Ultrasonic contrast agent with low side effect, preparation method and application

Abstract

The invention belongs to the technical field of ultrasonic imaging agents, and particularly relates to an ultrasonic contrast agent with low side effects, a preparation method and application. The ultrasonic contrast agent with low side effect provided by the invention is prepared from perfluorooctane, phospholipid, cholesterol, 0 cocamidopropyl amine oxide and dithiothreitol. Experimental data show that after being injected with the ultrasonic contrast agent, rats do not die and basically do not have toxic or side effect symptoms, after anatomy, visceral organs do not have obvious pathological changes, and the acute toxicity of the ultrasonic contrast agent is low.

Description

Technical Field

[0001] The invention belongs to the technical field of ultrasonic imaging agents, and in particular relates to an ultrasonic contrast agent with low side effects, a preparation method and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Ultrasound contrast agents are a type of contrast agent used to enhance the quality of ultrasound images. They are widely used in clinical medicine in the fields of vascular imaging, tumor diagnosis, cardiovascular disease detection, etc. Traditional ultrasound contrast agents are mainly composed of bubbles, microbubbles or other gas bubbles. These bubbles generate reflection signals under ultrasound irradiation, thereby enhancing the visualization of images such as blood flow dynamics and organ structure. With the continuous development of modern medical diagnostic technology, ultrasound contrast technology has occupied an important position in clinical diagnosis. Ultrasound contrast agents can enhance ultrasound scattering signals and improve image resolution and contrast, thereby helping doctors to detect and diagnose diseases more accurately, especially in the diagnosis and treatment monitoring of various diseases such as tumors and cardiovascular diseases. It has broad application prospects.

[0004] However, there are some problems with current ultrasound contrast agents. On the one hand, existing ultrasound contrast agents may cause a variety of side effects. Some components in traditional contrast agents may trigger an immune response in the body. For example, some patients may experience allergic reactions after using contrast agents, ranging from mild skin itching and rash to severe anaphylactic shock, which brings additional health risks to patients. In addition, some contrast agents may damage important organs such as the liver and kidneys during metabolism in the body, affecting the normal function of these organs. Especially for patients whose organ functions are already damaged to a certain extent, this potential hazard may further aggravate the condition. On the other hand, the stability of existing ultrasound contrast agents is also insufficient. During storage, some contrast agents are prone to aggregation, decomposition, etc., resulting in a decrease in their performance. During circulation in the body, some contrast agents have poor stability and may rupture prematurely or lose their contrast effect, and cannot accurately and continuously develop at the target site, affecting the accuracy of diagnosis.

[0005] The Chinese patent with publication number CN105833302A discloses a contrast agent and a method for making the same, which comprises, by weight: 2-4 parts of diphenylphosphoazide, 8-12 parts of dipalmitoylphosphatidylcholine, 3-5 parts of phospholipids, 2.5-3.5 parts of cholesterol, 1.5-2.5 parts of melanin and 8-15 parts of perfluoropropane, wherein the phospholipids are PEGylated phospholipids. The Chinese patent with publication number CN110639032A discloses a high-frequency ultrasound contrast agent, which is composed of a lipid shell and a gas core, wherein the lipid shell is composed of phospholipids, a stabilizer and sodium alginate, wherein the surface of the lipid shell is connected with superparamagnetic particles through an amide bond, wherein the phospholipids are one or more of carboxylated DSPC, DPPC, DSPE, DPPE and DSPE-PEG2000, and wherein the stabilizer is one or more of PEG4000, F68 and PEG40s. However, the problems existing in the above-mentioned ultrasound contrast agents have not been well solved. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the present invention provides a stable ultrasound contrast agent with low toxicity and side effects, which is composed of 2 parts by weight of perfluorooctane, 2 to 5 parts by weight of phospholipids, 0.5 to 1.5 parts by weight of cholesterol, 0.2 to 0.8 parts by weight of cocamidopropylamine oxide, and 0.2 to 0.8 parts by weight of dithiothreitol.

[0007] Furthermore, the phospholipid is selected from one or more of phosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylethanolamine, and sphingomyelin; preferably dipalmitoylphosphatidylcholine and sphingomyelin; more preferably, the mass ratio of dipalmitoylphosphatidylcholine and sphingomyelin is 1 to 3:1.

[0008] The preparation method of the ultrasound contrast agent with low side effects described in the present invention is:

[0009] A: Dissolve perfluorooctane, phospholipids and cholesterol in an appropriate amount of organic solvent, heat and rotary evaporate in a water bath at 30℃±5℃ to form a liposome film;

[0010] B: Dissolve cocamidopropylamine oxide and dithiothreitol in a buffer solution, add it to the film in step A to hydrate it, and continue to rotary evaporate to remove 1 / 3 of the volume of the solution;

[0011] C: High-pressure homogenization (pressure is 300-800 bar, time is 3-5 minutes, cycle 3-5 times), microfiltration, and you are done.

[0012] The organic solvent is a mixed solvent of chloroform and ethanol in a volume ratio of 4 to 6:1; the pH of the buffer solution is 4.5 to 6.5; and the buffer solution is selected from one of citric acid-sodium citrate buffer solution, lactic acid-sodium lactate buffer solution, and phosphoric acid-disodium hydrogen phosphate buffer solution.

[0013] Compared with the prior art, the technical effects of the present invention are:

[0014] The present invention utilizes liposome technology to encapsulate perfluorooctane to form an ultrasound contrast agent. The stability and drug loading of the ultrasound contrast agent are improved by optimizing the liposome material, ensuring that sufficient imaging substances are stably present in the contrast agent, so that the contrast between the tissue and the contrast agent in the ultrasound image is more obvious. In addition, the toxic and side effects of the ultrasound contrast agent of the present invention and the commercially available ultrasound contrast agent are compared through rat experiments. The experimental data show that after the rats are injected with the ultrasound contrast agent of the present invention, no death occurs, and the body temperature changes are within the normal range, indicating that the ultrasound contrast agent of the present invention has no serious adverse effects on the body temperature regulation mechanism of the rats, and there are no obvious pathological changes in the organs after dissection, and the acute toxicity of the ultrasound contrast agent of the present invention is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Drug loading and encapsulation efficiency of ultrasound contrast agents of Examples 1 to 5 and Comparative Examples 1 to 4.

[0016] Figure 2 : Drug loading and encapsulation efficiency of ultrasound contrast agents in each group.

[0017] Figure 3 : Statistics of poisoning symptoms in rats in each group.

[0018] Figure 4 : Changes of body temperature of rats in each group. DETAILED DESCRIPTION

[0019] In order to make the purpose and technical solution of the present invention clearer, the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0020] Example 1 Ultrasound contrast agent

[0021] formula:

[0022]

[0023] Preparation method:

[0024] A: Dissolve perfluorooctane, phospholipids and cholesterol in a mixed solvent of chloroform and ethanol in a volume ratio of 5:1, heat in a water bath at 30℃±5℃ and perform rotary evaporation to form a liposome film;

[0025] B: Dissolve cocamidopropylamine oxide and dithiothreitol in a phosphoric acid-disodium hydrogen phosphate buffer solution at pH 5.5, add it to the film in step A for hydration, and continue to rotary evaporation to remove 1 / 3 of the volume of the solution;

[0026] C: High pressure emulsification, microfiltration, and you are done.

[0027] Example 2 Ultrasound contrast agent

[0028] formula:

[0029]

[0030] Preparation method:

[0031] A: Dissolve perfluorooctane, phospholipids and cholesterol in a mixed solvent of chloroform and ethanol in a volume ratio of 4:1, heat in a water bath at 30℃±5℃ and perform rotary evaporation to form a liposome film;

[0032] B: Dissolve cocamidopropylamine oxide and dithiothreitol in a phosphoric acid-disodium hydrogen phosphate buffer solution at pH 5.5, add it to the film in step A for hydration, and continue to rotary evaporation to remove 1 / 3 of the volume of the solution;

[0033] C: High pressure emulsification, microfiltration, and you are done.

[0034] Example 3 Ultrasound contrast agent

[0035] formula:

[0036]

[0037] Preparation method:

[0038] A: Dissolve perfluorooctane, phospholipids and cholesterol in a mixed solvent of chloroform and ethanol in a volume ratio of 6:1, heat in a water bath at 30℃±5℃ and perform rotary evaporation to form a liposome film;

[0039] B: Dissolve cocamidopropylamine oxide and dithiothreitol in a phosphoric acid-disodium hydrogen phosphate buffer solution at pH 5.5, add it to the film in step A for hydration, and continue to rotary evaporation to remove 1 / 3 of the volume of the solution;

[0040] C: High pressure emulsification, microfiltration, and you are done.

[0041] Example 4 Ultrasound Contrast Agent

[0042] formula:

[0043]

[0044] Preparation method:

[0045] A: Dissolve perfluorooctane, phospholipids and cholesterol in a mixed solvent of chloroform and ethanol in a volume ratio of 5:1, heat in a water bath at 30℃±5℃ and perform rotary evaporation to form a liposome film;

[0046] B: Dissolve cocamidopropylamine oxide and dithiothreitol in a citric acid-sodium citrate buffer solution at pH 4.5, add it to the film in step A for hydration, and continue to rotary evaporate to remove 1 / 3 volume of the solution;

[0047] C: High pressure emulsification, microfiltration, and you are done.

[0048] Example 5 Ultrasound Contrast Agent

[0049] formula:

[0050]

[0051] Preparation method:

[0052] A: Dissolve perfluorooctane, phospholipids and cholesterol in a mixed solvent of chloroform and ethanol in a volume ratio of 5:1, heat in a water bath at 30℃±5℃ and perform rotary evaporation to form a liposome film;

[0053] B: Dissolve cocamidopropylamine oxide and dithiothreitol in a pH 6.5 lactic acid-sodium lactate buffer solution, add it to the film in step A for hydration, and continue to rotary evaporate to remove 1 / 3 of the volume of the solution;

[0054] C: High pressure emulsification, microfiltration, and you are done.

[0055] Comparative Example 1 Ultrasound Contrast Agent

[0056] formula:

[0057]

[0058] Preparation method:

[0059] A: Dissolve perfluorooctane, phospholipids and cholesterol in a mixed solvent of chloroform and ethanol in a volume ratio of 5:1, heat in a water bath at 30℃±5℃ and perform rotary evaporation to form a liposome film;

[0060] B: Dissolve dithiothreitol in a phosphoric acid-disodium hydrogen phosphate buffer solution at pH 5.5, add it to the film in step A to hydrate it, and continue to rotary evaporate to remove 1 / 3 of the volume of the solution;

[0061] C: High pressure emulsification, microfiltration, and you are done.

[0062] Comparative Example 2 Ultrasound Contrast Agent

[0063] formula:

[0064]

[0065] Preparation method:

[0066] A: Dissolve perfluorooctane, phospholipids and cholesterol in a mixed solvent of chloroform and ethanol in a volume ratio of 5:1, heat in a water bath at 30℃±5℃ and perform rotary evaporation to form a liposome film;

[0067] B: dissolve dodecyl dimethylamine oxide and dithiothreitol in a phosphoric acid-disodium hydrogen phosphate buffer solution at pH 5.5, add it to the film in step A for hydration, and continue to remove 1 / 3 of the volume of the solution by rotary evaporation;

[0068] C: High pressure emulsification, microfiltration, and you are done.

[0069] Comparative Example 3 Ultrasound Contrast Agent

[0070] formula:

[0071]

[0072] Preparation method:

[0073] A: Dissolve perfluorooctane, phospholipids and cholesterol in a mixed solvent of chloroform and ethanol in a volume ratio of 5:1, heat in a water bath at 30℃±5℃ and perform rotary evaporation to form a liposome film;

[0074] B: Dissolve cocamidopropylamine oxide in a phosphoric acid-disodium hydrogen phosphate buffer solution at pH 5.5, add it to the film in step A for hydration, and continue to rotary evaporate to remove 1 / 3 of the volume of the solution;

[0075] C: High pressure emulsification, microfiltration, and you are done.

[0076] Comparative Example 4 Ultrasound Contrast Agent

[0077] formula:

[0078]

[0079] Preparation method:

[0080] A: Dissolve perfluorooctane, phospholipids and cholesterol in a mixed solvent of chloroform and ethanol in a volume ratio of 5:1, heat in a water bath at 30℃±5℃ and perform rotary evaporation to form a liposome film;

[0081] B: Dissolve cocamidopropylamine oxide and dithiothreitol in a phosphoric acid-disodium hydrogen phosphate buffer solution at pH 5.5, add it to the film in step A for hydration, and continue to rotary evaporation to remove 1 / 3 of the volume of the solution;

[0082] C: High pressure emulsification, microfiltration, and you are done.

[0083] Drug loading and encapsulation efficiency of ultrasound contrast agents

[0084] Drug loading refers to the ratio of the mass of the drug loaded in the ultrasound contrast agent to the total mass of the contrast agent, usually expressed as a percentage. The calculation formula is: drug loading = drug mass / total mass of contrast agent × 100%.

[0085] The encapsulation rate refers to the ratio of the amount of drug encapsulated in the ultrasound contrast agent to the total amount of drug input, and the calculation formula is: encapsulation rate = encapsulated drug amount / total amount of drug input × 100%.

[0086] Figure 1 The drug loading and encapsulation efficiency of the ultrasound contrast agents of Examples 1 to 5 and Comparative Examples 1 to 4 are shown. It shows that the drug loading and encapsulation efficiency of the ultrasound contrast agents of Examples 1 to 5 of the present invention are relatively high, which means that more imaging substance perfluorooctane can be carried, ensuring that sufficient imaging substance exists stably in the contrast agent, making the contrast between the tissue and the contrast agent in the ultrasound image more obvious.

[0087] Particle size of ultrasound contrast agents

[0088] The ultrasound contrast agents of Examples 1 to 5 and Comparative Examples 1 to 4 were placed at a temperature of 40±2° C. and a relative humidity of 75%±5% for 30 days, and the particle sizes before and after the test were measured.

[0089] Table 1 Particle size of ultrasound contrast agents

[0090]

[0091] The particle size and standard deviation of the ultrasound contrast agents in Examples 1 to 5 of the present invention do not change significantly before and after placement, indicating that no obvious aggregation or fusion occurs during the storage process of the contrast agent. When the contrast agent is used in the ultrasound imaging process, its scattering and reflection characteristics in the ultrasound field are basically the same, and a stable enhanced signal can be provided, with stable imaging effects, high repeatability and reliability.

[0092] Single factor experimental study

[0093] In the process of screening solvents, it was found that not all solvents are suitable for this technical solution. The other parameters in the technical solution of Example 1 were used as quantitative parameters, and the variables were the type and ratio of organic solvents. Suitable organic solvents were screened by the drug loading and encapsulation rate of the ultrasound contrast agent.

[0094] A: Chloroform

[0095] B: Chloroform and ethanol in a volume ratio of 1:1

[0096] C: Chloroform and ethanol in a volume ratio of 2:1

[0097] D: Chloroform and methanol in a volume ratio of 5:1

[0098] E: Dichloromethane

[0099] Figure 2 The drug loading and encapsulation efficiency of each group of ultrasound contrast agents. In the experiment comparing the effects of different organic solvents on the drug loading of liposomes, it was found that chloroform can better encapsulate fat-soluble drugs in liposomes to a certain extent, because it can provide an environment conducive to the interaction between drugs and lipids, allowing the drugs to be embedded in the bilayer membrane of liposomes, thereby increasing the drug loading and encapsulation efficiency. However, the experiment found that the mixed solvent of chloroform and ethanol is more effective. Chloroform can interact with the fatty acid chain part, and ethanol can interact with the polar head, so that the drug is better dispersed in the solvent, increasing the chance of contact between the drug and the liposome material, which is beneficial to increase the drug loading and encapsulation efficiency.

[0100] Animal experiments

[0101] Acute toxicity test:

[0102] Experimental principle: By giving animals a high dose of ultrasound contrast agent in a short period of time, the side effects of the animals are observed.

[0103] Experimental procedures: SD rats, SPF grade, 180-220g, experimental animal license number: SYXK (Lu)

[0104] 20180008, randomly divided into Example 1 group (0.5ml, 2.0ml, 4.0ml), Example 2 group (0.5ml, 2.0ml, 4.0ml), Example 3 group (0.5ml, 2.0ml, 4.0ml), Example 4 group (0.5ml, 2.0ml, 4.0ml), Example 5 group (0.5ml, 2.0ml, 4.0ml), commercial group (0.5ml, 2.0ml, 4.0ml), blank group (injected with 0.5ml, 2.0ml, 4.0ml normal saline), 24 rats in each group. Different doses of ultrasound contrast agent were given by intravenous injection, and the symptoms of the animals after injection, such as activity status, respiratory rate, diet, etc., were recorded and observed for 14 days. Commercially available perfluorohexane liposome-micron ultrasound contrast agent was purchased from Xi'an Qiyue Biology.

[0105] Rat mortality

[0106] Table 2 Rat mortality rate (%)

[0107]

[0108] Statistics of rat poisoning symptoms

[0109] The poisoning symptoms included in the statistics include: allergic reaction, arrhythmia, blood pressure changes, wheezing, dyspnea, oliguria or anuria, and unsteady walking. The above symptoms are scored as 1 point. The cumulative score of each group of rats is divided by the number of rats for statistical analysis ( Figure 3 ).

[0110] During the experiment, rats in Example 1 group (0.5ml, 2.0ml, 4.0ml), Example 2 group (0.5ml, 2.0ml, 4.0ml), and Example 3 group (0.5ml, 2.0ml, 4.0ml) did not show obvious toxic symptoms, only a few rats showed mild symptoms (heart rate changes, unstable movements), no deaths occurred, and there were no obvious pathological changes in various organs after dissection, indicating that the acute toxicity of the ultrasound contrast agent of the present invention is low. The poisoning symptoms of the ultrasound contrast agents of Examples 2 to 5 are similar to those of the Example 1 group.

[0111] Side Effects - Temperature Change Statistics

[0112] Within 24 hours after intravenous injection, the body temperature of rats in each group with a dose of 4.0 ml was measured using a thermometer to observe the changes in body temperature ( Figure 4 ). As shown in the blank group, the body temperature of normal rats usually fluctuates within a certain range. After the injection of ultrasound contrast agent, the body temperature of rats in groups 1 to 5 of Examples 1 to 5 changed within the normal range, and no obvious increase or decrease in body temperature occurred, indicating that the ultrasound contrast agent of the present invention did not have a serious adverse effect on the temperature regulation mechanism of rats.

[0113] It should be noted that the present invention conducted a long-term toxicity experiment, that is, a low dose of ultrasound contrast agent was given to animals for a long time to observe its long-term effects on the physiological functions and tissues and organs of the animals. The results showed that there were no significant differences in various indicators of rats in each group of the present invention compared with those in the control group, and no obvious lesions were found in the pathological examination of the organs, indicating that the long-term toxicity of the ultrasound contrast agent of the present invention is low.

Claims

1. An ultrasound contrast agent with low side effects, characterized in that: The ultrasound contrast agent consists of 2 parts by weight of perfluorooctane, 2-5 parts by weight of phospholipids, 0.5-1.5 parts by weight of cholesterol, 0.2-0.8 parts by weight of cocamidopropylamine oxide, and 0.2-0.8 parts by weight of dithiothreitol.

2. The ultrasound contrast agent with low side effects according to claim 1, characterized in that: The phospholipid is selected from one or more of phosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylethanolamine and sphingomyelin.

3. The ultrasound contrast agent with low side effects according to claim 1, characterized in that: The phospholipids are dipalmitoylphosphatidylcholine and sphingomyelin.

4. The ultrasound contrast agent with low side effects according to claim 1, characterized in that: The phospholipids are dipalmitoylphosphatidylcholine and sphingomyelin in a mass ratio of 1 to 3:

1.

5. The ultrasound contrast agent with low side effects according to claim 1, characterized in that: The ultrasound contrast agent is composed of 2 parts by weight of perfluorooctane, 4 parts by weight of phospholipids, 1.0 parts by weight of cholesterol, 0.5 parts by weight of cocamidopropylamine oxide, and 0.5 parts by weight of dithiothreitol.

6. A method for preparing the ultrasound contrast agent with low side effects as claimed in claim 1, characterized in that: The preparation method is: A: Dissolve perfluorooctane, phospholipids and cholesterol in an appropriate amount of organic solvent, heat and rotary evaporate in a water bath at 30℃±5℃ to form a liposome film; B: Dissolve cocamidopropylamine oxide and dithiothreitol in a buffer solution, add it to the film in step A to hydrate it, and continue to rotary evaporate to remove 1 / 3 of the volume of the solution; C: High pressure emulsification, microfiltration, and you are done.

7. The preparation method according to claim 6, characterized in that: The organic solvent is a mixed solvent of chloroform and ethanol in a volume ratio of 4 to 6:

1.

8. The preparation method according to claim 6, characterized in that: The pH of the buffer solution is 4.5-6.

5.

9. The preparation method according to claim 6, characterized in that: The buffer solution is selected from one of citric acid-sodium citrate buffer solution, lactic acid-sodium lactate buffer solution, and phosphoric acid-disodium hydrogen phosphate buffer solution.

10. The preparation method according to claim 6, characterized in that: The organic solvent is a mixed solvent of chloroform and ethanol in a volume ratio of 5:1.

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