CN115607683A - Chitosan-deferoxamine composite nano suspension and preparation method and application thereof - Google Patents
Chitosan-deferoxamine composite nano suspension and preparation method and application thereof Download PDFInfo
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- CN115607683A CN115607683A CN202211303763.4A CN202211303763A CN115607683A CN 115607683 A CN115607683 A CN 115607683A CN 202211303763 A CN202211303763 A CN 202211303763A CN 115607683 A CN115607683 A CN 115607683A
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Abstract
The invention discloses a chitosan-deferoxamine composite nano suspension and a preparation method and application thereof, wherein a deferoxamine solution is added into a DMEM medium and fully and uniformly mixed to ensure that the final concentration of deferoxamine is 0.1-10mg/mL; adding a chitosan solution into the mixed solution to ensure that the final concentration of chitosan in the mixed solution is 0.5-15mg/mL; regulating the pH of the mixed solution to 7.0-7.6 by using a Tris solution; and placing the mixed solution in a carbon dioxide gas environment, and standing to obtain the chitosan-desferrioxamine composite nano suspension. The chitosan-deferoxamine composite nano particles in the chitosan-deferoxamine composite nano turbid liquid have stronger iron ion complexing capacity, can uniformly release deferoxamine drug molecules in an acid environment, and can remarkably inhibit myocardial poor reconstruction and promote repair of infarcted myocardium.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a chitosan-deferoxamine composite nano suspension, and a preparation method and application thereof.
Background
Cardiovascular diseases become a major public health problem in the global scope, and the Chinese cardiovascular health and disease report 2020 shows that the number of cardiovascular disease patients in China is up to 3.3 hundred million, and the number of cardiovascular disease deaths accounts for more than 40% of the number of disease deaths of residents in China. Myocardial infarction due to coronary artery occlusion is the most harmful of the cardiovascular diseases, accounting for nearly 80% of all cardiovascular death events. The occurrence of myocardial infarction can cause massive death of myocardial cells and a severe immune inflammatory response. Due to the lack of reproducible ability of cardiomyocytes, scar repair of the heart is mediated during the compensatory phase after infarction mainly by activation of cardiac fibroblasts, which can lead to poor remodeling of the heart, which in turn leads to the development of cardiac diastolic dysfunction and heart failure. Thus, inhibiting myocardial damage and necrosis after infarction can greatly improve the prognosis of patients with myocardial infarction.
Iron, an important essential element of the human body, plays an important role in the regulation of cardiac function. When myocardial infarction occurs, the expression of ferritin is obviously reduced, and the binding capacity of ferritin to free iron ions is obviously weakened; meanwhile, the acidic and highly reductive environment caused by continuous ischemia can degrade ferritin inside and outside cells, and a large amount of iron ions in the ferritin are released. The free iron in turn converts superoxide and hydrogen peroxide in the environment to hydroxyl radicals and hydroxide anions with stronger oxidizing properties by fenton's reaction. These free radical by-products can lead to mitochondrial damage and dysregulation of calcium homeostasis in cardiomyocytes, thereby exacerbating cardiac dysfunction. At present, researches show that the cardiac contractile function can be obviously improved, the cell activity is increased, and the cardiac remodeling is inhibited by targeted inhibition of iron overload during the cardiac ischemic injury. Deferoxamine is a natural siderophore, has strong affinity to iron ions, and can bind free iron ions in human body and iron ions in ferritin and ferrierite. Studies show that deferoxamine can inhibit free radical increase during myocardial ischemia reperfusion and relieve reperfusion injury. In addition, deferoxamine is also considered to be a stabilizer for expression of hypoxia inducible factor HIF-1 alpha, and further promotes expression of vascular endothelial growth factor, which makes it more potential as a clinical treatment drug for myocardial infarction. But because the deferoxamine is a water-soluble small-molecule drug, the deferoxamine has short circulation time in vivo and is easy to metabolize by the liver and the kidney. Related clinical studies have also found that continuous intravenous administration is required to exert the desired cardioprotective effect, which greatly limits its application in clinical practice.
With the development of nanotechnology, nanomaterial-based drug delivery systems have been widely used in the biomedical field. Nanoparticles have good physicochemical properties and modification ability, and are often used as delivery carriers of small molecule active drugs and the like in the field of cardiovascular research. Among them, chitosan is a natural cationic polysaccharide with good histocompatibility and biodegradability, and is considered as an ideal carrier for many drug delivery. And because the modified starch has the modifiable property, the application property and the function of the modified starch can be enhanced through chemical modification, and the application range of the modified starch is greatly expanded. More importantly, as various functional groups such as hydroxyl, amino, acetyl and the like are distributed on the molecular chain of the chitosan, the chitosan material has chelation and enrichment effects on metal ions such as iron ions. This suggests that the chitosan-based nanomaterial can be applied to the treatment of myocardial infarction by exerting its effect of chelating iron ions in the myocardial infarction region.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a chitosan-deferoxamine composite nano suspension and a preparation method and application thereof, the chitosan-deferoxamine composite nano particles are prepared by utilizing the self-assembly characteristic of chitosan, and the chitosan-deferoxamine composite nano suspension aims at reducing the iron ion content of myocardial cells in an infarct area to relieve the oxidative stress injury of the myocardial cells through the synergistic action of chitosan and deferoxamine, and improving HIF-1 alpha expression to promote the transcription of vascular endothelial growth factors so as to achieve the aim of promoting myocardial repair after infarction.
A preparation method of chitosan-deferoxamine composite nano suspension comprises the following steps:
adding a desferoxamine solution into a DMEM culture medium, and fully and uniformly mixing;
adding a chitosan solution into the mixed solution to ensure that the final concentration of chitosan in the mixed solution is 0.5-15mg/mL;
regulating the pH value of the mixed solution to 7.0-7.6 by using a Tris solution;
and placing the mixed solution in a carbon dioxide gas environment, and standing to obtain the chitosan-deferoxamine composite nano suspension.
Further, a deferoxamine solution was added to the DMEM medium such that the final concentration of deferoxamine was 0.1-10 mg/mL. Within this concentration range, the final concentration of the deferoxamine solution does not affect the morphology and electronegativity of the synthesized chitosan-deferoxamine composite nanoparticles.
Further, the final concentration of deferoxamine is 1mg/mL.
Further, the final concentration of chitosan was 1.25mg/mL.
Further, the pH of the mixture was adjusted to 7.4 with Tris solution.
Further, the concentration of the Tris solution is 0.1M.
The chitosan-deferoxamine composite nano suspension prepared by the method.
An application of chitosan-deferoxamine composite nano-suspension in preparing a myocardial repair medicament.
The invention has the following beneficial effects:
(1) Compared with a pure deferoxamine molecule and a pure chitosan nano material, the chitosan and the deferoxamine have a synergistic effect on complexing iron ions, and chitosan-deferoxamine composite nano particles in the chitosan-deferoxamine composite nano turbid liquid show stronger iron ion complexing capacity.
(2) The chitosan-deferoxamine composite nanoparticle has the characteristic of pH response release, can uniformly release deferoxamine drug molecules in an acidic environment, and obviously prolongs the drug release time.
(3) The chitosan-desferrioxamine composite nanoparticle can relieve hypoxia and oxidative stress injury of myocardial cells, has the effects of resisting oxidative stress, resisting apoptosis, resisting inflammation and promoting angiogenesis, can promote the tube forming capability of endothelial cells, can protect the cardiac function of mice after myocardial infarction, and can obviously inhibit poor reconstruction of cardiac muscle.
(4) The chitosan-deferoxamine composite nano-particles realize the targeted and efficient enrichment of deferoxamine in the heart, enhance the biological functions of the deferoxamine in inhibiting oxidative stress injury and promoting angiogenesis, and further promote the repair of infarcted myocardium. This provides new strategy and idea for clinical treatment of myocardial infarction.
Drawings
FIG. 1 is a graphical representation of chitosan-desferrioxamine composite nanoparticles; wherein, the picture (A) is a TEM picture of the chitosan-deferoxamine composite nano-particles; FIG. B shows the results of particle size analysis of chitosan-desferrioxamine composite nanoparticles; the graph (C) is a Zeta potential result of the chitosan-deferoxamine composite nano-particles; panel (D) is a spectral analysis of chitosan-desferrioxamine composite nanoparticles.
Fig. 2 is a structural characterization of chitosan-desferrioxamine composite nanoparticles; wherein, the figure (A) is the XRD analysis result of the chitosan-deferoxamine composite nano-particles; FIG. (B) shows the FT-IR analysis results of chitosan-deferoxamine composite nanoparticles; the figure (C) is the analysis result of carbon (C) element in XPS detection of the chitosan-desferrioxamine composite nanoparticle; and (D) is the analysis result of nitrogen (N) element in XPS detection of chitosan-desferrioxamine composite nanoparticles.
Fig. 3 is a graph showing the efficiency of the chitosan-desferrioxamine composite nanoparticle in releasing desferrioxamine in vitro, as detected by HPLC; wherein, the graph (A) is a fitting straight line of the concentration of the deferoxamine and the detection peak area constructed by HPLC; panel (B) is the release profile of deferoxamine at different pH in HPLC experiments (n = 3).
FIG. 4 is a graph of the effect of chitosan-desferrioxamine composite nanoparticles on cardiomyocyte activity; wherein, the graph (A) is a statistical graph of the CCK-8 experimental results after the deferoxamine with different concentrations is co-cultured with primary myocardial cells and H9C2 cells (n = 5); panel (B) is a statistical plot of CCK-8 experimental results after co-culture of chitosan-desferrioxamine composite nanoparticles at different concentrations with primary cardiomyocytes and H9C2 cells (n = 5).
Fig. 5 is a measurement of the ability of free chitosan, chitosan nanoparticles, deferoxamine, and chitosan-deferoxamine composite nanoparticles to complex iron, wherein the free chitosan group n =4 and the other groups n =5, representing p < 0.01.
FIG. 6 is a graph of the effect of chitosan-desferrioxamine composite nanoparticles on the post-myocardial function of mice; wherein, the image (A) is a representation image of the ultrasonic result of the heart of the mouse; FIG. B is a representative graph of ejection fraction, fractional shortening, diastolic left ventricular inside diameter and systolic left ventricular inside diameter of 7 th cardiac ultrasonography mice after myocardial infarction; FIG. C is a representative graph of the ejection fraction, the fractional shortening, the diastolic left ventricular inside diameter and the systolic left ventricular inside diameter of the hypercardiac examination mice on day 14 after myocardial infarction; FIG. D is a representative graph of the ejection fraction, the fractional shortening, the diastolic left ventricular inside diameter and the systolic left ventricular inside diameter of the hypercardiac examination mice on day 28 after myocardial infarction; in graphs (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,. Indicates p < 0.05,. Indicates p < 0.01). Experimental grouping description: sham (Sham group), MI (myocardial infarction group), nano-CS (chitosan nanoparticle-treated myocardial infarction group), DFO (free deferoxamine-treated myocardial infarction group), nano-CS/DFO (chitosan-deferoxamine composite nanoparticle-treated myocardial infarction group).
FIG. 7 is a graph showing the particle size distribution of the synthesized chitosan-deferoxamine composite nanoparticles at different concentrations of chitosan and different pH values.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments, and the objects and effects of the present invention will become more apparent, it being understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.
1. Detection of physicochemical Properties
Adding a deferoxamine solution into a DMEM medium, fully and uniformly mixing (the final concentration of deferoxamine is 1.0 mg/mL), adding a chitosan solution (the final concentration of chitosan is 1.25 mg/mL) into the mixed solution, adjusting the pH of the mixed solution to 7.4 by using a Tris solution, and placing the mixed solution in a carbon dioxide gas environment for standing to obtain the chitosan-deferoxamine composite nano suspension. Observing the morphology of the composite nanomaterial by a Transmission Electron Microscope (TEM); detecting the particle size and the Zeta potential of the composite nano particles by using a Dynamic Light Scattering (DLS); analyzing the composition of the nanoparticles by using an Energy Dispersive Spectrometer (EDS); the chemical composition and structure of the composite nanomaterial were analyzed using an X-ray diffraction (XRD), fourier transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS).
The experimental results are as follows:
TEM results show that the chitosan-deferoxamine composite nanoparticles in the system have good dispersibility, are spherical particles with rough surfaces, and have the particle size of about 80nm, as shown in (A) in figure 1; it was proved to have good dispersibility and to be electronegative.
The particle size and Zeta potential of the composite nanoparticles were measured by DLS. As a result of the particle size analysis, the diameter distribution of the composite nanoparticles was more concentrated, mainly between 30 and 100nm, as shown in FIG. 1 (B). The Zeta potential result shows that the free chitosan and the deferoxamine respectively have obvious positive electricity (9.720 mV) and negative electricity (-7.787 mV); in an acid solution environment with pH =6.2, the mixed solution of chitosan and deferoxamine has weak electronegativity (-1.178 mV); whereas in the environment of pH =7.4 solution, the synthesized chitosan-deferoxamine composite nanoparticles exhibited electronegativity (-4.400 mV) more similar to that of deferoxamine, as shown in (C) of fig. 1. The elemental composition of the chitosan-deferoxamine composite nanoparticles was demonstrated by EDS spectroscopy results analysis to be carbon (C), nitrogen (N) and oxygen (O), as shown in (D) of fig. 1.
The structure of the chitosan-deferoxamine composite nanoparticle was then determined by XRD, FT-IR and XPS. First, the chitosan-deferoxamine composite nanoparticle was observed to have a significantly increased and heightened peak by XRD, which represents that it exhibited an increase in the crystalline state, as shown in (a) of fig. 2. Secondly, a characteristic free hydroxyl peak in chitosan and deferoxamine and an associated hydroxyl peak of the chitosan-deferoxamine composite nanoparticle were observed, indicating that the chitosan and deferoxamine were chemically bonded during the chitosan self-assembly process, as shown in (B) of fig. 2. Finally, XPS found that the chitosan-deferoxamine composite nanoparticle was shifted in peak positions of carbon and nitrogen elements compared to the chitosan nanoparticle, indicating that chitosan and deferoxamine were chemically bonded between carbon and nitrogen elements, as shown in (C) and (D) of fig. 2. It is demonstrated that in the chitosan-deferoxamine composite nanoparticle, the chitosan and the deferoxamine have not only physical adsorption but also chemical bond function, and the chitosan and the deferoxamine together mediate the formation of the nanoparticle.
2. Evaluation of in vitro drug Release characteristics
Adding a deferoxamine solution into a DMEM medium, fully and uniformly mixing (the final concentration of deferoxamine is 1.0 mg/mL), adding a chitosan solution (the final concentration of chitosan is 1.25 mg/mL) into the mixed solution, adjusting the pH of the mixed solution to 7.4 by using a Tris solution, and placing the mixed solution in a carbon dioxide gas environment for standing to obtain the chitosan-deferoxamine composite nano suspension. Transferring 10mL of chitosan-desferrioxamine composite nanoparticle suspension into a Visking-MD34 dialysis bag, taking Tris-HCl with different pH values as release media, sampling at time points of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 and 18 hours under the condition of rotation speed of 50rpm/min, and detecting the in-vitro drug release characteristics of the composite nanomaterial by using a High Performance Liquid Chromatography (HPLC). The specific test parameters are as follows: column SHISEIDO CAPCELL PAK C18 (4.6 mM I.D. 250mm,5 μm), flow rate 1.0mL/min, detection wavelength 210nm, column temperature 50 ℃, mobile phase 10mM sodium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) -acetonitrile.
The experimental results are as follows:
in order to detect the in-vitro release efficiency of the chitosan-deferoxamine composite nanoparticles to deferoxamine, a high performance liquid chromatograph is used for detecting deferoxamine. 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 prepared with the deferoxamine concentration as the x-axis and the HPLC peak area as the y-axis, as shown in fig. 3 (a). Curve correlation coefficient R2=0.9967 was found to be reasonable for the chosen chromatographic conditions. And then transferring the chitosan-deferoxamine composite nanoparticles into a dialysis bag, respectively taking Tri-HCl with pH of 6.2 and 7.4 as release media, stirring at constant temperature of 37 ℃, sampling at different time points, and determining the deferoxamine content by an HPLC method. The curve of the content of deferoxamine with time is shown in figure 3 (B). Deferoxamine is released almost without release in a pH =7.4 environment, whereas in a weakly acidic environment with pH =6.2, the release starts from 0 hours and reaches about 80% by 18 hours. The result shows that the chitosan-deferoxamine composite nanoparticle has the characteristic of pH response, can be stably maintained in a neutral environment, and can be stably and uniformly released in an acidic environment.
3. Detection of iron ion complexing capacity of chitosan-desferrioxamine composite nanoparticles
mu.L of a free chitosan solution (concentration of 1.25 mg/mL), a chitosan nanoparticle solution (concentration of chitosan of 1.25mg/mL, preparation method as described above, equivalent volume of DMEM in the synthesis system instead of the deferoxamine solution), a deferoxamine solution (concentration of 1 mg/mL) and a chitosan-deferoxamine composite nanoparticle solution (concentration of chitosan of 1.25mg/mL, concentration of deferoxamine of 1mg/mL, preparation method as described above) were mixed with an iron ion solution (total amount of iron ions of 3. Mu. Mol) respectively, and left to stand at room temperature for 10 minutes. And (4) detecting the absorbance of different solutions by taking 430nm as a detection wavelength, and calculating the complexing ability of the solutions and iron ions.
The experimental results are as follows:
the complexing conditions of the chitosan, chitosan nanoparticles, deferoxamine and chitosan-deferoxamine composite nanoparticles with the same concentration and iron ions were respectively compared, and the results are shown in fig. 5. As can be seen from fig. 5, free chitosan can hardly be complexed with iron ions, while iron ions complexed by the nanosized chitosan particles are significantly increased, which indicates that the process of nanosized chitosan is crucial for the iron complexing ability of chitosan. More importantly, compared with a pure deferoxamine molecule, the iron ion complexing capacity of the chitosan-deferoxamine composite nanoparticle formed by self-assembly is greatly improved, and as shown in fig. 5, the iron complexing capacity of the chitosan-deferoxamine composite nanoparticle is 2.05 times that of a pure deferoxamine drug.
4. In vitro evaluation of drug safety
In 96-well plates at 1X 10 4 Inoculating primary myocardial cells or H9C2 cells at the cell density of each hole, and culturing in an incubator at 37 ℃; removing the culture medium, and adding sterile PBS to clean the cells; preparing a mixed solution of the CCK-8 reagent and a cell culture medium according to the proportion of 1; the mixture was added to a 96-well plate at 100. Mu.L/well, and 5 blank wells without cells were prepared and an equal amount of the mixture was added for blank control. The 96-well plate cells were incubated in an incubator at 37 ℃ for 4 hours, absorbance at 450nm was measured using a microplate reader, and cell activity was calculated. The formula is as follows: cell activity (%) = (OD 450 treatment-OD 450 blank)/(OD 450 control-OD 450 blank) × 100%.
The experimental results are as follows:
after the deferoxamine and chitosan-deferoxamine composite nanoparticles with different concentrations were co-cultured with primary cardiomyocytes and H9C2 cardiomyocyte line for 24 hours, respectively, it was found that both 1-1000 μ M of deferoxamine and chitosan-deferoxamine composite nanoparticles did not affect cardiomyocyte activity, as shown in fig. 4.
5. Mouse myocardial infarction model construction and myocardial point injection drug delivery
By constructing a mouse myocardial infarction model, the protective effect of the chitosan-desferrioxamine composite nanoparticles on mouse myocardial infarction and the related mechanism research are researched. After model construction, chitosan-desferrioxamine composite nanoparticles were administered by intramyocardial site injection to mice around infarcted myocardium. And then observing the release condition of the composite nano particles in the heart through an in vitro fluorescence imaging experiment. And then detecting the cardiac function of the mice by cardiac ultrasound at 7 days, 14 days and 28 days after the myocardial infarction of the mice to evaluate the protective effect of the chitosan-desferrioxamine composite nanoparticles on the cardiac function of the mice. The effect of the composite nanoparticles on myocardial remodeling was then analyzed by Masson staining. Then, the protective effect of the composite nano-particles on oxidative stress injury, apoptosis and inflammation of infarcted myocardium of the mice is analyzed by immunofluorescence staining in the acute stage after the myocardial infarction of the mice. Finally, in the chronic period after the myocardial infarction operation, the influence of the composite nano-particles on myocardial angiogenesis in the myocardial marginal area is observed by carrying out western blotting and immunofluorescence staining on the infarct area and the surrounding myocardial tissues.
The method comprises the following specific steps: the mice were first weighed for unhairing and were anesthetized by intraperitoneal injection using 0.3% sodium pentobarbital at a dose of 25 mL/kg. After the mice were anesthetized, the mice were fixed and maintained under anesthesia with isoflurane anesthesia. The neck and the chest of a mouse are disinfected by alcohol and an iodophor cotton ball in sequence, then the skin on two sides of the neck is separated to expose the trachea, a 19G needle head is inserted into the trachea of the mouse to the position above the bifurcation of the trachea, the respiratory state of the mouse is maintained by a MinVent mouse respirator, the respiratory ratio is set to be 1. Then, the skin and the muscle are separated layer by layer at the left anterior chest part 1cm above the mouse xiphoid process, and the beating heart in the chest wall can be observed. Then, the fourth and fifth intercostal spaces are searched according to the intercostal spaces, and the intercostal spaces are separated to expose the heart. The heart of the mouse is observed under a microscope, the left anterior descending branch of the mouse is bound by 6-0 polypropylene thread, and the color change of the myocardium of the mouse is observed through the microscope to judge whether the binding is successful. After the molding is successful, the injection is performed on corresponding points of the mice about 30 minutes, the administration dose is 40 mu L, and the injection is averagely performed on four points to the periphery of the infarcted area. After the spot injection is completed, the chest wall muscles and skin are restored layer by layer and the chest wall is sterilized. And (3) transferring the mouse to a 37 ℃ heat-insulating pad for resuscitation, and returning the mouse to an animal room after the mouse revives.
The experimental results are as follows:
in the experiment, a mouse left anterior descending ligation myocardial infarction model is firstly constructed, and chitosan-deferoxamine composite nanoparticles are administrated into myocardial tissues around an infarcted area in a myocardial point injection mode 30 minutes after the mouse myocardial infarction for repairing the infarcted myocardial tissues. Experiments show that the chitosan-deferoxamine composite nanoparticles entering myocardial tissues can reduce oxidative stress injury by complexing free ferric ions, and the free deferoxamine released from the chitosan-deferoxamine composite nanoparticles can stabilize the expression of HIF-1 sodium, further mediate the transcriptional increase of VEGF and promote angiogenesis.
Mechanically, the chitosan-desferrioxamine composite nanoparticles are point-injected, so that the active oxygen level of the mice 1 day after myocardial infarction is obviously reduced. The expression of inflammatory factors and apoptosis were also significantly inhibited 3 days after myocardial infarction. Subsequently, by means of macrophage immunofluorescence staining, the chitosan-desferrioxamine composite nanoparticles are found to inhibit infiltration of macrophages after myocardial infarction of mice. Finally, the composite nano-particle remarkably promotes the expression of HIF-1 alpha and VEGF in the myocardial infarction area, and can promote angiogenesis in the myocardial infarction marginal area.
After a mouse myocardial infarction model is successfully constructed, in-vitro fluorescence imaging experiments show that the chitosan-deferoxamine composite nanoparticles remarkably prolong the drug release time.
6. Mouse heart ultrasonic detection
Cardiac function was assessed in mice by the Vevo1100 mouse ultrasound imaging system on days 7, 14 and 28 post-myocardial infarction. The mice were depilated on their breasts prior to sonication, fully exposing the left precordial region. Mice were taken and transferred to a chamber filled with isoflurane for gas anesthesia. And after the mice are confirmed to be sufficiently anesthetized, transferring the mice to a detection table for fixation, and continuously maintaining the anesthetized state by using isoflurane through a breathing tube. Then, an ultrasonic probe of MS400C is used to reach the front of the left chest of the mouse, and B-type and M-type echocardiogram information of the long axis of the left chamber of the mouse is obtained for subsequent data analysis. And after the ultrasonic detection is finished, the mice are transferred to a constant temperature pad for resuscitation, and then transferred to a cage for continuous feeding. Through Vevo ultrasonic analysis software, cardiac function indexes such as left ventricular ejection fraction, left ventricular shortening fraction, left ventricular diastolic phase ventricular diameter and left ventricular systolic phase ventricular diameter are mainly statistically analyzed.
The experimental results are as follows:
to determine whether the chitosan-deferoxamine composite nanoparticle has a protective effect on myocardial infarction of mice, the change of cardiac function of the mice at 7, 14 and 28 days after the operation was first detected by cardiac ultrasound, and the result is shown in (a) of fig. 6. The cardiac ultrasound results showed that the Ejection Fraction (EF) and the shortening Fraction (FS) of the free deferoxamine group and chitosan-deferoxamine composite nanoparticle group mice were significantly increased compared to the myocardial infarction group 7 days after the operation, and the chitosan-deferoxamine composite nanoparticle treatment could significantly inhibit the expansion of the left ventricular systolic ventricular inner diameter of the mice after the myocardial infarction, as shown in (B) of fig. 6. However, at 14 days after the operation, the free deferoxamine group also had a significantly higher ejection fraction than the myocardial infarction group, but both the ejection fraction and the shortened fraction of the chitosan-deferoxamine nanoparticles were significantly higher than those of the free deferoxamine group, as shown in fig. 6 (C). At 28 days after the operation, there was no significant difference in the ejection fraction and the shortening fraction between the chitosan nanoparticle group and the free deferoxamine group and the myocardial infarction group, but at this time, the ejection fraction and the shortening fraction of the chitosan-deferoxamine nanoparticle group were both significantly higher than those of the free deferoxamine group, and the left ventricular systolic ventricular inside diameter was also significantly reduced, as shown in (D) of fig. 6. The above results suggest that the treatment by intramyocardial administration of free deferoxamine has a certain protective effect on the cardiac function of mice in the acute myocardial infarction stage, but cannot achieve a long-term protective effect. Compared with free chitosan treatment, the chitosan-desferrioxamine composite nanoparticle treatment can obviously improve the treatment effect, prolong the duration of cardiac function protection and obviously inhibit myocardial remodeling of mice after myocardial infarction.
7. Chitosan-deferoxamine composite nanoparticles synthesized under different concentrations of chitosan and different pH values
In the experimental process, the synthesis of chitosan-deferoxamine composite nanoparticles under different concentrations of chitosan (0.5-15 mg/mL) and different pH (7.0-7.6) is attempted. As shown in fig. 7, when the chitosan concentration is too low (0.5 mg/mL), the peak values in the particle size distribution curve are 352.1, 1720 and 5560nm, respectively, which indicates that the particle size distribution of the chitosan-deferoxamine composite nanoparticle is not uniform; when the chitosan concentration is too high (15 mg/mL), the peak value in the particle size distribution curve is about 2078nm, which indicates that the chitosan-deferoxamine composite nano particles are seriously agglomerated and have too large particle sizes; when the synthesis pH is too low (pH 7.0), no peak appears in the particle size distribution curve, which indicates that no chitosan-deferoxamine composite nano-particles are generated; when the synthesis pH is too high (pH 7.6), the peak values in the particle size distribution curve are 347.8, 720.6 and 821.2nm respectively, which indicates that the chitosan-deferoxamine composite nanoparticles are seriously agglomerated and have too large and non-uniform particle sizes.
Meanwhile, in the experimental process, the synthesis of the chitosan-deferoxamine composite nanoparticle under the condition of deferoxamine with different concentrations (0.1-10 mg/mL) is also tried. The peak values in the particle size distribution curve of the chitosan-deferoxamine composite nanoparticle are all about 50nm (refer to fig. 1B), which indicates that the concentration of deferoxamine does not affect the particle size distribution of the chitosan-deferoxamine composite nanoparticle.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and although the invention has been described in detail with reference to the foregoing examples, it will be apparent to those skilled in the art that various changes in the form and details of the embodiments may be made and equivalents may be substituted for elements thereof. All modifications, equivalents and the like which come within the spirit and principle of the invention are intended to be included within the scope of the invention.
Claims (8)
1. A preparation method of chitosan-deferoxamine composite nano-suspension is characterized by comprising the following steps:
adding a desferoxamine solution into a DMEM culture medium, and fully and uniformly mixing;
adding a chitosan solution into the mixed solution to ensure that the final concentration of chitosan in the mixed solution is 0.5-15mg/mL;
regulating the pH value of the mixed solution to 7.0-7.6 by using a Tris solution;
and 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 nano-suspension according to claim 1, wherein a deferoxamine solution is added into a DMEM medium so that the final concentration of deferoxamine is 0.1-10mg/mL; within this concentration range, the final concentration of the deferoxamine solution does not affect the morphology and electronegativity of the synthesized chitosan-deferoxamine composite nanoparticles.
3. The method for preparing a chitosan-deferoxamine composite nano-suspension according to claim 2, wherein the final concentration of deferoxamine is 1mg/mL.
4. The method for preparing a chitosan-deferoxamine composite nano-suspension according to claim 1, wherein the final concentration of chitosan is 1.25mg/mL.
5. The method of claim 1, wherein the pH of the mixture is adjusted to 7.4 with Tris solution.
6. The method of claim 1, wherein the Tris solution is at a concentration of 0.1M.
7. A chitosan-deferoxamine composite nano-suspension prepared by the method of any one of claims 1 to 6.
8. The use of the chitosan-deferoxamine composite nano-suspension of claim 7 in the preparation of a myocardial repair drug.
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| US20010033860A1 (en) * | 1999-12-30 | 2001-10-25 | Gwathmey Judith K. | Iron chelator delivery system |
| CN104856960A (en) * | 2015-04-27 | 2015-08-26 | 国家纳米科学中心 | Nanometer particles with iron chelators encapsulated in amphiphilic polymers and preparation method and application thereof |
| CN114569789A (en) * | 2022-03-23 | 2022-06-03 | 宁波市医疗中心李惠利医院 | Preparation method and application of BPN-DFO gel scaffold |
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| US20010033860A1 (en) * | 1999-12-30 | 2001-10-25 | Gwathmey Judith K. | Iron chelator delivery system |
| CN104856960A (en) * | 2015-04-27 | 2015-08-26 | 国家纳米科学中心 | Nanometer particles with iron chelators encapsulated in amphiphilic polymers and preparation method and application thereof |
| CN114569789A (en) * | 2022-03-23 | 2022-06-03 | 宁波市医疗中心李惠利医院 | Preparation method and application of BPN-DFO gel scaffold |
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