified substance M in a mixed solvent, mixing with corresponding ultrasonic substances, preparing into a nano-emulsion in a self-assembly mode, separating and purifying, and performing temperature-induced phase change or sound-induced phase change to prepare the phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent; wherein the modifying substance M is: providing the shell with a material comprising a PEG segment that avoids clearance by the immune system in vivo; the mixed solvent is a mixed solvent of tetrahydrofuran and methanol, or: a mixed solvent of chloroform and methanol.
Further, in the step (2), the method for preparing the phospholipid-like amphiphilic comb-shaped 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 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 comb-shaped graft copolymer-based ultrasonic contrast agent;
the second method comprises the following steps: carrying out ultrasonic action on the prepared phospholipid-like amphiphilic comb-shaped graft copolymer-based nanoemulsion suspension by using an ultrasonic therapeutic apparatus to obtain the phospholipid-like amphiphilic comb-shaped 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 glycolAlcohol 2000(DSPE-MPEG2000), distearoyl phosphatidyl ethanolamine-azido polyethylene glycol 5000(DSPE-PEG 5000-N)3) Distearoyl phosphatidyl ethanolamine-azido polyethylene glycol 2000(DSPE-PEG 2000-N)3) Distearoyl phosphatidyl ethanolamine-polyethylene glycol-sulfhydryl cross-linked substance (DSPE-PEG5000-SH), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-sulfhydryl cross-linked substance (DSPE-PEG2000-SH), distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-amino cross-linked substance (DSPE-PEG 5000-NH)2) Distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino cross-linked complex (DSPE-PEG 2000-NH)2) At least one of distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-carboxyl cross-linked complex (DSPE-PEG5000-COOH), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-carboxyl cross-linked complex (DSPE-PEG2000-COOH) or distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-hydroxyl cross-linked complex (DSPE-PEG 5000-OH).
Further, the method for preparing the phospholipid-like amphiphilic comb-shaped graft copolymer PCL-g-PMPC in the step (1) comprises the following steps: epsilon-Caprolactone (CL) and 2-Methacryloyloxyethyl Phosphorylcholine (MPC) are polymerized by an atom transfer radical polymerization (ARGET ATRP) method of an electron transfer regeneration catalyst to obtain the phospholipid-like amphiphilic comb-shaped graft copolymer PCL-g-PMPC.
Furthermore, the method for preparing the phospholipid-like amphiphilic comb graft copolymer PCL-g-PMPC in the step (1) comprises the following steps:
1) macromolecular backbone P (BrCL)p-co-CL)mThe preparation of (1):
firstly, cyclohexanone is taken as a raw material to react with liquid bromine to obtain alpha-bromocyclohexanone, then the alpha-bromocyclohexanone reacts with m-chloroperoxybenzoic acid to obtain alpha-bromo-epsilon-caprolactone (alpha BrCL), then the alpha BrCL and the epsilon-CL are taken as monomers, stannous octoate is taken as a catalyst, and ring-opening random polymerization is carried out to obtain macromolecular main chain P (BrCL)p-co-CL)mWherein, p is the number of alpha BrCL contained in the main chain of the copolymer on average, and m is the polymerization degree of the copolymer;
2) preparation of phospholipid-like amphiphilic comb graft copolymer PCL-g-PMPC:
using P (BrCL) obtained in the step 1)p-co-CL)mIs a macromolecular main chain2-Methacryloyloxyethyl Phosphorylcholine (MPC) is taken as a monomer, and an amphiphilic comb-shaped graft copolymer PCL-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 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 graft copolymer-based ultrasonic contrast agent prepared by the method has uniform particle size distribution (the average particle size distribution is about 5 mu m), has stability obviously superior to that of a phospholipid-based ultrasonic contrast agent (a commercial Sonowev SonoVue contrast agent), and is more suitable for ultrasonic diagnosis and treatment research.
(2) The phospholipid-like amphiphilic comb-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 graft copolymer PCL-g-PMPC to coat the phase-change type ultrasonic contrast agent prepared by liquid fluorocarbon (namely PCL-g-PMPC is a shell and the liquid fluorocarbon is a core), and explores the feasibility of serving as the ultrasonic contrast agent; in vitro and in vivo experiments show that the PCL-g-PMPC-based ultrasonic contrast agent has good echogenic characteristics under various ultrasonic parameter conditions. More importantly, the imaging time of the PCL-g-PMPC-based ultrasound contrast agent is longer than that of the phospholipid-based ultrasound contrast agent under the same ultrasound parameters and concentrations, and the PCL-g-PMPC-based ultrasound contrast agent has great potential as a novel contrast agent in ultrasound imaging.
(4) The amphiphilic comb-shaped graft copolymer in the phospholipid-like amphiphilic comb-shaped 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 route diagram of the phospholipid-like amphiphilic comb-shaped graft copolymer PCL-g-PMPC and a schematic diagram of the preparation of an ultrasonic contrast agent.
FIG. 2 shows the polymer P (BrCL) obtained in the example5-co-CL)40And PCL40-g-PMPC10×5Nuclear magnetic spectrum of (1).
FIG. 3 shows the polymer P (BrCL) obtained in example5-co-CL)40And PCL40-g-PMPC10×5An infrared spectrum of (1).
FIG. 4 shows the polymer PCL obtained in example80、P(BrCL5-co-CL)79、P(BrCL5-co-CL)40、PCL79-g-PMPC10×5、PCL79-g-PMPC5×5、PCL40-g-PMPC10×5And PCL40-g-PMPC5×5A DSC temperature increase curve (a) and a temperature decrease curve (b) of (a); wherein for a series of PCLm-g-PMPCn×pIn other words, m represents the Degree of Polymerization (DP) of P (BrCL-co-CL), n represents the DP of the PMPC side chains, and P represents the average number of PMPC side chains in the copolymer backbone.
FIG. 5 shows the phospholipid-like amphiphilic comb-graft copolymer PCL obtained in the example40-g-PMPC5×5、PCL40-g-PMPC10×5、PCL79-g-PMPC5×5And PCL79-g-PMPC10×5CLSM picture (a), size distribution map (b) and size and zeta data (c) based on ultrasound contrast agent.
FIG. 6 shows PCL obtained in example40-g-PMPC5×5、PCL40-g-PMPC10×5、PCL79-g-PMPC5×5And PCL79-g-PMPC10×5Contrast map of different ultrasound parameters in vitro of ultrasound-based contrast agent and corresponding grey value-frequency (a, d)Grey value-mechanical index MI (b, e) and grey value-density (c, f).
FIG. 7 shows the phospholipid-like amphiphilic comb-graft copolymer PCL obtained in the example40-g-PMPC5×5、PCL40-g-PMPC10×5、PCL79-g-PMPC5×5And PCL79-g-PMPC10×5Variation of the in vitro contrast effect of the base ultrasound contrast agent over time the ultrasound contrast map (a) and the corresponding grey value (b) are determined.
FIG. 8a shows the phospholipid-like amphiphilic comb-graft copolymer PCL obtained in the example40-g-PMPC5×5、PCL40-g-PMPC10×5、PCL79-g-PMPC5×5And PCL79-g-PMPC10×5The change of the contrast effect in the animal body of the ultrasonic contrast agent along with the time is an ultrasonic contrast image, and the corresponding gray value is shown in figure 8 b; wherein the red circles represent the kidney visualization area of the rabbits.
Detailed Description
The following further describes specific embodiments of the present invention in conjunction with examples, which are not intended to limit the invention thereto.
Example 1
1. Preparation of amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent
1) Preparation of PCL-g-PMPC copolymer:
firstly, synthesizing and characterizing alpha-bromocyclohexanone: cyclohexanone (31.00g,0.3159mol) and deionized water (200.0mL) were added to the flask (500mL) and stirred with a magnetic rotor; then, dropwise adding liquid bromine (50.61g, 0.3167 mol) in 5h, and controlling the temperature to be 25-30 ℃ during the period; after the completion of the dropwise addition, stirring was continued until the reaction mixture was colorless (about 1 h); separating the lower organic layer from the aqueous layer and using anhydrous MgSO4And (5) drying. Pure α -bromocyclohexanone (26.7g, 47% yield) was obtained by distillation;
② the synthesis and characterization of alpha-bromo-epsilon-caprolactone (alpha BrCL): 3-Chloroperoxybenzoic acid (36.80g, 0.1599mol, 75%) was added to the CH of alpha-bromocyclohexanone (26.70g, 0.1508mol)2Cl2(200.0mL) in solution; after stirring at room temperature for 48h,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. 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, collecting a second fraction; the solvent was removed by rotary evaporation and the white solid was dried under vacuum at room temperature overnight; yield: 20.20g (69%);
③P(BrCLp-co-CL)msynthesis and characterization of (2): synthesis of P (BrCL) by Ring opening polymerization of alpha BrCL and epsilon CLp-co-CL)m. Wherein p represents the average number of α BrCL in the copolymer backbone, and m represents the Degree of Polymerization (DP); there are two DP's for this copolymer, namely P (BrCL)5-co-CL)40And P (BrCL)5-co-CL)79(ii) a To synthesize P (BrCL)5-co-CL)40For example, α BrCL (0.9670g, 5.010mmol), ε CL (4.000g, 35.04mmol) and lauryl alcohol (0.1860g, 1.000mmol) were added to a Schlenk flask and stirred; then, stannous octoate catalyst (5.200mg, 0.1 wt%) was added to the previous mixture; after the deoxygenation operation, the mixture was in N2The reaction is carried out at 120 ℃ for 24 h. The crude product was dissolved in CH2Cl2And using cold methanol to form a precipitate; the final product P (BrCL)5-co-CL)405.118g, 93%) were dried in a vacuum oven at 35 ℃ for 24 h;
④PCLm-g-PMPCn×psynthesis and characterization of (2): synthesis of comb-shaped graft copolymer PCL by ARGET (Electron transfer regeneration activator) ATRP methodm-g-PMPCn×pM represents the Degree of Polymerization (DP) of P (BrCL-co-CL), n represents the DP of the PMPC side chains, and P represents the average number of PMPC side chains on the copolymer backbone; four comb copolymers in the examples, PCL40-g-PMPC5×5，PCL40-g-PMPC10×5，PCL79-g-PMPC5×5And PCL79-g-PMPC10×5(ii) a With PCL40-g-PMPC10×5For example, Me6TREN(20μL)，CuBr2(8.200mg) and a mixed solution of THF/MeOH (V/V ═ 6/6mL) were added to a Schlenk flask and stirred; then, in N2MPC (1.117g), P (BrCL) were added5-co-CL)40(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 ℃; the crude solution was dialyzed for 72 h. The resulting suspension was then freeze-dried and a pure comb copolymer PCL was obtained40-g-PMPC10×5(99.3％)。
2) Preparing an ultrasonic contrast agent:
3mg of PCL-g-PMPC copolymer and 1mg of DPPE-mPEG 5000 were weighed and dissolved in 567. mu.L of Tetrahydrofuran (THF) and 283. mu.L of methanol (MeOH) (2:1, v/v), and after being sufficiently dispersed and dissolved by ultrasonic treatment in a water bath, 2mL of PBS buffer (0.01M, pH 7.4) was added, and then 150. mu.L of perfluoropentane (PFP) was added; ultrasonically emulsifying by using a probe under an ice-water bath environment, wherein ultrasonic parameters are as follows: the frequency is 24KHz, the power is 35W, the ultrasonic is switched on for 3s and then switched off for 6s, the diameter of a sound vibration probe is 3mm, the processing time is 3min, a milky suspension is formed, then the suspension is centrifuged for 5min under the centrifugal force of 3000g, the supernatant is discarded, and 6mL of PBS is added for heavy suspension; and (3) carrying out water bath at 70 ℃ for 10min on the obtained copolymer-based nano-emulsion suspension to prepare the amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent.
2. Characterization of amphiphilic comb graft copolymers
FIG. 2 shows that Polymer P (BrCL) obtained in example 15-co-CL)40And PCL40-g-PMPC10×5The nuclear magnetic spectrum of (1) shows that characteristic peaks (delta is 4.1ppm, delta is 2.3ppm, delta is 1.7ppm, and delta is 1.4ppm) of segments of the PCL all appear on the nuclear magnetic spectrum, which indicates that the PCL is successfully synthesized, and the polymerization degree of the 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; as can be seen from fig. 3: 1728cm-1The peak at (A) is a stretching vibration peak of C ═ O, 2910cm-1Are continuous methylene absorption peaks; it can be seen that PCL-g-PMPC shows a characteristic absorption peak (1090 cm) related to the structure of phosphatidylcholine in an infrared spectrum-1And 1230cm-1，-POCH2-；970cm-1，N+(CH3)3) Successful synthesis of PCL-g-PMPC copolymer is also shown.
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; compared with PCL, the PCL-g-PMPC copolymer is introduced with an amorphous PMPC chain segment, and when the PCL is crystallized, the regular arrangement of the chain segment is hindered, so that various data are reduced.
3. Characterization of microvesicles
The PCL-g-PMPC copolymer solution added with PBS and PFP is changed into opaque milky white suspension after sound vibration treatment, and is still milky white suspension after the phase change of PFP is triggered by temperature after organic solvent is removed. The result of the particle size change of the copolymer-based ultrasound contrast agent after the phase transition is shown in fig. 5, and it can be seen from fig. 5 that the particle size distribution range of the PCL-g-PMPC copolymer and the ultrasound contrast agent is narrow, and the average particle size of the PCL-g-PMPC copolymer-based ultrasound contrast agent measured by a malvern nano-particle size analyzer 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 Sonowei SonoVue contrast agent) measured by Coulter are diluted, so that the copolymer-based ultrasonic contrast agent and the phospholipid-based ultrasonic contrast agent have the same concentration, and the copolymer-based ultrasonic contrast agent and the phospholipid-based ultrasonic contrast agent are placed in a refrigerator at 4 ℃ and are kept still for 24 hours and then observed under a sampling microscope, and the result shows 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.
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 ═ 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 nephelography
The method comprises the steps of fixing a male New Zealand white rabbit with the body weight of 2.5-3 kg on an experiment table, placing an indwelling needle into an ear edge vein, after unhairing of the back of the right waist, respectively injecting a commercial Sonowev contrast medium and a PCL-g-PMPC copolymer contrast medium by adopting an ear edge vein bolus injection method, and obtaining an ultrasonic contrast image of the kidney. 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. Significant contrast enhancement began to appear in the renal area following intravenous injection of SonoVue and PCL-g-PMPC copolymer contrast agents, respectively, compared to the PBS control group. Clear ultrasound images of the kidneys show that PCL-g-PMPC copolymer contrast agent successfully passes through the pulmonary capillaries during blood circulation, which is required for venous contrast agent safety performance. As can be seen from FIG. 8, the ultrasound signal intensity of the SonoVue contrast agent is substantially comparable to the contrast image intensity of the PCL-g-PMPC copolymer contrast agent at 20 s; however, at 40s, the ultrasonic signal of the SonoVue microbubble is rapidly reduced, and at 80s, the ultrasonic signal is basically disappeared, and the PCL-g-PMPC copolymer contrast agent still has a contrast signal which can be observed after 80 s; this shows that the PCL-g-PMPC phospholipid amphiphilic graft polymer nano ultrasonic contrast agent has longer in vivo duration compared with a SouoVue contrast agent, and proves that the PCL-g-PMPC phospholipid amphiphilic graft polymer nano ultrasonic contrast agent has great potential as an ultrasonic contrast agent with novel diagnosis and treatment effects.
Example 2
A method different from that of example 1 was used to prepare a PCL-g-PMPC contrast medium by a shear method.
The specific preparation process of the PCL-g-PMPC phospholipid amphiphilic graft polymer nano ultrasonic contrast agent prepared by the shearing method comprises the following steps:
3mg of PCL-g-PMPC copolymer and 1mg of DPPE-mPEG 5000 were respectively weighed and dissolved in 567. mu.L of Tetrahydrofuran (THF) and 283. mu.L of methanol (MeOH) (2:1, v/v), and after being sufficiently dispersed and dissolved by ultrasonic treatment in a water bath, 2mL of PBS buffer (0.01M, pH 7.4) was added, and then 150. mu.L of perfluoropentane (PFP) was added; under the condition of ice-water bath, adding liquid perfluoropentane, adopting an electric internally tangent homogenate method, wherein the homogenate rotating speed is 12000-30000rpm, and the homogenate time is 1-3min, and obtaining milky graft polymer mixed solution wrapping the perfluoropentane after homogenate.
The mixed solution is subjected to phase change by a therapeutic ultrasonic instrument to form the PCL-g-PMPC amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent, and the parameter of the ultrasonic instrument is 3W/cm2The duty ratio is 50%, and the action time is 3 min.
Example 3
The preparation method of the PCL-g-PMPC amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent by a freeze-drying method comprises the following specific preparation processes:
3mg of PCL-g-PMPC copolymer and 1mg of DPPE-mPEG 5000 were respectively weighed and dissolved in 567. mu.L of Tetrahydrofuran (THF) and 283. mu.L of methanol (MeOH) (2:1, v/v), and after being sufficiently dispersed and dissolved by ultrasonic treatment in a water bath, 2mL of PBS buffer (0.01M, pH 7.4) was added, and then 150. mu.L of perfluoropentane (PFP) was added; performing ultrasonic vibration under ice salt water bath condition, wherein the probe frequency is 24KHz, the power is 35W, the ultrasonic is turned on for 3s and turned off for 6s, the acting time is 3min, a milky suspension is formed, then centrifuging for 5min under the action of 3000g centrifugal force, discarding supernatant, and adding 6mL of sucrose solution containing 10% wt for resuspension. 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 prepared 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 1min at the oscillation frequency of 75Hz to obtain the PCL-g-PMPC amphiphilic comb-grafted copolymer-based ultrasonic contrast agent.
According to the three examples, the invention can be seen that the PCL-g-PMPC amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent prepared by different preparation methods is spherical in shape, good in dispersion, smooth and transparent in surface, uniform in size (see figure 5), and about 5 μm in average particle size distribution.
The mechanical index value (MI) has a great influence on the ultrasound contrast image, which substantially determines the intensity of the ultrasound waves, since the contrast agent may oscillate more strongly due to the ultrasound drive with higher energy, and when the MI is too large, it may cause the destruction of the contrast agent and the loss of the contrast effect. Compared with a phospholipid-based contrast agent (SonoVue contrast agent), the PCL-g-PMPC amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent can tolerate the action of higher ultrasonic mechanical index and keep an integral morphological structure.
The change of the PCL-g-PMPC copolymer-based ultrasound contrast signal intensity with time after ultrasound irradiation is shown in fig. 7 by acquiring ultrasound images of the PCL-g-PMPC copolymer-based ultrasound contrast agent and the SonoVue contrast agent at different time points, and analyzing the change of the average gray level of the copolymer-based ultrasound contrast agent and the SonoVue contrast image. As can be seen from the attached figure 7, 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; the brightness of the ultrasonic image of the PCL-g-PMPC copolymer-based ultrasonic contrast agent shows good stability, the brightness of the image slowly becomes dark within 1-3min, 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, decreasing to about 20% of its initial intensity in about 3min, with only 2.5% of the initial intensity at 20 min; however, the ultrasonic signal intensity of the PCL-g-PMPC copolymer-based ultrasonic contrast agent can be maintained for about 20min and is reduced to the initial strong intensity at the highest compared with the initial ultrasonic signal intensity23% of the intensity, and at the lowest, only to 50% of its initial intensity. The stronger stability of the PCL-g-PMPC copolymer-based ultrasound contrast agent is attributed to the fact that the PCL-g-PMPC copolymer can form a stable core-shell structure in an aqueous solution, which provides a strong barrier for keeping PFP gas from diffusing and dissolving in the inner core under ultrasound radiation, while the lipid shell of the SonoVue ultrasound contrast agent has a thickness of only about 4nm, and gas in the micro-bubble can diffuse out through the shell quickly under ultrasound radiation, so that the contrast capability of the SonoVue ultrasound contrast agent is reduced rapidly. Furthermore, gaseous PFP in PCL-b-PMPC copolymer-based ultrasound contrast agents compared to SF in SonoVue ultrasound contrast agents6Gas, with lower water solubility, which also helps to enhance the stability of PCL-g-PMPC copolymer based ultrasound contrast agents. This allows the PCL-b-PMPC copolymer-based ultrasound contrast agents to have longer cycle periods, providing a longer imaging time window for clinical diagnosis.
From the above, the amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent obtained by the invention is applied to in vitro model ultrasonic contrast imaging, and the concentration of microbubbles is set to be 5.0 × 108After the volume/mL and the mechanical index MI are 0.2, an ultrasonic contrast image is obtained in a frequency range of 4-10 MHz, the change of the average gray value of the contrast image of the copolymer ultrasonic contrast agent along with the frequency is analyzed, and due to the difference between the resonance frequency of the ultrasonic contrast agent and the ultrasonic driving frequency, the brightness of the ultrasonic contrast image of part of the PCL-g-PMPC copolymer-based ultrasonic contrast agent is increased along with the increase of the frequency from 4MHz to 5.71 MHz. When the frequency is increased by 6.67MHz, the brightness of all the ultrasonic contrast images is rapidly reduced; the brightness of the image remains substantially unchanged by 7.27 MHz. When the ultrasound frequency is chosen at 5.71MHz, the contrast image grey value of the ultrasound contrast agent is maximal. Copolymer-based ultrasound contrast agents have a longer duration than Sonovue contrast agents. The amphiphilic comb-shaped 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 compared with a phospholipid-based contrast agent, the amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent is high in contrastThe contrast time at mechanical index is extended.
Claims (13)
1. A phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent is characterized by comprising a shell and a core, wherein the shell is amphiphilic comb-shaped graft copolymer polycaprolactone-g-polymethacryloxyethylphosphocholine, the envelope further comprising a modifying substance M, the modifying substance M being a substance providing the envelope with PEG-containing segments which can avoid clearance by the immune system in vivo, 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-mercapto, at least one of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-sulfhydryl, distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-amino, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino, distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-carboxyl, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-carboxyl or distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-hydroxyl;
the inner core is an ultrasonic responder which is liquid perfluorocarbon or gaseous perfluorocarbon capable of changing phase;
wherein the amphiphilic comb-graft copolymer polycaprolactone-g-polymethacryloxyethylphosphocholine is prepared by the following method:
1) macromolecular backbone P (BrCL)p-co-CL)mThe preparation of (1):
firstly, cyclohexanone is used as a raw material to react with liquid bromine to obtain alpha-bromocyclohexanone, then the alpha-bromocyclohexanone reacts with m-chloroperoxybenzoic acid to obtain alpha-bromo-epsilon-caprolactone, then the alpha-bromo-epsilon-caprolactone and the epsilon-caprolactone are used as monomers, stannous octoate is used as a catalyst, and ring-opening random polymerization is carried out to obtain a macromolecular main chain P (BrCL)p-co-CL)mWherein p is alpha-bromo-epsilon-hexane in the main chain of the copolymerThe average number of lactones, m being the degree of polymerization of the copolymer;
2) phospholipid-like amphiphilic comb-graft copolymer polycaprolactone-gPreparation of polymethacryloxyethylphosphocholine:
using P (BrCL) obtained in the step 1)p-co-CL)mIs a macromolecular main chain, 2-methacryloyloxyethyl phosphorylcholine is a monomer, and the amphiphilic comb-shaped graft copolymer polycaprolactone-containing material is obtained by side chain polymerization of an atom transfer radical polymerization method of an electron transfer regenerated catalystg-polymethacryloxyethylphosphocholine.
2. The phospholipid-like amphiphilic comb-graft copolymer-based ultrasound contrast agent according to claim 1, wherein the core of the ultrasound contrast agent comprises at least one of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, or perfluoroheptane.
3. The phospholipid-like amphiphilic comb-graft copolymer-based ultrasound contrast agent as claimed in claim 1, wherein the phospholipid-like amphiphilic comb-graft copolymer-based ultrasound contrast agent is prepared from polycaprolactone-doped resin when the core of the ultrasound contrast agent is liquid perfluorocarbon capable of phase transitiongPreparing the polymethacryloxyethyl phosphorylcholine, the modified substance M and the ultrasonic response substance into the nano-emulsion in a self-assembly mode, and forming the phospholipid-like amphiphilic comb-shaped graft copolymer to wrap the ultrasonic contrast agent of the gaseous perfluorocarbon through phase change of the nano-emulsion.
4. The phospholipid-like amphiphilic comb-graft copolymer-based ultrasound contrast agent as claimed in claim 1, wherein the phospholipid-like amphiphilic comb-graft copolymer-based ultrasound contrast agent is prepared from polycaprolactone-when the core of the ultrasound contrast agent is gaseous perfluorocarbongThe polymethacryloxyethyl phosphorylcholine, the modified substance M and the ultrasonic responder are prepared into the ultrasonic contrast agent in a self-assembly mode.
5. The phospholipid-like amphiphilic comb-graft copolymer-based ultrasound contrast agent according to claim 4, wherein the self-assembly is in one of the following manners: high shear homogenization, high pressure homogenization, high speed oscillation or ultrasonic sound vibration.
6. The preparation method of the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent as defined in any one of claims 1 to 5, wherein the preparation method comprises the following steps: polycaprolactone-gThe poly (methacryloyloxyethyl phosphorylcholine), the modified substance M and the ultrasonic responder are prepared into the poly (caprolactone) -poly (caprolactone)g-an ultrasound contrast agent in which polymethacryloxyethylphosphocholine encapsulates an ultrasound responder.
7. The method for preparing the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent according to claim 6, wherein the self-assembly is performed in one of the following ways: high shear homogenization, high pressure homogenization, high speed oscillation or ultrasonic sound vibration.
8. The method for preparing the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent according to claim 6, wherein when the ultrasonic response substance in the ultrasonic contrast agent is gaseous perfluorocarbon, the method for preparing the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent comprises the following steps: polycaprolactone-gThe phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent is prepared from polymethacryloxyethyl phosphorylcholine, a modified substance M and an ultrasonic responder in a direct ultrasonic sound vibration or high-speed oscillation mode.
9. The method for preparing the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent as claimed in claim 6, wherein when the ultrasound-responsive substance in the ultrasonic contrast agent is liquid perfluorocarbon capable of phase transition, the method for preparing the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent comprises the following steps: firstly polycaprolactone-gPolymethacryloxyethylphosphocholine, modifying substances and ultrasound-responsive substancesPreparing the nano-emulsion in a self-assembly mode, and then carrying out temperature-induced phase change or sound-induced phase change on the obtained nano-emulsion to form the phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent.
10. The method for preparing the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent as claimed in claim 6, wherein when the ultrasonic response substance in the ultrasonic contrast agent is liquid perfluorocarbon capable of phase transition, the method comprises the following steps:
(1) preparation of phospholipid-like amphiphilic comb-graft copolymer polycaprolactone-g-polymethacryloxyethylphosphocholine;
(2) preparing a phospholipid-like amphiphilic comb graft copolymer-based ultrasonic contrast agent: dissolving the phospholipid-like amphiphilic comb-shaped 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 then performing temperature-induced phase change or sound-induced phase change to prepare the phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent; wherein the modifying substance M is: providing the shell with a material comprising a PEG segment that avoids clearance by the immune system in vivo; the mixed solvent is a mixed solvent of tetrahydrofuran and methanol, or: a mixed solvent of chloroform and methanol.
11. The method for preparing the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent according to claim 10, wherein in the step (2), the method for preparing the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent after the temperature-induced phase change or the acoustic-induced phase change of the separated and purified nano-emulsion comprises one of the following modes:
the first method is as follows: the prepared phospholipid-like amphiphilic comb-shaped graft copolymer-based nano-emulsion suspension is subjected to water bath at the temperature of 60-80 ℃ for 5-15 min to prepare a phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent;
the second method comprises the following steps: subjecting the prepared phospholipid-like amphiphilic comb-shaped graft copolymer-based nanoemulsion suspension to ultrasonic action by an ultrasonic therapeutic apparatusObtaining the phospholipid-like amphiphilic comb-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.
12. The method for preparing the phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent according to claim 11, wherein in the step (2), the volume ratio of the mixed solvent is 2:1 of tetrahydrofuran and methanol.
13. The phospholipid-like amphiphilic comb-graft copolymer-based ultrasonic contrast agent is used for in-vitro agarose model contrast imaging; wherein the phospholipid-like amphiphilic comb-shaped graft copolymer-based ultrasonic contrast agent is the phospholipid-like amphiphilic comb-shaped graft copolymer-based 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 12.
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