Ultrasound contrast agent and method for its preparation

Abstract

The application belongs to the technical field of biological medicine, and discloses an ultrasonic contrast agent and a preparation method thereof.The ultrasonic contrast agent comprises DPPA liposomes and gas wrapped in the DPPA liposomes; and the particle size of the ultrasonic contrast agent is 150-300 nm.The ultrasonic contrast agent provided by the application has few components, simple preparation process and short time consumption.The ultrasonic contrast agent is prepared by using the excellent carrier capacity of DPPA liposomes, adopting a nano precipitation method combined with ultrasonic oscillation method.The ultrasonic contrast agent has small size, can reach the nanometer level, can achieve good ultrasonic imaging effect at a low concentration, has excellent ultrasonic imaging effect, strong stability and high clinical application value.In addition, the ultrasonic contrast agent provided by the application has rapid cell uptake in vitro, deep tumor penetration and retention characteristics in vivo, has ultrasonic contrast capability, and has biological therapeutic activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an ultrasound contrast agent and its preparation method. Background Technology

[0002] Ultrasound contrast agents are liquids containing air bubbles a few micrometers in diameter. Utilizing the strong scattering property of air-bubbly liquids on ultrasound waves, these agents are injected into blood vessels in clinical practice to enhance the Doppler ultrasound signal of blood flow and improve the clarity and resolution of ultrasound images.

[0003] Contrast agents are primarily classified based on the type of gas encapsulated within the microbubbles. First-generation contrast agents contain air within the microbubbles, with the encapsulation typically consisting of polymers such as albumin or galactose. The physical characteristics of first-generation ultrasound contrast agents, including a thicker encapsulation, poor elasticity, and the high water solubility of the encapsulated air, limit their duration of action and susceptibility to rupture, thus restricting observation and diagnostic time in clinical applications. Second-generation ultrasound contrast agents consist of thin, flexible bubbles primarily encapsulating high-density inert gases (not easily soluble in water or blood), with diameters generally around 2-5 μm, exhibiting better vibration and echo characteristics. While ultrasound contrast agents have undergone several generations of evolution, most current ultrasound contrast agents still have micron-sized particles and require multiple components for synthesis, making the process relatively complex. Furthermore, most ultrasound contrast agents require high concentrations for use, exhibit poor stability, and have short continuous imaging times. This results in poor actual clinical efficacy and weak clinical translation capabilities for existing ultrasound contrast agents.

[0004] Therefore, there is an urgent need to provide an ultrasound contrast agent that has a small size and good stability, and can achieve good ultrasound imaging results at a low concentration, thus having high clinical application value. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes an ultrasound contrast agent and its preparation method. The ultrasound contrast agent prepared by the present invention has small size and good stability, and achieves good ultrasound imaging effects at low concentrations, thus having high clinical application value.

[0006] This invention provides an ultrasound contrast agent.

[0007] Specifically, an ultrasound contrast agent includes DPPA liposomes and a gas encapsulated within the DPPA liposomes; the particle size of the ultrasound contrast agent is 150-300 nm.

[0008] DPPA liposomes are synthesized by self-assembly of dipalmitoyl phosphatidyl acid (DPPA) in a solvent. The solvent is preferably water.

[0009] Preferably, the particle size of the ultrasound contrast agent is 150-250 nm.

[0010] Preferably, the gas is perfluorocarbon gas and / or sulfur fluoride gas; more preferably, the gas is perfluoropropane and / or sulfur hexafluoride. Octafluoropropane (C3F8) is chemically very stable, and its combination with DPPA liposomes is beneficial for improving ultrasound contrast imaging.

[0011] This invention provides a method for preparing an ultrasound contrast agent.

[0012] Specifically, a method for preparing an ultrasound contrast agent includes the following steps:

[0013] DPPA liposomes (NPs-DPPA) were synthesized by self-assembly via nanoprecipitation and then subjected to ultrasonic oscillation with gas to prepare an ultrasound contrast agent.

[0014] Preferably, the method for preparing the ultrasound contrast agent includes the following steps:

[0015] (1) Dissolve dipalmitoyl phosphatidyl acid (DPPA) in a low molecular weight alcohol, then add it dropwise to water, stir, and then purify to obtain DPPA liposome solution;

[0016] (2) Under ultrasonic oscillation, gas is introduced into the DPPA liposome solution to obtain a turbid, milky-white ultrasound contrast agent.

[0017] Preferably, in step (1), the low molecular weight alcohol is at least one of methanol, ethanol, n-butanol, or isopropanol; more preferably, the low molecular weight alcohol is methanol. Methanol not only dissolves dipalmitoyl phosphatidyl acid (DPPA) better, but also improves the uniformity of gas within DPPA liposomes, which is beneficial for reducing the size of ultrasound contrast agents and improving their stability.

[0018] Preferably, in step (1), the concentration of the DPPA liposome solution is 0.5-5 mg / mL. If the concentration of the DPPA liposome solution is too high, it will cause "overexposure" in the imaging effect.

[0019] Preferably, in step (1), the purification process is carried out in a filter, and the filtrate is removed by centrifugation to obtain the retentate, wherein the pore size of the filter is 100 kDa MW.

[0020] Preferably, in step (2), the volume ratio of the DPPA liposome solution to the gas is 1:(5-10); more preferably, the volume ratio of the DPPA liposome solution to the gas is 1:(6-10).

[0021] Preferably, in step (2), the power of the ultrasonic oscillation is 80-120W and the duration of the ultrasonic oscillation is 20-90s; more preferably, in step (2), the power of the ultrasonic oscillation is 90-110W and the duration of the ultrasonic oscillation is 30-60s.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) The ultrasound contrast agent provided by the present invention has few components, a simple preparation process, and a short preparation time. In the preparation process, dipalmitoyl phosphatidic acid (DPPA) is first self-assembled into DPPA liposomes (NPs-DPPA) by nanoprecipitation, and then combined with ultrasonic oscillation to encapsulate active gas, so that the prepared ultrasound contrast agent has no extra components other than DPPA liposomes and contrast gas.

[0024] (2) This invention utilizes the excellent carrier capacity of DPPA liposomes (NPs-DPPA) to prepare an ultrasound contrast agent using a combination of nanoprecipitation and ultrasonic oscillation. It is not only small in size, reaching the nanoscale, but also achieves good ultrasound imaging results at low concentrations, exhibiting excellent ultrasound imaging performance, strong stability, and high clinical application value.

[0025] (3) The ultrasound contrast agent provided by the present invention has rapid cell uptake in vitro and deep tumor penetration and preservation characteristics in vivo. It not only has ultrasound contrast capability, but also has biological therapeutic activity. Attached Figure Description

[0026] Figure 1 The image shows the appearance of the NPs-DPPA(C3F8) ultrasound contrast agent prepared according to an embodiment of the present invention.

[0027] Figure 2 This is a transmission electron microscope image of the NPs-DPPA(C3F8) ultrasound contrast agent prepared according to an embodiment of the present invention;

[0028] Figure 3 The particle size distribution diagram of the NPs-DPPA(C3F8) ultrasound contrast agent prepared in the embodiments of the present invention is shown.

[0029] Figure 4 The image shows the zeta potential distribution of the NPs-DPPA(C3F8) ultrasound contrast agent prepared according to an embodiment of the present invention.

[0030] Figure 5 This is a graph showing the particle size change of NPs-DPPA(C3F8) ultrasound contrast agent during placement.

[0031] Figure 6 In vitro ultrasound imaging results of ultrasound contrast agents of different concentrations of NPs-DPPA (C3F8);

[0032] Figure 7 In vitro ultrasound imaging results of NPs-DPPA(C3F8) ultrasound contrast agent at different dilution ratios;

[0033] Figure 8 Images of in vitro ultrasound imaging results of NPs-DPPA(C3F8) ultrasound contrast agent after being placed at 4°C for different numbers of days.

[0034] Figure 9 In vivo ultrasound imaging of a mouse subcutaneous tumor model after injection of NPs-DPPA (C3F8) ultrasound contrast agent;

[0035] Figure 10 Image showing cellular uptake of NPs-DPPA (C3F8) ultrasound contrast agent;

[0036] Figure 11 Image showing the size of the tumor after treatment with NPs-DPPA (C3F8) ultrasound contrast agent;

[0037] Figure 12 This is a statistical chart of tumor weight after treatment with NPs-DPPA (C3F8) ultrasound contrast agent. Detailed Implementation

[0038] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0039] In the following examples, C3F8 was purchased from Guangzhou Wolter Biotech Co., Ltd. (Guangzhou, China), 1,2-dipalmitoyltin glycerol 3-phosphate (DPPA) was purchased from Xi'an Ruixi Biotechnology Co., Ltd. (Xi'an, China), and methanol was purchased from Thermo Fisher Scientific (Massachusetts, USA). Unless otherwise specified, all other raw materials, reagents, or apparatus used were obtained from conventional commercial sources or by existing known methods.

[0040] Example

[0041] 1. Synthesis of NPs-DPPA (C3F8) ultrasound contrast agent

[0042] DPPA (20 mg / mL) was dissolved in methanol in a 65°C water bath and used fresh. Mixtures with final concentrations of 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, and 10 mg / mL were prepared (synthetic formulations are shown in Table 1). Then, the mixtures of each concentration were slowly added dropwise to glass vials containing 10 mL of water, continuously stirred at 1200 rpm, and kept at a constant temperature of 65°C. After maintaining the mixture under continuous stirring for 2 minutes, all solutions in the glass vials were transferred to an Amicon filter (100 kDa MW) for purification. After centrifugation, the NPs-DPPA remaining in the filter was aspirated and diluted to a final volume in sterile water. Prepare NPs-DPPA solutions with concentrations of 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, and 10 mg / mL, each with a total volume of 5 mL. Use a probe-type sonicator (Sonics Vibra-Cell, USA) at 75% power (total power: 130 W, frequency: 20 kHz) for continuous sonication for 45 seconds while simultaneously introducing 40 mL of C3F8 gas. This process yields ultrasound contrast agents of different liposome concentrations. Each ultrasound contrast agent is a turbid, milky-white liquid and is stored at 4°C for later use.

[0043] Table 1. Synthesis formulation of NPs-DPPA

[0044]

[0045] 2. Characterization of NPs-DPPA (C3F8) ultrasound contrast agent

[0046] The synthesized NPs-DPPA(C3F8) ultrasound contrast agent (liposome concentration of 3 mg / mL) was observed to have a cloudy, milky white appearance (as shown in the image). Figure 1 (As shown), the NPs-DPPA (C3F8) ultrasound contrast agent was then analyzed using transmission electron microscopy (TEM). 10 μL of fresh sample was dropped onto a copper grid and allowed to stand for 3 minutes. Then, the sample was blotted with filter paper to remove excess sample. Subsequently, 6 μL of 2% uranium acetate was added for negative staining for 3 minutes, followed by blotting with filter paper to remove excess sample. The sample was then allowed to air dry overnight in the dark. Observation using TEM revealed a diameter of approximately 200 nm (e.g., ...). Figure 2 (As shown). We also used a Malvern particle size analyzer (DLS) to detect the NPs-DPPA (C3F8) ultrasound contrast agent, and the particle size was approximately 220 nm (as shown). Figure 3 As shown), the potential is approximately -48.7mV (as shown). Figure 4(As shown). To ensure the usability of NPs-DPPA(C3F8) ultrasound contrast agent in clinical and biomedical applications, the long-term stability of NPs-DPPA(C3F8) ultrasound contrast agent at 4°C was further tested (from day 1, day 3, day 5, and day 7, respectively). The test results (as shown) Figure 5 As shown in the figure, DLS data analysis of NPs-DPPA(C3F8) ultrasound contrast agent indicates that the particle size of NPs-DPPA(C3F8) ultrasound contrast agent remained stable at around 200 nm within 7 days. These results suggest that NPs-DPPA(C3F8) ultrasound contrast agent can be used in a wide range of clinical and biomedical applications.

[0047] 3. In vitro imaging test of NPs-DPPA (C3F8) ultrasound contrast agent

[0048] Different concentrations (1 mL) of NPs-DPPA (C3F8) ultrasound contrast agent suspensions were prepared and placed in 1% agarose gels for in vitro ultrasound imaging. In both standard B-mode and contrast-enhanced modes, an ultrasound machine (V2100) with an MS400 probe was used. The main parameters were set with a mechanical index (MI) of 1.30 and a transducer frequency of 18 MHz. Ultrasound images were recorded at concentrations of 0 mg / mL, 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, and 10 mg / mL of the NPs-DPPA (C3F8) ultrasound contrast agent suspension. The in vitro imaging results are as follows: Figure 6 As shown, by Figure 6 It can be seen that NPs-DPPA(C3F8) ultrasound contrast agent at different concentrations exhibits good imaging effects in both conventional B mode and contrast-enhanced ultrasound (CEUS) mode. The acoustic signal of NPs-DPPA(C3F8) ultrasound contrast agent in both conventional B mode and CEUS mode increases with increasing concentration. It is noteworthy that when the concentration of NPs-DPPA(C3F8) ultrasound contrast agent reaches 5 mg / mL or higher, an "overexposure" phenomenon occurs in the imaging effect, which is due to the excessively high concentration of NPs-DPPA(C3F8) ultrasound contrast agent. To further support this argument, a 5 mg / mL concentration of NPs-DPPA(C3F8) ultrasound contrast agent was further diluted by 10 times, 100 times, 1000 times, 10000 times, and 100000 times, respectively, and then imaging tests were performed. The test results after dilution are as follows... Figure 7 As shown. By Figure 7 It can be seen that even when the NPs-DPPA(C3F8) ultrasound contrast agent is diluted 10,000 times, it still has very good ultrasound imaging effect in vitro.

[0049] To better evaluate the clinical translational potential and application value of NPs-DPPA (C3F8) ultrasound contrast agent, the stability of its long-term in vitro imaging capability was further assessed. The in vitro ultrasound imaging capability and stability of NPs-DPPA (C3F8) ultrasound contrast agent at a concentration of 3 mg / mL were evaluated on days 1, 3, 5, 7, 15, and 30. The evaluation results are as follows: Figure 8 As shown. By Figure 8 It is evident that the NPs-DPPA(C3F8) ultrasound contrast agent still possesses excellent in vitro imaging capabilities even on day 30. In summary, the NPs-DPPA(C3F8) ultrasound contrast agent synthesized in this invention not only exhibits superior imaging performance but also achieves this effect with only a low concentration, demonstrating high cost-effectiveness and clinical translation potential.

[0050] 4. In vivo imaging test of NPs-DPPA (C3F8) ultrasound contrast agent

[0051] One × 10⁶ 4T1 cells were suspended in 100 μL PBS and subcutaneously injected into Balb / c mice (4-5 weeks) to establish a triple-negative breast cancer xenograft model. Immediately afterwards, 100 μL of NPs-DPPA (C3F8) ultrasound contrast agent was injected into the tumor-bearing mice via the tail vein. Ultrasound imaging of the tumor site was observed at 5, 10, 30, and 60 minutes. Imaging results are shown below. Figure 9 As shown, the control group (Control) consists of ultrasound images of tumor sites without the injection of ultrasound contrast agent. Figure 9 It can be seen that mice exhibit good ultrasound contrast effects after injection of NPs-DPPA(C3F8) ultrasound contrast agent, with weak ultrasound imaging remaining even after 60 minutes. This also proves that the NPs-DPPA(C3F8) ultrasound contrast agent synthesized in this invention can remain in vivo for a relatively long time for ultrasound contrast imaging. Therefore, both in vivo and in vitro imaging tests verify that the NPs-DPPA(C3F8) ultrasound contrast agent synthesized in this invention has good ultrasound contrast capabilities, providing a solid foundation for future clinical translation.

[0052] 5. In vitro cell uptake assay of NPs-DPPA (C3F8) ultrasound contrast agent

[0053] First, NPs-DPPA(C3F8)[NPs-DPPA(C3F8)RhoB] loaded with DSPE-PEG-Rhodamine B (a polyethylene glycol-modified phospholipid fluorescent probe) was prepared. The preparation method was the same as that for the synthesis of NPs-DPPA(C3F8) ultrasound contrast agent, except that 25 μL of DSPE-PEG-Rhodamine B (concentration of 10 mg / mL) was added at a ratio of 1 mg DPPA to NPs-DPPA. 4T1 cells were seeded in glass-bottomed cell culture dishes. Nest (Wuxi, China). After 24 hours of cell adhesion, 50 μL of synthesized NPs-DPPA(C3F8)RhoB was added. After incubation at 37°C for 4 hours, the samples were washed three times with PBS, fixed with 4% (w / v) paraformaldehyde (PFA), and the nuclei and cell membranes were negatively stained with DAPI (nuclear dye) and WGA (cell membrane dye). The samples were then imaged using an Olympus Fluorescence View 1000 confocal microscope (Olympus Imaging Corporation, Tokyo, Japan). The NPs-DPPA(C3F8) cell uptake results are shown below. Figure 10 As shown, by Figure 10 It is known that NPs-DPPA(C3F8) ultrasound contrast agent can be well internalized by tumor cells.

[0054] 6. Evaluation of the biotherapeutic activity (antitumor) of NPs-DPPA (C3F8) ultrasound contrast agent

[0055] Cell line: 4T1 mouse breast cancer cell line. Animals: 10 Balb / c mice, 4-6 weeks old, weighing 13-20g, clean grade, purchased from the Animal Center of Sun Yat-sen University (North Campus). Groups: PBS group (5 mice) and NPs-DPPA (C3F8) ultrasound contrast agent treatment group (5 mice). 1×10 6 4T1 cells were suspended in 100 μL PBS and subcutaneously injected into Balb / c mice (4-6 weeks) to establish a triple-negative breast cancer xenograft model. The mice were allowed to develop a xenograft model when the subcutaneous tumor reached 100 mm in size. 3 At approximately 10:00 AM, mice were randomly divided into a PBS group (n=5 mice) and an NPs-DPPA (C3F8) ultrasound contrast agent treatment group (n=5 mice). The mice received three injections every other day, with tumor volume and mouse weight recorded every other day, until the tumor in the PBS group reached 1500 mm. 3 All mice were euthanized, and the tumors were dissected and their size and weight measured and recorded. Results are as follows: Figure 11 and Figure 12 As shown. By Figure 11 and Figure 12 It is known that the NPs-DPPA(C3F8) ultrasound contrast agent provided by the present invention has anti-tumor biological activity.

[0056] The NPs-DPPA(C3F8) ultrasound contrast agent provided by this invention possesses nanoscale dimensions and excellent stability, enabling it to be readily endocytosed by tumor cells. It exhibits excellent ultrasound imaging performance both in vivo and in vitro, and maintains stable imaging over a certain period. More importantly, this NPs-DPPA(C3F8) ultrasound contrast agent not only provides in vivo ultrasound imaging but also possesses biotherapeutic activity, exerting an anti-tumor growth effect. Furthermore, the NPs-DPPA(C3F8) ultrasound contrast agent provided by this invention has a simple manufacturing process, uses very few types of materials and requires minimal dosage, making it highly cost-effective and suitable for clinical translation.

Claims

1. An ultrasound contrast agent, characterized in that, It includes DPPA liposomes and gas encapsulated within the DPPA liposomes; the particle size of the ultrasound contrast agent is 150-300 nm; The preparation method of the ultrasound contrast agent includes the following steps: (1) Dissolve dipalmitoylphosphatidyl acid in a low molecular weight alcohol, then add it dropwise to water, stir, and then purify to obtain DPPA liposome solution; the low molecular weight alcohol is at least one of methanol, ethanol, n-butanol or isopropanol; (2) Under ultrasonic oscillation, a gas is introduced into the DPPA liposome solution to obtain a turbid milky white ultrasonic contrast agent; the gas is perfluorinated carbon gas and / or sulfur fluoride gas.

2. The ultrasound contrast agent according to claim 1, characterized in that, The particle size of the ultrasound contrast agent is 150-250 nm.

3. The ultrasound contrast agent according to claim 1, characterized in that, In step (1), the low molecular weight alcohol is methanol.

4. The ultrasound contrast agent according to claim 1, characterized in that, In step (1), the concentration of the DPPA liposome solution is 0.5-5 mg / mL.

5. The ultrasound contrast agent according to claim 1, characterized in that, In step (2), the volume ratio of the DPPA liposome solution to the gas is 1:(5-10).

6. The ultrasound contrast agent according to claim 1, characterized in that, In step (2), the power of the ultrasonic oscillation is 80-120W, and the duration of the ultrasonic oscillation is 20-90s.

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