CN110639032B - High-frequency ultrasonic contrast agent and preparation method thereof - Google Patents
High-frequency ultrasonic contrast agent and preparation method thereof Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
Abstract
The invention discloses a high-frequency ultrasonic contrast agent and a preparation method thereof. The high-frequency ultrasonic contrast agent consists of a lipid shell and an air inner core, wherein the lipid shell consists of phospholipid, a stabilizing agent and sodium alginate, the surface of the lipid shell is connected with superparamagnetic particles through amido bonds, the phospholipid is one or more of carboxylated DSPC, DPPC, DSPE, DPPE and DSPE-PEG2000, and the stabilizing agent is one or more of PEG4000, F68 and PEG40 s. The ultrasonic contrast agent has good response to the ultrasonic (20-50MHz) for high-frequency ultrasonic diagnosis, has long in-vivo circulation time, and can obtain obvious and clear in-vivo microvascular morphology and perfusion information by combining with a corresponding high-frequency ultrasonic imaging technology.
Description
Technical Field
The invention belongs to the field of ultrasonic imaging, and particularly relates to a high-frequency ultrasonic contrast agent and a preparation method thereof.
Background
Ultrasound imaging reflects physiological and pathological information inside tissues by using differences in the intensity of reflected and scattered signals of ultrasound waves in different tissues. Compared with other imaging modes, the ultrasonic imaging has the characteristics of invasiveness and no radioactivity, can provide real-time, rich and visual in-vivo information, and is widely popular among patients and doctors. The ultrasonic contrast agent is a microbubble with gas wrapped by phospholipid, protein, polymer and other membrane materials, and the particle size is 1-8 um. After the ultrasonic contrast agent is injected through a peripheral vein, the ultrasonic contrast agent can flow in a blood vessel along with blood, so that the blood can generate strong scattering, and an ultrasonic medical detection signal can be obviously enhanced. The ultrasonic contrast technique has the characteristics of real-time, dynamic and continuous display of visceral parenchyma, lesion vascular frameworks, tissue perfusion conditions and the like, and is widely applied to the diagnosis process of most diseases. Many types of ultrasound contrast agent products are in clinical use, mainly Definity, Optison, Sonovue, Sonazoid and the like are in international markets, and Sonovue and Xuenixin are ultrasound contrast agents approved for use in China. The microvesicles can improve blood echo, thereby improving signal-to-noise ratio, is used for improving Doppler signal-to-noise ratio of large blood vessels and small blood vessels and improving imaging effect of lesion blood vessel formation, and is mainly used for imaging liver, spleen and kidney superficial organs and heart cavities of kidney, pancreas, spleen, thyroid gland, mammary gland, blood vessel, tumor and the like. However, the current contrast technique has poor resolution and poor perfusion effect on the microvasculature, especially on the myocardial microcirculation and the microvasculature in the plaque, which greatly influences the morphology and perfusion information of the microvasculature observed by the doctor. However, it has to be mentioned that microvascular and cardiovascular diseases are closely related, for example studies have shown that the occurrence of myocardial infarction is associated with microangiopathies. In addition, the pathological changes of the superficial organs of the liver, the kidney and the spleen and the tumor require further understanding of the condition of the microvasculature, which is of great help for diagnosis and treatment of the disease. High frequency ultrasound imaging (> 15MHz) achieves high resolution imaging at the expense of lower penetration depth, making the technique particularly suitable for imaging small animal and human superficial organs. If matching high frequency ultrasound contrast microbubbles can be used, it is highly likely that microvascular morphology and perfusion information will be obtained, however the clinically Sonovue and cerinum contrast frequencies are low (3-5MHz) and are not satisfactory. Therefore, a high-frequency ultrasound contrast agent capable of displaying microvessels is urgently needed clinically.
Disclosure of Invention
The main purpose of the present invention is to solve the defects in the prior art, and provide a high-frequency ultrasound response contrast agent, which can obtain ultrasound echo signals under the stimulation of high-frequency ultrasound waves (20-50MHz), and a preparation method of the corresponding high-frequency ultrasound contrast agent, which can obtain an ultrasound contrast agent with long stabilization time and good contrast effect.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the high-frequency ultrasonic contrast agent consists of a lipid shell and an air inner core, wherein the lipid shell consists of phospholipid, a stabilizing agent and sodium alginate, the surface of the lipid shell is connected with superparamagnetic particles through amido bonds, the phospholipid is one or more of carboxylated DSPC, carboxylated DPPC, DSPE, DPPE and DSPE-PEG2000, the stabilizing agent is one or more of PEG4000, F68 and PEG40s, and the air inner core is a composition of inert gas and atmospheric pressure low-temperature plasma. The preparation method of the high-frequency ultrasonic contrast agent comprises the following steps: (1) dissolving phospholipid, a stabilizer and sodium alginate in chloroform according to the weight ratio of 3-10: 1-3: 1 to obtain a lipid mixed solution; (2) removing the trichloromethane through water bath negative pressure to enable the lipid mixed solution to form a uniform film on the tube wall; the temperature of the water bath is 40-60 ℃, and the vacuum degree of the negative pressure is 0.01-0.1 MPa; (3) after adding the hydration liquid, shaking to completely dissolve the film to obtain a suspension, and then subpackaging in containers; the hydration liquid consists of glycerol and phosphate buffer solution, and the volume ratio is 1-2: 10; (4) replacing air in the container with a composition of inert gas and atmospheric pressure low-temperature plasma, and oscillating to obtain an original microbubble suspension; (5) adding a calcium chloride solution with the mass fraction of 1% into the original microbubble suspension, and incubating at room temperature for 10min to obtain microbubble suspension; (6) adding superparamagnetic particles and initiators EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) into the microbubble suspension, and incubating at room temperature for 24 hours to obtain the high-frequency ultrasonic contrast agent. Wherein the temperature of the water bath in the step 2 is 40-60 ℃, and the vacuum degree of negative pressure is 0.01-0.1 MPa.
The high-frequency ultrasonic contrast agent and the preparation method thereof disclosed by the invention have the beneficial effects that: the invention adopts the amido bond and the network cross-linking system of the hydrogel to modify the microvesicles, which can obviously improve the stability of the microvesicles and prolong the circulation time of the microvesicles in blood. In addition, the elasticity of the shell of the microbubble can be adjusted by changing the thickness of the membrane material and the concentration of the superparamagnetic particles, so that the microbubble can generate a long-time stable ultrasonic enhanced signal under the high-frequency (20-50MHz) ultrasonic stimulation, and an obvious and clear in-vivo ultrasonic contrast image can be obtained.
Drawings
FIG. 1 is an image of a high frequency ultrasound contrast agent under a 400-fold optical microscope;
FIG. 2 is a high frequency ultrasound image of a high frequency ultrasound contrast agent in rabbit muscle.
Detailed Description
Example one
A high-frequency ultrasonic contrast agent comprises a lipid shell and a gas inner core, wherein the lipid shell comprises phospholipid, a stabilizing agent and sodium alginate, the surface of the lipid shell is connected with superparamagnetic particles through amido bonds, the phospholipid is carboxylated DSPC and DSPE-PEG2000, the stabilizing agent is F68 and PEG40s, the superparamagnetic particles are aminated superparamagnetic ferroferric oxide particles, the particle size is 30nm, and the gas inner core is perfluorobutane.
The preparation process of the high-frequency ultrasonic contrast agent of the embodiment is as follows:
(1) weighing 100mg of carboxylated DSPC, DSPE-PEG 200015 mg, F6820 mg, PEG40S 10mg and 10mg of sodium alginate into a beaker with the volume of 250ml, then adding 20ml of trichloromethane, fully and uniformly mixing, and clarifying and transparent the solution;
(2) removing the trichloromethane by water bath negative pressure for 30min, wherein the temperature of the water bath is 55 ℃, and the negative pressure is 0.05MPa, so that the phospholipid forms a uniform film on the tube wall;
(3) weighing 4.005g of NaCl, 0.097g of KCl and Na 2 HPO 4 ·H 2 O1.145g and KH 2 PO 4 0.096g, adding deionized water to the solution until the volume is 500ml, and preparing phosphate buffer solution for later use;
mixing 80ml of the phosphate buffer solution prepared in the step with 10ml of glycerol uniformly to prepare a hydration solution;
adding 50ml of the prepared hydration liquid into the step (2), shaking to completely dissolve the film to obtain suspension, and then subpackaging 3ml of the suspension in a 5ml container;
(4) replacing air in the container with inert gas perfluorobutane, sealing and oscillating to obtain original microbubble suspension;
(5) dropwise adding 2ml of calcium chloride solution with the mass fraction of 1% into 10ml of original microbubble suspension, and incubating at room temperature for 10min to obtain microbubble suspension;
(6) adding aminated superparamagnetic ferroferric oxide particles (0.2mg/mL), initiators EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) into 10mL of microbubble suspension, wherein the concentration of the initiators EDC is 0.4mg/mL, the concentration of the NHS is 0.3mg/mL, the pH value of the solution is adjusted to be between 5 and 7, and incubating at room temperature for 24 hours to obtain the high-frequency ultrasonic contrast agent.
Example two
A high-frequency ultrasonic contrast agent comprises a lipid shell and an air inner core, wherein the lipid shell comprises phospholipid, a stabilizing agent and sodium alginate, the surface of the lipid shell is connected with superparamagnetic particles through amido bonds, the phospholipid is carboxylated DPPC and DSPE-PEG2000, the stabilizing agent is F68 and PEG4000, the superparamagnetic particles are aminated superparamagnetic ferroferric oxide particles with the particle size of 5nm, and the air inner core is perfluoropropane.
The preparation process of the high-frequency ultrasonic contrast agent of the embodiment is as follows:
(1) weighing 100mg of carboxylated DPPC, DSPE-PEG 200015 mg, F6820 mg, PEG 400010 mg and 10mg of sodium alginate in a beaker with the volume of 250ml, then adding 20ml of trichloromethane, fully and uniformly mixing, and clarifying and transparent the solution;
(2) removing the trichloromethane by water bath for 30min under negative pressure, wherein the temperature of the water bath is 40 ℃, and the negative pressure is 0.01MPa, so that the phospholipid forms a uniform film on the tube wall;
(3) weighing 4.005g of NaCl, 0.097g of KCl and Na 2 HPO 4 ·H 2 O1.145g and KH 2 PO 4 0.096g, adding deionized water to the solution until the volume is 500ml, and preparing phosphate buffer solution for later use;
putting 80ml of the phosphate buffer solution prepared in the step and 10ml of glycerol into a 100ml volumetric flask, and uniformly mixing to obtain a hydration solution;
adding 50ml of the prepared hydration liquid into the step (2), shaking to completely dissolve the film to obtain suspension, and then subpackaging 3ml of the suspension in a 5ml container;
(4) replacing air in the container with inert gas perfluoropropane, sealing and oscillating to obtain original microbubble suspension;
(5) dropwise adding 2ml of calcium chloride solution with the mass fraction of 1% into 10ml of original microbubble suspension, and incubating at room temperature for 10min to obtain microbubble suspension;
(6) adding aminated superparamagnetic ferroferric oxide particles (0.2mg/mL) and initiators EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) into 10mL of microbubble suspension, wherein the concentration of the initiator EDC is 0.4mg/mL, the concentration of the NHS is 0.3mg/mL, adjusting the pH of the solution to be between 5 and 7, and incubating at room temperature for 24 hours to obtain the high-frequency ultrasonic contrast agent.
EXAMPLE III
A high-frequency ultrasonic contrast agent comprises a lipid shell and an air inner core, wherein the lipid shell comprises phospholipid, a stabilizing agent and sodium alginate, the surface of the lipid shell is connected with superparamagnetic particles through amido bonds, the phospholipid is carboxylated DSPC and DPPE, the stabilizing agent is F68 and PEG40s, the superparamagnetic particles are aminated superparamagnetic ferroferric oxide particles with the particle size of 100nm, and the air inner core is argon.
The preparation process of the high-frequency ultrasonic contrast agent of the embodiment is as follows:
(1) weighing 100mg of carboxylated DSPC, 15mg of DPPE, F6820 mg, 40mg of PEG40S 10 and 10mg of sodium alginate into a beaker with the volume of 250ml, then adding 20ml of trichloromethane, fully and uniformly mixing, and clarifying and transparent the solution;
(2) removing the trichloromethane by water bath negative pressure for 30min, wherein the temperature of the water bath is 60 ℃, and the negative pressure is 0.1MPa, so that the phospholipid forms a uniform film on the tube wall;
(3) weighing 4.005g of NaCl, 0.097g of KCl and Na 2 HPO 4 ·H 2 O1.145g and KH 2 PO 4 0.096g, adding deionized water to the solution until the volume is 500ml, and preparing phosphate buffer solution for later use;
putting 80ml of the phosphate buffer solution prepared in the step and 10ml of glycerol into a 100ml volumetric flask, and uniformly mixing to prepare a hydration solution;
adding 50ml of the prepared hydration liquid into the step (2), shaking to completely dissolve the film to obtain suspension, and then subpackaging 3ml of the suspension in a 5ml container;
(4) replacing air in the container with argon, sealing and oscillating to obtain original microbubble suspension;
(5) dropwise adding 2ml of calcium chloride solution with the mass fraction of 1% into 10ml of original microbubble suspension, and incubating at room temperature for 10min to obtain microbubble suspension;
(6) adding aminated superparamagnetic ferroferric oxide particles (0.2mg/mL) and initiators EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) into 10mL of microbubble suspension, wherein the concentration of the initiator EDC is 0.4mg/mL, the concentration of the NHS is 0.3mg/mL, adjusting the pH of the solution to be between 5 and 7, and incubating at room temperature for 24 hours to obtain the high-frequency ultrasonic contrast agent.
Example four
A high-frequency ultrasonic contrast agent comprises a lipid shell and a gas inner core, wherein the lipid shell comprises phospholipid, a stabilizing agent and sodium alginate, the surface of the lipid shell is connected with superparamagnetic particles through amido bonds, the phospholipid is carboxylated DSPC and DSPE, the stabilizing agent is F68 and PEG40s, the superparamagnetic particles are aminated superparamagnetic ferroferric oxide particles, the particle size is 60nm, and the gas inner core is a composition of perfluoropropane and atmospheric pressure low-temperature plasma.
The preparation process of the high-frequency ultrasonic contrast agent of the embodiment is as follows:
(1) weighing 100mg of carboxylated DSPC, 15mg of DSPE, F6820 mg, 40S 10mg of PEG and 10mg of sodium alginate into a beaker with the volume of 250ml, then adding 20ml of trichloromethane, fully and uniformly mixing, and clarifying and transparent the solution;
(2) removing the trichloromethane by water bath for 30min under negative pressure, wherein the temperature of the water bath is 50 ℃, and the negative pressure is 0.05MPa, so that the phospholipid forms a uniform film on the tube wall;
(3) weighing 4.005g of NaCl, 0.097g of KCl and Na 2 HPO 4 ·H 2 O 1.145g，KH 2 PO 4 0.096g, adding deionized water to the solution to a constant volume of 500ml to prepare phosphate buffer solution for later use;
putting 80ml of the phosphate buffer solution prepared in the step and 10ml of glycerol into a 100ml volumetric flask, and uniformly mixing to prepare a hydration solution;
adding 50ml of the prepared hydration liquid into the step (2), shaking to completely dissolve the film to obtain suspension, and then subpackaging 3ml of the suspension in a 5ml container;
(4) placing the air in a container by using a composition of inert gas perfluoropropane and atmospheric pressure low-temperature plasma, sealing and oscillating to obtain an original microbubble suspension;
(5) dropwise adding 2ml of calcium chloride solution with the mass fraction of 1% into 10ml of original microbubble suspension, and incubating at room temperature for 10min to obtain microbubble suspension;
(6) adding aminated superparamagnetic ferroferric oxide particles (0.2mg/mL), initiators EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) into 10mL of microbubble suspension, wherein the concentration of the initiators EDC is 0.4mg/mL, the concentration of the NHS is 0.3mg/mL, the pH value of the solution is adjusted to be between 5 and 7, and incubating at room temperature for 24 hours to obtain the high-frequency ultrasonic contrast agent.
EXAMPLE five
A high-frequency ultrasonic contrast agent comprises a lipid shell and an air inner core, wherein the lipid shell comprises phospholipid, a stabilizing agent and sodium alginate, the surface of the lipid shell is connected with superparamagnetic particles through amido bonds, the phospholipid is carboxylated DSPC and DSPE-PEG2000, the stabilizing agent is F68, and the superparamagnetic particles are aminated superparamagnetic ferroferric oxide particles with the particle size of 100 nm; the gas inner core is perfluorobutane.
The preparation process of the high-frequency ultrasonic contrast agent of the embodiment is as follows:
(1) weighing 100mg of carboxylated DSPC, DSPE-PEG 200015 mg, F6820 mg and 10mg of sodium alginate into a beaker with the volume of 250ml, then adding 20ml of trichloromethane, fully and uniformly mixing, and clarifying and transparent the solution;
(2) removing the trichloromethane by water bath negative pressure for 30min, wherein the temperature of the water bath is 55 ℃, and the negative pressure is 0.05MPa, so that the phospholipid forms a uniform film on the tube wall;
(3) weighing 4.005g of NaCl, 0.097g of KCl and Na 2 HPO 4 ·H 2 O 1.145g，KH 2 PO 4 0.096g, adding deionized water to the solution to a constant volume of 500ml to prepare phosphate buffer solution for later use;
putting 80ml of the phosphate buffer solution prepared in the step and 10ml of glycerol into a 100ml volumetric flask, and uniformly mixing to prepare a hydration solution;
adding 50ml of the prepared hydration solution into the step (2), shaking to completely dissolve the film to obtain a suspension, and then subpackaging 3ml of the suspension in a 5ml container;
(4) replacing air in the container with inert gas perfluorobutane, sealing and oscillating to obtain original microbubble suspension;
(5) dropwise adding 2ml of 1% calcium chloride solution into 10ml of original microbubble suspension, and incubating at room temperature for 10min to obtain microbubble suspension;
(6) adding aminated superparamagnetic ferroferric oxide particles (0.2mg/mL), initiators EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) into 10mL of microbubble suspension, wherein the concentration of the initiators EDC is 0.4mg/mL, the concentration of the NHS is 0.3mg/mL, the pH value of the solution is adjusted to be between 5 and 7, and incubating at room temperature for 24 hours to obtain the high-frequency ultrasonic contrast agent.
The microbubbles without being modified by the superparamagnetic particles are milky white, and the surfaces of the microbubbles carry the superparamagnetic particles after being modified by chemical bonds, so that the microbubbles are yellow overall, which proves that the superparamagnetic particles are modified on the surfaces of the microbubbles.
Fig. 1 is a figure showing the microbubble morphology of the high-frequency ultrasound contrast agent obtained by the invention, and it can be seen from fig. 1 that the microbubble is spherical, has a core-shell structure and uniform particle size distribution, most of the particle sizes of the microbubble are about 800nm, the particle size distribution is narrow, and the solution has no obvious impurities, so that the contrast requirement of the ultrasound contrast agent can be met.
Fig. 2 is a high-frequency ultrasonic development image of the high-frequency ultrasonic contrast agent obtained by the invention in rabbit muscle, a Japanese long-ear white rabbit is taken as an experimental object, a peripheral venous channel is established in the left ear of the rabbit through an ear vein, a three-way pipe is connected at the tail end of a catheter, one channel is used for injecting the ultrasonic contrast agent prepared by the invention, and the other channel is followed by physiological saline. Japanese big-ear white rabbits were anesthetized intraperitoneally with 3% sodium pentobarbital (40 mg/kg). After the rabbit is completely anesthetized, the right waist is carefully removed by using depilatory cream, ultrasonic examination without injection of contrast agent is carried out on the kidney of the rabbit, and an image in a basic state is recorded. The ultrasonic contrast agent (0.1ml/kg) prepared by the invention is injected into the ear vein of the rabbit in a bolus manner, immediately and 1ml of physiological saline is used for washing the pipeline, and the echo intensity enhancement condition of the superficial muscle tissue of the thigh of the rabbit is dynamically observed and recorded in real time in a high-frequency ultrasonic contrast mode. When the diagnosis and treatment integrated preparation is not injected, any echo signal can not be seen in the visual field, the blood vessels of the muscles are fully perfused within a few seconds by injecting the diagnosis and treatment integrated preparation, the boundaries of the blood vessels of the muscles are clearly developed, and the filling process from the large blood vessels to the micro blood vessels is also clearly visible.
Claims (2)
1. A high-frequency ultrasonic contrast agent is characterized in that the high-frequency ultrasonic contrast agent consists of a lipid shell and an air inner core, the lipid shell consists of phospholipid, a stabilizing agent and sodium alginate, the surface of the lipid shell is connected with superparamagnetic particles through amido bonds, the gas inner core is one of inert gas and atmospheric pressure low-temperature plasma, or the composition of inert gas and atmospheric pressure low-temperature plasma, the phospholipid is one or more of carboxylated DSPC, carboxylated DPPC, DSPE, DPPE and DSPE-PEG2000, the stabilizer is one or more of PEG4000, F68 and PEG40s, the weight ratio of the phospholipid to the stabilizer to the sodium alginate is 3-10: 1-3: 1, the surfaces of the superparamagnetic particles are modified with amino groups, the particle size of the superparamagnetic particles is 5-100 nm, and the saturation magnetization is 20-100 emu/g.
2. A preparation method of a high-frequency ultrasonic contrast agent is characterized by comprising the following steps:
(1) dissolving phospholipid, a stabilizer and sodium alginate in chloroform according to the weight ratio of 3-10: 1-3: 1 to obtain a lipid mixed solution, wherein the stabilizer is one or more of PEG4000, F68 and PEG40s, and the phospholipid is one or more of carboxylated DSPC, carboxylated DPPC, DSPE, DPPE and DSPE-PEG 2000;
(2) removing trichloromethane through water bath negative pressure to enable the lipid mixed solution to form a uniform film on the tube wall, wherein the temperature of the water bath is 40-60 ℃, and the vacuum degree of the negative pressure is 0.01-0.1 MPa;
(3) after adding a hydration solution, shaking to completely dissolve the film to obtain a suspension, wherein the hydration solution consists of glycerol and a phosphate buffer solution in a volume ratio of 1-2: 10;
(4) replacing air in a suspension container with one of inert gas and atmospheric pressure low-temperature plasma or a combination of inert gas and atmospheric pressure low-temperature plasma, sealing, and oscillating to obtain original microbubble suspension;
(5) adding a calcium chloride solution with the mass fraction of 1% into the original microbubble suspension, and incubating at room temperature for 10min to obtain microbubble suspension;
(6) adding superparamagnetic particles, initiator 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into the microbubble suspension, and incubating at room temperature for 24 hours to obtain the high-frequency ultrasonic contrast agent, wherein the particle size of the superparamagnetic particles is 5-100 nm, and the saturation magnetization is 20-100 emu/g.
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