CN118370721A - Application of liquid metal particles to delivery of tanshinone IIA for preparing medicines for treating myocardial infarction - Google Patents

Abstract

The invention discloses an application of liquid metal particles to delivering tanshinone IIA for treating myocardial infarction, which relates to the technical field of medicines and has the technical key points that: the invention uses a drug SA/LM/TA composed of Sodium Alginate (SA), liquid Metal (LM) and tanshinone IIA (TA), wherein the drug is used for stabilizing the release of the tanshinone IIA drug and the treatment of myocardial infarction; the drug is an injectable biological material, TA is a clinically verified myocardial infarction treatment drug, and SA/LM/TA has radioscopy due to the addition of LM particles; by embedding the TA in the SA carrier, the drug is released slowly and stably in the pericardial space, increasing the residence time of the drug in the heart.

Description

Application of liquid metal particles to delivery of tanshinone IIA for preparing medicines for treating myocardial infarction

Technical Field

The invention relates to the technical field of medicines, in particular to an application of liquid metal particles to delivering tanshinone IIA for medicines for treating myocardial infarction.

Background

Cardiovascular disease is one of the leading causes of death worldwide, and among various types of cardiovascular disease, myocardial Infarction (MI) is the leading cause of cardiovascular death. MI is a common cardiovascular disease characterized by irreversible myocardial necrosis caused by coronary ischemia and hypoxia. In large heart attacks, particularly when MI is caused, the patient may lose about 10 billion healthy cardiomyocytes. A large number of fibroblasts replace necrotic cardiomyocytes. At present, clinical methods for MI treatment, such as anticoagulants, percutaneous coronary intervention, bypass surgery, etc., only partially reduce the severity of the disease, but interventional registration statistics show that 1 year mortality in ST elevation MI (STEMI) patients still accounts for about 10%. Recently, regenerative therapies using living cells and proteins have also been used in experimental studies to reduce adverse cardiac remodeling and promote cardiac repair. However, these methods also have limitations such as difficulty in delivering drugs effectively to the heart, and efficacy is still controversial. Due to the complex heart structure, the blood supply is numerous, and achieving targeted and efficient drug delivery may be difficult. This may limit the effectiveness of certain therapeutic approaches, especially those that rely on pharmaceutical intervention. For the treatment of MI, the drugs commonly used in clinic are oral or intravenous. Intravenous injection is a convenient and safe method; however, the heart has poor drug retention.

Tanshinone IIA (TA) is an effective drug-derived compound extracted from Salvia Miltiorrhiza, and has been used in clinical practice for decades. Previous clinical studies have shown that TA is capable of reducing the occurrence of Major Adverse Cardiac Events (MACEs) such as malignant arrhythmias, recurrent Heart Failure (HF), recurrent myocardial infarction and death in acute MI patients following Percutaneous Coronary Intervention (PCI). Possible mechanisms are associated with inhibition of thrombosis and improvement of coronary circulation. Furthermore, it has been found that TA can improve cardiac function in STEMI patients. However, pharmacokinetic results in mice indicate that TA has a short half-life and rapidly clears circulating bodies. Thus, the development of potential drug carriers to achieve sustained release and to extend the residence time of the drug at the site of cardiac injury would be a viable and urgent treatment.

For this reason, the present invention aims to provide the use of liquid metal microparticles to deliver tanshinone IIA for the treatment of myocardial infarction, in order to solve the above-mentioned problems.

Disclosure of Invention

The invention aims to solve the problems, provides application of liquid metal particles to deliver tanshinone IIA for treating myocardial infarction, shows a strategy for treating myocardial infarction through medicines containing LM particles, namely, injecting SA/LM/TA through pericardial cavity, simultaneously expands the application field of LM biological materials in disease treatment, is beneficial to facilitating auxiliary application of the LM biological materials in diagnosis and treatment of cardiovascular diseases in the future, and is not limited to the prior tumor treatment. The medicine is expected to be widely applied to myocardial infarction treatment and accurate medicine transmission and release.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

the invention provides an application of liquid metal particles in delivering tanshinone IIA for treating myocardial infarction, wherein the drug is SA/LM/TA, and the drug is used for stabilizing the release of the tanshinone IIA drug and treating myocardial infarction; the drug is an injectable biomaterial.

The medicine is prepared by the following steps:

S1, melting 75.5% of gallium and 24.5% of indium at 180 ℃ and mixing in a continuous stirrer to obtain liquid metal LM;

S2, adding the liquid metal LM into a sodium alginate solution, performing ultrasonic treatment in an ice-water bath for 10 minutes, and obtaining SA/LM through an ultrasonic crusher;

s3, mixing the SA/LM with tanshinone IIA to obtain the SA/LM/TA.

In the scheme, SA/LM/TA loaded with Liquid Metal (LM) and tanshinone IIA (TA) is an injectable biological material and is used for stabilizing drug release and treating myocardial infarction. TA is a clinically verified myocardial infarction treatment drug which can be slowly released at a focus after being embedded in a Sodium Alginate (SA) carrier. As LM particles are added, SA/LM/TA has radioscopy. SA/LM/TA has low viscosity and can be injected smoothly by a syringe. The SA/LM/TA has excellent biocompatibility and blood compatibility as shown by in vitro and in vivo experiments. SA/LM/TA has low toxicity and biological safety in vivo as demonstrated by mouse blood test. In the rat model of myocardial infarction, the injection of SA/LM/TA in the pericardial space has proven to be a biosafety and effective route for delivery of carriers containing LM particles and TA drugs. After SA/LM/TA drug injection, the drug is released slowly and stably in the pericardial cavity, and the residence time of the drug in the heart is increased. After 7 days of surgery and treatment, M-mode echocardiography measurements showed improved cardiac function, including LV-EF and LV-FS, in rats compared to the control and TA direct injection groups. After 7 days of intrapericardiac injection of SA/LM/TA, the infarct area calculated from TTC test and the fibrosis area calculated from H & E staining were significantly reduced compared to the control group. In addition, serum levels of myocardial injury biomarkers including cTnT and cTnI, and myocardial enzymes including CK, CK-MB and LDH were reduced in the SA/LM/TA group. The SA/LM/TA group was able to significantly reduce the levels of the pro-inflammatory cytokines TNF- α and TGF- β while increasing the levels of the anti-inflammatory cytokines IL-4 and IL-10. According to the test results, the synergistic effect achieved by the use of SA/LM/TA exceeded the effect of TA given alone.

Compared with the prior art, the beneficial effect of this scheme:

1. the SA/LM/TA medicine has excellent biocompatibility and blood compatibility;

2. the SA/LM/TA medicine has low toxicity and biological safety in vivo;

3. the SA/LM/TA medicine is a biological safe and effective way for conveying a carrier containing LM particles and TA medicine; after injection, the drug is released slowly and stably in the pericardial cavity, increasing the residence time of the drug in the heart.

Drawings

FIG. 1 is a representation of a set of SA/LM/TA of an embodiment of the invention, wherein A is a photograph and TEM image of SA/LM/TA; b is a DLS analysis chart of LM particles, SA/LM and SA/LM/TA; c is a viscosity analysis chart of water, SA solution, SA/LM and SA/LM/TA; d is a schematic representation of Zeta potential (n=3) of TA, SA solution, SA/LM and SA/LM/TA at pH 7.4; e is an ion concentration schematic diagram in SA/LM/TA leaching solution;

Fig. 2 is a schematic set of in vitro cytotoxicity and drug release results in an example of the invention, wherein a is the cell viability (n=3, ns p >0.05, < p < 0.05) of H9C2 cells after different sample contacts; b is a CLSM image of H9C2 cells after different samples are contacted, living cells are green, and dead cells are red; c is a flow cytometry analysis plot of H9C2 cells after exposure to different samples; d is a plot of inhibition activity for TA and SA/LM/TA (n=3); e is a schematic of cumulative release results of TA in different solvents, including saline and 5% glucose; f is a schematic representation of the hemolysis assay result (n=3, ns p > 0.05);

FIG. 3 is a schematic set of serum biochemical tests of SA/LM/TA in vivo in biocompatible mice injected with SA/LM, wherein A is ALT and AST (n=3), according to the examples of the present invention; b is CREA and UREA (n=3); c is ALT and AST (n=3); d is CREA and UREA (n=3); e is a histological photograph of major organ tissue 4 weeks after SA/LM/TA injection;

fig. 4 is a schematic set of central function monitoring results according to an embodiment of the present invention, wherein a is a representative echocardiogram image showing LV wall motion of the rat heart, echocardiogram measurements of cardiac function including BLV-EF, C LV-FS post-treatment (n=6), and D pre-operative and post-operative electrocardiography of the rat;

Fig. 5 is a schematic diagram of a TTC staining and cardiac injury biomarker detection result set in an embodiment of the present invention, where a is a TTC staining image showing infarct area, B is myocardial infarction area, and C-G is cardiac injury biomarkers cTnT (n=3), cTnI (n=3), CK-MB (n=3), and LDH (n=3), respectively;

FIG. 6 is a schematic set of H & E staining, masson staining and inflammatory cytokine detection results in an example of the invention, wherein A is representative H & E staining and Masson stained myocardial sections showing 7 days after various treatments; b is the myocardial slice fibrosis area based on Masson staining (n=3); c is TNF- α (n=10); d is IL-4 (n=10); e is IL-10 (n=10); f is TGF- β (n=10).

Detailed Description

In order that those skilled in the art will better understand the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, wherein it is to be understood that the illustrated embodiments are merely exemplary of some, but not all, of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.

Example 1:

Preparation and characterization of SA/LM/TA

SA/LM/TA is a mixture of Liquid Metal (LM) and tanshinone IIA (TA) drug dispersed in Sodium Alginate (SA) solution by ultrasound. LM is radio-transparent under X-ray or CT and is liquid at room temperature. Thus, the addition of LM can confer SA/LM/TA radio-transparency without affecting its injectability. The surface morphology of SA/LM/TA was observed by Transmission Electron Microscopy (TEM). As shown in fig. 1A, LM particles are uniformly dispersed in SA solution and maintain a stable spherical structure without agglomeration. The LM particles are in a regular shape, and the surfaces of the LM particles are covered with a SA film carrying TA. From the TEM images, it was determined that the LM particles were about 800 nm in diameter (fig. 1A). The size of the LM particles and SA/LM/TA was assessed using Dynamic Light Scattering (DLS). As shown in FIG. 1B, the LM particle size dispersed in water is about 600 nm, while the SA/LM/TA size is about 800 nm. This also demonstrates that the LM particle surface is covered with a SA film. To demonstrate the injectability of SA/LM/TA and ensure that it can be injected smoothly by syringe, the viscosity of SA/LM/TA at a constant shear rate (100 s-1) was measured. As shown in FIG. 1C, the viscosity of SA/LM/TA is higher than that of water. This viscosity is sufficient to meet the injection requirements and SA/LM/TA can be injected smoothly through the syringe. Furthermore, due to the flexibility of LM, the addition of LM particles does not affect the viscosity of SA/LM/TA compared to SA solutions. From the Zeta potential value, the addition of TA does not affect the SA/LM/TA potential (FIG. 1D). To verify the release of metal ions from SA/LM/TA in different solvents, SA/LM/TA was immersed in water, physiological saline and 5% glucose, respectively, for 48 hours, and then the ion concentration of the exudates was measured. As shown in fig. 1E, almost no gallium and indium ions were released from SA/LM/TA among the three solvents. This also ensures the biosafety of the SA/LM/TA during subsequent use.

Example 2:

In vitro cytotoxicity and drug release assessment

To assess the cytotoxicity of the biological material, CCK-8 cell viability tests were performed by H9C2 cells. The results show the viability of the cells after direct contact with these samples within 48 hours. As shown in FIG. 2A, SA/LM/TA has no obvious cytotoxicity, and the in vitro biosafety is proved. And also showed that other samples containing SA or LM were less toxic in the cell experiments. However, TA not encapsulated in a drug carrier has an impact on cell viability. Confocal Laser Scanning Microscopy (CLSM) images of H9C2 cells also demonstrated low toxicity of SA/LM/TA (fig. 2B). For flow cytometry analysis of H9C2 cells in fig. 2C, SA/LM was biosafety for cells compared to control. However, the proportion of viable cells in the SA/LM/TA and TA groups was slightly decreased due to the addition of the drug. The effect of drug concentration on cell viability was further studied, and the inhibitory activity of TA and SA/LM/TA was tested using H9C2 cells. As shown in FIG. 2D, the half-lethal concentration (IC 50) of SA/LM/TA was slightly higher than TA (0.0766 mg/mL). This suggests that SA-encapsulated TAs are still effective in inhibiting activity on H9C2 cells. In addition, TA drug release profile of SA/LM/TA was assessed. As shown in FIG. 2E, in saline or 5% glucose, the drug release rate of SA/LM/TA was slower than that of direct TA. In saline, the drug release of SA/LM/TA was similar to that in 5% glucose. In order to confirm the blood compatibility of SA/LM/TA, an in vitro hemolysis test was performed. As shown in FIG. 2F, the hemolysis values of LM, SA/LM and SA/LM/TA were lower than those reported previously for biological materials. The whole blood was used for a hemolysis test in contact with SA/LM/TA to evaluate its excellent blood compatibility.

Example 3:

Biocompatibility of SA/LM/TA in vivo

To verify the biocompatibility of SA/LM and SA/LM/TA in vivo, serum biochemical tests were performed after subcutaneous injections of SA/LM and SA/LM/TA injected mice. SA/LM and SA/LM/TA were injected into mice for 1,2, 3,4 weeks, respectively. Serum biochemical test results of SA/LM and SA/LM/TA groups were compared with control groups. Alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) are major biomarkers of liver function, while Creatinine (CREA) and UREA (UREA) are major biomarkers of kidney function, according to clinical biochemical parameters of serum. These four parameters were used as indicators to reflect the effects of SA/LM and SA/LM/TA on liver and kidney function in vivo. The results for the SA/LM set are shown in FIG. 3A, B. These four parameters did not show a significant increase over 4 weeks after SA/LM injection compared to control mice. As for AST in fig. 3A, its value increases slightly after 4 weeks. The results for the SA/LM/TA group are shown in FIG. 3C, D. These four parameters were not increased over 4 weeks after SA/LM/TA injection compared to the control group. At 2 weeks post injection, AST values increased slightly but then began to decline. 3 weeks after injection, AST values had recovered to normal levels. This indicates that SA/LM/TA is less toxic to mice in vivo. SA/LM and SA/LM/TA injected mice are in good health. In addition, SA/LM/TA injected mice were subjected to histological photographs of major organs including heart, liver, spleen, lung and kidney, after 4 weeks of injection. As shown in fig. 3E, they all had healthy tissue structure, demonstrating the biosafety of SA/LM/TA. Injection of SA/LM/TA did not affect major organs and tissues.

Example 4:

treatment of Myocardial Infarction (MI) using SA/LM/TA injection

Prior to treatment, the rat left anterior descending coronary artery was ligated while leaving the pericardium, confirming successful construction of the MI model. And demonstrates that a pericardial cavity injection can be performed on rats during open chest surgery. Rats subjected to MI surgery alone were used as control groups. Other rats subjected to MI surgery were injected with TA, SA/LM and SA/LM/TA, respectively, in the pericardial space. After 7 days of treatment, cardiac function was assessed using M-mode echocardiography. As shown in fig. 4A, the images show Left Ventricular (LV) wall movement after various treatments. Direct injection of TA into the pericardial space showed no improvement in recovery of cardiac function compared to the control group. Fig. 4B, C shows the same results, revealing that direct injection of TA has no significant effect on improving left ventricular ejection fraction (LV-EF) and left ventricular end-diastole volume (LV-ES). Injection of SA/LM and SA/LM/TA proved to have a positive effect on LV-EF and LV-ES compared to the control and TA groups (FIG. 4B, C). Notably, SA/LM/TA injection showed significant and statistically significant improvement in LV-EF and LV-ES. This suggests that direct injection of TA into the pericardial cavity can rapidly diffuse, affecting drug absorption, while TA release in SA/LM/TA encapsulated by SA/LM carrier is slow. The slow down of the diffusion of the drug prolongs the action time of the drug at the damaged part. In addition, rats were allowed to recover and continue to be observed for 7 days after surgery. Successful open chest surgical induction of MI was assessed by elevation of ST segment on Electrocardiogram (ECG) after 7 days. Electrocardiogram of SA/LM/TA group rats showed no abnormalities. Representative ECG showed that elevation of ST segment was gradually restored to normal 7 days after surgery in TA and SA/LM/TA group rats compared to control group (fig. 4D), indicating that cardiac function was gradually restored to normal level. Thus, echocardiography and ECG measurements indicate that the injection of SA/LM/TA therapy promotes cardiac function. Taken together, these data demonstrate that drug delivery using SA/LM/TA injection in biological materials is effective and safe for cardiac repair.

Example 5:

Evaluation of therapeutic Effect of SA/LM/TA injection

Rats were sacrificed 7 days after intracardiac injection of TA, SA/LM/TA for The Triphenyltetrazolium Chloride (TTC) test. TTC staining was performed to assess infarct size of the heart. As shown in fig. 5A, the study found that TA or SA/LM/SA injections showed reduced infarct size compared to the control group. In particular, by SA/LM/TA injection, the SA/LM/TA group showed a significant reduction in infarct size (FIG. 5B). To assess myocardial damage, serum was collected and levels of cardiac troponin T (cTnT), cardiac troponin I (cTnI), creatine Kinase (CK), creatine kinase MB (CK-MB) and Lactate Dehydrogenase (LDH) were measured at 12 hours, 24 hours and 48 hours after MI and injection treatment, respectively. As shown in fig. 5C, D, both cardiac injury biomarkers cTnT and cTnI were reduced in the TA, SA/LM and SA/LM/TA groups compared to the control group. In particular for the SA/LM/TA group, the values of cTnT and cTnI decreased over time. In addition, the levels of the myocardial enzymes CK, CK-MB and LDH in the serum were measured simultaneously. The results indicate that the SA/LM/TA group showed significantly reduced levels of the myocardial enzymes CK, CK-MB and LDH at 48 hours. These indicators indicate that there is evidence of improved cardiac function after MI following an intracavitary injection of SA/LM/TA.

Example 6:

Exhibiting therapeutic effects of MI

Rat heart morphology and fibrosis were shown 7 days after various treatments using heme and eosin (H & E) staining and Masson staining (fig. 6A). H & E staining demonstrated morphology of myocardial tissue and local cellular inflammatory infiltrates. Whereas Masson staining showed that SA/LM/TA injection reduced fibrosis of myocardial tissue. As shown in fig. 6B, the area of fibrosis following SA/LM/TA treatment was significantly reduced compared to the control group. In addition, the levels of inflammatory cytokines including tumor necrosis factor alpha (TNF-. Alpha.), interleukin 4 (IL-4), interleukin 10 (IL-10), and transforming growth factor beta (TGF-. Beta.) were measured. The results indicate that the SA/LM/TA group was able to significantly reduce the levels of the pro-inflammatory cytokines TNF- α (FIG. 6C) and TGF- β (FIG. 6F) while increasing the levels of the anti-inflammatory cytokines IL-4 (FIG. 6D) and IL-10 (FIG. 6E). The synergistic effect achieved by the use of SA/LM/TA exceeds the effect of TA administration alone.

Example 7:

experimental part:

Materials: gallium (Ga) and indium (In) were purchased from beijing local corporation. Sodium Alginate (SA) is from Sainofil chemical Co., ltd. Shanghai, china. Tanshinone IIA (TA) and sodium thiotanshinone injection (5 mg/mL,2 mL) were purchased from Shanghai medical control Inc. Du's modified eagle sperm cell Medium (DMEM) was from Weison (China). Saline, 5% dextrose, phosphate Buffered Saline (PBS), fetal Bovine Serum (FBS), and penicillin/streptomycin (P/S) were purchased from Thermo FISHER SCIENTIFIC Corporation (China). H9C2 cells were supplied by American Type Culture Collection (ATCC) and obtained from local company. Cell counting kit-8 (CCK-8) was obtained from the dao-qiong technology laboratory (China). Acridine Orange (AO) and Ethidium Bromide (EB) were purchased from Solarbio (china). Laboratory mice and rats were supplied by beijing viterbi laboratory animal technologies limited (beijing, china). These animals were housed in the university of Qinghai laboratory animal research center, which is approved by the International Association for laboratory animal assessment and authentication (AAALAC). The animal protocol used in this study was approved by the Institutional Animal Care and Use Committee (IACUC). The mice and rats experiments were conducted in accordance with the protocol approved by the ethical committee of the university of Qinghai, contract number 18-LJ1.

Preparation of LM, SA/LM and SA/LM/TA: LM of composition LM was prepared from gallium and indium containing 75.5% (Ga) and 24.5% (In) by weight. The weighed gallium and indium were melted at 180 ℃ and mixed in a continuous stirrer. For SA/LM preparation, bulk LM was added to the SA solution and sonicated. SA/LM was obtained by a ultrasonic crusher (BRANSON, USA). The sonication process was carried out in an ice-water bath for 10 minutes. SA/LM/TA was prepared by mixing TA with SA/LM.

Characterization of LM, SA/LM and SA/LM/TA: the surface morphology of SA/LM/TA was observed by means of a transmission electron microscope (TEM, H-7650B, japan). The dimensions of the LM particles, SA/LM and SA/LM/TA were measured using a dynamic light scattering instrument (DLS, dynaPro NanoStar, USA). The viscosities of water, SA solution, SA/LM and SA/LM/TA were measured at room temperature with a rheometer (MCR 301, austria) at a constant shear rate (100 s-1). Zeta potentials of TA, SA/LM and SA/LM/TA were measured at pH 7.4 using Malvern Zetasizer Nano ZS (UK). The leakage was obtained by immersing SA/LM/TA in different solvents (water, saline, 5% glucose) for 48 hours. The leakage was then removed for gallium and indium ion concentration testing (Thermo FisherX Series II, usa).

In vitro cytotoxicity test: the cytotoxicity of samples (SA solution, LM, TA solution, SA/LM and SA/LM/TA) on H9C2 cells was studied using a cell counting kit-8 (CCK-8) experiment. H9C2 cells were cultured in a humidified 5% CO ₂ atmosphere at 37℃in the presence of complete medium (89% DMEM,10% FBS and 1% P/S). Cells were placed in 96-well plates, each well containing cells (density 5000 cells per well) supplemented with medium (100 μl). Cells were cultured for 24 hours for adhesion. The medium was then removed and medium (100. Mu.L) containing the different samples was added separately. The cells were cultured for an additional 48 hours. Finally, the medium was removed and medium containing CCK-8 solution (100. Mu.L) was added to each well. Cells were incubated for 2 hours. Cell viability was determined by absorption of 450nm microwell reader (Thermo, varioskan Flash, usa).

H9C2 cells were seeded in glass dishes at a density of 10000 cells per well. After 12 hours, media containing TA solution, SA/LM and SA/LM/TA were added to the petri dishes, respectively. Cells were cultured in an incubator for 48 hours. Carefully remove the media and wash the cells with PBS. Subsequently, the cells were treated as AO-EB solution, wherein living cells were stained green and dead cells were stained red. Cell status was observed by confocal laser scanning microscopy (CLSM, zeiss LSM710, china). Flow cytometry analysis of H9C2 cells was performed using a flow cytometer (BD FACSCalibur, usa). H9C2 cells were seeded in 6-well plates, 105 cells per well, and contacted with TA solution, SA/LM and SA/LM/TA for 48 hours. Thereafter, the cells were rinsed with PBS and immediately subjected to flow cytometry testing.

According to the concentrations of TA, A/LM/TA and sodium thiotanshinone injection, respectively diluting into solutions with different concentrations. The H9C2 cells were then contacted with solutions of different concentrations for 48 hours. Their cell viability was tested and half inhibition concentration (IC 50) was calculated.

SA/LM/TA in vitro drug Release: drug release of TA in SA/LM/TA in vitro was analyzed by dialysis of samples containing TA solution and SA/LM/TA in saline and 5% glucose. SA/LM/TA and sodium thiotanshinone injection were placed in dialysis bags and dialyzed against 100mL saline and 5% glucose at 37℃for 60 hours, respectively. At specific time intervals, 500. Mu.L of the solution was removed. And an equal amount of fresh solution was added to maintain a constant volume. The TA concentration released from the SA/LM/TA was measured by a microwell reader (Thermo, varioskan Flash, USA) and by absorbance at 460nm (OD 460 nm).

SA/LM/TA in vitro and in vivo biocompatibility: a hemolysis test was performed to verify the in vitro hemolysis of SA/LM/TA. LM, SA/LM and SA/LM/TA were placed in 1.5mL tubes, respectively. Citrate blood was diluted 50-fold into saline solution. An equal amount of diluted blood was added to the tube. Diluted saline-containing blood was used as a negative control and deionized water (DI water) blood was used as a positive control. All samples were incubated at 37℃for 2 hours. These samples were then centrifuged and the supernatant transferred to a 96-well plate. The data were tested by a microwell reader. The haemolysis rate was calculated by the following formula: hemolysis ratio (%) = (a sample-a negative)/a positive x 100%, where a sample is absorbance of the sample at 545nm, a negative is absorbance of the negative control, and a positive is absorbance of the positive control. Three replicates were run for each group.

Female mice 8 weeks old were used for in vitro and in vivo biocompatibility experiments. Mice were injected with 100. Mu.L SA/LM and SA/LM/TA, respectively, in subcutaneous tissue. Their blood was collected weekly after injection. The biochemical index of blood was analyzed by clinical laboratory at the university of Qinghai. 300 μl of whole blood was centrifuged at 4deg.C to obtain serum for liver and kidney function tests, including ALT, AST, CREA and UREA. The results of blood tests prior to SA/LM or SA/LM/TA injection in mice served as controls. Over 4 weeks, these mice were sacrificed in the SA/LM/TA group by standard euthanasia methods. Tissue of heart, liver, spleen, lung and kidney was removed separately. These tissues were immersed in formalin for 1 day and fixed with paraffin. These samples were then sectioned and stained with H & E methods. They were observed under a microscope (Nikon Eclipse 90 i).

Rat Myocardial Infarction (MI) model and intrapericardiac injection: before the start of the experiment, these rats were kept in a 12 hour light/12 hour dark environment. The temperature is 25 ℃ and the humidity is 40-60%. Rat model preparation of myocardial infarction was performed according to the previously reported protocol. Rats were anesthetized by intraperitoneal injection, and then subjected to ventilation and chest windowing. Next, the left anterior descending coronary artery was ligated with 6-0 lines while leaving the pericardium. Infarcts were confirmed when the top area was observed to be pale. After myocardial infarction model surgery, TA, SA/LM and SA/LM/TA were carefully injected into the pericardial cavity, respectively. Their injection volume was 100. Mu.L. After injection was completed, the chest of the rat was closed.

Heart function measurement: cardiac function was assessed ON day 7 after pericardial cavity injection using the Vevo 2100 high resolution imaging system (Visual sonic, toroto, ON, canada). The heart of the rat was scanned using an MS250 probe. The M-mode echocardiography is used to measure the inside diameter of the left ventricle and the anterior/posterior wall thickness of the left ventricle during diastole/systole. Left ventricular ejection fraction (LV-EF) and left ventricular short axis contraction fraction (LV-FS) were calculated to assess cardiac function in rats.

Electrocardiogram (ECG) of rats: the electrocardiogram of the rats was monitored using an electrocardiogram processor (SP 2006, softron, china). First, anesthetized rats were fixed on a heating table and the hairs of the limbs were cleaned. Four injection needles were then inserted through the skin of the rat, but without puncturing the muscle of the rat. Next, clip conductors are attached to the respective pins according to the labels on the amplifiers. Setting the a/D converter to X1, CE-01 is selected as the amplifier setting. Electrocardiogram signals of rats were collected and recorded by the instrument.

Triphenyltetrazolium salt (TTC) test: rats were sacrificed on day 7 post-pericardial intra-luminal injection. Saline was infused retrograde through the aortic arch to eliminate blood, followed by two washings. After removal of the heart, it was frozen at-80℃for 24 hours. A heart mold was used to obtain cross sections of hearts, each producing 5-6 slices. The fragments were immersed in physiological saline and then gently shaken at room temperature in a pre-heated 1% TTC phosphate buffer for 10 minutes at 55 ℃. Next, the fragment was transferred to an incubator at 37℃for 15 minutes, and then washed with physiological saline. Fragments were fixed overnight in 4% paraformaldehyde. The right ventricle of the fixed heart slice was carefully removed with forceps and scissors. Photographs were taken by a stereomicroscope (ME 30-Z, MSHOT, china) to determine the areas showing signs of infarcted tissue. Infarct size was quantified using Image J software by dividing the white area by the red white infarct area within the range of the hazard zone. These experiments are important to evaluate the effect and potential therapeutic effect of SA/LM/TA in the rat myocardial infarction model. The influence of the medicine on the heart function and the infarcted area can be comprehensively evaluated through heart function measurement, electrocardiogram monitoring and TTC test, and important data support is provided for further research.

Tissue sections, H & E staining and Masson staining: hearts were removed from sacrificed rats and fixed in 4% paraformaldehyde overnight. After washing with running water, the heart was dehydrated through a series of alcohol of gradual concentration, treated clearly, embedded in paraffin, and sectioned. The sections were then de-waxed and stained using hematoxylin and eosin (H & E) staining methods. After another round of clearing, the sections were mounted under a microscope for observation. Morphological changes in rat hearts were observed under a microscope (nikoneclipse 90i, japan). The dewaxed sections were dehydrated in water and stained with hematoxylin for 5-10 minutes. Then, the mixture was separated in a hydrochloric acid-alcohol solution, treated with running water and blue, washed with distilled water, and stained with an eosin acidophilic solution for 5 to 8 minutes. Then, washing with distilled water, dyeing with 1% phosphomolybdic acid solution for 1-3 minutes, directly immersing in aniline blue solution without washing with water for 5 minutes, washing with water, air-drying in an incubator at 60 ℃, cleaning with xylene, and then mounting.

Cardiac injury biomarker and inflammatory factor detection: blood was collected from rats under anesthesia and centrifuged at 3000 rpm for 15 minutes at 4 ℃. Myocardial injury biomarkers cardiac troponin T (cTnT) and cardiac troponin I (cTnI) were detected using ELISA kit (SED 232Ra and SEA478 Ra). Serum levels of the cardiomyoenzymes glutamyl transferase (CK), creatine kinase MB isozyme (CK-MB), lactate Dehydrogenase (LDH) were measured by an automatic analyzer (Chemray, rayto, china). The inflammatory factors tumor necrosis factor alpha (TNF-alpha), interleukin 4 (IL-4), interleukin 10 (IL-10) and transforming growth factor beta (TGF-beta) were detected using ELISA kits (EK 382/3-02, EK304/2-01, EK310/2-01 and EK 981-01).

Statistical analysis: data analysis was performed using GRAPHPAD PRISM software. All data are presented as mean plus minus Standard Deviation (SD). The sample size (n) is labeled in the legend. Data analysis used unpaired two-tailed t-test. The significance of the differences between groups at p <0.05 is expressed as follows: ns represents p >0.05, p <0.01, p <0.001.

The above specific embodiments are provided for illustrative purposes only and are not intended to limit the invention, and modifications, no inventive contribution, will be made to the embodiments by those skilled in the art after having read the present specification, as long as they are within the scope of the patent statutes.

Claims (3)

1. The application of liquid metal particles to delivering tanshinone IIA for treating myocardial infarction is characterized in that: the medicine is SA/LM/TA, and is used for stabilizing tanshinone IIA medicine release and treating myocardial infarction.

2. The method for preparing the liquid metal microparticle delivery tanshinone IIA medicament for treating myocardial infarction as set forth in claim 1, which is characterized in that: the medicine is prepared by the following steps:

S1, melting 75.5% of gallium and 24.5% of indium at 180 ℃ and mixing in a continuous stirrer to obtain liquid metal LM;

S2, adding the liquid metal LM into a sodium alginate solution, performing ultrasonic treatment in an ice-water bath for 10 minutes, and obtaining SA/LM through an ultrasonic crusher;

s3, mixing the SA/LM with tanshinone IIA to obtain the SA/LM/TA.

3. Use of liquid metal particles as claimed in claim 1 for delivering tanshinone IIA for the manufacture of a medicament for the treatment of myocardial infarction, characterized by: the drug is an injectable biomaterial.