CN115607683B - A kind of chitosan-deferoxamine composite nano-suspension and its preparation method and application - Google Patents

Abstract

The invention discloses a chitosan-deferoxamine composite nano-suspension and its preparation method and application. Deferoxamine solution is added to DMEM culture medium, and after being fully mixed evenly, the final concentration of deferoxamine is 0.1- 10mg/mL; add chitosan solution to the mixed solution so that the final concentration of chitosan in the mixed solution is 0.5-15mg/mL; then use Tris solution to adjust the pH of the mixed solution to 7.0-7.6; place the mixed solution in carbon dioxide In a gas environment, let it stand to obtain a chitosan-deferoxamine composite nano-suspension. The chitosan-deferoxamine composite nanoparticles in the chitosan-deferoxamine composite nano-suspension of the present invention have stronger iron ion complexing ability and can uniformly release deferoxamine drug molecules in an acidic environment. It can significantly inhibit adverse myocardial remodeling and promote the repair of infarcted myocardium.

Description

A kind of chitosan-deferoxamine composite nano-suspension and its preparation method and application

Technical field

The invention belongs to the technical field of biomedical materials, and specifically relates to a chitosan-deferoxamine composite nano suspension and its preparation method and application.

Background technique

Cardiovascular disease has become a major public health issue worldwide. The "China Cardiovascular Health and Disease Report 2020" shows that the number of patients with cardiovascular disease in my country is as high as 330 million, and the number of cardiovascular disease deaths accounts for 40% of the number of deaths from disease among residents in our country. %above. Myocardial infarction due to coronary artery obstruction is the most harmful type of cardiovascular disease, accounting for nearly 80% of all cardiovascular deaths. The occurrence of myocardial infarction will cause massive death of myocardial cells and severe immune-inflammatory response. Due to the lack of regenerative capacity of cardiomyocytes, cardiac scar repair is mainly mediated by cardiac fibroblast activation during the compensatory period after infarction, which will lead to adverse cardiac remodeling, thereby causing cardiac diastolic dysfunction and heart failure. . Therefore, inhibiting myocardial damage and necrosis after infarction can greatly improve the prognosis of patients with myocardial infarction.

As an important essential element for the human body, iron plays an important role in regulating heart function. When myocardial infarction occurs, the expression of ferritin will be significantly down-regulated, and its ability to bind free iron ions will be significantly weakened; at the same time, the acidic and highly reducing environment caused by continued ischemia will lead to the degradation of ferritin inside and outside the cells, and the ferritin in ferritin will be degraded. Iron ions are released in large quantities. Free iron in turn converts superoxide and hydrogen peroxide in the environment into more oxidative hydroxyl radicals and hydroxide anions through the Fenton reaction. These free radical by-products can cause mitochondrial damage and calcium homeostasis disorders in cardiomyocytes, thereby exacerbating cardiac dysfunction. Current studies have found that by targeting the inhibition of iron overload during cardiac ischemic injury, cardiac contractile function can be significantly improved, cell vitality increased, and cardiac remodeling inhibited. Deferoxamine is a natural siderophore with a strong affinity for iron ions and can bind free iron ions in the human body as well as iron ions in ferritin and hemosiderin. Some studies have found that deferoxamine can inhibit the increase of free radicals during myocardial ischemia and reperfusion and reduce reperfusion injury. In addition, deferoxamine is also considered to be a stabilizer of the expression of hypoxia-inducible factor HIF-1α, thereby promoting the expression of vascular endothelial growth factor, which also makes it more potential to become a clinical treatment drug for myocardial infarction. However, because deferoxamine is a water-soluble small molecule drug, it has a short circulation time in the body and is easily metabolized by the liver and kidneys. Relevant clinical studies have also found that continuous intravenous administration is required to achieve the ideal cardioprotective effect, which greatly limits its application in clinical practice.

With the development of nanotechnology, drug delivery systems based on nanomaterials have been widely used in the biomedical field. Nanoparticles have good physical and chemical properties and the ability to be modified, and are often used as delivery vehicles for small molecule active drugs in the field of cardiovascular research. Among them, chitosan is a natural cationic polysaccharide with good tissue compatibility and biodegradability, and is considered an ideal carrier for many drug delivery. And because of its modifiable properties, its application properties and functions can be enhanced through chemical modification, greatly expanding its application scope. More importantly, since the chitosan molecular chain is distributed with various functional groups such as hydroxyl, amino and acetyl groups, the chitosan material itself has a chelating and enriching effect on metal ions such as iron ions. This suggests that chitosan-based nanomaterials can be used in the treatment of myocardial infarction by chelating overloaded iron ions in the myocardial infarction area.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention proposes a chitosan-deferoxamine composite nano-suspension and its preparation method and application. The self-assembly properties of chitosan are used to prepare chitosan-deferoxamine composite nanoparticles. , aiming to use the synergistic effect of chitosan and deferoxamine to, on the one hand, reduce the iron ion content of myocardial cells in the infarct area and alleviate their oxidative stress damage, and on the other hand, increase the expression of HIF-1α to promote the transcription of vascular endothelial growth factor. In this way, the purpose of promoting myocardial repair after infarction is achieved.

A method for preparing chitosan-deferoxamine composite nano-suspension, including:

Add deferoxamine solution to DMEM medium and mix thoroughly;

Add chitosan solution to the mixed solution so that the final concentration of chitosan in the mixed solution is 0.5-15mg/mL;

Then use Tris solution to adjust the pH of the mixture to 7.0-7.6;

The mixed solution is placed in a carbon dioxide gas environment and allowed to stand to obtain a chitosan-deferoxamine composite nanometer suspension.

Further, deferoxamine solution is added to the DMEM medium so that the final concentration of deferoxamine is 0.1 to 10 mg/mL. Within this concentration range, the final concentration of deferoxamine solution does not affect the morphology and electronegativity of the synthesized chitosan-deferoxamine composite nanoparticles.

Further, the final concentration of deferoxamine is 1 mg/mL.

Further, the final concentration of chitosan is 1.25 mg/mL.

Further, use Tris solution to adjust the pH of the mixed solution to 7.4.

Further, the concentration of the Tris solution is 0.1M.

A chitosan-deferoxamine composite nano-suspension prepared by the above method.

Application of a chitosan-deferoxamine composite nano-suspension in the preparation of myocardial repair drugs.

The beneficial effects of the present invention are as follows:

(1) Compared with simple deferoxamine molecules and simple chitosan nanomaterials, chitosan and deferoxamine have a synergistic effect in complexing iron ions. In the chitosan-deferoxamine composite nanomaterial suspension The chitosan-deferoxamine composite nanoparticles showed stronger iron ion complexing ability.

(2) Chitosan-deferoxamine composite nanoparticles have the characteristics of pH-responsive release, which can uniformly release deferoxamine drug molecules in an acidic environment and significantly extend the drug release time.

(3) Chitosan-deferoxamine composite nanoparticles can reduce hypoxia and oxidative stress damage in cardiomyocytes, have anti-oxidative stress, anti-apoptosis, anti-inflammation and promote angiogenesis, and can promote endothelial cells. The tube-forming ability can protect the cardiac function of mice after myocardial infarction and significantly inhibit adverse myocardial remodeling.

(4) Chitosan-deferoxamine composite nanoparticles achieve targeted and efficient enrichment of deferoxamine in the heart, and enhance the biological function of deferoxamine in inhibiting oxidative stress damage and promoting angiogenesis, thereby promoting infarction. Repair of myocardium. This provides new strategies and ideas for the clinical treatment of myocardial infarction.

Description of the drawings

Figure 1 shows the morphology characterization of chitosan-deferoxamine composite nanoparticles; Figure (A) is the TEM image of chitosan-deferoxamine composite nanoparticles; Figure (B) is the chitosan-deferoxamine composite nanoparticles The particle size analysis results of composite nanoparticles; Figure (C) shows the Zeta potential results of chitosan-deferoxamine composite nanoparticles; Figure (D) shows the energy spectrum analysis of chitosan-deferoxamine composite nanoparticles.

Figure 2 shows the structural characterization of chitosan-deferoxamine composite nanoparticles; Figure (A) shows the XRD analysis results of chitosan-deferoxamine composite nanoparticles; Figure (B) shows chitosan-deferoxamine composite nanoparticles FT-IR analysis results of amine composite nanoparticles; Picture (C) shows the analysis results of carbon (C) element in XPS detection of chitosan-deferoxamine composite nanoparticles; Picture (D) shows chitosan-deferoxamine composite nanoparticles Analysis results of nitrogen (N) element in XPS detection of amine composite nanoparticles.

Figure 3 shows the HPLC method for detecting the efficiency of deferoxamine release from chitosan-deferoxamine composite nanoparticles in vitro; Figure (A) is the fitting straight line between the deferoxamine concentration and the detection peak area constructed by HPLC; Figure ( B) is the release curve of desferrioxamine at different pH in HPLC experiment (n=3).

Figure 4 shows the effect of chitosan-deferoxamine composite nanoparticles on cardiomyocyte activity; Figure (A) shows the statistics of CCK-8 experimental results after different concentrations of deferoxamine were co-cultured with primary cardiomyocytes and H9C2 cells. Picture (n=5); Picture (B) is a statistical diagram of CCK-8 experimental results after co-culture of different concentrations of chitosan-deferoxamine composite nanoparticles with primary cardiomyocytes and H9C2 cells (n=5).

Figure 5 shows the determination of the iron complexing ability of free chitosan, chitosan nanoparticles, deferoxamine and chitosan-deferoxamine composite nanoparticles. Among them, the free chitosan group n=4, and the other groups n=5 , ** represents p<0.01.

Figure 6 shows the effect of chitosan-deferoxamine composite nanoparticles on cardiac function in mice after myocardial infarction; Figure (A) is a representative image of the mouse cardiac ultrasound results; Figure (B) is the 7th day after myocardial infarction. Representative images of the ejection fraction, fractional shortening, left ventricular diameter during diastole and left ventricular diameter during systole detected by echocardiography in mice; Figure (C) shows the ejection fraction, fractional shortening, and left ventricular diameter in mice detected by echocardiography on the 14th day after myocardial infarction. Representative diagrams of left ventricular diameter in diastole and left ventricular diameter in systole; Figure (D) shows the ejection fraction, fractional shortening, diastolic left ventricular diameter and systolic left ventricular diameter in mice measured by echocardiography on the 28th day after myocardial infarction. Representative figures; in Figures (B), (C) and (D), Sham group n=10, MI group n=7, Nano-CS group n=8, DFO group n=8, Nano-CS/DFO group n= 9, * represents p<0.05, ** represents p<0.01). Experimental group description: Sham (sham operation group), MI (myocardial infarction group), Nano-CS (chitosan nanoparticles treatment of myocardial infarction group), DFO (free deferoxamine treatment of myocardial infarction group), Nano-CS/DFO (Chitosan-deferoxamine composite nanoparticles for the treatment of myocardial infarction group).

Figure 7 is a characterization diagram of the particle size distribution of chitosan-deferoxamine composite nanoparticles synthesized at different concentrations of chitosan and different pH values.

Detailed ways

The present invention will be described in detail below based on the accompanying drawings and preferred embodiments. The purpose and effects of the present invention will become more clear. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

1. Physical and chemical performance testing

Add deferoxamine solution to DMEM medium and mix thoroughly (the final concentration of deferoxamine is 1.0 mg/mL), add chitosan solution to the mixture (the final concentration of chitosan is 1.25 mg/mL) , use Tris solution to adjust the pH of the mixed solution to 7.4, and place the mixed solution in a carbon dioxide gas environment to obtain a chitosan-deferoxamine composite nano-suspension. Observe the morphology of composite nanomaterials through transmission electron microscopy (TEM); use dynamic light scattering (DLS) to detect the particle size and Zeta potential of composite nanoparticles; use energy dispersive spectrometer , EDS) to analyze the composition of nanoparticles; use X-ray diffraction (XRD), Fourier transform infrared spectroscopy (Fourier transforminfrared spectroscopy, FT-IR) and XPS) to analyze the chemical composition and structure of composite nanomaterials.

The experimental results are as follows:

The TEM results show that the chitosan-deferoxamine composite nanoparticles in this system have good dispersion and appear as spherical particles with a rough surface and a particle size of about 80 nm, as shown in (A) in Figure 1; Proof It has good dispersion and is electronegative.

The particle size and Zeta potential of composite nanoparticles were detected by DLS. The particle size analysis results found that the diameter distribution of the composite nanoparticles was relatively concentrated, mainly between 30-100 nm, as shown in (B) in Figure 1. Zeta potential results show that free chitosan and deferoxamine have obvious positive electricity (9.720mV) and negative electricity (-7.787mV) respectively; while in the acidic solution environment with pH=6.2, chitosan and deferoxamine The mixed solution of deferoxamine was weakly electronegative (-1.178mV); while in the environment of a pH=7.4 solution, the synthesized chitosan-deferoxamine composite nanoparticles showed a negative electronegativity (-) similar to that of deferoxamine. 4.400mV), as shown in (C) in Figure 1. Analysis of EDS energy spectrum results proves that the elemental composition of chitosan-deferoxamine composite nanoparticles is carbon (C), nitrogen (N) and oxygen (O), as shown in (D) in Figure 1.

The structure of chitosan-deferoxamine composite nanoparticles was then determined by XRD, FT-IR and XPS. First, it was observed through XRD that the peaks of the chitosan-deferoxamine composite nanoparticles increased significantly, which represented an increase in the crystalline state, as shown in Figure 2 (A). Secondly, the characteristic free hydroxyl peaks in chitosan and deferoxamine and the associated hydroxyl peaks of chitosan-deferoxamine composite nanoparticles were observed, indicating that chitosan and deferoxamine during the self-assembly process of chitosan Chemical bonding occurs, as shown in (B) in Figure 2. Finally, through XPS, it was found that the peak positions of carbon and nitrogen elements of chitosan-deferoxamine composite nanoparticles were shifted compared with chitosan nanoparticles, indicating that chitosan and deferoxamine are in the gap between the two elements of carbon and nitrogen. Chemical bonding occurs, as shown in (C) and (D) in Figure 2. It was demonstrated that in chitosan-deferoxamine composite nanoparticles, chitosan and deferoxamine are not only physically adsorbed, but also have chemical bonds, which jointly mediate the formation of nanoparticles.

2. Evaluation of drug release characteristics in vitro

Add deferoxamine solution to DMEM medium and mix thoroughly (the final concentration of deferoxamine is 1.0 mg/mL), add chitosan solution to the mixture (the final concentration of chitosan is 1.25 mg/mL) , use Tris solution to adjust the pH of the mixed solution to 7.4, and place the mixed solution in a carbon dioxide gas environment to obtain a chitosan-deferoxamine composite nano-suspension. Move 10 mL of chitosan-deferoxamine composite nanoparticle suspension into the Visking-MD34 dialysis bag, use Tris-HCl with different pH as the release medium, and rotate at 0, 1, 2, 3, and 50 rpm at a rotation speed of 50 rpm/min. Samples were taken at time points of 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, and 18 hours, and high performance liquid chromatography (HPLC) was used to detect the in vitro drug release characteristics of the composite nanomaterials. The specific test parameters are: chromatographic column SHISEIDO CAPCELL PAK C18 (4.6mmI.D.*250mm, 5μm), flow rate 1.0mL/min, detection wavelength 210nm, column temperature 50℃, mobile phase 10mM sodium dihydrogenphosphate (use phosphoric acid to adjust pH to 3.0)-acetonitrile.

The experimental results are as follows:

In order to detect the release efficiency of deferoxamine from chitosan-deferoxamine composite nanoparticles in vitro, deferoxamine was detected by high-performance liquid chromatography. First, in order to verify the rationality of the selected chromatographic conditions, a series of deferoxamine solutions with different concentrations were prepared, and a standard curve was made with the deferoxamine concentration as the x-axis and the HPLC peak area as the y-axis, as shown in Figure 3 ( A) shown. It was found that the curve correlation coefficient R2=0.9967, indicating that the selected chromatographic conditions were reasonable. Then, the chitosan-deferoxamine composite nanoparticles were moved into the dialysis bag, using Tri-HCl with pH 6.2 and 7.4 as the release medium respectively, stirring at a constant temperature of 37°C, and taking samples at different time points to measure deferoxamine by HPLC. content. The change curve of deferoxamine content over time is shown in (B) in Figure 3 . Deferoxamine was almost not released in the environment of pH=7.4, but in the weakly acidic environment of pH=6.2, it started to be released from 0 hours, and by 18 hours, the release amount was about 80%. This shows that chitosan-deferoxamine composite nanoparticles have pH-responsive characteristics and can remain stable in a neutral environment and achieve stable and uniform release in an acidic environment.

3. Detection of the ability of chitosan-deferoxamine composite nanoparticles to complex iron ions

Add 250 μL of free chitosan solution (concentration of 1.25 mg/mL) and chitosan nanoparticle solution (chitosan concentration of 1.25 mg/mL). The configuration method is as mentioned above. An equal volume of DMEM is used to replace deferoxamine in the synthesis system. solution), deferoxamine solution (concentration is 1 mg/mL) and chitosan-deferoxamine composite nanoparticle solution (chitosan concentration is 1.25 mg/mL, deferoxamine concentration is 1 mg/mL, the configuration method is as mentioned above ) were mixed with the iron ion solution (the total amount of iron ions was 3 μmol) and left to stand at room temperature for 10 minutes. Using 430nm as the detection wavelength, detect the absorbance in different solutions and calculate their complexing ability with iron ions.

The experimental results are as follows:

The complexing conditions of free chitosan, chitosan nanoparticles, deferoxamine and chitosan-deferoxamine composite nanoparticles with iron ions were compared at the same concentration. The results are shown in Figure 5. As can be seen from Figure 5, free chitosan can hardly complex with iron ions, while the number of iron ions complexed by nanosized chitosan particles increases significantly, which shows that the nanonization process has a significant impact on the complexation of iron ions by chitosan. Competence is crucial. More importantly, compared with pure deferoxamine molecules, the ability of self-assembled chitosan-deferoxamine composite nanoparticles to complex iron ions has been greatly improved. As shown in Figure 5, chitosan-deferoxamine composite nanoparticles The iron complexing capacity of ferrioxamine composite nanoparticles is 2.05 times that of deferoxamine alone.

4. In vitro evaluation of drug safety

Sow primary cardiomyocytes or H9C2 cells in a 96-well plate at a cell density of 1×10 ⁴ cells/well, and culture them in a 37°C incubator; discard the culture medium, add sterile PBS to wash the cells; add CCK Prepare a mixture of -8 reagent and cell culture medium at a ratio of 1:9; add the above mixture into a 96-well plate at 100 μL/well, and prepare 5 cell-free blank wells to add an equal amount of the mixture for blank control. Place the cells in the 96-well plate and incubate them in a 37°C incubator for 4 hours. Use a microplate reader to measure the absorbance at 450 nm and calculate the cell viability. The formula is: cell activity (%) = (OD 450 treated group - OD 450 blank group) / (OD 450 control group - OD 450 blank group) × 100%.

The experimental results are as follows:

Different concentrations of deferoxamine and chitosan-deferoxamine composite nanoparticles were co-cultured with primary cardiomyocytes and H9C2 cardiomyocyte line for 24 hours, and it was found that 1-1000 μM deferoxamine and chitosan-deferoxamine None of the composite nanoparticles affected cardiomyocyte activity, as shown in Figure 4.

5. Construction of mouse myocardial infarction model and myocardial point injection administration

By constructing a mouse myocardial infarction model, the protective effect and related mechanisms of chitosan-deferoxamine composite nanoparticles on myocardial infarction in mice were studied. After the model was constructed, chitosan-deferoxamine composite nanoparticles were administered to the periphery of the infarcted myocardium of mice through intramyocardial injection. Subsequently, the release of the composite nanoparticles in the heart was observed through in vitro fluorescence imaging experiments. Then, the cardiac function of the mice was detected by cardiac ultrasound at 7, 14 and 28 days after myocardial infarction to evaluate the protective effect of chitosan-deferoxamine composite nanoparticles on the cardiac function of the mice. The effect of the composite nanoparticles on myocardial remodeling was then analyzed through Masson staining. Subsequently, the protective effect of the composite nanoparticles on oxidative stress damage, apoptosis and inflammation in the infarcted myocardium of mice was analyzed by immunofluorescence staining in the acute phase after myocardial infarction in mice. Finally, in the chronic phase after myocardial infarction surgery, western blotting and immunofluorescence staining were performed on the infarct area and surrounding myocardial tissue to observe the effect of the composite nanoparticles on angiogenesis in the border zone of myocardial infarction.

The specific steps are as follows: First, the mice were weighed and hairless, and the mice were anesthetized by intraperitoneal injection using 0.3% sodium pentobarbital at a dose of 25 mL/kg. After the mice were anesthetized, they were fixed and anesthetized with isoflurane to maintain anesthesia. Disinfect the mouse's neck and chest with alcohol and iodophor cotton balls, then separate the skin on both sides of the neck to expose the trachea, insert a 19G needle into the mouse's trachea to above the tracheal bifurcation, and use a MinVent mouse ventilator Maintain the respiratory state of the mouse, set the inhalation-to-exhalation ratio to 1:2, the ventilation volume to 200 μL, and the respiratory rate to 200 times/min. Finally, the neck muscles and skin are sutured and disinfected. Then, the skin and muscles were separated layer by layer on the left chest part about 1cm above the xiphoid process of the mouse. At this time, the beating heart in the chest wall could be observed. Then search for the fourth and fifth intercostal spaces according to the intercostal spaces, and separate the intercostal spaces to expose the heart. The mouse heart was observed under a microscope, and the left anterior descending branch of the mouse was ligated with 6-0 polypropylene suture. The color change of the mouse myocardium was observed through the microscope to determine whether the ligation was successful. About 30 minutes after the successful modeling, the mice were given corresponding point injection treatment. The dosage was 40 μL, and the injection was divided into four points evenly around the infarction area. After the point injection is completed, the chest wall muscles and skin are restored layer by layer, and the chest wall is disinfected. Transfer the mice to a 37°C warming pad for recovery, and return them to the animal room after they wake up.

The experimental results are as follows:

In this part of the experiment, a mouse myocardial infarction model with left anterior descending artery ligation was first constructed. 30 minutes after myocardial infarction, chitosan-deferoxamine composite nanoparticles were administered to the infarct area through myocardial point injection. In the surrounding myocardial tissue, it is used to repair the infarcted myocardial tissue. Through experiments, it was found that chitosan-deferoxamine composite nanoparticles entering myocardial tissue can reduce oxidative stress damage by complexing free iron ions, and the free deferoxamine released from chitosan-deferoxamine composite nanoparticles Amine can stabilize the expression of HIF-1, thereby mediating the increased transcription of VEGF and promoting angiogenesis.

Mechanistically, point injection of chitosan-deferoxamine composite nanoparticles significantly reduced the level of reactive oxygen species in mice 1 day after myocardial infarction. The expression of inflammatory factors and cell apoptosis 3 days after myocardial infarction were also significantly inhibited. Subsequently, through macrophage immunofluorescence staining, it was found that chitosan-deferoxamine composite nanoparticles inhibited the infiltration of macrophages after myocardial infarction in mice. Finally, the composite nanoparticles significantly promoted the expression of HIF-1α and VEGF in the myocardial infarction area and promoted angiogenesis in the myocardial infarction edge area.

After successfully constructing a mouse myocardial infarction model, through in vitro fluorescence imaging experiments, it was found that chitosan-deferoxamine composite nanoparticles significantly prolonged the drug release time.

6. Mouse cardiac ultrasound detection

On days 7, 14, and 28 after myocardial infarction in mice, the cardiac function of the mice was evaluated using the Vevo1100 small animal ultrasound imaging system. Before ultrasound, the mouse chest was depilated to fully expose the left anterior chest area. The mice were transferred to a chamber filled with isoflurane for gas anesthesia. After it is confirmed that the mouse is fully anesthetized, it is transferred to the testing table and fixed, and the anesthesia state is continued to be maintained with isoflurane through the breathing tube. Then, use the MS400C ultrasound probe to the left chest of the mouse to obtain the B-mode and M-mode echocardiographic information of the long axis of the mouse's left ventricle for subsequent data analysis. After the ultrasound examination, the mice were transferred to a constant-temperature pad for recovery, and then transferred to a cage for continued rearing. Through Vevo ultrasound analysis software, cardiac function indicators such as left ventricular ejection fraction, left ventricular shortening fraction, left ventricular diastolic inner diameter, left ventricular inner ventricular diameter during systole, etc. were mainly statistically analyzed.

The experimental results are as follows:

In order to clarify whether chitosan-deferoxamine composite nanoparticles have a protective effect on myocardial infarction in mice, the changes in cardiac function of mice on postoperative days 7, 14, and 28 were first detected by cardiac ultrasound. The results are shown in Figure 6 As shown in (A). Cardiac ultrasound results showed that 7 days after surgery, the ejection fraction (EF) and fractional shortening (FS) of mice in the free deferoxamine group and chitosan-deferoxamine composite nanoparticles group were significantly higher than those in the myocardial infarction group. , and chitosan-deferoxamine composite nanoparticle treatment can significantly inhibit the expansion of the ventricular diameter during left ventricular systole in mice after myocardial infarction, as shown in Figure 6 (B). However, at 14 days after surgery, although the ejection fraction of the free deferoxamine group was significantly higher than that of the myocardial infarction group, the ejection fraction and shortening fraction of chitosan-deferoxamine nanoparticles were significantly higher than those of the free deferoxamine group. The deferoxamine group is shown in (C) in Figure 6 . At 28 days after surgery, there was no significant difference in ejection fraction and fractional shortening between the chitosan nanoparticles group, the free deferoxamine group and the myocardial infarction group, but at this time the ejection fraction of the chitosan-deferoxamine nanoparticles group was significantly lower than that of the myocardial infarction group. The fraction and shortening fraction were significantly higher than those in the free deferoxamine group, and the inner ventricular diameter during systole of the left ventricle was also significantly reduced, as shown in (D) in Figure 6 . The above results suggest that although intramyocardial administration of free deferoxamine has a certain protective effect on the cardiac function of mice in the acute phase of myocardial infarction, it cannot achieve long-term protective effects. Compared with free chitosan treatment, chitosan-deferoxamine composite nanoparticle treatment can significantly improve the therapeutic effect, prolong the duration of cardiac function protection, and significantly inhibit myocardial remodeling after myocardial infarction in mice. .

7. Chitosan-deferoxamine composite nanoparticles synthesized under different concentrations of chitosan and different pH

During the experiment, attempts were made to synthesize chitosan-deferoxamine composite nanoparticles under different concentrations of chitosan (0.5-15 mg/mL) and different pH (7.0-7.6). As shown in Figure 7, when the chitosan concentration is too low (0.5 mg/mL), the peaks in the particle size distribution curve are 352.1, 1720, and 5560 nm respectively, indicating the particle size distribution of chitosan-deferoxamine composite nanoparticles. Non-uniform; when the concentration of chitosan is too high (15mg/mL), the peak value in the particle size distribution curve is approximately 2078nm, indicating that the chitosan-deferoxamine composite nanoparticles are seriously agglomerated and the particle size is too large; when the synthesis pH is too high When the pH is low (pH 7.0), there is no peak in the particle size distribution curve, indicating that no chitosan-deferoxamine composite nanoparticles are produced; when the synthesis pH is too high (pH 7.6), the peaks in the particle size distribution curve are 347.8. , 720.6 and 821.2nm, indicating that the chitosan-deferoxamine composite nanoparticles are seriously agglomerated and the particle size is too large and uneven.

At the same time, during the experiment, we also tried to synthesize chitosan-deferoxamine composite nanoparticles at different concentrations of deferoxamine (0.1-10 mg/mL). The peaks in the particle size distribution curve of chitosan-deferoxamine composite nanoparticles are all around 50nm (refer to Figure 1B), indicating that the concentration of deferoxamine does not affect the particle size distribution of chitosan-deferoxamine composite nanoparticles .

Those of ordinary skill in the art can understand that the above are only preferred examples of the invention and are not intended to limit the invention. Although the invention has been described in detail with reference to the foregoing examples, those skilled in the art can still The technical solutions recorded in the foregoing examples are modified, or some of the technical features are equivalently replaced. All modifications, equivalent substitutions, etc. that are within the spirit and principle of the invention shall be included in the protection scope of the invention.

Claims (7)

1. The preparation method of the chitosan-deferoxamine composite nano suspension is characterized by comprising the following steps of:

adding a deferoxamine solution into a DMEM culture medium, and fully and uniformly mixing to ensure that the final concentration of deferoxamine is 0.1-10mg/mL; within the concentration range, the final concentration of the deferoxamine solution does not influence the morphology and electronegativity of the synthesized chitosan-deferoxamine composite nano-particles;

adding chitosan solution into the mixed solution to make the final concentration of chitosan in the mixed solution be 0.5-15mg/mL;

regulating the pH value of the mixed solution to 7.0-7.6 by using Tris solution;

placing the mixed solution in a carbon dioxide gas environment, and standing to obtain the chitosan-deferoxamine composite nano suspension.

2. The method for preparing a chitosan-deferoxamine composite nanosuspension according to claim 1, wherein the final concentration of deferoxamine is 1mg/mL.

3. The method for preparing a chitosan-deferoxamine composite nanosuspension according to claim 1, wherein the final concentration of chitosan is 1.25mg/mL.

4. The method for preparing chitosan-deferoxamine composite nanosuspension according to claim 1, wherein the pH of the mixed solution is adjusted to 7.4 with Tris solution.

5. The method for preparing a chitosan-deferoxamine composite nanosuspension according to claim 1, wherein the concentration of the Tris solution is 0.1M.

6. A chitosan-deferoxamine composite nanosuspension prepared by the method of any one of claims 1 to 5.

7. Use of the chitosan-deferoxamine composite nano-suspension according to claim 6 in the preparation of a myocardial repair drug.