A chitosan-based Cu 2-x Se microneedle and preparation method thereof

Abstract

The present invention relates to the field of microneedle technology, and in particular to a chitosan-based Cu 2‑x Se microneedle and preparation method thereof, including synthesis of Cu 2‑x Se and Cu 2‑x Se-chitosan microneedles. Cu-chitosan disclosed in the present invention 2‑x Se microneedle is a patch loaded with biocompatible nanozymes. Nanozymes are composed of selenium and copper, which are trace elements required by the human body. They are prepared using PVP-assisted water precipitation to form Cu 2‑ x Se nanocrystals. Then, Cu 2‑x Se nanocrystals are dissolved in a chitosan matrix to prepare a nanozyme microneedle patch, which is then inserted into the residual tumor. The patch can retain its sustained effect in the surgical wound for more than 10 days, and the microneedle can greatly reduce the recurrence rate of the residual tumor model and inhibit the growth of the re-inoculated tumor. The microneedle also has excellent biocompatibility. The method of the present invention provides a simple and effective preparation for convenient and safe tumor vaccination.

Description

Technical Field

[0001] The present invention relates to the field of microneedle technology, and in particular to a chitosan-based Cu 2-x Se microneedles and preparation methods thereof. Background Art

[0002] The construction of in situ tumor vaccines requires the use of drugs that activate the immune system. However, existing drug molecules that regulate the immune system often cause serious side effects. Even in the case of local intratumoral administration, serious toxicity cannot be ignored, such as lymphopenia and anemia. The use of hydrogels in the resection cavity, whether sprayed or injectable, can only be applied to the surface of the residual tumor. If the tumor invades the surrounding normal tissue, it is difficult for the drug to penetrate effectively. Therefore, it is difficult for normal hydrogels to penetrate the inner layer of tumor tissue and achieve sustained release of drugs. Therefore, it is very attractive to develop a formulation with tumor-specific immune activation, deep and sustained action, and easy application for more effective in situ vaccination.

[0003] Microneedle patches are transdermal drug delivery systems composed of an array of 25-2000 μm needles that have been widely reported in the treatment of various skin diseases. Microneedle patches help enhance drug delivery in the dermis, maximizing the efficacy of drugs loaded a few centimeters beneath the superficial skin. In addition, microneedle patches are easy to design in morphology and matrix, making them compatible with adhesion to different surfaces. Therefore, microneedle patches are an ideal delivery system for targeting residual tumors during surgery to overcome the above limitations of traditional hydrogels. However, no studies have yet used microneedle patches for in situ vaccination of residual tumors during tumor resection.

[0004] In recent years, the ability of nanozymes to specifically modulate the tumor immune environment to activate antitumor immune responses has been recognized. For example, nanozymes with glutathione oxidase (GSHOx), peroxidase (POD) or oxidase (OXD) activities can generate reactive oxygen species (ROS) by utilizing abnormally high concentrations of glutathione (GSH) and H2O2 in tumor areas, promote immunogenic cell death (ICD) and activate immune cell functions. Therefore, nanozymes have the potential to modulate the tumor immune environment in response to tumor characteristics, thereby replacing traditional immune activators with high toxicity risks for specific in situ tumor vaccination. However, most nanozymes with dual activity for H2O2 and GSH are usually prepared in organic solvents at high temperatures or involve elements that are not needed by the human body. In addition, since nanozyme-induced ICD is limited by the short lifespan and easy inactivation of ROS, existing studies use multiple functional units to achieve tumor elimination and immune activation, such as phototherapy and chemotherapy drugs, but this complicates the formulation and is difficult to clinically translate. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a chitosan-based Cu 2-x Se microneedles and preparation methods thereof.

[0006] The purpose of the present invention is achieved through the following technical scheme: a chitosan-based Cu 2-x The preparation method of Se microneedle comprises the following steps:

[0007] S1. Synthesis of Cu 2-x Se: Selenium powder is added to hydrogen peroxide, nitrogen is introduced and stirred to expel the air in the system; sodium borohydride solution is then added to react for 1.5 to 2.5 hours; a mixed solution of anhydrous copper chloride and PVP is then added to the system, and the reaction is continued for 50 to 90 minutes. The solution after the reaction is dialyzed in a 100,000D dialysis bag;

[0008] S2. Preparation of Cu 2-x Se-chitosan microneedles: Take the Cu prepared in step S1 2-x Se, to a concentration of 500-1000 μg / ml, Cu 2-x The Se solution was slowly added to the stirring chitosan gel and continued to stir for 9-11 hours. After standing to expel the air in the gel, it was poured into the microneedle mold and centrifuged at 2-6°C. After drying, the Cu 2-x Se-chitosan microneedles.

[0009] Furthermore, the mass volume ratio of the selenium powder and the hydrogen peroxide is 1 mg:0.8-2 ml, and the concentration of the sodium borohydride solution is 25-30 mg / ml.

[0010] Furthermore, in the mixed solution of anhydrous copper chloride and PVP, the concentration of anhydrous copper chloride is 60-75 mg / ml, and the concentration of PVP is 15-25 mg / ml.

[0011] Furthermore, the volume ratio of the hydrogen peroxide solution of selenium powder, the sodium borohydride solution, the anhydrous cupric chloride and the PVP mixed solution is 20-30:1:1.

[0012] Furthermore, the dialysis method is to first boil the dialysis bag in boiling water for 10 to 20 minutes, then add the reacted solution into the dialysis bag and dialyze it in clean water for 45 to 52 hours, changing the water once in the middle.

[0013] Furthermore, the Cu 2-x The volume ratio of Se solution to chitosan gel is 1:5-10.

[0014] Furthermore, the centrifugal condition is 2300-2700 rpm for 12-18 min.

[0015] Furthermore, the preparation method of the chitosan gel is as follows: chitosan powder is dissolved in an acetic acid aqueous solution, stirred for 6 to 8 hours until the powder is completely dissolved, and then dialyzed using a 3500D dialysis bag for 46 to 52 hours, with the water replaced every 24 hours, and the dialysate after the dialysis is dried in an oven at 55 to 65°C to 23 to 28% of the original solution.

[0016] Furthermore, the volume percentage concentration of the acetic acid aqueous solution is 1%, and the mass volume ratio of the chitosan powder to the acetic acid aqueous solution is 1g:45-55ml.

[0017] The chitosan-based Cu prepared by the above method 2-x Se microneedle.

[0018] The present invention has the following advantages: The present invention discloses a chitosan-based Cu 2-x Se microneedle is a patch loaded with biocompatible nanozymes, which is applied to residual tumors to construct a whole tumor vaccine in situ. Through continuous and in-depth in situ catalytic therapy, nanozymes can more fully release their immune activation ability. Nanozymes are composed of selenium and copper, trace elements required by the human body, and are prepared using PVP-assisted water precipitation to form Cu 2-x Se nanocrystals. Then, Cu 2-x Se nanocrystals are dissolved in a chitosan matrix to prepare a nanozyme microneedle patch, which is then inserted into the residual tumor. The patch can retain its sustained effect in the surgical wound for more than 10 days, and the microneedle can greatly reduce the recurrence rate of the residual tumor model and inhibit the growth of the re-inoculated tumor. The microneedle also has excellent biocompatibility. The method of the present invention provides a simple and effective preparation for convenient and safe tumor vaccination. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Cu 2-x Characterization of Se nanoparticles and microneedle patches. In the figure, ab.Cu 2-x Transmission electron microscopy characterization of Se nanoparticles and photos of powder and solution; c. Selected area diffraction and element mapping data of nanoparticles; d. Powder diffraction data; eg. XPS energy spectrum data; h. Particle size distribution data; i. Photos and scanning electron microscopy data of microneedle patches.

[0020] Figure 2 The effect of the microneedle of the present invention on the mouse model, in the figure, (1) the drug administration and evaluation process of the mouse residual tumor model; (2)-(4) the tumor recurrence rate of mice in different groups; (5) the growth of the distal tumor inoculated again in normal mice; (6) Cu 2-x The growth of distal tumors after re-inoculation with Se microneedle patches; (7) The process from surgical tumor resection to monitoring recurrence. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The protection scope of the present invention is not limited to the following: Example 1: A Cu based on chitosan 2-x The preparation method of Se microneedle comprises the following steps:

[0022] S1. Synthesis of Cu 2-x Se: Selenium powder was added to hydrogen peroxide, the mass volume ratio of selenium powder to hydrogen peroxide was 1mg:0.8ml, nitrogen was introduced and stirred to expel the air in the system; then sodium borohydride solution with a concentration of 25mg / ml was added to react for 1.5h; then a mixed solution of anhydrous copper chloride and PVP was added to the system, and the reaction was continued for 50min, and the solution after the reaction was dialyzed in a 100000D dialysis bag, and the dialysis method was first to boil the dialysis bag in boiling water for 10min, and then the solution after the reaction was added to the dialysis bag and dialyzed in clean water for 45h, with water changed once in the middle;

[0023] The concentration of anhydrous copper chloride in the mixed solution of anhydrous copper chloride and PVP is 60 mg / ml, and the concentration of PVP is 15 mg / ml; the volume ratio of the hydrogen peroxide solution of selenium powder, the sodium borohydride solution, the anhydrous copper chloride and the PVP mixed solution is 20:1:1

[0024] S2. Preparation of Cu 2-x Se-chitosan microneedles: Take the Cu prepared in step S1 2-x Se, to a concentration of 500 μg / ml, Cu 2-x Se solution was slowly added to the stirring chitosan gel. 2-x The volume ratio of Se solution to chitosan gel was 1:5, and the mixture was stirred for 9 h. After the air in the gel was removed, the mixture was poured into the microneedle mold and centrifuged at 2300 rpm for 12 min at 2 °C. After drying, the Cu solution was demoulded to obtain Cu 2-x Se-chitosan microneedles.

[0025] Wherein, the preparation method of the chitosan gel is:

[0026] Chitosan powder was dissolved in 1% acetic acid aqueous solution by volume, the mass volume ratio of chitosan powder to acetic acid aqueous solution was 1g:45ml, stirred for 6h until the powder was completely dissolved, then dialyzed for 46h using a 3500D dialysis bag, with water replaced every 24h. After the dialysis was completed, the dialysate was dried in a 55°C oven to 23% of the original solution.

[0027] Example 2: A chitosan-based Cu 2-x The preparation method of Se microneedle comprises the following steps:

[0028] S1. Synthesis of Cu2-x Se: Selenium powder was added to hydrogen peroxide, the mass volume ratio of selenium powder to hydrogen peroxide was 1mg:2ml, nitrogen was introduced and stirred to expel air from the system; sodium borohydride solution with a concentration of 30mg / ml was then added to react for 2.5h; a mixed solution of anhydrous copper chloride and PVP was added to the system, and the reaction was continued for 90min. The solution after the reaction was dialyzed in a 100000D dialysis bag, and the dialysis method was first to boil the dialysis bag in boiling water for 20min, and then the solution after the reaction was added to the dialysis bag and dialyzed in clean water for 52h, with water changed once in the middle;

[0029] The concentration of anhydrous copper chloride in the mixed solution of anhydrous copper chloride and PVP is 75 mg / ml, and the concentration of PVP is 25 mg / ml; the volume ratio of hydrogen peroxide solution of selenium powder, sodium borohydride solution, anhydrous copper chloride and PVP mixed solution is 30:1:1

[0030] S2. Preparation of Cu 2-x Se-chitosan microneedles: Take the Cu prepared in step S1 2-x Se, to a concentration of 1000 μg / ml, Cu 2-x Se solution was slowly added to the stirring chitosan gel. 2-x The volume ratio of Se solution to chitosan gel was 1:10, and the mixture was stirred for 9-11 h. After the air in the gel was removed, the mixture was poured into the microneedle mold and centrifuged at 6 °C and 2700 rpm for 18 min. After drying, the Cu solution was demolded to obtain Cu 2-x Se-chitosan microneedles.

[0031] Wherein, the preparation method of the chitosan gel is:

[0032] Chitosan powder was dissolved in 1% acetic acid aqueous solution with a mass volume ratio of chitosan powder to acetic acid aqueous solution of 1g:55ml. The mixture was stirred for 8h until the powder was completely dissolved, and then dialyzed using a 3500D dialysis bag for 52h. The water was changed every 24h. After the dialysis was completed, the dialysate was dried in an oven at 65°C to 28% of the original solution.

[0033] Example 3: A chitosan-based Cu 2-x The preparation method of Se microneedle comprises the following steps:

[0034] S1. Synthesis of Cu 2-xSe: Selenium powder was added to hydrogen peroxide, the mass volume ratio of selenium powder to hydrogen peroxide was 1mg:1.2ml, nitrogen was introduced and stirred to expel air from the system; then sodium borohydride solution with a concentration of 27mg / ml was added to react for 2h; then a mixed solution of anhydrous copper chloride and PVP was added to the system, and the reaction was continued for 60min, and the solution after the reaction was dialyzed in a 100000D dialysis bag, and the dialysis method was first to boil the dialysis bag in boiling water for 13min, and then the solution after the reaction was added to the clean water in the dialysis bag and dialyzed for 48h, with water changed once in the middle;

[0035] The concentration of anhydrous copper chloride in the mixed solution of anhydrous copper chloride and PVP is 65 mg / ml, and the concentration of PVP is 18 mg / ml; the volume ratio of the hydrogen peroxide solution of selenium powder, the sodium borohydride solution, the anhydrous copper chloride and the PVP mixed solution is 24:1:1

[0036] S2. Preparation of Cu 2-x Se-chitosan microneedles: Take the Cu prepared in step S1 2-x Se, to a concentration of 600 μg / ml, Cu 2-x Se solution was slowly added to the stirring chitosan gel. 2-x The volume ratio of Se solution to chitosan gel was 1:6, and the mixture was stirred for 10 h. After the air in the gel was removed, the mixture was poured into the microneedle mold and centrifuged at 4 °C and 2500 rpm for 14 min. After drying, the Cu solution was demolded to obtain Cu 2-x Se-chitosan microneedles.

[0037] Wherein, the preparation method of the chitosan gel is:

[0038] Chitosan powder was dissolved in 1% acetic acid aqueous solution with a mass volume ratio of chitosan powder to acetic acid aqueous solution of 1g:48ml. The mixture was stirred for 7h until the powder was completely dissolved, and then dialyzed using a 3500D dialysis bag for 48h. The water was changed every 24h. After the dialysis was completed, the dialysate was dried in an oven at 58°C to 25% of the original solution.

[0039] Example 4: A chitosan-based Cu 2-x The preparation method of Se microneedle comprises the following steps:

[0040] S1. Synthesis of Cu 2-xSe: Selenium powder was added to hydrogen peroxide, the mass volume ratio of selenium powder to hydrogen peroxide was 1mg:1.8ml, nitrogen was introduced and stirred to expel the air in the system; then sodium borohydride solution with a concentration of 28mg / ml was added to react for 2.3h; then a mixed solution of anhydrous copper chloride and PVP was added to the system, and the reaction was continued for 80min, and the solution after the reaction was dialyzed in a 100000D dialysis bag, and the dialysis method was first to boil the dialysis bag in boiling water for 18min, and then the solution after the reaction was added to the clean water in the dialysis bag and dialyzed for 50h, with water changed once in the middle;

[0041] The concentration of anhydrous copper chloride in the mixed solution of anhydrous copper chloride and PVP is 72 mg / ml, and the concentration of PVP is 22 mg / ml; the volume ratio of the hydrogen peroxide solution of selenium powder, the sodium borohydride solution, the anhydrous copper chloride and the PVP mixed solution is 28:1:1

[0042] S2. Preparation of Cu 2-x Se-chitosan microneedles: Take the Cu prepared in step S1 2-x Se, the concentration is 800μg / ml, Cu 2-x Se solution was slowly added to the stirring chitosan gel. 2-x The volume ratio of Se solution to chitosan gel was 1:9, and the mixture was stirred for 10 h. After the air in the gel was removed, the mixture was poured into the microneedle mold and centrifuged at 5 °C and 2600 rpm for 16 min. After drying, the Cu solution was demolded to obtain Cu 2-x Se-chitosan microneedles.

[0043] Wherein, the preparation method of the chitosan gel is:

[0044] Chitosan powder was dissolved in 1% acetic acid aqueous solution with a mass volume ratio of chitosan powder to acetic acid aqueous solution of 1g:53ml. The mixture was stirred for 7.5h until the powder was completely dissolved. Then, it was dialyzed for 50h using a 3500D dialysis bag, with water replaced every 24h. After the dialysis, the dialysate was dried in an oven at 63°C to 26% of the original solution.

[0045] The beneficial effects of the present invention are described below by experiments:

[0046] 1.Cu 2-x Se synthesis steps, dialysis and storage

[0047] 1) Weigh 39.48 mg of selenium powder and pour it into a dry three-necked bottle, taking care to avoid the selenium powder sticking to the bottle wall, then add 48 ml of ddH2O, add a rotor, place it on a magnetic stirrer and pass nitrogen for 30 minutes to remove air from the system.

[0048] 2) Weigh 56.7 mg of sodium borohydride, dissolve it in 2 ml of ddH2O, add it along the wall using a 2.5 ml syringe, and react for 2 hours until the system becomes clear and colorless.

[0049] 3) Weigh 134 mg of anhydrous copper chloride and 40 mg of PVP (completely mixed), dissolve in 2 ml of ddH2O, and ultrasonicate for 3 min (until the mixed liquid is completely clear and transparent), then quickly inject into the system using a 2.5 ml syringe, and stop the reaction after 1 h.

[0050] 4) Take a small amount of liquid to dilute and see if it is clear. If it is clear, it is the synthesized nanoparticles

[0051] 5) Dialysis: Boil the 100000D dialysis bag for 15 minutes, then 2-x Se was added into the dialysis bag and dialyzed in clean water for 48 hours, with the water needing to be changed once in the middle.

[0052] 6) Freeze-dried storage: After dialysis is completed, place in a 50 ml centrifuge tube and freeze-dry in a freeze dryer. After freeze-drying is completed, place in a desiccator for storage.

[0053] 2 Preparation method of chitosan gel

[0054] Weigh 6g chitosan powder and dissolve it in 300ml 1% (v / v) acetic acid aqueous solution, stir until the powder is completely dissolved (about 6-8h), then dialyze it using a 3500D dialysis bag for 48h, changing the water every 24h. After dialysis, pour it into a beaker and dry it in a 60℃ oven to 25% of the original solution, and store it at 4℃.

[0055] 3.Cu 2-x Preparation method of Se-chitosan microneedles

[0056] 1) Pour 7 ml of the prepared chitosan gel into a beaker and add a rotor;

[0057] 2) Weigh Cu 2-x Se nanoparticles were added to 1 ml of ddH2O and sonicated to dissolve completely (the final Cu 2-x The concentrations of Se nanoparticles were 500 μg / ml, 800 μg / ml, and 1000 μg / ml, respectively. When preparing blank microneedles, 1 ml of ddH2O without nanoparticles was added to the chitosan gel to ensure the same gel viscosity);

[0058] 3) Use a 200 μl pipette tip to slowly add the chitosan gel to the stirring process several times. After stirring for 10 h, let it stand to expel the air in the gel.

[0059] 4) Pour into the mold and centrifuge in a refrigerated centrifuge at 4°C, 2500 rpm, for 15 min to ensure that each needle tip has Cu2-xSe-chitosan gel;

[0060] 5) Place at room temperature or in a 37°C oven until the microneedles are completely dry, then remove from the mold and store at 4°C.

[0061] 2. Product Characterization

[0062] Prepared by 1 Cu 2-x After freeze drying, the Se nanoparticles can be stored as dry powder for a long time. Figure 1 In Figure 1 a. Cu was observed by transmission electron microscopy (TEM) 2-x Se appears as dispersed ultra-small dots. High-resolution TEM (HRTEM) shows Cu 2-x The lattice distance of Se is 0.33 nm, corresponding to Cu 2-x Se (111) crystal plane, such as Figure 1 In Figure 1 b. Selected area electron diffraction (SAED) pattern ( Figure 1 c) also proved that the interplanar spacing matches the (111) plane of the cubic structure well. Powder X-ray diffraction (XRD) further confirmed that Cu 2-x The crystal structure of Se matches the characteristic peaks of JCPDS 06-0680 ( Figure 1 d). X-ray photoelectron spectroscopy (XPS) measurement spectrum ( Figure 1 e) shows four characteristic peaks of copper (Cu 2p), oxygen (O 1s), carbon (C 1s), and selenium (Se). Then, the valence states of Cu and Se were examined by high-resolution XPS, indicating the presence of Cu + , Cu 2+ and Se 2- ( Figure 1 f and g). Its hydrated particle size is 27.9±4.21nm. Then, we 2-x Se was loaded into a chitosan matrix and a therapeutic patch was fabricated in a micro-mold with a 12 × 12 needle array, a length of 2000 μm, and a needle tip spacing of 1500 μm. 2-x Se microneedle patch, see Figure 1 .Cu 2-x The detailed morphology of the Se patch is visualized by scanning microscopy with a conical structure and tip ( Figure 1 i, lower row). Unlike most reports on microneedle patches for transdermal drug delivery, which used needles less than 1000 μm in length, Cu 2-x The Se microneedle patch has longer needles. This design is to facilitate the Cu2-x Se can be transported across tumors and its retention time in tumors can be prolonged. 2-x The main task of the Se microneedle patch is to maximize the effect of Cu by enhancing the permeability and sustained action in the tumor environment. 2-x Catalytic activity of Se. Since the surgery was performed under anesthesia, the pain caused by microneedle insertion was acceptable.

[0063] 4.Cu 2-x Application and Effect of Se-chitosan Microneedles

[0064] In order to explore Cu 2-x Se microneedle patch inhibits residual tumors in breast cancer model mice, using Cu 2-x Se microneedle patch was implanted into the residual tumor site and the recurrence of tumor in mice was observed. For mice without recurrence, the other side of the tumor was inoculated to observe the immune activation effect in the mice.

[0065] Experimental groups: surgical resection only group, blank microneedle group, Cu 2-x Se microneedle patch set.

[0066] The experimental results are as follows Figure 2 As shown, the product's treatment process and experimental results are as follows Figure 2 (1) After the wound is sutured, observe whether the residual tumor recurs. Figure 2 The curve in (2) records the volume change of each residual tumor in 7 mice treated only by surgical resection. All mice in this group experienced postoperative tumor recurrence, with a recurrence rate of 7 / 7. Similarly, Figure 2 (3) For postoperative use, no Cu loading 2-x The relapse rate of 8 mice treated with blank microneedles containing Se nanoparticles was 8 / 8; Figure 2 (4) To accept Cu 2-x In mice treated with Se microneedle vaccine, tumor recurrence was effectively suppressed after surgery, with the recurrence rate being only 1 / 8. 2-x After Se microneedle patch treatment, mice developed a significant immune memory effect. Figure 2 (5) is a mouse that has not been immunotherapy treated. After inoculation of tumor cells, significant tumor growth was observed in all mice. After the second inoculation of tumor cells in mice that were cured by the above immunotherapy, no tumor growth was observed, as shown in 2(6). Figure 2 (7) The process from surgical tumor resection to monitoring for recurrence is intuitively demonstrated.

[0067] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A chitosan-based Cu 2-x The method for preparing Se microneedles is characterized in that: The Cu 2-x Se microneedle implantation at the residual tumor site involves the following steps: S1. Synthesis of Cu 2-x Se: Selenium powder is added to ddH2O, nitrogen is introduced and stirred to expel the air in the system; sodium borohydride solution is then added to react for 1.5-2.5 hours; a mixed solution of anhydrous copper chloride and PVP is then added to the system, and the reaction is continued for 50-90 minutes. The solution after the reaction is dialyzed in a 100000D dialysis bag; The mass volume ratio of the selenium powder and ddH2O is 1mg:0.8-2ml, and the concentration of the sodium borohydride solution is 25-30mg / ml; the concentration of the anhydrous copper chloride in the mixed solution of anhydrous copper chloride and PVP is 60-75mg / ml, and the concentration of PVP is 15-25mg / ml; the volume ratio of the ddH2O solution of selenium powder, the sodium borohydride solution, the anhydrous copper chloride and the PVP mixed solution is 20-30:1:1; S2. Preparation of Cu 2-x Se-chitosan microneedles: Take the Cu prepared in step S1 2-x Se, to a concentration of 500~1000μg / ml, Cu 2-x Se solution was slowly added into the stirring chitosan gel, Cu 2-x The volume ratio of Se solution to chitosan gel was 1:5-10, and the mixture was stirred for 9-11 hours. After the air in the gel was removed, the mixture was poured into the microneedle mold and centrifuged at 2-6°C. After drying, the Cu was removed from the mold. 2-x Se-chitosan microneedles.

2. A chitosan-based Cu according to claim 1 2-x The method for preparing Se microneedles is characterized in that: The dialysis method comprises first boiling the dialysis bag in boiling water for 10 to 20 minutes, then adding the reacted solution into the dialysis bag and dialysing it in clean water for 45 to 52 hours, changing the water once in the middle.

3. A chitosan-based Cu according to claim 1 2-x The method for preparing Se microneedles is characterized in that: The centrifugal condition is 2300-2700 rpm for 12-18 min.

4. The chitosan-based Cu prepared by any one of the methods described in claims 1 to 3 2-x Se microneedle.

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