A method for preparing a nanoscale ultrasound contrast agent based on hydrogen gas

Abstract

The application discloses a preparation method of a nanoscale ultrasonic contrast agent based on hydrogen. The method comprises the following steps: adding DPPA, DSPE-mPEG2000, DPPE and DBPC in a specific ratio into PG, heating at 80 DEG C and ultrasonicating for 8-10 minutes to prepare a lipid solution; mixing glycerol and a PBS buffer in a ratio of 15:10:75 and preheating; adding the preheated mixed solution into the lipid solution, ultrasonicating at room temperature for 8-15 minutes to form a nanobubble suspension; transferring the suspension into a headspace vial, filling with hydrogen for 5 minutes and sealing; oscillating at 200-300 rpm for 30-60 seconds; and finally, centrifuging at 50 rcf for 3-5 minutes for purification. The nanoscale ultrasonic contrast agent prepared by the method has good biocompatibility, high hydrogen encapsulation capacity and stability, has the potential of diagnosis and treatment integration, can be used for improving myocardial injury induced by a chemotherapy drug, and is expected to play a role in the diagnosis and treatment of heart diseases.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically to a method for preparing a hydrogen-based nanoscale ultrasound contrast agent. Background Technology

[0002] Chemotherapy drugs, such as doxorubicin, play a crucial role in cancer treatment. However, these drugs are often accompanied by severe cardiotoxicity, which has become a major factor limiting their clinical application and affecting patient prognosis. Chemotherapy-induced myocardial injury, characterized by excessive oxidative stress and myocardial fibrosis, can lead to various cardiac problems, including heart failure, severely threatening patients' quality of life. Currently, effective prevention and treatment methods for this type of myocardial injury remain limited in clinical practice, and significant challenges exist in early diagnosis and intervention.

[0003] Existing ultrasound contrast agents are mostly micron-sized bubbles. While they can effectively enhance ultrasound signals, their circulation time in the body is short, and they generally lack therapeutic functions, making it difficult to integrate diagnosis and treatment. Furthermore, traditional hydrogen delivery methods, such as inhaling hydrogen or drinking hydrogen-rich water, suffer from problems such as short hydrogen retention time in the body, poor targeting, and difficulty in precisely controlling concentration, limiting their application in the treatment of specific diseases, especially in scenarios requiring localized high concentrations of hydrogen to exert antioxidant and anti-fibrotic effects.

[0004] Therefore, the main problem facing current technology is:

[0005] 1. There is a lack of strategies to effectively prevent and treat chemotherapy-induced myocardial damage.

[0006] 2. There is a lack of a stable, efficient, and targeted hydrogen delivery system to overcome the disadvantages of hydrogen in vivo, such as easy volatility, difficulty in storage, and short circulation time.

[0007] 3. Existing ultrasound contrast agents are usually limited to diagnostic functions and cannot provide therapeutic effects at the same time, making it difficult to achieve "integrated diagnosis and treatment".

[0008] 4. In the existing technology, there are still technical bottlenecks in preparing nanoscale ultrasound contrast agents with good biocompatibility, stability and high hydrogen encapsulation rate.

[0009] Therefore, it is necessary to propose a method for preparing hydrogen-based nanoscale ultrasound contrast agents to solve the above problems. Summary of the Invention

[0010] The purpose of this invention is to address the lack of effective integrated diagnostic and therapeutic methods in the existing technology to deal with chemotherapy drug-induced myocardial damage.

[0011] To achieve the above objectives, the present invention specifically adopts the following technical solution:

[0012] A method for preparing a hydrogen-based nanoscale ultrasound contrast agent includes the following steps:

[0013] a. Preparation of lipid solution: Sodium 1,2-dipalmitoyl-sn-3-phosphate (DPPA), distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), 1,2-dipalmitoyl-sn-3-phosphate ethanolamine (DPPE) and 1,2-bis(eicosicosicosyl-Sn-glycerol-3-phosphatidylcholine) (DBPC) were added to propylene glycol (PG), heated at 80°C and sonicated for 8 to 10 minutes at a frequency of 30 to 40 kHz and a power of 150 to 180 W to completely dissolve the powdered lipid reagents and form a lipid solution;

[0014] b. Preparation of the mixture: Glycerol and PBS buffer are mixed and preheated at 80°C for 5 minutes to obtain the preheated mixture;

[0015] c. Formation of nanobubble suspension: The preheated mixture obtained in step b is added to the lipid solution obtained in step a, and ultrasonic treatment is performed at room temperature for 8 to 15 minutes at an ultrasonic frequency of 30 to 40 kHz and an ultrasonic power of 150 to 180 W to form a milky white nanobubble suspension.

[0016] d. Hydrogen filling and sealing: Transfer the nanobubble suspension obtained in step c to a headspace vial, fill it with hydrogen for 5 minutes, and then seal the vial with a rubber stopper and an aluminum cap;

[0017] e. Shaking treatment: Place the sealed vial obtained in step d in a benchtop constant temperature shaker and shake at a speed of 200 to 300 rpm for 30 to 60 seconds to obtain a preliminary milky white hydrogen nanobubble suspension.

[0018] f. Purification: The preliminary hydrogen nanobubble suspension obtained in step e is transferred to a microcentrifuge tube, centrifuged at 50 rcf for 3 to 5 minutes at room temperature, washed and concentrated to remove unencapsulated liposomes or impurities, and finally a hydrogen-based nanoscale ultrasound contrast agent is obtained.

[0019] Further, in step a, the mass percentage of DBPC is 50% to 70%, the mass percentage of DPPA is 5% to 15%, the mass percentage of DPPE is 10% to 25%, and the mass percentage of DSPE-mPEG2000 is 5% to 15%.

[0020] Furthermore, in step b, the volume ratio of propylene glycol, glycerol, and PBS buffer is 15:10:75.

[0021] Furthermore, in step a, the heating temperature is 80°C.

[0022] Furthermore, in step a, the ultrasonic treatment duration is 8 to 10 minutes, the ultrasonic frequency is 30 to 40 kHz, and the ultrasonic power is 150 to 180 W.

[0023] Furthermore, in step c, the ultrasonic treatment duration is 8 to 15 minutes, the ultrasonic frequency is 30 to 40 kHz, and the ultrasonic power is 150 to 180 W.

[0024] Furthermore, in step d, the hydrogen filling time is 5 minutes.

[0025] Furthermore, in step e, the oscillation speed is 200 to 300 rpm, and the oscillation duration is 30 to 60 seconds.

[0026] Furthermore, in step f, the centrifugation speed is 50 rpm and the centrifugation time is 3 to 5 minutes.

[0027] Furthermore, the hydrogen-based nanoscale ultrasound contrast agent prepared by the method has a core-shell structure and an initial average diameter of approximately 265.1 ± 26 nm.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. This invention employs a method combining self-assembly and ultrasonic treatment, which is simple to operate, easy to implement, and has high preparation efficiency.

[0030] 2. The hydrogen-based nanoscale ultrasound contrast agent prepared in this invention has a uniform nanoscale size with an average diameter of approximately 265.1 ± 26 nm, which is beneficial for its circulation and targeted delivery in vivo. Simultaneously, the selected lipid material exhibits good biocompatibility and low toxicity, meeting the requirements for biomedical applications.

[0031] 3. This invention, through a unique lipid formulation and preparation process, effectively improves the encapsulation efficiency and in vivo stability of hydrogen, significantly prolonging the hydrogen release time and enabling it to exert a sustained therapeutic effect. Compared with ordinary hydrogen water, the nanobubbles prepared by this method have a higher hydrogen loading capacity and a longer hydrogen storage time.

[0032] 4. This hydrogen nanoscale ultrasound contrast agent can inhibit the PI3K / AKT pathway and the TGF-β / Smad pathway, thereby inhibiting oxidative stress and the subsequent pathological process of fibrosis, and exerting a protective effect on cardiomyocytes. Attached Figure Description

[0033] Figure 1 A: Shows the particle size distribution of unfilled hydrogen nanoparticles (a1) and hydrogen-based nanoscale ultrasound contrast agents (HNBs) at different time points (0 min (a2), 30 min (a3), 60 min (a4), 90 min (a5), 120 min (a6)). B: Shows a line graph of particle size variation over time for hydrogen-based nanoscale ultrasound contrast agents (HNBs) and unfilled hydrogen nanoparticles (NBs).

[0034] Figure 2 Image A: Transmission electron microscopy (TEM) image of hydrogen-free nanoparticles, showing their shape and structure, scale bar 200 nm. Image B: Transmission electron microscopy (TEM) image of hydrogen-based nanoscale ultrasonic contrast agents (HNBs), showing their shape and structure, scale bar 200 nm.

[0035] Figure 3 A: Shows a schematic diagram of each titration state when measuring hydrogen content by chemical titration, including the state before titration, after adding methylene blue-platinum reagent, after shaking, and at the reaction endpoint. B: A histogram of hydrogen content for hydrogen-based nanoscale ultrasound contrast agents (HNBs) and hydrogen-water, used to compare their hydrogen loading capacity.

[0036] Figure 4 In the image: AC shows the cell survival rates at 24 hours (A), 48 hours (B), and 72 hours (C) after treatment with different concentrations of hydrogen-based nanoscale ultrasound contrast agents, as determined by the CCK8 assay.

[0037] Figure 5 A: Shows in vivo fluorescence imaging images of mice, including the control group (a1) and images of the hydrogen-based nanoscale ultrasound contrast agent at 0.5 hours (a2), 1 hour (a3), 2 hours (a4), and 4 hours (a5) after injection, used to assess its distribution in mice. B: Shows a line graph of fluorescence intensity in the cardiac region over time.

[0038] Figure 6 In the middle: AC: This shows the expression levels of antioxidant stress-related genes SOD2 (A), GPX-1 (B), and NRF2 (C) after co-incubation of doxorubicin (Dox) with hydrogen-based nanoscale ultrasound contrast agents (HNBs) as detected by RT-qPCR.

[0039] Figure 7 A: Shows a schematic diagram of the doxorubicin (Dox)-induced mouse myocardial injury model. B: Shows representative echocardiographic images of mice used to assess cardiac function.

[0040] Figure 8In the middle: AC: This shows the relative mRNA expression levels of PIK3CA (A), PIK3CB (B), and AKT1 (C) in the PI3K / AKT signaling pathway as detected by RT-qPCR.

[0041] Figure 9 In the middle: AF: This shows the relative mRNA expression levels of TGFB1 (A), TGFBR1 (B), SMAD1 (C), SMAD2 (D), SMAD3 (E) and SMAD4 (F) in the TGF-β / Smad signaling pathway as detected by RT-qPCR.

[0042] Figure 10 In the middle: A: Shows representative Western blot images of the PI3K / AKT signaling pathway. BC: Shows the quantitative analysis results of the p-PI3K / PI3K (B) and p-AKT / AKT (C) ratios. Detailed Implementation

[0043] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] Please see Figures 1 to 10 A method for preparing a hydrogen-based nanoscale ultrasound contrast agent.

[0045] The solvent system should be maintained at a ratio of approximately PG:glycerol:PBS solution = 15:10:75 to ensure appropriate viscosity and membrane toughness.

[0046] The mass ratio range and molar ratio range of each lipid component, as well as their effects on nanobubble stability, hydrogen encapsulation efficiency, and biocompatibility, are as follows:

[0047] Components mass ratio range molar ratio range Impact on stability Effect on hydrogen encapsulation efficiency Impact on biocompatibility DBPC 50%-70% 40%-60% +++(skeleton) +++ (Core Barrier) ++ (Neutral) DPPA 5%-15% 10%-20% ++(charge repulsion) +(Reduce defects) -(negative charge) DPPE 10%-25% 15%-30% +++ (Eliminate defects) ++ (Patching vulnerabilities) - / + (potential immunity) DSPE-mPEG2000 5%-15% 2%-8% +++(Spatial resistance) --(When in excess) +++ (long loop)

[0048] Note: + indicates positive promotion, - indicates negative impact, and the quantity indicates the degree.

[0049] In the preparation, 1,2-dipalmitoyl-sn-3-phosphate sodium salt (DPPA), distearate phosphatidyl ethylamine alcohol-polyethylene glycol 2000 (DSPE-mPEG2000), 1,2-dipalmitoyl-sn-3-phosphate ethanolamine (DPPE) and 1,2-bis(eicosicosicosyl-Sn-glycerol-3-phosphatidylcholine) (DBPC) are first added to propylene glycol (PG), and heated at 80°C while sonicating for 8-10 min (frequency 30-40 kHz, power 150-180 W) to completely dissolve the above powdered lipid reagent.

[0050] After mixing glycerol (Glyphosate) and PBS buffer, preheat the mixture at 80°C for 5 min. Then add the preheated mixture to the lipid solution obtained in the first step and sonicate at room temperature for 8-15 min (frequency 30-40 kHz, power 150-180 W).

[0051] Transfer the 1ml solution obtained in the above steps to a 3ml headspace vial, purge with hydrogen for 5 minutes (using a Medhart hydrogen-oxygen integrated machine), then seal with a rubber stopper and aluminum cap, and finally seal with a vial curler. Use a benchtop thermostatic shaker (25mm track diameter) to shake at 200-300 rpm for 30-60 seconds to obtain a milky white nanobubble suspension. Then, place the micro / nanobubble suspension into a 1.5ml or 2.2ml microcentrifuge tube (EP tube) and centrifuge at 50rcf for 3-5 minutes at room temperature. Wash and concentrate the nanobubbles to remove unencapsulated liposomes or impurities.

[0052] Characterization of hydrogen-based nanoscale ultrasound contrast agents:

[0053] In an in vitro simulated physiological environment stability study, the particle size of HNBs and unfilled hydrogen nanoparticles was monitored using NTA. The results showed that the initial particle size of HNBs was relatively large, with an average diameter of 265.1 ± 26 nm. During continuous observation, the particle size gradually decreased over time. Figure 1 To further illustrate this dynamic process, we plotted a line graph showing the evolution of particle size over time based on data obtained from NTA ( .A). Figure 1 (B) Through this line graph, we can see more clearly that the particle size of HNBs gradually decreases within 2 hours, reflecting the gradual release of hydrogen.

[0054] To delve deeper into the microstructure of HNBs, we used TEM for high-resolution observation of their morphology. The results showed that the hydrogen-free nanoparticles exhibited a uniform, solid spherical structure. Figure 2 In contrast, HNBs exhibit significantly different morphological characteristics: they possess a typical core-shell structure (A). Figure 2The outer shell is a continuous, dense coating layer assembled from lipid or polymer materials, while the interior shows distinct low-electron-density regions corresponding to the encapsulated hydrogen core. This structure indicates that HNBs are a precisely designed nanocarrier system, with the outer shell providing stability and protection, thereby achieving efficient loading and delivery of hydrogen at the nanoscale.

[0055] To quantitatively assess the actual hydrogen loading capacity of HNBs, the hydrogen content was precisely determined using the methylene blue reduction method (chemical titration). Each measurement was independently repeated three times, and the average value was taken to improve data reliability. To better evaluate the hydrogen storage performance of HNBs, the hydrogen content of hydrogen-water of the same volume was simultaneously measured as a control. The results showed that, under the same treatment conditions, the hydrogen content of the HNBs system was higher than that of ordinary hydrogen-water, indicating that its encapsulation structure effectively improved the hydrogen storage efficiency and concentration. Furthermore, based on the measured hydrogen content and nanoparticle concentration, the average hydrogen loading capacity of HNBs was estimated to be approximately 1.9 mg / L using the hydrogen loading capacity calculation formula. Figure 3 (AB). This data quantitatively confirms that HNBs, as a highly efficient nanocarrier, can achieve a higher concentration of hydrogen gas loading per unit volume than traditional dissolution methods.

[0056] To assess the biological safety of HNBs, the CCK8 assay was used to systematically examine the effects of different concentrations of HNBs on cell viability at three time points: 24 h, 48 h, and 72 h. The results showed that within the concentration range of 200–600 μg / mL, the cell viability of the HNB-treated groups remained consistently high. Figure 4 (A–C)

[0057] To clarify the distribution characteristics and targeting accumulation capacity of HNBs in mice, fluorescently labeled HNBs were injected via the tail vein, and their dynamic distribution in mice was monitored in real time using a small animal in vivo fluorescence imaging system. Figure 5 The results showed that HNBs rapidly entered the systemic circulation after injection and exhibited a significant tissue-specific accumulation trend over time. Further quantitative analysis of the fluorescence signal in the cardiac region was performed, and fluorescence intensity-time curves were plotted. Figure 5 (B). The curve shows that the fluorescence intensity in the cardiac region reached its peak approximately one hour after injection, indicating that HNBs had accumulated in large quantities in the myocardial tissue at this time. The signal remained high in the cardiac region for the next four hours, demonstrating that HNBs could maintain a high local concentration and a long retention time in the myocardial tissue.

[0058] The therapeutic effects of hydrogen-based nanoscale ultrasound contrast agents:

[0059] To evaluate the potential protective effect of HNBs against Dox-induced cardiotoxicity, a cell model of Dox injury was first established and co-incubated with HNBs. The cells were divided into a Control group, a Dox group, and a Dox+HNBs group. The Control group was supplemented with 100 μL of PBS, the Dox group was supplemented with 0.5 μM of Dox, and the Dox+HNBs group was supplemented with 0.5 μM of Dox and 100 μL of 500 μg / ml HNBs.

[0060] Figure 6 RT-qPCR results of (AC) showed increased expression of antioxidant stress-related genes such as NRF2, SOD2, and GPX-1 in the Dox+HNBs group.

[0061] A mouse cardiomyopathy model induced by doxorubicin ( Figure 7 (A) To evaluate the reversal effect of hydrogen-infused sodium bubbles on a mouse model of myocardial injury. Seven- to eight-week-old male mice were used in this study and underwent a one-week acclimatization period before the experiment. The experiment was conducted under a 12-hour light / 12-hour dark cycle (6:00 AM to 6:00 PM). Mice were divided into a Control group, a Dox group, and a Dox+HNBs group. The Control group received saline via tail vein injection; the Dox group received Dox (5 mg / kg) once a week for 4 weeks; the Dox+HNBs group received 100 µl of hydrogen-infused sodium bubbles three times a week via tail vein injection in addition to doxorubicin injection for 4 weeks.

[0062] Four weeks after Dox injection, cardiac function was assessed in vivo by transthoracic echocardiography. Mice were first shaved, then anesthetized with 2% isoflurane, and their body temperature was maintained at 37°C. Echocardiography was performed using a VevoF2 device while cardiac rate was monitored by electrocardiography. Figure 7 B-mode and M-mode ultrasound images of the parasternal left ventricle were taken. Preliminary findings suggest that its mechanism of action is closely related to the PI3K / AKT and TGF-β / Smad pathways.

[0063] Figure 8 (AC) The relative mRNA expression levels of PIK3CA (A), PIK3CB (B) and AKT1 (C) in the PI3K / AKT pathway were determined by RT-qPCR.

[0064] Figure 9 (AF) The relative mRNA expression levels of TGFB1 (A), TGFBR1 (B), SMAD1 (C), SMAD2 (D), SMAD3 (E) and SMAD4 (F) in the TGF-β / Smad pathway were determined by RT-qPCR.

[0065] Figure 10(A) Representative protein blot images of the PI3K / AKT pathway. (BC) Quantitative analysis of the p-PI3K / PI3K (B) and p-AKT / AKT (C) ratios.

[0066] In summary, the contrast agent prepared by this invention can synergistically inhibit the PI3K / AKT and TGF-β / SMAD signaling pathways, thereby effectively alleviating myocardial oxidative stress and fibrosis caused by chemotherapy drugs such as doxorubicin, improving cardiac function, and its good contrast function holds promise for achieving integrated diagnosis and treatment.

[0067] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogen-based nanoscale ultrasound contrast agent, characterized in that, Includes the following steps: a. Preparation of lipid solution: Sodium 1,2-dipalmitoyl-sn-3-phosphate (DPPA), distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), 1,2-dipalmitoyl-sn-3-phosphate ethanolamine (DPPE) and 1,2-bis(eicosicosicosyl-Sn-glycerol-3-phosphatidylcholine) (DBPC) were added to propylene glycol (PG), heated at 80°C and sonicated for 8 to 10 minutes at a frequency of 30 to 40 kHz and a power of 150 to 180 W to completely dissolve the powdered lipid reagents and form a lipid solution; b. Preparation of the mixture: Glycerol and PBS buffer are mixed and preheated at 80°C for 5 minutes to obtain the preheated mixture; c. Formation of nanobubble suspension: The preheated mixture obtained in step b is added to the lipid solution obtained in step a, and ultrasonic treatment is performed at room temperature for 8 to 15 minutes at an ultrasonic frequency of 30 to 40 kHz and an ultrasonic power of 150 to 180 W to form a milky white nanobubble suspension. d. Hydrogen filling and sealing: Transfer the nanobubble suspension obtained in step c to a headspace vial, fill it with hydrogen for 5 minutes, and then seal the vial with a rubber stopper and an aluminum cap; e. Shaking treatment: Place the sealed vial obtained in step d in a benchtop constant temperature shaker and shake at a speed of 200 to 300 rpm for 30 to 60 seconds to obtain a preliminary milky white hydrogen nanobubble suspension. f. Purification: The preliminary hydrogen nanobubble suspension obtained in step e is transferred to a microcentrifuge tube, centrifuged at 50 rcf for 3 to 5 minutes at room temperature, washed and concentrated to remove unencapsulated liposomes or impurities, and finally a hydrogen-based nanoscale ultrasound contrast agent is obtained.

2. The method for preparing the hydrogen-based nanoscale ultrasound contrast agent according to claim 1, characterized in that, In step a, the mass percentage of DBPC is 50% to 70%, the mass percentage of DPPA is 5% to 15%, the mass percentage of DPPE is 10% to 25%, and the mass percentage of DSPE-mPEG2000 is 5% to 15%.

3. The method for preparing the hydrogen-based nanoscale ultrasound contrast agent according to claim 1, characterized in that, In step b, the volume ratio of propylene glycol, glycerol, and PBS buffer is 15:10:

75.

4. The method for preparing the hydrogen-based nanoscale ultrasound contrast agent according to claim 1, characterized in that, In step a, the heating temperature is 80°C.

5. The method for preparing the hydrogen-based nanoscale ultrasound contrast agent according to claim 1, characterized in that, In step a, the ultrasonic treatment lasts for 8 to 10 minutes, the ultrasonic frequency is 30 to 40 kHz, and the ultrasonic power is 150 to 180 W.

6. The method for preparing the hydrogen-based nanoscale ultrasound contrast agent according to claim 1, characterized in that, In step c, the ultrasonic treatment lasts for 8 to 15 minutes, the ultrasonic frequency is 30 to 40 kHz, and the ultrasonic power is 150 to 180 W.

7. The method for preparing the hydrogen-based nanoscale ultrasound contrast agent according to claim 1, characterized in that, In step d, the hydrogen filling time is 5 minutes.

8. The method for preparing the hydrogen-based nanoscale ultrasound contrast agent according to claim 1, characterized in that, In step e, the oscillation speed is 200 to 300 rpm, and the oscillation duration is 30 to 60 seconds.

9. The method for preparing the hydrogen-based nanoscale ultrasound contrast agent according to claim 1, characterized in that, In step f, the centrifugation speed is 50 rpm and the centrifugation time is 3 to 5 minutes.

10. The method for preparing the hydrogen-based nanoscale ultrasound contrast agent according to claim 1, characterized in that, The hydrogen-based nanoscale ultrasound contrast agent prepared by the method has a core-shell structure and an initial average diameter of approximately 265.1 ± 26 nm.

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