Natural polysaccharide drug controlled release system with multi-therapy synergistic effect, preparation method and application

Abstract

The invention discloses a natural polysaccharide drug controlled release system with a multi-therapy synergistic effect, a preparation method and application, and belongs to the technical field of material synthesis and biological medicine. The invention provides a natural polysaccharide drug controlled release system, which comprises hydrogel generated by Schiff base reaction of carboxymethyl chitosan and oxidized hyaluronic acid, and anti-cancer drugs-copper and glucose oxidase loaded in the hydrogel. The invention further provides a preparation method of the natural polysaccharide drug controlled release system and application of the natural polysaccharide drug controlled release system or the preparation method in preparation of anti-cancer drugs. According to the drug controlled release system, slow release of adriamycin-copper and glucose oxidase is achieved through decomposition of hydrogel in a weak acid environment, and therefore the synergistic effect of chemotherapy, chemodynamic therapy and hunger therapy is achieved. The drug controlled release system is simple to prepare and high in biocompatibility, and can be widely applied to the field of biological medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of material synthesis and biomedicine, and specifically relates to a multi-therapy synergistic natural polysaccharide drug controlled release system, a preparation method and an application thereof. Technical Background

[0002] Cancer has long been a threat to human health. Breast cancer is one of the most common cancers. Currently, chemotherapy is still one of the main ways to treat breast cancer, but it often causes serious side effects, such as nausea, vomiting, and fever. And due to the drug resistance of tumors, it is difficult to achieve successful treatment of cancer with a single chemotherapy. Therefore, it is urgent to develop a multi-therapy combined targeted drug controlled release system.

[0003] Doxorubicin is an anthracycline drug widely used in chemotherapy for malignant tumors, and has a highly effective therapeutic effect on cancer. However, due to its non-specificity to cancer tissues, doxorubicin can cause serious toxic side effects, among which the most limiting factor in its clinical application is that it can cause irreversible cardiac toxicity.

[0004] Chemodynamic therapy is an emerging treatment method that uses transition metals to catalyze endogenous hydrogen peroxide in the tumor microenvironment to produce reactive oxygen species under acidic conditions through the Fenton reaction or Fenton-like reaction, thereby killing cancer cells and treating tumors. However, the low concentration of endogenous hydrogen peroxide has become an important factor limiting chemodynamic therapy.

[0005] Glucose oxidase is an enzyme that catalyzes the reaction of glucose and oxygen to produce gluconic acid and hydrogen peroxide. In the tumor microenvironment, cancer cells need a large amount of glucose to provide energy for differentiation and growth. The presence of glucose oxidase can consume glucose in the tumor site, thereby limiting the energy input of cancer cells and performing synergistic treatment with starvation therapy; at the same time, it can also provide more endogenous hydrogen peroxide in the tumor microenvironment to enhance the effect of chemodynamic therapy.

[0006] Hyaluronic acid is a linear polysaccharide present in the human body, which is composed of alternating units of D-glucuronic acid and N-acetyl D-glucosamine. Due to its good biocompatibility, it is widely used in biomedicine and other fields. The use of sodium periodate to oxidize hyaluronic acid can oxidize the hydroxyl groups of hyaluronic acid into aldehyde groups. Oxidized hyaluronic acid containing aldehyde groups can be cross-linked with amino-containing polymers through Schiff base reaction to form hydrogels, avoiding the potential biosafety risks of using cross-linking agents.

[0007] Carboxymethyl chitosan is a water-soluble derivative of chitosan. The introduction of carboxymethyl groups into the chitosan structure greatly improves the solubility of chitosan in neutral and alkaline environments without affecting its properties. Compared with chitosan, carboxymethyl chitosan has improved water solubility and better mechanical strength, and plays a wider role in biomedicine, tissue engineering and other fields. Summary of the invention

[0008] In view of the problems existing in the above-mentioned prior art, the object of the present invention is to design and provide a kind of multi-therapy synergistic natural polysaccharide drug controlled release, and oxidized hyaluronic acid, carboxymethyl chitosan, anticancer drug-metal (such as doxorubicin-copper) and glucose oxidase are combined together. The amino group on carboxymethyl chitosan and the aldehyde group on oxidized hyaluronic acid undergo Schiff base reaction to generate hydrogel, and anticancer drug-metal (such as doxorubicin-copper) and glucose oxidase are loaded into the hydrogel to achieve the synergistic effect of chemotherapy, chemodynamic therapy and starvation therapy. The present invention forms doxorubicin-copper by combining doxorubicin with metal copper ions, and utilizes its coordination bond to the weak acid sensitivity in the tumor environment and the redox sensitivity of metal ions to achieve the controlled release of doxorubicin.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] On the one hand, the present invention provides a multi-therapeutic synergistic natural polysaccharide drug controlled-release system, comprising a hydrogel generated by a Schiff base reaction of carboxymethyl chitosan and oxidized hyaluronic acid, and anticancer drugs-metal and glucose oxidase loaded in the hydrogel; the metal ions can form a coordination bond with doxorubicin that is sensitive to pH and can be reduced by glutathione, and can undergo a Fenton-like reaction with hydrogen peroxide.

[0011] Preferably, the anticancer drug is doxorubicin;

[0012] Preferably, the metal is copper, iron or manganese.

[0013] In a second aspect, the present invention provides a method for preparing the multi-therapy synergistic natural polysaccharide drug controlled release system, comprising the following steps:

[0014] (1) Weighing an anticancer drug, dispersing it in water, stirring until dissolved, and adding a metal salt solution to obtain an anticancer drug-metal;

[0015] (2) weighing oxidized hyaluronic acid and adding it to the anticancer drug-metal obtained in the above step (1) to obtain oxidized hyaluronic acid / anticancer drug-metal;

[0016] (3) weighing carboxymethyl chitosan, dispersing it in the glucose oxidase solution, and stirring until dissolved to obtain carboxymethyl chitosan / glucose oxidase;

[0017] (4) The oxidized hyaluronic acid / anticancer drug-copper obtained in step (2) and the carboxymethyl chitosan / glucose oxidase obtained in step (3) are fully mixed and allowed to stand to obtain an oxidized hyaluronic acid / carboxymethyl chitosan / anticancer drug-metal / glucose oxidase drug controlled release system.

[0018] In the preparation method, the mass volume ratio of the anticancer drug, water and metal salt solution in step (1) is 1-2 mg: 1-2 mL: 1-2 mL;

[0019] The metal salt solution is 0.05 to 0.1 g / mL;

[0020] The metal salt solution is a cupric chloride solution.

[0021] The preparation method, the preparation method of the oxidized hyaluronic acid in step (2) is: weigh sodium hyaluronate, add it into water and stir to dissolve, add sodium periodate, stir to react in the dark, add ethylene glycol, stir to react, place it in a dialysis bag for dialysis, and then freeze-dry to obtain oxidized hyaluronic acid.

[0022] The preparation method, the stirring reaction time is 12 hours;

[0023] The dialysis time is 3 to 5 days;

[0024] The molecular cutoff of the dialysis bag is 3500 to 10000;

[0025] The mass volume ratio of the sodium hyaluronate, water, sodium periodate and ethylene glycol is 0.9-1.1 g:90-110 mL:1.0-1.2 g:3-5 mL.

[0026] In the preparation method, the mass volume ratio of oxidized hyaluronic acid to anticancer drug-metal in step (2) is 0.1-0.2 g:2-4 mL.

[0027] In the preparation method, the mass volume ratio of carboxymethyl chitosan to glucose oxidase solution in step (3) is 0.2-0.4 g:2-4 mL;

[0028] The concentration of the glucose oxidase solution is 40-60 μg / mL.

[0029] In the preparation method, in step (4), the volume ratio of oxidized hyaluronic acid / anticancer drug-metal to carboxymethyl chitosan / glucose oxidase is 2-4:2-4;

[0030] The standing time is 5 to 10 minutes.

[0031] The natural polysaccharide drug controlled release system or the natural polysaccharide drug controlled release system obtained by any one of the preparation methods described above is used in the preparation of anticancer drugs.

[0032] An anticancer drug comprises the natural polysaccharide drug controlled release system, or the natural polysaccharide drug controlled release system obtained by any one of the preparation methods.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The multi-therapy synergistic natural polysaccharide drug controlled release system of the present invention can slowly decompose in a weakly acidic environment and in the presence of glutathione, and the decomposition of the hydrogel realizes the slow release of anticancer drugs-metals (such as doxorubicin-copper) and glucose oxidase, thereby realizing long-term drug treatment. The present invention is based on the Fenton-like reaction of metal ions (such as copper ions) to produce hydroxyl radicals to realize chemodynamic therapy; the consumption of glucose can realize starvation therapy and chemodynamic therapy, thereby achieving a synergistic effect with higher efficiency and lower side effects. The controlled release system of the present invention is simple to prepare and has high biocompatibility, and can be widely used in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the visible spectrum of doxorubicin-copper in Example 1;

[0036] Figure 2 ] are infrared spectra of oxidized hyaluronic acid, hyaluronic acid, doxorubicin-copper, carboxymethyl chitosan, glucose oxidase, oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase in Example 1, wherein A is the infrared spectra of oxidized hyaluronic acid and hyaluronic acid prepared in Example 1, and B is the infrared spectra of doxorubicin-copper, carboxymethyl chitosan, glucose oxidase, and oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase;

[0037] Figure 3 is a scanning electron micrograph of oxidized hyaluronic acid / carboxymethyl chitosan / adriamycin-copper / glucose oxidase in Example 1;

[0038] Figure 4 It is a drug release curve diagram of the oxidized hyaluronic acid / carboxymethyl chitosan / adriamycin-copper / glucose oxidase drug controlled release system in different environments under different conditions in Example 1;

[0039] Figure 5 The UV-visible spectrum of 2-nitro-5-thiobenzoic acid for evaluating the glutathione consumption capacity of the oxidized hyaluronic acid / carboxymethyl chitosan / adriamycin-copper / glucose oxidase drug controlled release system in Example 1 and Comparative Example 1;

[0040] Figure 6The visible spectrum of methylene blue used to evaluate the ability of the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase controlled drug release system to generate hydroxyl radicals under different conditions in Example 1;

[0041] Figure 7 The in vitro cytotoxicity graphs are of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase in Example 1, oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper in Comparative Example 2, and oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin in Comparative Example 3. DETAILED DESCRIPTION

[0042] The present invention will now be further described in conjunction with specific embodiments. The following embodiments are intended to illustrate the present invention rather than to further limit the present invention.

[0043] Embodiment 1:

[0044] 1. A method for preparing a multi-therapy synergistic natural polysaccharide gel drug controlled release system, comprising the following steps:

[0045] (1) Weigh 1 mg of doxorubicin and disperse it in 2 mL of deionized water. Stir until dissolved, and add 2 mL of 0.1 g / mL copper chloride solution to obtain doxorubicin-copper.

[0046] The visible spectra of the prepared doxorubicin-copper and doxorubicin are shown in Figure 1 As shown in Figure 1, the characteristic peak of doxorubicin is at 480 nm when pH = 7.4. After the complexation of copper ions, it red-shifts to 512 nm. This is because the copper ions bind to the carbonyl and hydroxyl groups on the anthraquinone ring of doxorubicin through coordination bonds, resulting in the π-π * At pH = 5, in the presence of 10 mM glutathione, the characteristic peak of doxorubicin-copper in the visible spectrum returned to 480 nm, indicating that the destruction of the coordination bond by the acidic environment and the reduction of copper ions by glutathione converted doxorubicin-copper into free doxorubicin.

[0047] (2) Weigh 1.0 g of sodium hyaluronate and disperse it in 100 mL of deionized water, stir until dissolved, add 1 g of sodium periodate, stir for 12 hours in the dark, add 5 mL of ethylene glycol, stir for 3 hours in the dark, place the solution after the reaction in a dialysis bag with a molecular cutoff of 3500 and dialyze for 3 days. After dialysis, freeze-dry the solution for 3 days to obtain oxidized hyaluronic acid.

[0048] The infrared spectra of the prepared oxidized hyaluronic acid and hyaluronic acid are as follows Figure 2 As shown in Figure A, compared with the infrared spectrum of hyaluronic acid, the infrared spectrum of oxidized hyaluronic acid at 1735 cm -1 The extra peak at 878 cm-1 The extra peak at belongs to the hemiacetal structure formed by the aldehyde group and the adjacent hydroxyl group, proving the successful oxidation of hyaluronic acid.

[0049] (3) After vacuum drying, 0.2 g of oxidized hyaluronic acid was dissolved in 4 mL of doxorubicin-copper to obtain oxidized hyaluronic acid / doxorubicin-copper.

[0050] (4) Dissolve 0.4 g of carboxymethyl chitosan in 4 mL of a 50 μg / mL glucose oxidase solution to obtain carboxymethyl chitosan / glucose oxidase.

[0051] (5) 4 mL of the oxidized hyaluronic acid / doxorubicin-copper solution and 4 mL of the carboxymethyl chitosan / glucose oxidase solution were thoroughly mixed and allowed to stand for 5 minutes until gelation was completed to obtain the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system.

[0052] Infrared spectra of doxorubicin-copper, carboxymethyl chitosan, glucose oxidase and oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase Figure 2 As shown in B, doxorubicin-copper is at 2923 cm -1 The characteristic peak at 722 cm is the stretching vibration peak of -CH -1 The characteristic peak at 1649 cm is the out-of-plane bending vibration of -NH2. -1 The characteristic peak at 1408 cm-1 is the out-of-plane bending vibration of NH. -1 The characteristic peak at 1735cm is the stretching vibration of -COOH. In the infrared spectrum of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase, the above characteristic peaks of doxorubicin-copper, carboxymethyl chitosan and glucose oxidase can be observed. -1 878cm -1 The characteristic peaks of the aldehyde group and hemiacetal structure at 1620 cm -1 The characteristic peak of C=N bond was observed at , proving that the oxidized hyaluronic acid and carboxymethyl chitosan were cross-linked into a hydrogel through the C=N bond formed by the Schiff base reaction to successfully load doxorubicin-copper and glucose oxidase.

[0053] The field emission scanning electron microscopy image of the prepared oxidized hyaluronic acid / carboxymethyl chitosan / adriamycin-copper / glucose oxidase is shown in Figure 3 As shown, it can be clearly seen from the figure that oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase presents a three-dimensional network structure with a large cavity.

[0054] 2. The release effect of doxorubicin at different pH values ​​and at different times of the drug controlled release system of the present invention

[0055] 2 mL of the prepared oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system was taken and placed in a dialysis bag with a molecular cutoff of 3500, and the dialysis bag was placed in 50 mL of phosphate buffer solutions with a glutathione concentration of 10 mM and pH values ​​of 5.0, 6.5 and 7.4, respectively, and a phosphate buffer solution with no glutathione added and pH value of 5.0, respectively. The drug was released in vitro under constant temperature of 37° C. with magnetic stirring. During the drug release process, 4 mL of the solution was taken out at regular intervals to determine the amount of doxorubicin released, and 4 mL of fresh phosphate buffer solution was added at the same time. The absorption peak intensity of doxorubicin at 480 nm was determined using an ultraviolet-visible spectrophotometer, and its concentration was calculated, thereby calculating the cumulative percentage of doxorubicin released at different times at different pH values.

[0056] Drug release curves of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase controlled release system at different pH values Figure 4 As shown in the figure, the cumulative release of doxorubicin increases with the decrease of pH in the environment containing glutathione. This is because under acidic conditions, the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system will release doxorubicin-copper due to the breakage of the C=N bond between oxidized hyaluronic acid and carboxymethyl chitosan. At the same time, under the action of acidic conditions and glutathione, the coordination bond between doxorubicin-copper will be destroyed, releasing doxorubicin. In the environment with pH=5.0 but without glutathione, the cumulative release rate of doxorubicin is significantly reduced compared with that under the condition containing glutathione. This is because the lack of glutathione and copper ion redox reaction improves the stability of the coordination bond between copper and doxorubicin. The above results show that the release of doxorubicin has dual responsiveness to pH and glutathione. When the release reaches 60 hours in the presence of 10 mM glutathione, the cumulative release percentages at pH values ​​of 5.0, 6.5 and 7.4 are 85.2%, 71.0% and 60.3%, respectively. In the absence of glutathione, the cumulative release percentage at pH 5.0 is 45.8%.

[0057] 3. Test on the glutathione consumption capacity of the drug controlled release system of the present invention

[0058] (1) In order to evaluate the glutathione consumption capacity of the material, 0.5 mL of 50 μg / mL 5,5'-dithiobis(2-nitrobenzoic acid), 1.5 mL of phosphate buffer solution with a pH value of 5.0 and 10 mM glutathione were placed in a 5 mL centrifuge tube, and 2 mL of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system and 2 mL of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin / glucose oxidase prepared in Comparative Example 1 were added to two centrifuge tubes respectively. After reacting for 12 hours in a dark environment at 37°C, centrifugation was performed, and the supernatant was measured and compared with the absorption peak intensity of 2-nitro-5-thiobenzoic acid, the reaction product of glutathione and 5,5'-dithiobis(2-nitrobenzoic acid), at 412 nm for qualitative analysis.

[0059] The results are as follows Figure 5 As shown, it is shown that compared with the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin / glucose oxidase prepared in Comparative Example 1, the absorption intensity of the drug controlled release system of Example 1 at 412nm is significantly reduced due to the addition of doxorubicin-copper. This is because the reduction in glutathione content leads to a decrease in 2-nitro-5-thiobenzoic acid content, which proves that the presence of copper ions can effectively consume glutathione in the environment.

[0060] 4. Test on the ability of oxidized hyaluronic acid / carboxymethyl chitosan / adriamycin-copper / glucose oxidase drug controlled release system to produce hydroxyl radicals

[0061] Specifically, 0.5 mL of 100 μg / mL methylene blue solution, 0.5 mL of 1% hydrogen peroxide by volume, 2 mL of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system and 10 mM glutathione were placed in a 5 mL centrifuge tube, and 1.5 mL of phosphate buffer solution with a pH value of 5.0, 6.5 or 7.4 was added respectively. After reacting in a dark environment at 37°C for 12 hours, the mixture was centrifuged, and the supernatant was measured and compared with the absorption peak intensity of methylene blue at 664 nm using a UV-visible spectrophotometer.

[0062] Visible spectra of different systems are shown in Figure 2. Figure 6As shown. The results show that as the pH value continues to decrease, the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system prepared in Example 1 can produce more hydroxyl radicals, thereby continuously reducing the absorption peak intensity of methylene blue at 664nm. This is because a lower pH environment will promote the faster cleavage of the carbon-nitrogen double bond between oxidized hyaluronic acid / carboxymethyl chitosan, and will also make the coordination bond of doxorubicin-copper more easily destroyed, releasing more doxorubicin and copper, and then more copper ions will undergo Fenton-like reactions; at the same time, since Fenton-like reactions will also be promoted under acidic conditions, more hydroxyl radicals will be produced.

[0063] 5. Cytotoxicity of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system

[0064] Breast cancer 4T1 cells and mouse L929 cells were placed in a humidified incubator at 37°C and 5% CO2 in DMEM medium. The cells were then digested, counted, and prepared into 1×10 5 Cell suspensions of 100 μg / mL were inoculated in 96-well plates and cultured in a 37°C, 5% CO2 incubator for 24 h. 10 μL of materials of different concentrations were added to each well and cultured for 24 h. Finally, 10 μL of CCK-8 solution was added to each well. The plate was placed in a cell culture incubator and incubated for 3 h. The absorbance was measured at 450 nm using an ELISA reader and the cell survival rate was calculated.

[0065] The results are as follows Figure 7 As shown, when the concentrations of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase were 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL and 512 μg / mL, the cell viability rates were 70.9%, 64.0%, 35.7%, 29.5%, 24.9% and 15.4%, respectively, proving that the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system has significant cytotoxicity to 4T1 breast cancer cells.

[0066] Embodiment 2:

[0067] A method for preparing a multi-therapy synergistic natural polysaccharide gel drug controlled release system comprises the following steps:

[0068] (1) Weigh 2 mg of doxorubicin and disperse it in 1 mL of deionized water. Stir until dissolved, and add 1 mL of 0.1 g / mL copper chloride solution to obtain doxorubicin-copper.

[0069] (2) Weigh 1.0 g of sodium hyaluronate and disperse it in 100 mL of deionized water, stir until dissolved, add 1 g of sodium periodate, stir and react in the dark for 12 hours, add 5 mL of ethylene glycol, stir and react in the dark for 3 hours, place the reaction solution in a dialysis bag with a molecular cutoff of 3500 and dialyze for 3 days. After dialysis, freeze-dry the solution for 3 days to obtain oxidized hyaluronic acid.

[0070] (3) After vacuum drying, 0.1 g of oxidized hyaluronic acid was dissolved in 2 mL of doxorubicin-copper to obtain oxidized hyaluronic acid / doxorubicin-copper.

[0071] (4) Dissolve 0.2 g of carboxymethyl chitosan in 2 mL of 40 μg / mL glucose oxidase solution to obtain carboxymethyl chitosan / glucose oxidase.

[0072] (5) 2 mL of the oxidized hyaluronic acid / doxorubicin-copper solution and 2 mL of the carboxymethyl chitosan / glucose oxidase solution were fully mixed and allowed to stand for 5 minutes until gelation was completed to obtain an oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system, the structure of which was similar to that of the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system obtained in Example.

[0073] Embodiment 3:

[0074] A method for preparing a multi-therapy synergistic natural polysaccharide gel drug controlled release system comprises the following steps:

[0075] (1) Weigh 2 mg of doxorubicin and disperse it in 2 mL of deionized water. Stir until dissolved, and add 2 mL of 0.1 g / mL copper chloride solution to obtain doxorubicin-copper.

[0076] (2) Weigh 1.0 g of sodium hyaluronate and disperse it in 100 mL of deionized water, stir until dissolved, add 1 g of sodium periodate, stir and react in the dark for 12 hours, add 5 mL of ethylene glycol, stir and react in the dark for 3 hours, place the reaction solution in a dialysis bag with a molecular cutoff of 3500 and dialyze for 3 days. After dialysis, freeze-dry the solution for 3 days to obtain oxidized hyaluronic acid.

[0077] (3) After vacuum drying, 0.1 g of oxidized hyaluronic acid was dissolved in 4 mL of doxorubicin-copper to obtain oxidized hyaluronic acid / doxorubicin-copper.

[0078] (4) Dissolve 0.2 g of carboxymethyl chitosan in 4 mL of a 60 μg / mL glucose oxidase solution to obtain carboxymethyl chitosan / glucose oxidase.

[0079] (5) 4 mL of the oxidized hyaluronic acid / doxorubicin-copper solution and 4 mL of the carboxymethyl chitosan / glucose oxidase solution were fully mixed and allowed to stand for 5 minutes until gelation was completed to obtain an oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system, the structure of which was similar to that of the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase drug controlled release system obtained in Example.

[0080] Comparative Example 1:

[0081] (1) Disperse 1 mg of doxorubicin in 2 mL of deionized water and stir until dissolved.

[0082] (2) Dissolve 0.2 g of oxidized hyaluronic acid in 4 mL of doxorubicin solution to obtain oxidized hyaluronic acid / doxorubicin.

[0083] (3) Dissolve 0.4 g of carboxymethyl chitosan in 4 mL of a 50 μg / mL glucose oxidase solution to obtain carboxymethyl chitosan / glucose oxidase.

[0084] (4) 4 mL of the oxidized hyaluronic acid / doxorubicin-copper solution and 4 mL of the carboxymethyl chitosan / glucose oxidase solution were thoroughly mixed and allowed to stand for 5 minutes until gelation was completed to obtain the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin / glucose oxidase.

[0085] (5) The oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin / glucose oxidase prepared in Comparative Example 1 was tested using the same glutathione consumption test method as in Example 1. The results were compared with the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase prepared in Example 1 for their glutathione consumption capacity. Figure 5 As shown in the figure, the absorption intensity of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin / glucose oxidase at 412 nm is significantly higher than that of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase, which proves that the presence of copper ions can effectively consume glutathione in the environment.

[0086] Comparative Example 2:

[0087] (1) Weigh 1 mg of doxorubicin and disperse it in 2 mL of deionized water. Stir until dissolved, and add 2 mL of 0.1 g / mL copper chloride solution to obtain doxorubicin-copper.

[0088] (2) Dissolve 0.2 g of oxidized hyaluronic acid in 4 mL of doxorubicin-copper to obtain oxidized hyaluronic acid / doxorubicin-copper.

[0089] (3) Dissolve 0.4 g of carboxymethyl chitosan in 4 mL of deionized water to obtain a carboxymethyl chitosan solution.

[0090] (4) 4 mL of the oxidized hyaluronic acid / doxorubicin-copper solution and 4 mL of the carboxymethyl chitosan solution were fully mixed and allowed to stand for 5 minutes to complete gelation to prepare the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper.

[0091] (5) The cytotoxicity of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper to breast cancer 4T1 cells in Comparative Example 2 is shown in Table 2. Figure 7 As shown, when the concentrations of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper were 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL and 512 μg / mL, the cell survival rates were 92.4%, 85.7%, 71.8%, 57.0%, 48.6%, and 34.8%, respectively. The toxicity to breast cancer cells was significantly lower than that of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase in Example 1, proving that the starvation therapy of glucose oxidase has better synergistic effect.

[0092] Comparative Example 3:

[0093] (1) Weigh 1 mg of doxorubicin and disperse it in 2 mL of deionized water. Stir until dissolved.

[0094] (2) Dissolve 0.2 g of oxidized hyaluronic acid in 4 mL of doxorubicin solution to obtain oxidized hyaluronic acid / doxorubicin.

[0095] (3) Dissolve 0.4 g of carboxymethyl chitosan in 4 mL of deionized water to obtain a carboxymethyl chitosan solution.

[0096] (4) 4 mL of the oxidized hyaluronic acid / doxorubicin solution and 4 mL of the carboxymethyl chitosan solution were mixed thoroughly and allowed to stand for 5 minutes until gelation was complete to obtain the oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin.

[0097] (5) The cytotoxicity of oxidized hyaluronic acid / carboxymethyl chitosan / adriamycin to breast cancer 4T1 cells in Comparative Example 3 is as follows Figure 7 As shown in the figure, when the concentrations of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin were 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL and 512 μg / mL, the cell viability was 99.8%, 99.7%, 97.1%, 93.0%, 86.2% and 64.8%, respectively. The toxicity to breast cancer cells was significantly lower than that of oxidized hyaluronic acid / carboxymethyl chitosan / doxorubicin-copper / glucose oxidase in Example 1, proving the synergistic effect of chemotherapy, copper ion-guided chemodynamic therapy and glucose oxidase-guided starvation therapy.

Claims

1. A multi-therapy synergistic natural polysaccharide drug controlled release system, characterized in that: A hydrogel comprising carboxymethyl chitosan and oxidized hyaluronic acid subjected to a Schiff base reaction, and anticancer drugs-metal and glucose oxidase loaded in the hydrogel; Preferably, the anticancer drug is doxorubicin; Preferably, the metal is copper, iron or manganese.

2. The method for preparing a multi-therapy synergistic natural polysaccharide drug controlled release system according to claim 1, characterized in that: The following steps are involved: (1) Weighing an anticancer drug, dispersing it in water, stirring until dissolved, and adding a metal salt solution to obtain an anticancer drug-metal; (2) weighing oxidized hyaluronic acid and adding it to the anticancer drug-metal obtained in the above step (1) to obtain oxidized hyaluronic acid / anticancer drug-metal; (3) weighing carboxymethyl chitosan, dispersing it in the glucose oxidase solution, and stirring until dissolved to obtain carboxymethyl chitosan / glucose oxidase; (4) The oxidized hyaluronic acid / anticancer drug-copper obtained in step (2) and the carboxymethyl chitosan / glucose oxidase obtained in step (3) are fully mixed and allowed to stand to obtain an oxidized hyaluronic acid / carboxymethyl chitosan / anticancer drug-metal / glucose oxidase drug controlled release system.

3. The preparation method according to claim 2, characterized in that: The mass volume ratio of the anticancer drug, water and metal salt solution in step (1) is 1-2 mg: 1-2 mL: 1-2 mL; The metal salt solution is 0.05 to 0.1 g / mL; The metal salt solution is a cupric chloride solution.

4. The preparation method according to claim 2, characterized in that: The preparation method of the oxidized hyaluronic acid described in step (2) is: weigh sodium hyaluronate, add it into water and stir to dissolve, add sodium periodate, stir to react in the dark, add ethylene glycol, stir to react, place it in a dialysis bag for dialysis, and then freeze-dry to obtain oxidized hyaluronic acid.

5. The preparation method according to claim 4, characterized in that: The stirring reaction time is 12 hours; The dialysis time is 3 to 5 days; The molecular cutoff of the dialysis bag is 3500 to 10000; The mass volume ratio of the sodium hyaluronate, water, sodium periodate and ethylene glycol is 0.9-1.1 g:90-110 mL:1.0-1.2 g:3-5 mL.

6. The preparation method according to claim 2, characterized in that: In step (2), the mass volume ratio of oxidized hyaluronic acid to anticancer drug-metal is 0.1-0.2 g:2-4 mL.

7. The preparation method according to claim 2, characterized in that: In step (3), the mass volume ratio of carboxymethyl chitosan to glucose oxidase solution is 0.2-0.4 g: 2-4 mL; The concentration of the glucose oxidase solution is 40-60 μg / mL.

8. The preparation method according to claim 2, characterized in that: In step (4), the volume ratio of oxidized hyaluronic acid / anticancer drug-metal to carboxymethyl chitosan / glucose oxidase is 2-4:2-4; The standing time is 5 to 10 minutes.

9. Use of the natural polysaccharide drug controlled release system as claimed in claim 1, or the natural polysaccharide drug controlled release system obtained by the preparation method as claimed in any one of claims 2 to 8 in the preparation of anticancer drugs.

10. An anticancer drug, characterized in that: The invention comprises the natural polysaccharide drug controlled release system as claimed in claim 1, or the natural polysaccharide drug controlled release system obtained by the preparation method as claimed in any one of claims 2 to 8.

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