C-COS-OHA hydrogel as well as preparation method and application thereof

Abstract

The invention discloses C-COS-OHA hydrogel as well as a preparation method and application thereof, the C-COS-OHA hydrogel is prepared from a carboxylated chitosan oligosaccharide solution, an oxidized hyaluronic acid solution, an enzyme active component solution and an agar solution through cross-linking reaction, and the enzyme active component is natural catalase or Fe < 3 + > / AMP coordination polymer. According to the invention, carboxylated chitosan oligosaccharide and oxidized hyaluronic acid are cross-linked for preparation, so that the stability and injectability of the material are ensured; wherein natural catalase or Fe < 3 + > / AMP coordination polymer is added, so that controllable release of oxygen is realized, and an anoxic microenvironment is relieved; in-vivo and in-vitro experiments prove that the compound has excellent functions of resisting oxidation, promoting macrophage M2 type polarization and promoting angiogenesis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a C-COS-OHA hydrogel and its preparation method and application. Background Art

[0002] Myocardial infarction (MI) is a serious cardiovascular disease widely existing globally, and its high fatality rate and disability rate pose a major threat to human health and life safety. As a sudden cardiovascular event, the pathogenesis of myocardial infarction is complex and involves multiple pathophysiological processes, among which inflammatory response, oxidative stress, and irreversible necrosis of cardiomyocytes are the main pathological mechanisms. Inflammatory response can lead to local vascular inflammation and thrombosis, further aggravating myocardial ischemia; oxidative stress can damage the cell membrane and mitochondria of cardiomyocytes, resulting in cell dysfunction and death; and the irreversible necrosis of cardiomyocytes is the most direct and serious consequence of myocardial infarction, leading to damage and dysfunction of myocardial tissue. These pathological processes are intertwined, jointly leading to damage and dysfunction of myocardial tissue, seriously affecting the quality of life and prognosis of patients.

[0003] Traditional treatment methods, such as drug treatment and interventional surgery, although can relieve symptoms to a certain extent, cannot effectively reverse the already occurred myocardial damage. Drug treatment mainly relieves symptoms by means of dilating blood vessels, inhibiting platelet aggregation, and improving myocardial metabolism, etc., but it is powerless for the already necrotic cardiomyocytes; interventional surgeries such as coronary artery stent implantation and coronary artery bypass grafting, although can restore blood supply to the myocardium, cannot repair the already damaged myocardial tissue. Therefore, the cardiac function of patients often continues to decline, and may eventually lead to serious consequences such as heart failure. Heart failure is a chronic progressive disease, and patients will present symptoms such as dyspnea, edema, fatigue, etc., seriously affecting the quality of life and having a poor prognosis.

[0004] In view of this, the medical community and the field of materials science are urgently in need of developing a new type of biomaterial to meet the needs of myocardial infarction treatment. This new type of biomaterial should have good biocompatibility, be able to coexist harmoniously with human tissues, and not cause immune reactions or other adverse reactions. At the same time, this material should also have targeting, be able to precisely act on the myocardial injury area, so as to achieve effective repair and functional recovery of the damaged myocardium. Through targeted repair, the interference with normal tissues can be minimized to improve the treatment effect. To sum up, developing a new type of biomaterial with good biocompatibility and targeting is of great significance for the treatment of myocardial infarction, and is expected to bring new hope and better prognosis for patients. Summary of the Invention

[0005] The object of the present invention is to overcome the defects of the prior art and provide a C-COS-OHA hydrogel.

[0006] Another object of the present invention is to provide a preparation method of the above-mentioned C-COS-OHA hydrogel.

[0007] Another object of the present invention is to provide the use of the above-mentioned C-COS-OHA hydrogel.

[0008] The technical solution of the present invention is as follows:

[0009] A C-COS-OHA hydrogel is prepared by cross-linking reaction of a carboxylated chitosan (C-COS) solution at 75-85 mg / mL, an oxidized hyaluronic acid (OHA) solution at 55-65 mg / mL, an enzyme active ingredient solution at 0.8-1.2 mg / mL, and an agar solution at 0.8-1.2 wt%. The enzyme active ingredient is natural catalase (CAT) or Fe 3+ / AMP coordination polymer.

[0010] In a preferred embodiment of the present invention, the molecular weight of the carboxylated chitosan in the carboxylated chitosan solution is 1000-5000 Da, and the degree of deacetylation is ≥90%.

[0011] In a preferred embodiment of the present invention, the oxidized hyaluronic acid in the oxidized hyaluronic acid solution is prepared by oxidizing hyaluronic acid with sodium periodate, and the oxidation degree is 40-50%.

[0012] In a preferred embodiment of the present invention, the Fe 3+ / AMP coordination polymer is prepared by coordinating ferric chloride hexahydrate with 5'-adenosine monophosphate disodium salt.

[0013] In a preferred embodiment of the present invention, the volume ratio of the carboxylated chitosan solution, the oxidized hyaluronic acid solution, the natural catalase solution and the agar solution is 1:1:0.25:0.5, or the volume ratio of the carboxylated chitosan solution, the oxidized hyaluronic acid solution, the Fe 3+ / AMP coordination polymer solution and the agar solution is 1:1:0.2:0.5.

[0014] The preparation method of the above-mentioned C-COS-OHA hydrogel includes the following steps:

[0015] (1) Carboxylate chitosan by laccase-TEMPO system to prepare carboxylated chitosan;

[0016] (2) Oxidize hyaluronic acid with sodium periodate to prepare oxidized hyaluronic acid;

[0017] (3) Mix the carboxylated chitosan oligosaccharide solution, oxidized hyaluronic acid solution, agar solution, and enzyme active ingredient solution, stir evenly at room temperature, and then let stand for crosslinking reaction until a gel is formed, and that's it.

[0018] The application of the above C-COS-OHA hydrogel in the preparation of a drug for treating myocardial infarction.

[0019] In a preferred embodiment of the present invention, the dosage form of the drug for treating myocardial infarction is an injection.

[0020] A drug for treating myocardial infarction, having the above C-COS-OHA hydrogel.

[0021] In a preferred embodiment of the present invention, its dosage form is an injection.

[0022] The beneficial effects of the present invention are:

[0023] 1. The present invention is prepared by crosslinking carboxylated chitosan oligosaccharide (C-COS) and oxidized hyaluronic acid (OHA) to ensure the stability and injectability of the material.

[0024] 2. In the present invention, natural catalase (CAT) or Fe3+ / AMP coordination polymer is added to achieve the controllable release of oxygen and relieve the hypoxic microenvironment.

[0025] 3. The present invention has been proven to have excellent antioxidant, macrophage M2 polarization promotion, and angiogenesis promotion functions in in vitro and in vivo experiments. Description of the Drawings

[0026] Figure 1 It is the nuclear magnetic resonance ( 13 C NMR) spectrum of COS and C-COS in Example 5 of the present invention.

[0027] Figure 2 It is the FTIR spectrum of COS and C-COS in Example 5 of the present invention.

[0028] Figure 3 It is the nuclear magnetic resonance (1H NMR) spectrum of HA and OHA in Example 6 of the present invention.

[0029] Figure 4 It is the FTIR spectrum of HA and OHA in Example 6 of the present invention.

[0030] Figure 5 It is the morphological characterization of the Fe 3+ / AMP coordination polymer in Example 7 of the present invention: (a) particle size distribution; (b) Zeta potential; (c) TEM image.

[0031] Figure 6Showing in Example 8 of the present invention: (a) Gelation diagram of C-COS-OHA hydrogel; (b) Injectability effect diagram of C-COS-OHA hydrogel.

[0032] Figure 7 FTIR diagram of C-COS-OHA series hydrogels in Example 8 of the present invention.

[0033] Figure 8 Showing in Example 9 of the present invention: (a) Equilibrium swelling ratio of C-COS-OHA series hydrogels; (b) In vitro degradation rate of C-COS-OHA series hydrogels.

[0034] Figure 9 Showing in Example 10 of the present invention: Rheological behavior of C-COS-OHA series hydrogels in frequency sweep mode (frequency range from 0.1 to 10 Hz): (a) C-COS-OHA hydrogel; (b) C-COS-OHA-CAT hydrogel; (c) C-COS-OHA-Fe3+ / AMP hydrogel.

[0035] Figure 10 Showing the shear thinning and self-healing properties of C-COS-OHA series hydrogels in Example 10 of the present invention: (a) Variation of hydrogel viscosity with shear rate, from 0.01 to 1000 s -1 ; and continuous alternating strain sweep (γ = 1% - 20%) at a fixed frequency (1 Hz): (b) C-COS-OHA hydrogel; (c) C-COS-OHA-CAT hydrogel; (d) C-COS-OHA-Fe 3+ / AMP hydrogel.

[0036] Figure 11 Showing the cell viability of HUVEC cells in the extract of C-COS-OHA series hydrogels in Example 11 of the present invention.

[0037] Figure 12 Live / dead staining diagram of HUVEC cells in the extract of C-COS-OHA series hydrogels in Example 11 of the present invention.

[0038] Figure 13 Showing the hemolytic effect of C-COS-OHA series hydrogels in Example 11 of the present invention: (a) White light diagram of the hemolysis experiment of the hydrogel; (b) Statistical analysis diagram of the hemolysis rate of the hydrogel.

[0039] Figure 14 Showing the antioxidant property and enzyme activity of C-COS-OHA series hydrogels in Example 12 of the present invention: (a) Hydroxyl radical scavenging rate; (b) Organic oxygen radical scavenging rate; (c) Catalase activity.

[0040] Figure 15 Showing the detection of the angiogenesis-promoting ability of the C-COS-OHA series hydrogels in the ROS microenvironment in Example 12 of the present invention: (a) Migration results of HUVEC cells in each group at 24 h; (b) Luminal formation of HUVEC cells in each group at 6 h; (c) Statistical chart of migration results; (d) Statistical chart of the number of vascular junctions.

[0041] Figure 16 Showing the ROS scavenging and oxygen production in the infarcted area of mice with myocardial infarction treated with the C-COS-OHA series hydrogels in Example 13 of the present invention: (a) Photoacoustic blood oxygen saturation images of mice at 1 day and 3 days after surgery; (b) Statistical chart of blood oxygen saturation in the anterior wall of the heart in the photoacoustic images (n = 6); (c) Statistical chart of DHE levels in the infarcted area of the heart after 3 days of treatment (n = 3).

[0042] Figure 17 Showing the comparison of TUNEL staining in the myocardial infarction areas of each group in Example 13 of the present invention, with green being apoptotic cells and blue being cell nuclei.

[0043] Figure 18 Showing the quantitative analysis of apoptotic cardiomyocytes in Example 13 of the present invention.

[0044] Figure 19 Showing the echocardiogram of mice for detecting cardiac function in Example 13 of the present invention: (a) M-mode image of echocardiogram of the heart 28 days after myocardial infarction; (b) Statistical charts of LVEF, LVFS, LVIDD, and LVIDS of mice at 14 and 28 days after modeling. Detailed implementation manners

[0045] The technical solutions of the present invention will be further described and illustrated below by specific implementation manners in conjunction with the accompanying drawings.

[0046] Example 1 Preparation of C-COS

[0047] (1) Add 0.08 g of TEMPO to 0.2 M sodium acetate buffer (pH = 4.8), and perform ultrasonic oscillation to completely dissolve it;

[0048] (2) Add 4 g of COS to the material obtained in step (1), and stir until it is completely dissolved;

[0049] (3) Put the material obtained in step (2) into a water bath, continue to stir under the reaction condition of 30 °C for 20 min, add 400 μL of laccase, and carry out the reaction by passing oxygen for 18 h;

[0050] (4) Add an appropriate amount of sodium carbonate to the material obtained in step (3), adjust the pH to 7.0, rotary evaporate to 20 mL, then add 400 mL of absolute ethanol, and let it stand for 1 h;

[0051] (5) Use a ground glass core barrel-shaped suction filtration funnel to perform vacuum suction filtration on the reaction product precipitated in step (4), and repeatedly wash the suction filtration product with anhydrous ethanol until the washing liquid is clear and transparent;

[0052] (6) Put the washed suction filtration product obtained in step (5) into a vacuum drying oven for drying, and after grinding, C-COS powder with a relative molecular mass of 1000 Da and a degree of deacetylation of 90.5% can be obtained.

[0053] Example 2 Preparation of OHA

[0054] (1) Add 1.072 g of sodium periodate (NaIO4) to 2.5 mL of deionized water, and after vortex dissolution, obtain a 0.5 mol / L NaIO4 solution;

[0055] (2) Add 2 g of HA to 200 mL of deionized water to prepare a uniform solution of 10 mg / mL;

[0056] (3) Dropwise add the NaIO4 solution obtained in step (1) into the material obtained in step (2), and react in the dark for 2 h; then add 1 mL of ethylene glycol, continue to react for 1 h, take an appropriate amount of the reaction solution and add it to a dialysis bag, and dialyze for at least 3 days;

[0057] (4) Freeze-dry the material obtained in step (3) to obtain OHA with an oxidation degree of 46.6%.

[0058] Example 3 Preparation of Fe 3+ / AMP coordination polymer

[0059] (1) Dissolve 0.1352 g of ferric chloride hexahydrate and 0.196 g of 5'-adenosine monophosphate disodium salt (AMP) in 100 mL of deionized water respectively;

[0060] (2) After mixing the two solutions obtained in step (1), add 80 mL of HEPES buffer solution and let it stand at room temperature for 30 min;

[0061] (3) Centrifuge (12000 rpm, 10 min) and repeatedly wash with deionized water 5 times to remove the uncoordinated components in the material obtained in step (2) to obtain Fe 3+ / AMP coordination polymer.

[0062] Example 4 Preparation of C-COS-OHA series hydrogels

[0063] (1) Solution preparation: The C-COS powder obtained in Example 1, the OHA obtained in Example 2, and the Fe 3+ / AMP coordination polymer and natural catalase (CAT) were respectively dissolved in HEPES buffer to obtain a C-COS solution of 80 mg / mL, an OHA solution of 60 mg / mL, a Fe 3+ / AMP solution of 1 mg / mL and a CAT solution of 1 mg / mL; agar was dissolved in deionized water, and stirred and heated to obtain a 1 wt% agar solution.

[0064] (2) Preparation of hydrogel: The above solutions were mixed according to the ratio in the following table, stirred evenly at room temperature, and then left standing at room temperature for 5 - 10 s to crosslink and form a gel-like substance, thus obtaining the C-COS-OHA series hydrogels.

[0065]

[0066] Example 5 Magnetic Resonance and FTIR Analyses of C-COS-OHA Series Hydrogels

[0067] For magnetic resonance analysis, COS (chitosan oligosaccharide) and C-COS were respectively dissolved in heavy water to prepare heavy aqueous solutions of COS and C-COS with a concentration of 250 mg / mL, and 13C NMR nuclear magnetic resonance scans were performed at room temperature. HA and OHA were respectively dissolved in heavy water to prepare heavy aqueous solutions of HA (hyaluronic acid) and OHA with a concentration of 10 mg / mL, and 1H NMR nuclear magnetic resonance scans were performed at room temperature. For FTIR analysis, a small amount of COS, C-COS, HA powder, and the freeze-dried OHA and C-COS-OHA series hydrogels obtained in Example 4 were placed on the sample cell of the FTIR, the sampling background was set to air, and the attenuated total reflection (ATR) method was used to detect the FTIR spectrum in the range of 500 cm -1 ~4000 cm -1 -1.

[0068] As Figure 1 shown, compared with the 13 13C NMR spectrum of COS, a new resonance peak corresponding to the carboxylate group (-COO-) appeared at a chemical shift of 164 ppm in C-COS, proving the success of the carboxylation modification of COS.

[0069] As Figure 2 shown, the infrared spectra of COS and C-COS are roughly the same, but compared with COS, the absorption band of C-COS in the range of 3400 cm -1 to 3200 cm -1 -1 is significantly broadened, indicating an enhanced signal of the hydroxyl and amino groups in C-COS; in addition, C-COS has absorption peaks at 1588 cm -1 and 1409 cm -1A characteristic peak corresponding to the asymmetric stretching vibration of C=O appeared, indicating that carboxyl or aldehyde groups were newly formed after the modification of COS. In summary, according to the results of nuclear magnetic resonance and FTIR, COS has been successfully modified to C-COS.

[0070] Example 6 Nuclear magnetic resonance and FTIR analysis of Example 2

[0071] According to the experimental method in Example 5, nuclear magnetic resonance and FTIR characterization were carried out on HA and OHA, and the experimental results are as Figure 3 and Figure 4 shown. Although the 1 1H NMR and FTIR results of the two are similar, a new characteristic peak corresponding to the aldehyde group (-C=O) appears at 4.9 - 5.0 ppm in the 1 1H NMR of OHA, and a new characteristic peak corresponding to the aldehyde group also appears at 1732 cm -1 in FTIR, indicating that HA has been successfully oxidized and modified to OHA.

[0072] Example 7 Microscopic morphology characterization of Example 3

[0073] After centrifugal washing, the Fe 3+ / AMP coordination polymer was vacuum dried, the powder was collected, deionized water was added, and it was ultrasonically dissolved into a 1 mg / mL suspension. Subsequently, 10 μL of the suspension was dropped on a copper mesh. After the deionized water evaporated and dried at room temperature, its morphological structure was analyzed by TEM. Then, an appropriate amount of the above suspension was added to a centrifuge tube, and it was oscillated for several minutes with an ultrasonic cleaner to make it evenly dispersed. At room temperature, 1.5 mL of the treated suspension was taken and added to a sample dish, and a dynamic light scattering experiment (Dynamic Light Scattering, DLS) was carried out using a nanoparticle size and zeta potential analyzer to measure its particle size and zeta potential. After three repeated tests, the average value was taken.

[0074] As Figure 5 shown, the particle size of the Fe 3+ / AMP coordination polymer is mostly between 150 nm and 200 nm, the particle size distribution is uniform, and it has a negative charge of -16.33 mV, proving that the Fe 3+ / AMP coordination polymer has been successfully synthesized. In addition, it is obtained by transmission electron microscopy that the nanoparticles are evenly distributed, showing a complex structure, and the average particle size is about 175 nm, which is consistent with the DLS result. This particle size is more conducive to exerting its catalase-like activity.

[0075] Example 8 Injectability of C-COS-OHA hydrogel

[0076] The injectable hydrogel is a liquid precursor before injection and forms a hydrogel only after entering the body. However, to ensure the therapeutic effect, it is necessary to reasonably control its gelation time. If the time is too long, it is easy to release the toxicity of the raw materials; if the time is too short, it is easy to block the needle. Therefore, in this embodiment, it is very necessary to regulate the proportion of raw materials to obtain a suitable gelation time and gel state.

[0077] The C-COS-OHA hydrogel can be prepared by quickly and fully mixing the C-COS solution, OHA solution and agar solution in Example 4. A reversible imine bond is formed through the Schiff base reaction between the aldehyde group on OHA and the primary amino group on C-COS to synthesize the hydrogel. By adjusting the concentration and volume ratio (V C-COS :V OHA :V 琼脂 ) of the three solutions, hydrogels with different gelation times and gel states can be prepared.

[0078] In this embodiment, the gelation attempt of the C-COS-OHA hydrogel is carried out according to the volume ratio schemes such as V C-COS :V OHA :V 琼脂 =9:1:1, 7:3:5, 1:1:0.5, etc. The results show that when the volume ratio of the C-COS solution is too large, only part of the flocculent colloid cannot form a gel, mainly because there are too many carboxyl groups in the hydrogel and only part of the components undergo the Schiff base reaction; when the volume ratio of the agar solution is too large, the hydrogel can form a gel, but its colloid is too hard and loses its injectable property.

[0079] Through continuous debugging, this embodiment finally determines to prepare the C-COS-OHA hydrogel with a volume ratio of V C-COS :V OHA :V 琼脂 =1:1:0.5, and respectively add natural catalase and the Fe 3+ / AMP coordination polymer prepared in Example 3 to prepare the C-COS-OHA-CAT and C-COS-OHA-Fe 3+ / AMP hydrogels with both oxygen-producing and ROS-scavenging functions. Hereinafter, the three hydrogels of C-COS-OHA, C-COS-OHA-CAT and C-COS-OHAFe 3+ / AMP are collectively referred to as the C-COS-OHA series of hydrogels for performance characterization.

[0080] As Figure 6As shown, C-COS, OHA and agar solution were fully mixed at room temperature, and the gelation time was measured by the test tube pouring method. The results showed that all C-COS-OHA series hydrogels could gel within 2 min. The prepared gel was aspirated with a syringe with an outer diameter of 0.26 mm, and the hydrogel could be smoothly injected, demonstrating its injectability. After freeze-drying the synthesized C-COS-OHA series hydrogels, Fourier transform infrared spectroscopy characterization ( Figure 7 ) showed that a new characteristic peak corresponding to the imine bond appeared at 1622 cm -1 for all C-COS-OHA series hydrogels, proving that gelation was achieved through the Schiff base reaction.

[0081] Example 9 Swelling and in vitro degradation of C-COS-OHA series hydrogels

[0082] The C-COS-OHA series hydrogels were freeze-dried, weighed separately, and then immersed in PBS at 37 °C with pH = 7.4. At regular intervals, they were taken out, weighed again, and the PBS immersion solution was replaced until the mass of the hydrogel no longer changed, at which point swelling equilibrium was reached. W t and W d are the wet weight of the hydrogel after reaching swelling equilibrium and the mass at freeze-drying, respectively. The equilibrium swelling ratio (ESR) = (W t - W d ) / W d

[0083] The C-COS-OHA series hydrogels were immersed in PBS, taken out and weighed at the same time point every day, and the PBS immersion solution was replaced. The degradation of the hydrogels was understood by recording the weight changes of the hydrogels within 7 days. The initial weight of the hydrogel was (W s ), and the weight at each weighing was (W i ).

[0084] The remaining weight ratio = 1 - 100% * (W s - W i ) / W s

[0085] As Figure 8 (a) shows, the C-COS-OHA series hydrogels basically reached the equilibrium swelling ratio in about 1 h. The equilibrium swelling ratio of the C-COS-OHA hydrogel was 228% ± 40%, and that of the C-COS-OHA-CAT hydrogel was 107% ± 13%. The C-COS-OHA-Fe 3+The equilibrium swelling ratio of the C-COS-OHA hydrogel was 73% ± 15%. The swelling ratios of the three hydrogels decreased in turn. The above results were related to the microstructures of the three hydrogels and the hydrophilic interactions of the groups. Compared with the other two gels added with enzymes, the C-COS-OHA hydrogel contained abundant hydrophilic groups such as hydroxyl, amino, and carboxyl groups, making it easier to produce hydration, and its loose network structure was more conducive to water absorption. In addition, Figure 8 (b) Recorded the degradation of the C-COS-OHA series of hydrogels in PBS at 37°C for 7 days. Among them, the C-COS-OHA hydrogel degraded the fastest, with 50% degraded in 7 days, followed by C-COS-OHA-CAT, and C-COS-OHA-Fe + / AMP hydrogel degraded the slowest, but all three degraded slowly and uniformly. It can also be seen from the figure that the degradation processes of the three hydrogels were roughly the same in the first 2 days, and then the degradation rate of the C-COS-OHA hydrogel began to accelerate. At 7 days, the remaining weight ratios of the C-COS-OHA, C-COS-OHA-CAT, and C-COS-OHA-Fe 3+ / AMP hydrogels were 33.36% ± 3.02%, 45.51% ± 1.60%, and 52.80% ± 2.50% respectively. This was because the crosslinking density of the C-COS-OHA hydrogel was small, the exposed contact surface of the polymer chain became larger, and the hydrogel was more easily degraded. For the C-COS-OHA-Fe 3+ / AMP hydrogel, with the addition of Fe 3+ / AMP, metal chelation occurred, increasing the crosslinking density of the hydrogel, so the degradation slowed down. Since the C-COS-OHA series of hydrogels in this example adopted the intramyocardial injection route of administration, and the degradation time should cover the treatment window period as much as possible and consider the slow release of enzymes, the degradation time was preferably between 1 week and 6 weeks. At the same time, to avoid aggravating the blockage degree of blood vessels in the myocardial infarction area after injection, the C-COS-OHA series of hydrogels should have a low swelling ratio. And the above results showed that the C-COS-OHA series of hydrogels could degrade slowly and uniformly, the degradation time was greater than 1 week, and they had a low swelling ratio, so they were suitable for myocardial infarction treatment.

[0086] Example 10 Rheological property test of the C-COS-OHA series of hydrogels

[0087] Place the synthesized C-COS-OHA series hydrogels on the test bench. First, in the frequency sweep test, use 1% strain and rheological properties from 0.1 Hz to 10 Hz as test parameters to obtain the variation of G' and G" with frequency to compare the stability of each group of hydrogels. Set the shear rate to 0.01 / s to 1000 / s to test the shear thinning properties of each group of hydrogels. Subsequently, fix the frequency at 1 Hz and continuously alternate the strain between 1% and 20% to test the self-healing properties of each group of hydrogels.

[0088] Evaluate the mechanical properties of the C-COS-OHA series hydrogels through rheological tests. When the storage modulus (G') > loss modulus (G"), the hydrogel is in a gel state, and when G' < G", it is in a sol state. As Figure 9 shown, the frequency sweep indicates that within the range of 1% strain and 0.1 Hz to 10 Hz, the G' of the hydrogel is always higher than G", and it changes little with frequency, indicating the successful formation of the gel. The G' value of the C-COS-OHA-Fe 3+ / AMP hydrogel is the highest, and the G' value of the C-COS-OHA hydrogel is the lowest, indicating that the C-COS-OHA-Fe 3+ / AMP hydrogel has a high cross-linking density and a more stable structure compared to the C-COS-OHA hydrogel, which is consistent with the previous analysis results of the microscopic morphology of the hydrogel. Although the overall G' value of the C-COS-OHA series hydrogels is low, when used as an injectable hydrogel for myocardial repair, mechanical strength is not the most important factor, and a lower G' value is beneficial, indicating that the prepared hydrogel is soft and easy to flow, which is conducive to promoting the transmission of mechanical signals and coordinating with the pulsation of myocardial tissue, thus promoting the reconstruction of cardiac function.

[0089] Under a certain time and temperature program, along a certain direction, set the shear rate to 0.01 / s to 1000 / s to measure the shear thinning characteristics of the hydrogel. Figure 10 (a) It can be seen that the viscosity of each group of gels decreases uniformly with the increase of the shear rate, verifying the injectability of the hydrogel. Fix the frequency at 1 Hz and perform continuous alternating strain scanning on the hydrogel, switching from a large strain of 20% to a small strain of 1% to test its self-healing property. As Figure 10As shown in (b)-(d), the hydrogel was damaged at high strain (20%) (G'<G"), and immediately self-healed at low strain (1%) (G'>G"). The above results indicate that the C-COS-OHA series of hydrogels have self-healing properties, which may be due to the dynamic chemical bonds - imine bonds in the C-COS-OHA series of hydrogels. These bonds are broken at high strain, and the primary amine groups and aldehyde groups enter the free state. As the structural test bench rotates continuously, more opportunities are created for the free ions to recombine. Therefore, they will recombine into imine bonds at low strain, showing a higher storage modulus.

[0090] Example 11 Biocompatibility and Hemolysis Detection of C-COS-OHA Series Hydrogels

[0091] The hydrogel was mixed with serum-free DMEM medium at a ratio of 1:3 and soaked for 24 h to prepare a hydrogel extract. HUVECs were seeded on a 96-well plate at a cell density of 1×10 4 cells / well, cultured overnight in an incubator. The next day, the medium was replaced with the hydrogel extract and cultured for another 24 h and then removed. After preparing a CCK-8 detection working solution by mixing serum-free medium and CCK-8 at a ratio of 10:1, 110 μL of the above working solution was added to each well and incubated in the dark for 1.5 h. During the detection process, a blank control group (wells containing only the CCK-8 detection working solution) and a control group (cells cultured only with complete medium) were set up. After setting the wavelength of the microplate reader to 450 nm, the absorbance was measured.

[0092] Cell survival rate (%) = (OD 实验组 - OD 空白组 ) / (OD 对照组 - OD 空白组 ) × 100%

[0093] Calcein and PI dyes can effectively distinguish between live cells and dead cells and visually evaluate the survival status of cells under different treatment conditions. Specifically, cells were first seeded on a 24-well plate at a density of 1×105 HUVEC cells / well. After culturing for 24 h until the cells adhered to the wall, the medium was replaced with a serum-free extract of the C-COS-OHA series of hydrogels and cultured for another 24 h. After gently washing the cells 1-2 times with PBS buffer, according to the instructions, Calcein-AM and PI dyes were diluted to an appropriate concentration, and stained at 1 mL / well in the dark for 30 min. Then, the fluorescence signals of the cells were photographed and recorded using a fluorescence microscope.

[0094] Fresh blood was drawn from the orbits of mice and placed in an anticoagulant tube and mixed well; centrifuged at 1200 rpm for 5 min at low temperature, the supernatant floating above was removed, and the precipitated blood cells were left. PBS was added again and centrifuged again. This step was repeated 2 - 3 times until the supernatant was clear; the whole red blood cells and PBS were diluted at a ratio of 1:10 to obtain a 10% blood cell suspension. 500 μL of the PBS leaching solution of each group of hydrogels and 500 μL of the 10% blood cell suspension were added to the centrifuge tube, where

[0095] the negative control was 500 μL of PBS and 500 μL of the 10% blood cell suspension, and the positive control was 50 μL of whole red blood cells plus 950 μL of distilled water; after waiting for 1 h in a 37°C incubator, centrifuged at 3500 rpm for 5 min, after photographing and recording; 100 μL / well of the supernatant was added to a 96-well plate, and the absorbance was measured at a wavelength of 540 nm, repeated three times and recorded.

[0096] Hemolysis rate (%) = (OD 样本组 - OD 阴性组 ) / (OD 阳性组 - OD 阴性组 ) * 100%

[0097] The results of CCK-8 are as Figure 11 shown. The cell survival rates of C-COS-OHA and C-COS-OHA-Fe 3+ / AMP hydrogels are both greater than 100%, indicating that both can promote cell growth. Similarly, when adding enzymes, compared with the C-COS-OHA-Fe 3 + / AMP hydrogel with nanozyme added, the cell survival rate of the C-COS-OHA-CAT hydrogel with natural enzyme added is only 65%. This may be because the loose structure of the hydrogel causes CAT to be released slightly faster, and coupled with the ROS scavenging ability of C-COS itself, the ROS generated during the normal aerobic metabolism process inside the cells is over-scavenged and too much oxygen is produced, resulting in oxidative stress damage, which reduces the cell survival rate corresponding to this hydrogel. The live / dead cell staining method can more intuitively show the survival situation of cells. Among them, AM will stain live cells green, and PI will stain dead cells red. Figure 12 In the C-COS-OHA-CAT hydrogel group in 3+The / AMP was the lowest, which was consistent with the CCK-8 detection results. However, no large red fluorescence signals were observed in the C-COS-OHA series hydrogels, indicating good biocompatibility within an acceptable range. The blood cells were mixed with the extract of the C-COS-OHA series hydrogels and placed in an incubator at 37 °C for 1 h to simulate the contact between the hydrogel and blood cells in vivo and evaluate the blood compatibility of the hydrogel. As Figure 13 shown in (a), compared with the control group, the supernatant of the hydrogel group was clear and no obvious red color was observed, indicating that hemolysis did not occur between the hydrogel and red blood cells. Figure 13 The statistical results in (b) showed that the hemolysis rates of the C-COS-OHA series hydrogels were all lower than 5%, further indicating that the possibility of hemolysis of this hydrogel was small. Based on the results of the above three experimental detections, it can be proved that the C-COS-OHA series hydrogels have good cytocompatibility. Except that the C-COS-OHA-CAT hydrogel may cause excessive scavenging of normal intracellular ROS, resulting in a slightly lower cell survival rate.

[0098] Example 12 Evaluation of the blood vessel promoting and antioxidant properties of the C-COS-OHA series hydrogels

[0099] A. Detection of the scavenging ability of the C-COS-OHA series hydrogels against hydroxyl radicals:

[0100] First, prepare the following solutions:

[0101] ① 2 mM FeSO4 solution: Add 0.0139 g of FeSO4 to water and make up the volume to 25 mL;

[0102] ② 6 wt% H2O2 solution: Add 5 mL of 30% H2O2 to 20 mL of water;

[0103] ③ 360 μg / mL saffron solution: Dissolve 0.018 g of saffron powder in water and make up the volume to 50 mL.

[0104] ④ Extract of the C-COS-OHA series hydrogels: Immerse the hydrogel in PBS at a volume ratio of 1:3 for 24 h.

[0105] Repeat the following experiment 3 times. In the experimental group, 500 μL of deionized water, 600 μL of FeSO4 solution, and 500 μL of saffron solution were successively added to 500 μL of the extract, shaken and mixed well. After standing for 15 min, 800 μL of 6 wt% H2O2 solution was added, and the reaction was carried out in a water bath at 55 °C for 30 min. In the control group, deionized water was used instead of the extract, and in the blank group, deionized water was used instead of the extract and 6 wt% H2O2, and the order and dosage of other reagents remained unchanged. After the reaction, take a 96-well plate and add the reaction solution of each group at 200 μL / well, and measure the absorbance value at 492 nm.

[0106] Hydroxyl radical scavenging ability = (OD 样品组 - OD 空白组 ) / (OD 对照组 - OD 空白组 ) × 100%

[0107] Detection of the scavenging ability of B.C-COS-OHA series hydrogels against organic oxygen radicals:

[0108] First, prepare a 0.2 mM DPPH solution by dissolving 0.00315 g of DPPH in 20 mL of absolute ethanol and making up the volume to 40 mL.

[0109] In the experimental group, 100 μL of hydrogel extract was added to 100 μL of DPPH solution. In the blank group, 100 μL of hydrogel extract was added to 100 μL of absolute ethanol. In the control group, 100 μL of absolute ethanol was added to 100 μL of DPPH solution. After waiting for 30 min in the dark, the absorbance was measured at 517 nm.

[0110] Scavenging ability of organic oxygen radicals = [1 - (OD 实验组 - OD 空白组 ) / OD 对照组 × 100%

[0111] Detection of catalase activity of C.C-COS-OHA series hydrogels:

[0112] First, place the prepared CAT detection working solution in a water bath at 37 °C for 20 min. Add 10 μL of the PBS extract of the hydrogel and 190 μL of the CAT detection working solution to each well of a 96-well plate. Immediately mix well and then measure the absorbance A0 at 240 nm. Measure the absorbance A1 again after 1 min. ΔA = A0 - A1.

[0113] CAT (U / mL) = [ΔA × V × (ε × L) × 106] / (v × t) = 764.5 × ΔA

[0114] V: Total volume of the system, 2 × 10 -4 L; ε: Molar extinction coefficient of H2O2, 43.6 L / mol / cm; L: Optical path of the 96-well plate: 0.6 cm; v: Volume of the sample added, 0.01 mL; t: Reaction time, 1 min.

[0115] D. Tube formation assay: HUVEC cells within 10 passages were cultured with HUVEC-specific medium. When the cell density reached 95%, 100 μM H2O2 solution was added, and after 1 h of light-avoiding treatment in the incubator, it could be used for the tube formation assay. Related experimental consumables such as Matrigel and 96-well plates were pre-cooled in advance. First, take out the pre-cooled 96-well plate and place it on ice, then quickly add 50 μL of Matrigel to each well, gently shake it to make it evenly cover the bottom of the well, and then carefully and steadily place it in the incubator. After waiting for 1 h, take out the 96-well plate and add 4 the cells treated with H2O2 solution at a density of 1×10

[0116] per well. In the experimental group, 100 μL of hydrogel extract was added, and in the positive control group, serum-free medium was used instead of the extract. Three replicates were set for each group. After culturing for 6 h, take pictures under the microscope for recording, and use Image J for data analysis. Figure 14 As shown in 3+ , among the C-COS-OHA series of hydrogels, C-COS-OHA-CAT hydrogel showed the highest hydroxyl radical scavenging ability, reaching 63.33% ± 1.83%. For the scavenging ability of DPPH, the C-COS-OHA hydrogel had the highest scavenging rate of organic oxygen radicals, followed by C-COS-OHA-Fe Figure 14 (c) shows that C-COS-OHA-Fe 3+ / AMP had the strongest enzyme activity, followed by C-COS-OHA-CAT, and C-COS-OHA was the weakest, indicating that the addition of the enzyme did improve the hydrogen peroxide scavenging ability of C-COS-OHA hydrogel (P < 0.05). In addition, although the enzyme activity of the nanozyme-like Fe 3+ / AMP was slightly higher than that of natural catalase, the difference was not significant (P > 0.05). In summary, it shows that the three hydrogels in the C-COS-OHA series have different emphases on the scavenging ability of different ROS.

[0117] As shown in Figure 15As shown in (a) and (c), compared with the positive control group, all C-COS-OHA series hydrogels could significantly improve the cell migration ability under the oxidative stress microenvironment (P<0.0001). Among them, the ability of C-COS-OHA-CAT hydrogel to promote endothelial cell migration was slightly weaker, at 14.45%±0.46%, while the C-COS-OHA-Fe 3+ / AMP hydrogel group migrated about 26.93%±0.89% within 24 h, showing significant differences from both the C-COS-OHA group (P<0.0001), indicating that the addition of nanozymes could significantly improve the cell migration ability under the oxidative stress microenvironment. However, due to its strong hydroxyl radical scavenging ability, CAT caused oxidative stress damage to some cells, resulting in a slightly lower cell migration rate than the C-COS-OHA group. Subsequently, in this example, the ability of endothelial cells to respond to angiogenesis signals was detected through a lumen experiment. As can be seen from Figure 15 (b), there were obvious morphological differences among the groups. The HUVECs in the hydrogel extract began to gradually aggregate to form lumen-like structures, while only scattered cells were visible in the control group treated with H2O2 alone, and no reticular structure was formed. Through statistical analysis of the lumen structures cultured in the hydrogel extract for 6 h, as can be seen from Figure 15 (d), the C-COS-OHA group had the strongest angiogenesis-promoting ability, and there was no significant difference between the C-COS-OHA-Fe 3+ / AMP and C-COS-OHA-CAT groups (P>0.05). Combining the structural characteristics of the C-COS-OHA series hydrogels and the relevant research conclusions of chitosan-hyaluronic acid hydrogels, the main reason for this difference can be obtained as follows: The main component that plays an angiogenesis-promoting role in the C-COS-OHA series hydrogels is hyaluronic acid, and C-COS-OHA degrades the fastest within 24 h, releasing more hyaluronic acid components. Although the addition of enzymes can reduce the impact of oxidative stress on angiogenesis, its impact on angiogenesis promotion is not obvious in the short term.

[0118] Example 13 Application of C-COS-OHA series hydrogels in the treatment of myocardial infarction

[0119] Evaluation of the antioxidant and oxygen-producing effects of AC-COS-OHA series hydrogels: Photoacoustic (PA) technology is a new imaging technology for in vivo detection. Photoacoustics provides anatomical information through ultrasound imaging, and provides specific information, such as blood oxygen saturation, through the characteristics of tissues absorbing light energy to generate ultrasound waves. The MX250s probe of the small animal in vivo ultrasound photoacoustic multimodal imaging system was used to monitor the blood oxygen saturation of the anterior wall of the heart of mice with myocardial infarction treated with C-COS-OHA series hydrogels on the 1st and 3rd days, and the system's own software was used for semi-quantitative analysis to evaluate the cardiac blood perfusion of C-COS-OHA series hydrogels in the infarct area and the longitudinal changes in the oxygen-producing function of the hydrogels. Figure 16 As shown in (a), there is almost no red signal on the anterior cardiac wall in the PBS group, indicating that the blood oxygen saturation is low and myocardial infarction has occurred. The oxygen saturation signals of the anterior cardiac wall in the three treatment groups of the C-COS-OHA series hydrogel are stronger than those in the PBS group, indicating that the C-COS-OHA series hydrogel can alleviate hypoxia in the MI area. On the first day of treatment, the blood oxygen saturation in the anterior cardiac wall area of ​​the C-COS-OHA-CAT hydrogel group was the highest, and on the third day of treatment, the blood oxygen saturation in the anterior cardiac wall area of ​​the C-COS-OHA-Fe 3+ The blood oxygen saturation of the / AMP hydrogel group was the highest in the anterior cardiac wall area. Figure 16 (b) Quantitative statistical results showed that the blood oxygen saturation of the C-COS-OHA-CAT group showed a trend of first high and then low during the treatment, while the C-COS-OHA-Fe 3+ The / AMP group showed a trend of first low and then high, while the C-COS-OHA group had a very uniform blood oxygen saturation of the anterior cardiac wall during the treatment. 3+ The reason why the AMP group showed this trend is that this may be due to C-COS-OHAFe 3+ / AMP has a stronger gel structure and the release rate of nanoenzymes is slower than that of CAT, so it starts to work on the third day; on the other hand, the main types of ROS produced by myocardial infarction are hydroxyl free radicals and superoxide anions, and C-COS-OHA-CAT has a strong hydroxyl free radical scavenging ability, so it may have cleared more ROS on the first day. On the third day, the CAT group may have almost cleared ROS, so the oxygen production effect began to decrease, and the blood oxygen saturation of the anterior wall of myocardial infarction also decreased accordingly. Figure 16 (a) also shows that C-COS-OHA-Fe 3+ / AMP hydrogel has abundant oxygen supply in the entire heart area, indicating that the oxygen production range of this hydrogel is wider.

[0120] To quantitatively evaluate the effect of the oxygen-producing function of the hydrogel on ROS scavenging ability, the apex tissues of mice treated for 3 days were taken, and the ROS levels in the myocardial infarction area were obtained through DHE fluorescent probes. As Figure 16 (c) shows, 3 days after the injection treatment, obvious DHE signals could be detected in the PBS group, indicating a large accumulation of ROS in the infarcted area, while the DHE fluorescence of the C-COS-OHA-CAT and C-COS-OHA-Fe 3+ / AMP hydrogel groups was significantly reduced compared with C-COS-OHA (P<0.05), indicating that the addition of enzymes could further improve the antioxidant properties of the hydrogel. In addition, there was no significant difference in the ROS levels on the third day of C-COS-OHA-CAT and C-COS-OHA-Fe 3+ / AMP treatment (P>0.05), indicating that the order of oxygen production did not affect the ROS scavenging effect. In summary, the above results confirmed that the C-COS-OHA hydrogel has antioxidant and oxygen-producing effects, and the addition of enzymes further enhanced its above properties. And within about three days after treatment, although the order of oxygen production of Fe 3+ / AMP was different from that of CAT, it did not affect the overall ROS scavenging effect.

[0121] B. TUNEL assessment of cell apoptosis in the myocardial infarction area of C-COS-OHA series hydrogels: In this patent, the TUNEL method was used to detect the cell apoptosis in the myocardial infarction area of mice modeled for 3 days to evaluate the effects of the antioxidant and oxygen-producing functions of the C-COS-OHA series hydrogels on cell survival in the myocardial infarction area. As Figure 17 shown, blue granules represent the distribution of cell nuclei, and green granules represent apoptotic cardiomyocytes. TUNEL-positive stained cells were massively aggregated in the infarcted area of the PBS group, while there were fewer positive cells in the C-COS-OHA series hydrogels. Through further quantitative analysis of the TUNEL staining results ([[]]END]] Figure 18 ), the degree of cell apoptosis in the COS-OHA-CAT group and the C-COS-OHA-Fe 3+ / AMP group was alleviated compared with the C-COS-OHA group, but the difference among the three was not significant. The above results indicate that the addition of enzymes not only improved the antioxidant and oxygen-producing effects of the hydrogel, but also further promoted macrophage polarization, reduced oxidative stress and inflammatory responses in the myocardial infarction area, improved the hypoxia condition of cells in the infarcted area, and reduced the number of apoptotic cells in the infarcted area.

[0122] C. Echocardiogram assessment of the improvement of cardiac function in mice by C-COS-OHA series hydrogels: To evaluate the improvement of cardiac function in myocardial infarction mice by the hydrogel, echocardiograms were taken on the 14th and 28th days after the hydrogel injection treatment. Figure 19(a) The M-mode echocardiogram images of the mouse hearts at 28 days after surgery are shown. In the sham group, the anterior and posterior walls of the mouse heart beat rhythmically and powerfully, and the cardiac systolic function was normal. In the PBS group, the cardiac structure changed to a balloon shape, the anterior wall of the left ventricle did not show obvious contraction, and the interventricular septum became larger, indicating cardiac function decline and ventricular dilation. Compared with the PBS group, in the C-COS-OHA series hydrogel treatment groups, the anterior and posterior walls of the left ventricle beat rhythmically, and the interventricular septum was smaller, indicating that the hydrogel treatment could restore the systolic function of the anterior wall of the left ventricle to a certain extent in mice. After myocardial infarction, a large area of cell necrosis and scar hyperplasia occur, and the cardiac ejection function of the mouse begins to decline, and the LVEF and LVFS will decrease significantly. Therefore, an LVEF value less than 40% can prove successful myocardial infarction modeling. In addition, the ventricular wall of the mouse will gradually become thinner, dilate and deform, so the LVIDD and LVIDS will increase significantly. According to Figure 19 (b) Quantitative analysis of echocardiograms showed that 14 days after treatment, the LVEF and LVFS values of the three groups of hydrogels were significantly higher than those of the PBS group (P < 0.0001), and the LVIDD and LVIDS were significantly lower than those of the PBS group (P < 0.0001). The LVEF values of PBS, C-COS-OHA, C-COS-OHA-CAT, and C-COS-OHAFe 3+ / AMP were 22.16 ± 5.3%, 51.02 ± 3.5%, 54.07 ± 5.8%, and 56.14 ± 3.1% respectively. It can be seen that the LVEF values of the treatment groups were all greater than 40%, indicating that the infarction condition had improved. Although there were slight differences among the three groups of hydrogel treatment groups, they were not significant (P > 0.05). 28 days after treatment, the differences among the treatment groups began to appear. Further analysis showed that the addition of enzymes could indeed make the C-COS-OHA hydrogel more significantly improve the ventricular ejection fraction and shortening fraction of myocardial infarction mice (P < 0.05). C-COS-OHA-Fe 3+ / AMP may have a stable structure and slightly higher catalase activity, making its various cardiac function indexes better than those of the C-COS-OHA-CAT group at 28 days (P < 0.05). In summary, the C-COS-OHA series hydrogels can all improve the cardiac function of myocardial infarction mice, and the C-COS-OHA-Fe 3+ / AMP hydrogel showed the best treatment effect 28 days after treatment.

[0123] As described above, it is only the preferred embodiment of the present invention, and thus the scope of implementation of the present invention cannot be limited thereby. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention.

Claims

1. A C-COS-OHA hydrogel, characterized in that: A carboxylated chitosan oligosaccharide solution with a concentration of 75 - 85 mg / mL, An oxidized hyaluronic acid solution at 55 - 65 mg / mL, an enzyme active ingredient solution at 0.8 - 1.2 mg / mL, and an agar solution at 0.8 - 1.2 wt% are prepared by crosslinking reaction, and the enzyme active ingredient is natural catalase or Fe 3+ / AMP coordination polymer.

2. The C-COS-OHA hydrogel according to claim 1, characterized in that: The molecular weight of the carboxylated chitosan oligosaccharide in the carboxylated chitosan oligosaccharide solution is 1000 - 5000 Da, and the degree of deacetylation is ≥90%.

3. The C-COS-OHA hydrogel according to claim 1, wherein: The oxidized hyaluronic acid in the oxidized hyaluronic acid solution is prepared by oxidizing hyaluronic acid with sodium periodate, and the degree of oxidation is 40 - 50%.

4. The C-COS-OHA hydrogel according to claim 1, wherein: The described Fe 3+ / AMP coordination polymer is prepared by coordinating ferric chloride hexahydrate with 5'-adenosine monophosphate disodium salt.

5. A C-COS-OHA hydrogel according to any one of claims 1 to 4, characterized in that: The volume ratio of the carboxylated chitosan oligosaccharide solution, oxidized hyaluronic acid solution, natural catalase solution and agar solution is 1:1:0.25:0.5, or the volume ratio of the carboxylated chitosan oligosaccharide solution, oxidized hyaluronic acid solution, Fe 3+ / AMP coordination polymer solution and agar solution is 1:1:0.2:0.

5.

6. A method for preparing a C-COS-OHA hydrogel according to any one of claims 1 to 5, characterized in that: It includes the following steps: (1) Carboxylate chitosan oligosaccharide by laccase - TEMPO system to prepare carboxylated chitosan oligosaccharide; (2) Oxidize hyaluronic acid with sodium periodate to prepare oxidized hyaluronic acid; (3) Mix the carboxylated chitosan oligosaccharide solution, oxidized hyaluronic acid solution, agar solution and enzyme - active ingredient solution evenly at room temperature, and then let it stand for cross - linking reaction until it forms a gel state, then it is ready.

7. Use of the C - COS - OHA hydrogel according to any one of claims 1 to 5 in the preparation of a medicament for treating myocardial infarction.

8. The application according to claim 7, wherein: The dosage form of the medicament for treating myocardial infarction is an injection.

9. A drug for treating myocardial infarction, characterized in that: It has the C - COS - OHA hydrogel according to any one of claims 1 to 5.

10. A myocardial infarction treatment drug according to claim 9, characterized in that Its dosage form is an injection.

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