CN107233583B

CN107233583B - Ultrasonic contrast agent with ultra-long duration and preparation method thereof

Info

Publication number: CN107233583B
Application number: CN201610188537.4A
Authority: CN
Inventors: 戴志飞; 刘仁发; 梁晓龙; 王金锐
Original assignee: Beijing Fei Rui Medical Technology Co Ltd
Current assignee: Beijing Fei Rui Medical Technology Co Ltd
Priority date: 2016-03-29
Filing date: 2016-03-29
Publication date: 2020-04-24
Anticipated expiration: 2036-03-29
Also published as: CN107233583A

Abstract

The invention relates to the field of medicines, and particularly discloses an ultrasonic contrast agent with ultra-long duration and a preparation method thereof. The contrast agent has the advantages that the contrast duration of the liver, the kidney, the spleen and other organs reaches more than 2h, the maximum duration can reach more than 6h, and the contrast agent is the contrast agent with the longest ultrasound contrast duration in the microbubble ultrasound contrast agents reported at present. In addition, the preparation process of the contrast agent is simple and controllable, large-scale production is easy to carry out, and the production raw materials are all approved injectable pharmaceutical excipients at present, so that the contrast agent has a very wide clinical application prospect.

Description

Ultrasonic contrast agent with ultra-long duration and preparation method thereof

Technical Field

The invention relates to the field of medicines, in particular to an ultrasonic contrast agent with ultra-long duration and a preparation method thereof.

Background

Ultrasound contrast agents are typically lipid, polymer or protein encapsulated gas microbubbles with diameters below 10 microns that can be used in ultrasound imaging to enhance the echo signal at the site of detection. The ultrasonic contrast agent has great advantages in the aspects of perfusion detection and imaging of blood flow of human body tiny blood vessels and tissues. In addition, recent studies show that ultrasound contrast agents have a wide application prospect in the aspects of targeted imaging and drug delivery of tumors or inflammatory sites, and therefore, ultrasound contrast agents are expected to become indispensable agents in clinical ultrasound diagnosis or treatment in the near future.

At present, a plurality of products of the ultrasonic contrast agent enter clinical application, such as Definity, Optison, Sonazoid, Sonovue and the like in international markets, and Sonovue and Xueliexin in domestic markets can be used. Although the ultrasound contrast agent has many application advantages in ultrasound diagnosis, the ultrasound contrast agent is rarely used by the general public in domestic clinical diagnosis at present, and is generally used in more than three hospitals. One important reason for this is that the contrast agent is very expensive, for example, the price of Sonovue per bottle is about 700 rmb, the price of xuerixin is more than 900 rmb per bottle, and common people are difficult to afford, thus greatly limiting the popularization of the ultrasound contrast agent. In addition, the imaging time of the existing ultrasonic contrast agents is short (the effective time of Sonovue is 4-8 minutes, and the Xuerioxin is less than 2 minutes), so that the requirement of a complete ultrasonic examination is difficult to meet. Particularly with the development of interventional ultrasound and like techniques, the need for contrast agents with long duration is more acute.

In addition, with the development of molecular ultrasound imaging and interventional therapy, submicron microbubbles (i.e., ultrasound microbubbles containing a certain proportion of nanobubbles) begin to show great potential for use. The nano-bubbles have better stability under high-frequency ultrasound and better ability of penetrating blood vessels, the ultrasound contrast enhancement effect of the micron-sized micro-bubbles is better, and the submicron micro-bubbles combine the advantages of the nano-bubbles and the micron-sized micro-bubbles to show better application value. For example, it was found that Definity has better sonothrombolysis effect due to the proportion of nano-bubbles, while albumin microbubbles under the same condition have no such effect due to the micron-sized micro-bubbles.

Disclosure of Invention

Based on the above needs and problems, the present invention provides a method for preparing an ultrasound contrast agent with an ultra-long duration, which is simple to operate, and the microbubble coating material can be dispersed under very mild conditions to obtain a uniform and stable solution, can be filtered for sterilization, and is easy for industrial production. The ultrasonic contrast agent is a freeze-dried preparation, in order to prepare the freeze-dried preparation, a suspension of an ultrasonic microbubble coating material needs to be prepared first, and components in the suspension are an amphiphilic coating material, a freeze-drying protective agent and a solvent.

The technical scheme of the invention is as follows:

a method for preparing an ultrasound contrast agent with an ultra-long duration, comprising the steps of:

1) preparing a microbubble coating material suspension, wherein the solvent of the suspension is a mixed solution of tert-butyl alcohol and water;

2) freeze-drying the suspension to obtain microbubble freeze-dried powder;

3) filling gas into the microbubble freeze-dried powder, wherein the filling gas is mixed gas.

The envelope material is an amphiphilic envelope material, the main component of the envelope material is phospholipid with negative charges, the length of a hydrophobic chain is preferably 18 carbons, and suitable phospholipid comprises one of distearoyl-phosphatidyl glycerol, distearoyl-phosphatidyl serine, distearoyl-phosphatidic acid and the like. Researches find that the phospholipids have negative net charges under physiological conditions, so that the microbubbles are ensured to have stronger negative charges, and the stability is greatly improved. And the longer hydrophobic chain can ensure that a stable membrane structure can be formed to prevent the gas in the micro-bubbles from overflowing. In addition, the structures of phosphoric acid, glycerol, serine and the like in the hydrophilic head of the phospholipid can form a stable hydrophilic layer through hydrogen bonds or electrostatic interaction, so that the stability of the microbubble is greatly improved.

The amphiphilic envelope material of the present invention may be incorporated with some other amphiphilic film-forming material in addition to the aforementioned negatively charged phospholipids, but in order to ensure the stability of the microvesicles, the weight ratio of the aforementioned negatively charged phospholipids (i.e., distearoyl-phosphatidylglycerol, distearoyl-phosphatidylserine, and distearoyl-phosphatidic acid) should be 70% or more, preferably 90% or more, and more preferably 100%. Other amphiphilic materials include various natural, semi-synthetic phospholipid compounds such as lecithin, lecithin phosphatidylglycerol, hydrogenated soybean phospholipid, hydrogenated egg yolk phospholipid, hydrogenated phosphatidylserine, hydrogenated phosphatidylglycerol, and the like. Also included are various synthetic phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and the like, with various fatty acid chains. Modified phospholipids, such as pegylated phospholipids, are also included. Also included are various synthetic cationic lipids such as hydroxyethylethylene diamine-based phospholipids, derivatives of ethylphosphatidylcholine (e.g., 1, 2-distearoyl-sn-glycero-3-ethylcholine phosphate), ammonium salts with alkyl chains (e.g., stearyl ammonium chloride, dodecyl ammonium bromide, etc.), and the like. Also included are various surfactants such as span, tweens, pegylated fatty acids, glyceryl monostearate, sucrose monostearate, hydrogenated castor oil, ascorbyl palmitate, and the like. Also included are steroid compounds (e.g., cholesterol, lanosterol, sitosterol, stigmasterol, ergosterol) and derivatives thereof, vitamin E and derivatives thereof, fatty acids (e.g., stearic acid, palmitic acid, arachidic acid, lauric acid, myristic acid, oleic acid, etc.).

The research shows that the amphiphilic envelope material of the present invention is best to use a single phospholipid, namely, one of distearoyl-phosphatidyl glycerol, distearoyl-phosphatidyl serine and distearoyl-phosphatidic acid is selected as the microbubble envelope material. On one hand, the single phospholipid avoids the problem of poor microbubble uniformity and repeatability caused by uneven mixing of multiple phospholipids. On the other hand, the adoption of a single phospholipid is beneficial to the formation of a uniform microbubble envelope structure by the phospholipid, and the stability is greatly improved.

In the process of preparing the microbubble coating material suspension, a freeze-drying protective agent is also required to be added.

The lyoprotectant adopted in the invention is polyethylene glycol, the average molecular weight is preferably 1000-10000, and the preferred polyethylene glycol has the average molecular weight of about 4000. The main function of the polyethylene glycol is that the suspension can be used as a film forming material for supporting during freeze drying, so that the suspension is kept in a stable dispersion state in a solution and cannot collapse or aggregate, and the freeze-dried powder can be easily dispersed to obtain a uniform and stable solution during hydration. In addition, polyethylene glycol can be attached to phospholipid on the surface of the microbubble membrane in water in a large amount through hydrogen bonding, the stability of the microbubble can be greatly improved through the attachment of the polymer, and the probability of identifying the microbubble in vivo can be reduced, so that the circulation time of the microbubble is prolonged.

In order to obtain a uniform and stable suspension of the microbubble coating material, a solvent is required to uniformly mix and disperse the microbubble coating material (and the lyoprotectant). In addition, in order to obtain microbubble lyophilized powder, the solvent also needs to have a proper melting point and saturated vapor pressure.

Water is the most commonly used solvent in the freeze drying process, but amphiphilic materials are easily self-assembled into structures such as micelles or liposomes in water, and the uniform mixing on the molecular level is difficult to realize by directly dispersing a plurality of amphiphilic materials in water.

The most preferred non-aqueous solvent that can be used for freeze-drying at present is t-butanol, which is a very significant cost saving because of its high melting point (25.7 ℃) and saturated vapor pressure (26.8mmHg, 20 ℃) and because of the fact that freeze-drying is accomplished with ordinary freeze-drying equipment. In addition, the lower toxicity also makes tert-butanol more suitable for freeze-drying of pharmaceutical preparations. Although pure t-butanol can solubilize most amphiphilic materials and polyethylene glycols of different molecular weights, it does not solubilize most lyoprotectants. In addition, microvesicles are usually prepared by adding a certain proportion of negatively charged phospholipids such as phosphatidylserine, phosphatidylglycerol or phosphatidic acid, which have poor solubility in t-butanol.

The solvent used for preparing the suspension of the microbubble coating material in the invention is a mixed solution of tert-butyl alcohol and water. Preferably, the mass content of the tertiary butanol in the mixed solution of tertiary butanol and water is 1% to 99%, more preferably 30% to 70%.

The film forming material adopted in the invention is mainly phospholipid with negative charges, the phospholipid is difficult to disperse in water, and the solubility of the phospholipid in most organic solvents such as chloroform, methanol, normal hexane, tertiary butanol, ethanol and the like is poor. And in addition to ensuring that the phospholipid is well dispersed, it is also necessary to ensure that the suspension can be lyophilised in conventional freeze-drying equipment. We have found through a number of experiments that only a mixture of t-butanol and water is satisfactory, that negatively charged phospholipids can be easily dissolved in this solvent, that the solvent in the suspension can be easily removed by freeze-drying, and that the biotoxicity of this solvent is low and that trace amounts of residual solvent toxicity can be neglected.

In addition, the research of the invention finds that the mixed solution of tertiary butanol and water is used as the solvent to dissolve most of the amphiphilic coating material and the freeze-drying protective agent simultaneously, the dissolving process does not need too high temperature, the solution can be dissolved at about 40 ℃ or even lower temperature, and the solution can not be separated out after being cooled to room temperature. The resulting suspension is clear, uniform, stable, and readily sterilized by filtration. In addition, when the freeze drying is carried out by using a mixed solution of tert-butyl alcohol and water as a solvent, the tert-butyl alcohol can form needle crystals in water, and the freeze drying period is greatly shortened.

The suspension of the microbubble coating material of the invention is prepared and then usually needs to be filtered, and a filter membrane with the thickness of 220nm is preferred to be filtered. The coating material can be easily dissolved by using tert-butyl alcohol and water as solvents to obtain a clear and transparent solution, and the solution can be easily filtered. The purpose of filtration is on the one hand to remove possible traces of impurities and on the other hand to filter-sterilize the suspension.

After the microbubble coating material suspension is prepared (for example, the suspension is subpackaged into penicillin bottles for pharmaceutical preparations), a solvent (namely a mixed solution of tert-butyl alcohol and water) is removed by a freeze drying method to obtain microbubble freeze-dried powder, and the microbubble freeze-dried powder can stably exist and can be stored for a long time at normal temperature. The freeze-drying may be carried out by a method commonly used in the art.

The method also comprises the step of filling gas into the prepared microbubble freeze-dried powder. For example, after freeze-drying is finished, the penicillin bottle is filled with filling gas. The filling gas is a component of the inner core of the ultrasonic microbubble, and in order to improve the stability of the ultrasonic microbubble, the filling gas should have low solubility in water and be safe and nontoxic to organisms. The filling gas of the commonly used ultrasound contrast agent is a fluorocarbon-based gas, but our studies have found that when a fluorocarbon-based gas such as octafluoropropane is used as the filling gas, the finally prepared ultrasound microbubbles are large in size, more microbubbles are above 10 microns in size, and such microbubbles are difficult to be safely used in living bodies because the inner diameter of the finest blood vessels of the lungs in a human body is around 10 microns. On the other hand, microbubbles prepared using a gas having a higher solubility in water, such as air or nitrogen, as the filling gas have extremely poor stability in spite of their small size.

The fill gas used in the present invention is a mixed gas containing two types of gases: gas a and gas B. The gas A is characterized by small molecular weight (<100), and the gas which can be used comprises one or a mixture of air, oxygen, nitrogen, carbon dioxide, helium, neon and argon. The gas B is characterized by having a relatively high molecular weight (>100) and containing fluorine elements, and the usable gas includes one or a mixture of several of carbon fluoride gases such as octafluoropropane, decafluorobutane, octafluorocyclobutane, perfluoropentane, perfluorohexane, perfluorooctane and the like, and sulfur fluoride gases such as sulfur hexafluoride and the like.

The volume proportion of the gas a in the filling gas (i.e., the mixed gas) in the present invention is 1% to 99%, and the preferable proportion is 95% to 99%. The use of the mixed gas can ensure that the micro-bubbles have good stability on one hand, and can remarkably reduce the sizes of the micro-bubbles so that the sizes of most micro-bubbles are below 7 microns and a plurality of nano-bubbles exist on the other hand.

The gas filling process in the present invention may be carried out outside the freeze dryer using a specific gas filling apparatus. The method for industrial production is more preferably that the gas filling and sealing processes are all carried out in a freeze-drying device. After the suspension is subpackaged in a penicillin bottle, the final ultrasonic contrast agent (microbubble freeze-dried powder) finished product can be obtained after freeze-drying, gas filling, sealing and aseptic treatment.

In the above-mentioned preparation method of the present invention,

step 1) adding the coating material and a freeze-drying protective agent into a mixed solution of tert-butyl alcohol and water, and heating and dissolving to obtain a microbubble coating material suspension; then filtering and sterilizing;

the weight ratio of the coating material to the freeze-drying protective agent is 1: 200-1: 5; preferably 1:50 to 1: 10.

The total weight of the coating material and the freeze-drying protective agent accounts for 1-50% of the weight of the solvent (namely the mixed solution of tert-butyl alcohol and water); preferably 5 to 20 percent;

the mass content of the tertiary butanol in the tertiary butanol and water mixed solution is 1-99%, preferably 30-70%.

The heating and dissolving temperature is 25-70 ℃; preferably 35 ℃ to 50 ℃.

The filtration sterilization is preferably performed by using a filter membrane with the wavelength of 220 nm.

Specifically, the preparation method of the ultrasonic contrast agent with the ultra-long duration comprises the following steps:

1) adding the coating material and the freeze-drying protective agent into a mixed solution of tert-butyl alcohol and water, and heating and dissolving to obtain a microbubble coating material suspension; then filtering and sterilizing by adopting a filter membrane of 220 nm;

2) freeze-drying the microbubble coating material suspension subjected to the filtration sterilization in the step 1) to obtain microbubble freeze-dried powder;

3) filling the microbubble freeze-dried powder prepared in the step 2) with mixed filling gas;

in the above-mentioned preparation method, the first step,

the coating material is distearoyl-phosphatidyl glycerol, distearoyl-phosphatidyl serine or distearoyl-phosphatidic acid;

the freeze-drying protective agent is polyethylene glycol with the average molecular weight of 4000;

the weight ratio of the coating material to the freeze-drying protective agent is 1: 50-1: 10;

the total weight of the coating material and the freeze-drying protective agent accounts for 5-20% of the weight of the solvent;

the heating and dissolving temperature is 35-50 ℃;

the mixed gas contains two types of gases: gas A and gas B; wherein the molecular weight of gas a is < 100; gas B has a molecular weight >100 and contains elemental fluorine;

the gas A comprises one or more of air, oxygen, nitrogen, carbon dioxide, helium, neon and argon;

the gas B comprises one or more of carbon fluoride gas and sulfur fluoride gas; wherein the fluorocarbon gas comprises octafluoropropane, decafluorobutane, octafluorocyclobutane, perfluoropentane, perfluorohexane and perfluorooctane; the sulfur fluoride-based gas includes sulfur hexafluoride.

The invention also discloses the ultrasonic contrast agent prepared by the method.

When the ultrasonic contrast agent (microbubble freeze-dried powder) is used, a hydration liquid needs to be injected into the ultrasonic contrast agent, and the ultrasonic contrast agent suspension is prepared through oscillation.

Specifically, when the ultrasound contrast agent (microbubble lyophilized powder) provided by the invention is used, a hydration liquid needs to be injected into a packaging container (such as a penicillin bottle), and the amount of the hydration liquid preferably accounts for 10% -80% of the volume of the packaging container (such as a penicillin bottle). The aqueous solution should be selected to be completely non-toxic and to be injectable intravenously. Pure water and most of clinically used injections can be used as hydration solutions, such as physiological saline, phosphate buffer, glycerol injection, glucose injection, amino acid injection, and the like. Or physiological saline or phosphate buffer solution added with a certain amount of glycerol and/or propylene glycol, wherein the addition of the glycerol can improve the viscoelasticity and stability of the microvesicle, and the propylene glycol can promote the dispersion of the amphiphilic coating material.

After the ultrasonic contrast agent (microbubble freeze-dried powder) is hydrated, the microbubbles are preferably activated by a mechanical oscillator to obtain microbubble suspension which can be used for ultrasonic imaging contrast. The mechanical oscillator used here is basically consistent with the structural principle of the silver mercury harmonizer used in dentistry in clinical practice. Preferably, the oscillation mode is reciprocating type, the oscillation frequency is 2000-7000 rpm, the oscillation amplitude is 0.5-3 cm, and the oscillation time is 30-180 s.

The preparation method is simple, the components of the microvesicle can be dispersed under very mild conditions to obtain a uniform and stable solution, the uniform mixing at a molecular level is achieved, the components are not easy to degrade in the preparation process, the sample freeze-drying efficiency is high, the sample is uniform and stable, the filtration sterilization can be carried out, the repeatability is high, the large-scale production is easy, and the wide clinical application prospect is shown. The invention solves the technical problem that the stability and the safety of the product are influenced because the lysophospholipid is easily degraded under the high-temperature condition or in the aqueous solution for a long time in the traditional method. The preferred method of the invention adopts a single negatively charged phospholipid as a film forming material, and improves the uniformity and repeatability of the product. The product is efficient and safe, and the method repeatability is high.

The contrast agent in the invention (for example, liver, kidney, spleen and other organs) has contrast duration of more than 2-6 hours in vivo, and is the contrast agent with the longest ultrasonic contrast duration in microbubble ultrasonic contrast agents reported at present. Breaks through the traditional cognition of poor stability of the gas microbubble ultrasonic contrast agent. The contrast agent contains many nano-scale bubbles besides micro-scale bubbles. In addition, the contrast agent has simple components and low price, the main raw materials are injectable pharmaceutic adjuvants approved by FDA, the preparation process is simple, the repeatability is high, the large-scale production is easy to carry out, and the expected production cost of each bottle after the industrialization is only about 2 RMB. The ultrasonic contrast agent has good application value due to good ultrasonic contrast effect and low price, is expected to be beneficial to popularization and application of the ultrasonic contrast agent, and has wide clinical application prospect.

Drawings

FIG. 1 is a photograph showing suspensions of the microbubble coating materials prepared in example 1 and comparative example 1.

FIG. 2 is a photomicrograph of a microbubble suspension prepared according to example 1.

FIG. 3 is a photomicrograph of microbubbles of the microbubble suspension obtained in comparative example 2.

FIG. 4 is a graph showing the distribution of the particle size of microbubbles in the suspension of microbubbles prepared in example 1.

FIG. 5 is a graph of the effect of ultrasound contrast agents (microbubble suspensions) prepared in example 1 on the renal and hepatic parenchyma of New Zealand white rabbits at different time points. A, before microbubble injection; b, 30 seconds after microbubble injection; c microbubble injection 30 minutes later; d, 1 hour after microbubble injection; e, 3 hours after microbubble injection; f, 5 hours post microbubble injection.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1

128mg distearoyl-phosphatidylglycerol and 1872mg PEG4000 were weighed accurately, added with a solvent (i.e., a mixture of 9g deionized water and 9g t-butanol), heated to 45 deg.C to dissolve to give a suspension of microbubble coating material as a clear solution (see FIG. 1), and then cooled to room temperature. The dissolved solution was filtered through a 220nm filter and then dispensed into 2mL vials (actual volume: about 3.5mL) each containing 250mg of penicillin. And (3) placing the subpackaged penicillin bottles on a clapboard of a freeze dryer, quickly cooling to-40 ℃, and freeze-drying overnight. And after the freeze-drying is finished, keeping the vacuum state of the freeze dryer, filling mixed gas of nitrogen and octafluoropropane with the volume ratio of the nitrogen being 98%, pressing down a rubber plug on the penicillin bottle after the vacuum degree is recovered to the normal pressure, and sealing the penicillin bottle to obtain the ultrasonic contrast agent.

Before the ultrasonic contrast agent (freeze-dried powder preparation) is used, 1mL of physiological saline is injected into a Xilin bottle, the Xilin bottle is placed on a chuck of a medical silver-mercury blending machine, the oscillation frequency is 4000rpm, and the microbubble suspension is obtained after oscillation is carried out for 45 s.

Example 2

An ultrasound contrast agent, which is prepared by the method different from example 1 only in that the distearoyl-phosphatidylglycerol component is replaced with: 128mg distearoyl-phosphatidylserine.

Example 3

An ultrasound contrast agent, which is prepared by the method different from example 1 only in that the distearoyl-phosphatidylglycerol component is replaced with: 128mg distearoyl-phosphatidic acid.

Example 4

An ultrasonic contrast medium was prepared by a method different from that of example 1 only in that the solvent was a mixture of 12.6g of deionized water and 5.4g of t-butanol.

Example 5

An ultrasonic contrast medium was prepared by a method different from that of example 1 only in that the solvent was a mixture of 5.4g of deionized water and 12.6g of t-butanol.

Example 6

An ultrasonic contrast agent which was prepared by a method different from that of example 1 only in that a mixed gas of perfluoropropane and nitrogen having a nitrogen content of 95% by volume was filled.

Example 7

An ultrasonic contrast agent which was prepared by a method different from that of example 1 only in that a mixed gas of perfluoropropane and air having an air volume content of 98% was filled.

Example 8

An ultrasonic contrast agent which was prepared by a method different from that of example 1 only in that a mixed gas of perfluorobutane and air having an air volume content of 98% was filled.

Example 9

An ultrasonic contrast agent which was prepared by a method different from that of example 1 only in that a mixed gas of perfluorobutane and nitrogen having a nitrogen content of 98% by volume was filled.

Example 10

An ultrasonic contrast agent which was prepared by a method different from that of example 1 only in that a mixed gas of perfluoropropane and oxygen having an oxygen content of 98% by volume was filled.

Comparative example 1

An ultrasonic contrast agent, the preparation method of which is different from that of example 1 only in that water is used as a solvent to prepare a microbubble coating material suspension.

During the preparation process, when the suspension of the microbubble coating material is prepared, the suspension in example 1 can be dissolved by heating at 45 ℃ for about 5 minutes to obtain clear and transparent suspension, while in comparative example 1, many insoluble substances in particles can be still seen after the suspension is dissolved by heating at 45 ℃ for 30 minutes, and the suspension is turbid and opaque (figure 1). When the suspension was filtered using a conventional 220nm PVDF membrane needle filter (25 mm diameter), the suspension in example 1 encountered no significant resistance to filtration after 100mL, whereas the suspension in comparative example 1 experienced very significant resistance to filtration, and after only 2mL had been filtered the filter was essentially completely blocked and could not be continued.

Comparative example 2

An ultrasound contrast agent is prepared by a method different from that of example 2 only in that the gas to be filled is perfluoropropane single gas.

Comparative example 3

An ultrasound contrast agent, the preparation method of which is different from that of example 2 only in that the filled gas is air single gas.

Comparative example 4

An ultrasound contrast agent, which is prepared by a method different from that of example 2 only in that the filling gas is nitrogen gas alone.

Experimental example 1

To obtain an image of the microbubbles under an optical microscope; the specific operation steps are as follows: the microbubble suspensions prepared in example 1 and comparative examples 2 to 4 were diluted with the same volume of physiological saline, 10 μ L of the microbubble suspension was pipetted onto a clean glass slide, the glass slide was carefully covered, and the size, morphology and dispersion of microbubbles were observed with a microscope under different objective lens magnification and photographed. FIG. 2 is a photomicrograph of the microbubbles obtained in example 1, and it can be seen that the size of the microbubbles is mostly below 2 μm and that many nanobubbles can be clearly seen. Fig. 3 is a photomicrograph of the microbubbles obtained in comparative example 2, which shows that many microbubbles with sizes of more than 10 μm are easy to block blood vessels when being used in vivo, and the safety is not guaranteed, so that the microbubbles cannot be used in vivo ultrasonic experiments. The microbubbles in comparative examples 3 and 4 are mostly smaller than 2 μm and have a small size, but the microbubbles are very unstable and completely disappear after being placed at normal temperature for about 30 minutes.

Experimental example 2

The size distribution and concentration of the novel microbubbles were measured using a coulter counter. The operation steps are as follows: 20 μ L of the freshly prepared microbubble suspension from example 1 was added to 20mL of physiological saline and mixed well with gentle shaking. The analysis volume was set to 20. mu.L, and the particle size distribution and concentration of microbubbles were tested. The sample testing was repeated three times and the average was taken. FIG. 4 shows a representative result, and it can be seen that the size distribution of microbubbles is narrow, mostly below 2 μm, consistent with the photomicrograph.

Experimental example 3

A new Zealand white rabbit is taken as an experimental object, a peripheral venous channel is established on the left ear of the rabbit through an ear vein, the tail end of a catheter is connected with a three-way pipe, one channel is used for injecting an ultrasonic contrast agent, and the other channel is followed by physiological saline. New Zealand white rabbits were anesthetized with 3% sodium pentobarbital by intravenous injection (30mg/kg) supplemented with 0.2% heparin sodium for anticoagulation. After the rabbits were completely anesthetized, they were fixed in a rabbit bed in a supine position. The right abdominal hair was carefully removed with depilatory cream, and the rabbit liver and kidney were examined ultrasonically without injection of contrast media and images recorded under basal conditions. After the images are satisfied, the ultrasonic contrast agent microbubble suspensions in the embodiment 1, the comparative example 3 and the comparative example 4 are injected into the rabbit ear vein in a bolus mode according to the dose of 20 mu L/kg, the pipeline is immediately flushed with 2.0mL of physiological saline, and the enhancement conditions of the echo intensities of the liver and the kidney of the rabbit and the enhancement conditions of the Doppler blood flow signals of the energy of the kidney of the rabbit are dynamically observed and recorded in real time.

FIG. 5 shows the results of the rabbit liver and kidney imaging with the microbubbles of example 1, and it can be seen that the contrast signal has a significant contrast enhancement effect in the liver and kidney, and can last for a long time, and a strong signal can still be seen 5 hours after injection. At 7 hours post-injection, a significant enhancement signal was seen at the liver site, although the renal signal had essentially completely disappeared.

Whereas the contrast agents of comparative examples 3 and 4 showed substantially no signal of contrast enhancement after injection into the animal.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for preparing an ultrasound contrast agent with an ultra-long duration, comprising the steps of:

1) preparing a microbubble coating material suspension; comprises adding coating material and PEG4000 into mixed solution of tert-butyl alcohol and water, heating and dissolving to obtain microbubble coating material suspension; wherein the mass content of the tertiary butanol in the mixed solution of the tertiary butanol and the water is 50 percent; the coating material is distearoyl-phosphatidyl glycerol; the mass ratio of the coating material to PEG4000 is 128: 1872; the mass ratio of the coating material to the mixed solution of the tert-butyl alcohol and the water is 128: 18000;

2) freeze-drying the suspension to obtain microbubble freeze-dried powder;

3) filling gas into the microbubble lyophilized powder; the filling gas is a mixed gas of nitrogen and octafluoropropane, wherein the volume ratio of the nitrogen is 98%.

2. The method of claim 1, comprising the steps of:

1) adding the coating material and PEG4000 into the mixed solution of tert-butyl alcohol and water, and heating and dissolving to obtain microbubble coating material suspension; then filtering and sterilizing by adopting a filter membrane of 220 nm; the heating and dissolving temperature is 35-50 ℃;

3) filling the filling gas into the microbubble lyophilized powder prepared in the step 2).

3. The method according to claim 1, wherein step 1) comprises accurately weighing 128mg distearoyl-phosphatidylglycerol and 1872mg PEG4000, adding a solvent, and heating to 45 ℃ to dissolve to obtain a suspension of the microbubble coating material; the solvent is a mixture of 9g of deionized water and 9g of tert-butanol.

4. An ultrasound contrast agent prepared by the method of any one of claims 1 to 3.